DRAFT

Invertebrate Two

- Generation ISR May 28 , 2013 

### Validation of the Mysid Two

- generation Tox icity Test and Harpacticoid

Copepod De velopment 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 2013DRAFT

Invertebrate Two

- Generation ISR ii May 2 8 , 2013 

### Contents 1

Page 2

EXECUTIVE SUMMARY

................................ ................................ ................................ ........ VI 3

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

an aquatic arthropod test to evaluate chemical effects on invertebrate 5

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

Tier 2 test to be used in EPA’s EDSP.

................................ ................................ . viii 7

OBJECTIVE

................................ ................................ ................................ ................................ . 1 8

1 INTRODUCTION

................................ ................................ ................................ ............. 1 9

1.1 Endocrine Disruptor Screening Program

................................ ................................ 2 10

1.2 Validation

................................ ................................ ................................ ................ 3 11

2 HISTORICAL OVERVIEW OF THE INVERTEBRATE TESTS

............................. 6 12

2.1 Purpose of Invertebrate Testing in EDSP

................................ ............................... 6 13

2.2 Invertebrate Life

- cycle and Multi

- generational Toxicity Tests for the 14

Detection of Possible Endocrine Disruption

................................ ........................... 8 15

2. 2.1 Relevance of the Mysid Test Model

................................ ........................... 8 16

2.2.2 Relevance of the Copepod Test Model

................................ ..................... 10 17

3 MYSID AND COPEPOD TE ST PROTOCOL DEVELOPM ENT

............................ 12 18

3.1 Summary of the Mysid Two

- Generation Toxicity Test (MTTT) Protocol

.......... 12 19

3.1.1 Principle of the Test

................................ ................................ .................. 12 20

3.1. 2 Endpoints

................................ ................................ ................................ .. 14 21

3.1.3 Statistical Analyses

................................ ................................ ................... 17 22

3.2 Summary of the Harpacticoid Copepod Development and Reproduction 23

Test (HCDRT) Protocol

................................ ................................ ........................ 18 24

3.2.1 Principle of the Test

................................ ................................ .................. 18 25

3.2. 2 Endpoints

................................ ................................ ................................ .. 20 26

3.2.3 Statistical Analyses

................................ ................................ ................... 21 27

4 DEMONSTRATION AND OP TIMIZATION OF THE M YSID AND 28

COPEPOD TEST METHODS

................................ ................................ ...................... 23 29

4.1 Mysid Two

- Generation Toxicity Test (MTTT) Guideline Performance

.............. 23 30

4.1.1 Demonstration Studies Using the 2004 Draft MTTT Guideline

.............. 23 31

4.1.2 Optimization Studies Using the 2004 Draft Guideline

............................. 35 32

4.1.3 Lessons Learned from Demonstration and Optimization Studies 33

Using the 2004 Draft MTTT Guidel ine

................................ .................... 38 34

4.2 Proposed Harpacticoid Copepod Development and Reproduction Test 35

Protocol Performance

................................ ................................ ............................ 44 36

4.2.1 Demonstration and Optimization Studies Using Proposed HCDRT 37

Protocol

................................ ................................ ................................ ..... 44 38

4.2.2 Lessons Learned from Demonstration and Optimization Studies 39

Using Proposed HCDRT Protocol

................................ ............................ 47 40

5 INTERLABORATORY EVAL UATION OF MYSID AND COPEPOD 41

TEST METHODS

................................ ................................ ................................ ........... 48 42

5.1 Lindane

................................ ................................ ................................ ................. 49 43DRAFT

Invertebrate Two

- Generation ISR iii May 2 8 , 2013

5.1.1 Exposure Levels

................................ ................................ ........................ 49 1

5.1.2 Sens itive Endpoints

................................ ................................ ................... 53 2

5.1.3 Conclusions

................................ ................................ ............................... 55 3

5.2 Atrazine

................................ ................................ ................................ ................. 55 4

5.2.1 Sensitive Endpoints

................................ ................................ ................... 56 5

5.2.2 Conclusions

................................ ................................ ............................... 56 6

5.3 Vinclozolin

................................ ................................ ................................ ............ 57 7

5.3.1 Exposure Levels

................................ ................................ ........................ 57 8

5.3.2 Sensitive Endpoints

................................ ................................ ................... 58 9

5.3.3 Conclusions

................................ ................................ ............................... 60 10

5.4 4

- tert

- octylphenol

................................ ................................ ................................ .. 60 11

5.4.1 Exposure Levels

................................ ................................ ........................ 60 12

5.4.2 Sens itive Endpoints

................................ ................................ ................... 61 13

5.4.3 Conclusions

................................ ................................ ............................... 62 14

6 DISCUSSION AND SUMMA RY OF THE MTTT AND H CDRT METHODS

....... 64 15

6.1 Reproducibility of the Test Methods

................................ ................................ .... 64 16

6.2 Data Interpretation

................................ ................................ ................................ 66 17

6.3 Strengths and Limitations of Each Test

................................ ................................ 67 18

6.3.1 Strengths and Limitations of the MTTT

................................ ................... 67 19

6.3.2 Strengths and Limitations of HCDRT

................................ ...................... 69 20

6.4 Rationale for the Use of Mysid Versus Copepod Test

................................ ......... 70 21

7 REFERENCES

................................ ................................ ................................ ................ 72 22

8 LIST OF APPENDICES

................................ ................................ ................................ 88 23

APPENDIX A. VALIDATI ON OF SCREENING AND TESTING ASSAYS 24

PROPOSED FOR THE EDS P (DRAFT JULY 2007)

................................ ................. 88 25

APPENDIX B. DETAILED REVIEW PAPER ON AQUA TIC ARTHROPODS IN 26

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

.............................. 88 27

APPENDIX C. REPORTS ON THE INTER

- LABORATORY VALIDATIO N 28

TRIALS FOR THE MITT

................................ ................................ ............................. 88 29

APPENDIX D. REPORTS ON THE INTER

- LABORATORY VALIDATIO N 30

TRIALS FOR THE HCDRT

................................ ................................ ......................... 88 31

D.6 COPEPO D STUDY WITH LINDANE (C5069), CEFAS, 2011

................................ . 89 32

APPENDIX E. MYSID TW O

- GENERATION TEST GUID ELINE (DRAFT 33

PROPOSAL FOR A STAND ARDIZED PRO TOCOL AS AN IN VIVO 34

TIER 2 TEST), APRIL 2013

................................ ................................ .......................... 89 35

APPENDIX F. HARPACTI COID COPEPOD DEVELOP MENT AND 36

REPRODUCTION TEST WI TH AMPHIASCUS TENUIREMIS 37

(STANDARDIZED OECD D RAFT PROTOCOL FOR TH E HCDRT AS A 38

POTENTIAL ALTERNATIV E TO THE MTTT), MAY 2009

................................ .. 89 39

APPENDIX G. INTRA

- LA BORATORY STATISTICAL ANALYSIS REPORTS 40

FOR TWO

- GENERATION INVERTEBR ATE STUDIES

................................ ........ 89 41

42DRAFT

Invertebrate Two

- Generation ISR iv May 2 8 , 2013 

### Table s 1

2

Table 1

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

.............. 3 3

Table 3

- 1. Test Conditions for the Mysid Two

- Generation T est (MTTT) Guideline.

........... 14 4

Table 3

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

Reproduction Test (HCDRT) guideline.

................................ ............................... 19 6

Table 4

- 1. Results of the EPA demonstration study of the MTTT with fenoxycarb.

............ 25 7

Table 4

- 2. Results of the EPA demonstration study of the MTTT with 3,5

- DCP.

................ 27 8

Table 4

- 3. Results of the EPA demonstration study of the MTTT with fipronil.

.................. 28 9

Table 4

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

............. 30 10

Table 4

- 5. Results of the EPA demonstration study of the MT TT with flutamide.

............... 32 11

Table 4

- 6. Results of the EPA demonstration study of the MTTT with ketoconazole.

......... 33 12

Table 4

- 7. Results of the EPA demonstration study of the MTTT with 4

- tert

- Octylphenol. 34 13

Table 4

- 8. Results of the EPA optimization study of the MTTT with lindane.

..................... 36 14

Table 4

- 9. Results of the EPA optimization study of the MTTT with prochloraz.

................ 37 15

Table 4

- 10. Results of the EPA optimization study of the MTTT with atrazine.

.................... 38 16

Table 4

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

perfluorodecanoic acid.

................................ ................................ ......................... 39 18

Table 4

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

with fipronil.

................................ ................................ ................................ ......... 44 20

Table 4

- 13. Results of the Bejarano and Chandler demonstration study of the HCD RT 21

with atrazine.

................................ ................................ ................................ ......... 45 22

Table 4

- 14. Results of the OECD demonstration study of the HCDRT with 3,5

- DCP.

.......... 46 23

Table 5

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

of the MTTT with lindane.

................................ ................................ .................... 50 25

Table 5

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

MTTT with lindane.

................................ ................................ .............................. 51 27

Table 5

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

with lindane.

................................ ................................ ................................ .......... 52 29

Table 5

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

test with lindane.

................................ ................................ ................................ ... 53 31

Table 5

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

with atrazine.

................................ ................................ ................................ ......... 56 33

Table 5

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

with vinclozolin.

................................ ................................ ................................ ... 57 35

Table 5

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

vinclozolin.

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

Invertebrate Two

- Generation ISR v May 2 8 , 2013 Table 5

- 8. Results of the EPA val idation study of the HCDRT with vinclozin.

.................... 59 1

Table 5

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

using 4

- tert

- Octylphenol.

................................ ................................ ...................... 61 3

Table 5

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

using 4 tert

- Octylphenol.

................................ ................................ ...................... 62 5

Table 6

- 1. Ranges of coefficient of variation (CV) in the F 0 and F 1 generations 6

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

chemicals (see Section 5).

................................ ................................ ..................... 65 8

9

10Invertebrate Two

- Generation ISR vi May 2 8 , 2013



### EXECUTIVE SUMMARY 1

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

Disruptor Screening Program (EDSP) Tier 2 Mysid Two

- generation Toxicity Test (MTTT) and 3

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

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

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

- depth 6

review completed by the Organisation for Economic Co

- Operation and Development (OECD) in 7

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

MTTT. Results from an inter

- laboratory test provid e insights into sources of variability 9

likely to arise in the conduct of a multi

- generation test at different testing laboratories. The 10

MTTT was used in an inter

- laboratory validation study consisting of six studies carried out at 11

four laboratories with th ree representative endocrine

- active chemicals (i.e., lindane, vinclozolin, 12

and 4

- tert

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

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

laborato ries were able to successfully execute the proposed MTTT 15

 Laboratory

- to

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

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

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

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

participated in the inter

- laboratory validation study, indicating difficulties in 20

transferability of the MTTT between these labs 21

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

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

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

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

4

- tert

- octylphenol), dose

- response relationships were not generally consistent (based 26

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

affected endpoints amo ng the laboratories. 28

 Endpoint responses between the F 0 and F 1 generations were not consistent and were 29

significantly different in several instances. 30

Advantages of the MTTT 31

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

du e to its long

- standing use as a standard test model for regulatory toxicity testing 33

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

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

eva luating endocrine toxicity 36

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

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

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

adequate statistical power 40

 Common population modeling approaches can be employed

. 41Invertebrate Two

- Generation ISR vii May 2 8 , 2013

Limitations of the MTTT 1

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

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

other endpoints 4

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

pairing of mature males/females, etc. are time

- consuming and resource

- intensive 6

 Significant variability was observed in endpoint respon ses between different 7

laboratories, resulting in reduced power to detect significant differences 8

 Some of the endpoints were consistently non

- responsive 9

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

required to perform th e MTTT

. The value of the additional information obtained from 11

the second generation was not obvious based on the results of the inter

- laboratory 12

validation studies

. 13

HCDRT

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

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

. A 15

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

- led 16

inter

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

 Based o n the pre

- validation study (OECD 2007), in which two harpacticoid ( N. 18

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

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

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

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

an exi sting ASTM method 23

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

the EPA

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

OECD validation study

. Laboratories in the OECD validation study had difficulties 26

obtaining the desired chemical concentrations

. Lindane generally caused reduced 27

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

observed in the concentrations at which these effects were reported by differe nt labs

. 29

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

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

validation study

. One of the laboratories was not able to achieve the req uired mating 32

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

within acceptable guidelines and reported consistent biological responses 34

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

concentr ations with none of the other endpoints significantly affected). 36

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

. While the 37

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

concentrations were undetected

. 39

Advantages of HCDRT 40

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

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

comparative data 43Invertebrate Two

- Generation ISR viii May 2 8 , 2013

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

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

 Microplate test allows for easy (individual

- based) tracking of endpoints and high 3

replication resulting in strong statistical power 4

 Relatively consistent c oncentration

- dependent delays in development rates, increased 5

naupliar

- specific mortality, mating

- success depressions, and reduction of fecundity 6

were observed during the lindane interlaboratory validation study 7

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

- only and 8

sediment:water matrices 9

Limitations of the HCRDT 10

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

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

as an issue with the HCDRT 13

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

quality and difficulties producing consistently accurate and precise measured 15

concentrations 16

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

concentrations 18

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

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

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

 Manpower required in the study 22

 Limited knowledge of the copepod endocrine system

. 23

24

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

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

survival. Therefore, the MTTT is proposed as a Tier 2 test to be used in EPA’s EDS P

. 27Invertebrate Two

- Generation ISR 1 May 2 8 , 2013



### OBJECTIVE 1

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

validation process for the Mysid Two

- generation Toxicity Test (MTTT) and the Harpacticoid 3

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

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

accompanying charge questions

. 6



### 1 INTRODUCTION 7

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

US EPA to: 9

10

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

scientifically relevant information, to determine whether certain substances may 12

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

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

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

16

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

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

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

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

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

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

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

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

. 24

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

the Organisation for Economic Co

- operation and Development (OECD) established the 26

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

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

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

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

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

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

considered as a potential alternative or option

. These tests are the subject of this r eport

. This 33

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

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

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

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

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

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

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

reproducibility, reliability, and protocol transferability. 41Invertebrate Two

- Generation ISR 2 May 2 8 , 2013



### 1.1 Endocrine Disruptor Screening Program 1

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

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

it recommended to the EPA a conceptual two

- tiered approach that involved screening and t esting 4

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

. 5

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

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

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

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

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

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

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

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

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

consequences resulting from possible endocrine disruption and establishment of dose

- response 15

relationships for hazard identification using Tier 2 te sting. 16

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

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

 be relatively fast and efficient 19

 be standardized and validated 20

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

rate of false positives 22

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

possible 24

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

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

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

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

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

estrogenic response). This redundancy allows for a weight

- of

- evidence approach, as 30

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

definitive, test ing. 32

The assays listed in Table 1

- 1 , recommended by EDSTAC, have undergone validation 33

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

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

modes of action. 36

37Invertebrate Two

- Generation ISR 3 May 2 8 , 2013

Table 1

- 1

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

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 steroidogen esis 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 t he 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 scre ening assay An in vivo assay for detection chemicals that interfere with the

HPG axes.

2

A weight

- of

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

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

sufficiently endocrine active to prompt requirem ents for Tier 2 testing (US EPA 2011)

. The 5

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

EDSP Tier 2: 7

 Two

- generation Rat Reproduction Test 8

 Medaka Multi

- generation Test 9

 Japanese Quail Two

- generation Test 10

 Mysid Two

- generation Toxicity Test 11

 Larval Amphibian Growth and Development Test. 12

The Two

- generation Rat Reproduction Test (OCSPP 870.3800) has already been adopted

. 13

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

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



### 1.2 Validation 16

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

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

. 18

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

laboratories. 20Invertebrate Two

- Generation ISR 4 May 2 8 , 2013

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

(OECD 1996)

. For Tier 1 EDSP assays, relevance can be defined as t he ability of an assay to 2

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

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

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

acceptance of these methods (OECD 2005a)

. The criteria used to guide the validation process for 6

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

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

and the OECD (OECD 2005a)

. These criteria as stated by ICCVAM ( NIEHS 1997) are as 9

follows: 10

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

statement of its proposed use, should be available. 12

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

biologic effect and toxicity of interest must be addressed. 14

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

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

include data analysis and decision criteria. 17

4. Within

- test, intra

- laboratory and inter

- laboratory variability and how these 18

parameters vary with time should have been evaluated. 19

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

reference chemicals preferably coded to exclude bias. 21

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

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

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

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

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

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

preferably, published in an independent, peer

- reviewed publication. 28

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

App endix A (EDSP 2007 ) which were considered to have been met in the review of th e Tier 1 30

assays

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

not replacement assays (Validation Criterion No. 6)

. Many of them are novel assays; 32

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

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

In general, the EPA uses a five

- part or stage validation process outlined by ICCVAM 35

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

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

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

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

alternatives to other assays

. Nonetheless, the stages of the process outlined by the ICCVAM are 40

as follows: 41

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

culminated in an initial protocol

. As part of this phase, EPA drafted a Detailed Review Paper 43

(DRP) to explain the purpose of the test method, the context in which it will be used, and the 44Invertebrate Two

- Generation ISR 5 May 2 8 , 2013

scientific bas i s upon which the assay’s protocol, endpoints, and re levance rest ( Appendix B )

. The 1

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

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

purpose, costs, and other practical co nsiderations

. The DRP also identified the developmental 4

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

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

validation, standardization and optimization

. During standardization and optimization, studies 7

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

assessing protocol transferability and performance

. In inter

- laboratory vali dation, studies were 9

conducted in several independent laboratories with the refined protocol

. The results of these 10

studies were used to determine inter

- laboratory variability and to set or cross

- check performance 11

criteria

. The report s on the inter

- laborato ry trials for the MTTT are provided in Appendix C

. The 12

report s on the inter

- laboratory trials for the HCDRT are provided in Appendix D

. Inter

13

laboratory validation is followed by peer review, an independent scientific review by qualified 14

experts, and by regulatory acceptance, adoption for regulatory use by an agency

. EPA has 15

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

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

decisions (US EPA 2006)

. 18

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

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

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

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

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

24Invertebrate Two

- Generation ISR 6 May 2 8 , 2013



### 2 HI STORICAL OVERVIEW OF THE INVERTEBRATE TESTS 1

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

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

in regulatory testing ; h owever

, few have been designed with endocrine

- specific endpoints in 4

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

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

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

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

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

deFur et al. 1999; Subramoniam 2000)

. E cdys teroids a re steroid hormone s that regulate growth 10

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

estrogen

. 12

It has been reported that vertebrate steroids

, such as the androgen testosterone , can 13

interfere with ecdysteroid regulation in a crustacean (Mu and LeBlanc 2002a)

. Also, the 14

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

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

in a crustacean (Mu and LeBlanc 2002b)

. Still, reported effects of vertebrate

- active hormones on 17

crustaceans are limited

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

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

1999a, 1999b; Breitholtz and Bengtsson 2001 in OECD 2006)

. Many crustacean species hold 20

key ecological functions and several crustacean species are of economic importance

. Further, 21

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

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

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

( Oehlmann and Schulte

- Oehlmann 2003 ; LeBlanc 200 7 )

. G iven that endocrine disruption has 25

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

evaluating potential effects of EDCs is relevant. 27



### 2.1 Purpose of Invertebrate Testing in EDSP 28

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

larger percentage of the Earth’s total animal abundance

. H ormones are important in controlling 30

phys iological processes in invertebrates (Lafont 2000 ; Oehlmann & Schulte

- Oehlmann 2003)

. 31

However, until recently

, invertebrates received little attention regarding potential endocrine 32

disruption, the main exception being studies of imposex in gastropods

. Inve rtebrate endocrine 33

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

vertebrates

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

Schulte

- Oehlmann 2003)

. Because many invertebrate e ndocrine systems are not very well 36

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

or may not be measurable (Oehlmann and Schulte

- Oehlmann 2003)

. However, the situation is 38

improving

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

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

endocrinology (Environment Agency 2007)

. Nonetheless, to gain a full understanding of the 41

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

(Vandenbergh et al. 2003). 43Invertebrate Two

- Generation ISR 7 May 2 8 , 2013

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

through riverine and estuarine pipelines (Nimmo and Hamaker 1982)

. Since the mid

- 1990s, there 2

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

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

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

and Cheek 2001)

. Although early concern over EDCs focused on vertebrates, attention has 6

broadened to include invertebrates

, because they are ecologically important and have distinctive 7

endocrine systems that diffe r from those of vertebrates

. Estuaries, which are intrinsically and 8

commercially important ecosystems, are among the earliest recipients of EDCs

. Among the 9

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

good c andidates for study of potential impacts

. Attention here is focused on aquatic arthropods 11

(insects and crustaceans)

, because they are often among the most abundant organisms in 12

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

known to be susceptible to the effects of EDCs. 14

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

specifically a daphnid or mysid, in the endocrine screening program

. The principal objection 16

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

daphnids

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

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

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

ecdysteroid o r juvenile hormone activity

. Hutchinson et al. (2000) also expressed concern that 21

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

results might be confounded by cannibalism of offspring by parents

. However, t hese three 23

concerns can be addressed

. 24

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

links between hor mones and physiological processes in crustaceans are increasingly established 26

(Verslycke et al. 2007)

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

that ecdysteroids regulate the embryonic development of D aphni a and that this developm ent 28

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

. Testosterone 29

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

occupy the ecdysteroid receptor (Mu and LeBlanc 2002a)

. F enarimol acted by reducing body 31

levels of ecdysone in individual daphnids (Mu and LeBlanc 2002b)

. Vandenbergh et al. (2003) 32

found that the synthetic estrogen 17α

- ethinylestradiol altered sexual development in individuals 33

of the amphipod Hyalella azteca

. Ma les did not develop secondary sex features, reproductive 34

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

. Further

, 35

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

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

in a freshwater prawn (Nagaraju et al. 2003)

. Also , driven by increased availability of crustacean 38

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

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

increasing (Dhadialla et al. 1998; Tarrant et al

. 2011)

. 41

Second, f

emale and male mysids have anatomical differences that can be used to indicate 42

maturity a nd gender

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

one indication of gender and maturity

. While m aturity is more difficult to recognize in males, 44

mature males have one pair of elongated abdominal pleopods that is modified t o assist in 45Invertebrate Two

- Generation ISR 8 May 2 8 , 2013

copulation and that can be used to identify gender

. Males of the most commonly used mysids test 1

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

. Molt 2

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

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

. Ultimately, 4

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

Third, t he susceptibility of young to being eaten by adult mysids is also well

- known 6

(e.g., Johnston and Ritz 2001 ; Quirt and Lasenby 2002)

. Premolt newborns are particularly 7

susceptible

. Testing protocols call for newly released young to be remove d from the tanks as 8

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

be exposed to a potential toxicant

. Mysids are also typically fed liberal quantities of food 10

( Artemia nauplii) to lessen the likelihood that adults will eat the young

. Taken together

, 11

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

regarding the use of mysids for testing of potential EDCs

. 13

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

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

those eliciting effects during acute exposures

. This emphasizes the importance of 16

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

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

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

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

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

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

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

gender

- specific responses (e.g., Hahn and Schulz 200 2 ) 24

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

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

- controlled 26

processes (e.g., Anders e n et al. 2001 ) 27

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

caused by non

- EDC stressors 29

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

actions of the individual components (e.g., Mu and LeBlanc 2004)

. In nature, most 31

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

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

evaluation program

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

at a minimum the indiv idual impacts and mechanisms of action of the component 35

chemicals need to be understood. 36



### 2.2 Invertebrate L ife

- cycle and M ulti

- generational T oxicity T ests for the 37



### D etection of P ossible E ndocrine D isruption 38

2.2.1 Relevance of the Mysid T est M odel 39

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

30 years

. Standard testing protocols have been developed for some species

. Despite that little is 41

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

few direct links between potential EDCs, beyond certain insect growth regulators ( IGRs ) , and 43Invertebrate Two

- Generation ISR 9 May 2 8 , 2013

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

hormonal control of crustacean molting (Cuzin

- Roudy and Saleuddin 1989)

. 2

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

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

application

. Crustaceans, which along with inse cts are in the phylum Arthropoda, could also be 5

sensitive to these compounds

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

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

1982; Nimmo et al. 1981)

. One study focused on exposure of Americamysis bahia to methoprene, 8

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

. The 9

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

cause problems for nontarget organisms

. The results showed a significant effect during the mysid 11

life cycle test

. Total lethality occurred at 125 μg/L in a 4

- day test

. Similar concentrations caused 12

significant mortality in the larvae of an estuarine crab and shrimp

. Mysids grown at sublethal 13

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

and produced fewer young per female than control animals

. McKenney and Celestial (1996) 15

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

methoprene. 17

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

which uses hormones that act like JH

. Retarded growth rates were also accompanied by 19

bioenergetic d isruption, resulting in lower net growth efficiency values

. This suggested that 20

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

production (McKenney 1982, 1985)

. The delays in mysid first brood production could be the 22

result of slowing sexual maturity and/or embryogenesis

. Diminished reproductive success could 23

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

successful embryogenesis

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

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

2006a, b ; Ghekiere et al. 2007 )

. Verslycke et al. (2002, 2003a, 2003b, 2004b) studied 27

testosterone metabolism in Neomysis integer and ex amined the changes in energy and 28

testosterone metabolism after exposure to potential EDCs

. F urther work is required with the 29

mysid to determine a more conclusive cause

- and

- effect relationship between potential EDCs and 30

their effects, as observed by test me asurement endpoints. 31

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

acute and chronic life

- cycle testing

. Nimmo and Hamaker compared acute and chronic toxicity 33

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

including grass shrimp, an estuarine fish, and 17 freshwater species

. Nimmo and Hamaker 35

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

generations, in part because the life cycle of mysids is short

. This role is being fulfilled by the 37

inclusion of mysids in an EDC evaluation program

. Verslycke et al. (2004a , 2007 ) evaluated the 38

potential for using mysids in an endocrine disruption evaluation program

. They reviewed aspect s 39

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

literature on toxicity studies done using mysids

. Their review also included a discussion of many 41

endpoints that may be useful when using mysids to test for endo crine disruption

. Verslycke et al. 42

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

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

estuarine invertebrates and co uld serve as a surrogate for other crustacean species

. 45Invertebrate Two

- Generation ISR 10 May 2 8 , 2013

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

. Its primary 1

advantages include its widespread availability and ease of culture

. Because animals can be 2

obtained from commer cial cultures, the likelihood of misidentifying the species is very low

. Its 3

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

17 to 20 days)

. The species is widely used in toxicological testing, and ap propriate test 5

conditions are well known

. Standardized life cycle test protocols have been developed ( OCSPP 6

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

1991; McKenney and Celestial 1996) or evaluated (Lussier et al. 1999)

. These standardized test 8

protocols contain several EDC

- related endpoints; others appear in peer

- reviewed publications 9

(Ingersoll et al. 1999)

. McKenney (1998) synthesized the results of several of his earlier studies 10

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

life

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

sensitive than survival

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

included standard and multi

- generational tests using A. bahia , could be used to extrapolate from 14

laboratory to population effects (Kuhn et al. 2000; 2001)

. McKenney (2005) also demonstrated 15

that A. bahia can be used to evaluate multi

- generational toxicity in a two

- generational exposure 16

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

disruptor evaluations, favored A. bahia as a reasonable test species

. One criticism of the 18

widespread use of A. bahia in toxicological test ing is that because it is a warm

- temperate or 19

subtropical species, it may not be ecologically relevant to colder

- water materials testing

. A 20

proposed Mysid Two

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

Section 3.1

. 22

2.2.2 Relevance of the C opepod T

est M odel 23

Copepods occur widely in almost every marine, brackish

, and freshwater ecosystem. 24

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

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

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

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

water and sediments (Chandler and Green 2001)

. Copepod development is characterized by the 29

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

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

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

and Coull 198 3 ) . The time to develop for copepods from hatch to adult as well as t he generation 33

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

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

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

and Green 1996)

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

each egg sac

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

species (Hicks and Coull 1983). 39

Harpacticoid copepods comprise more than 3

, 000 species, are mostly free

- living benthic 40

organisms

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

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

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

described toxic effects of single substances, oils, complex effluents

, etc. on harpacticoid 44

copepods, such as A. tenuiremis (Chand ler et al. 2004a, b; Bejarano and Chandler 2003), Tisbe 45Invertebrate Two

- Generation ISR 11 May 2 8 , 2013

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

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

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

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

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

important link between phytoplankton and higher communities (e.g.

, fish) in the pelagic food 6

chain (e.g.

, Størrup et al. 1986)

. For instance, Acartia tonsa has been widely used for 7

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

laboratory (Bushong et al. 1990; Kusk and Petersen 1997)

. A sensitive larval inhibition test has 9

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

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

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

copepodite stage (after 5 to 8 days depending on temper ature)

. 13

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

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

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

copepod Acartia tonsa were included

. The proposed guidelines were for evaluati ng long

- term 17

effects (incl uding endocrine

- disruptive effects) of various types of chemicals to aquatic 18

invertebrates (OECD 2007)

. In 2007, based on resul ts of a first intercalibration study

, it was 19

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

(OECD 2011)

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

is discussed further in Section 3.2

. 22

23Invertebrate Two

- Generation ISR 12 May 2 8 , 2013



### 3 MYSID AND COPEPOD TE ST PROTOCOL DEVELOPM ENT 1

As discussed in Section 2

, the focus of the ISR is on life

- cycle and multi

- generational 2

studies conducted using the mysid and copepod test model

. This section describes the 3

recommended Mysid Two

- Generation Toxicity Te st (MTTT) guideline and the recommended 4

Harpacticoid Copepod Development and Reproduction Test (HCDRT)

. Full versions of the 5

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

below. 7



### 3.1 Summary of the Mysid Two

- Generation To xicity Test (MTTT) P rotocol 8

3.1.1 Principle of the T est 9

The MTTT protocol describes a two

- generation toxicity test with an estuarine mysid that 10

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

exposure

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

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

toxicants (Verslycke et al

. 2004 a )

. This guideline is intended to be appli cable to the mysid, A. 14

bahia, but could be adapted to other mysid species

. The MTTT is a relatively long

- term assay 15

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

generations ( F 0 and F 1 )

. The MTTT is an extens ion of existing standard practice for conducting a 17

mysid life

- cycle test (ASTM 200 4 ; McKenney 1986, 1998; and Nimmo et al. 1977, 1978). 18

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

collecting definitive concentration

- r esponse information on adverse effects suitable for use in 20

ecological risk assessment

. Specifically, the design enables the collection of invertebrate 21

hormone

- regulated endpoint data (e.g., molt

- related endpoints) and information concerning 22

various aspects of the reproductive biology and life

- stage viability

. The former is useful as a 23

basis for better understanding comparative toxicological relationships across biological groups 24

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

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

McKenney 2005a,b). 27

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

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

. 29

The MTTT is initiated with newly released <24 hours old mysids ( F 0 generation) that are grown 30

to maturity (~13 to 16 days) and then their offspring are collected ( F 1 generation)

. The F 1 31

generation is also grown to maturity and their offspring enumerated (F 2 generation)

. In general

, 32

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

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

and opt ional biochemical measurements)

. W ith the appearance of a marsupial pouch on F 0 35

females (sexual maturity) , mature females are isolated as pairs with F 0 males (smaller individuals 36

without a visual marsupial pouch) in separate brood cups

. Optimally, s even breeding pairs are 37

selected in each replicate reproduction basket

. The exposure of these F 0 mysids is terminated 38

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

controls

. Toxicant and control treatments are deliv ered to the mysids in water via a continuous 40

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

intermittent flow (e.g., proportional diluter) (McKenney 1998)

. O ffspring ( F 1 ) from the parental 42Invertebrate Two

- Generation ISR 13 May 2 8 , 2013

generation ( F 0 ) are collected each morning from the brood cups and transferred immediately to 1

baskets, which are housed within separate tanks for each of the F 0 exposure conditions

. 2

The F 1 generation is begun with newly hatched offspring

. Offspring are collected each 3

mo r ning from the brood cups and tr ans ferred immediately to baskets (labeled with the date), 4

which are housed within separate tanks for each of the F 0 exposure conditions

. Optimally, two 5

groups of 15 F 1 juvenile mysids are selected and maintained within two separate baskets for each 6

of the F 0 exposure conditions (one basket housing animals for reproductive observations and a 7

second basket for growth or optional biochemical measurements)

. With the appeara nce of a 8

marsupial pouch on F 1 females (sexual maturity), mature females are isolated as pairs with F 1 9

males in separate brood cups

. Optimally, s even breeding pairs are selected in each replicate 10

reproduction basket

. The exposure of these F 1 mysids is term inated after the second br oo d release 11

is complete (or 7 days past the median second brood release in the control group)

. F 1 adults may 12

be discarded or save d for subsequent measurements (e.g., if stage

- specif ic survival is measured 13

for population modeling) after release of their second brood

. Offspring (F2) from the parental 14

generation ( F 1 ) are collected each morning from the brood cups and enumerated

. 15

Measurements are made of a number of endpoints in both F 0 and F 1 generations reflective 16

of functional endo crine regulation of development, growth, reproduction, and survival

. They 17

include sex ratio

, time to matur i ty , time to brood release for the first two broods , and interbrood 18

duration between the first two broods

. Observations are recorded daily for each individual on 19

mortality, sex determination, reproductive condition of female

, (marsupial with or without 20

developing embryos) , and number of young released in each of the first two broods

. A total of 45 21

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

. These mysids are 22

separated into nine groups of five mysids for weight determination

. At two time intervals, Day 7 23

and Day 14, animals from the growth baskets are sub sampled to determine length and weight

. 24

An overview of the recomm ended MTTT protocol is provided in Table 3

- 1

. The full 25

recommended MTTT protocol ( Appendix E )

, includes d etails regarding test animals ( i.e., care, 26

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

selection (i.e., range

- finding and definitive testing )

, test substance administration and analytical 28

determination, performance of the test and performance criteria, data reporting

, and exposure

. 29

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

three categories of b iological endpoints ( i.e., reproduction, survival, growth)

, which are 31

described further below

. Changes incorporated into the recommended MTTT protocol since the 32

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

section (Section 4 )

. 34

35Invertebrate Two

- Generation ISR 14 May 2 8 , 2013

Table 3

- 1

. Test Conditions for the Mysid Two

- Generation Test (MTTT) Guideline

. 1

Recommended species Mysid (Americamysis bahi a)

Test type Continuous or intermittent flow

- through

Water temperature 26 ± 1 o C

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 lit ers (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

- flo w system with self

- starting excurrent siphons (McKenney

1986)

Age of test organisms at

initiation < 24 hours

No. of mysids per test vessel F 0 : 40 ( min. 30 )/replicate; 20 ( min. 15 )/retention chamber; 2 (1 male & 1 female)/

brood chamber

F 1 : 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 conce ntration; 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 (F 0 only)

Two

- generation test: 7 days after median day of release of second brood in F 1

controls

Primary endpoints (Annex 4 of

MTTT) Survival (F 0 , F 1 );

Growth

- length & dry weight, (Days 0, 7 and 14 for F 0 and F 1 );

Time to maturation (F 0 , F 1 )

Time to first brood release (F 0 , F 1 )

Time to second brood release (F 0 , F 1 )

Interbrood duration (F 0 , F 1 )

Number of young per female in each of the first two broods (F 0 , F 1 )

Number of total young in the first two broods in each replicate population (F 0 , F 1 )

Sex ratio (F 0 , F 1 )

Percentage of females that are reproductively active (F 0 , F 1 )

Optional endp oints 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

3.1.2 Endpoints 2

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

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

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

Disruptive Effects (OECD 2006)

. The MTTT has more than 20 endpoints

, as shown in Annex 4 6Invertebrate Two

- Generation ISR 15 May 2 8 , 2013

of the MTTT protocol

– Appendix E )

. Some of the endpoints are recorded per mysid or 1

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

tank

. Endpoints measured in the MTTT are pres ented in more detail in the following subsection

. 3

Survival ( F

0 , F 1 ) 4

S urvival is assessed daily for F 0 and F 1 individuals throughout the MTTT to provide a 5

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

mortality

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

, and termination 7

(reproduction survival)

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

animals is measured at days 7, 14

, and termination (total survival)

. Survival rates should be 9

adjusted for non

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

removed from growth baskets for growth measurements

. Unless acceptable water quality 11

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

chemical exposure phase of the MT TT is generally <25%

. There should be at least 70% survival 13

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

. 14

Growth ( F

0 , F 1 ) 15

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

of the exoskeleton

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

other endpoints that are examined in the MTTT

. Because growth can only occur as a result of 18

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

. 19

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

signaling molecules to regulate molting, reproduction

, and development (Huberman 2000)

. The 21

actions of ecdysteroids are mediated by a ligand

- depende nt transcription factor, the ecdysteroid 22

receptor, which is present only in invertebrates

. The insect ecdysteroid receptor is known to play 23

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

, and 24

this regulatory func tion is intentionally targeted by many pesticides

. A similar central role for the 25

ecdysteroid receptor is expected, but presently unknown, in crustacean s

, making ecdysteroid 26

signaling a potential effective and unintended target of environmental chemicals

. While the 27

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

Hirano 2010 ), its functional role needs to be further elu c i dated (Verslycke et al. 2007)

. 29

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

crustaceans, dry weight and body length

. The most common response to exposure to toxicants is 31

reduced growth

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

. 32

Reduced growth has strong implications for r eproductive success in mysids

, as several studies 33

(e.g., Mees et al. 1994; Turpen et al

. 1994 ; Winkler and Greve, 2002 ) have shown that fecundity 34

is directly related to female body size

. 35

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

(to the nearest 0.1 mm) in the F 0 and F 1 generation

. Growth observations should be recorded 37

separately for males, females, and immature individuals

. Data analyses should also be conducted 38

relative to each sex

. 39

Reproducti on ( F 0 , F 1 ) 40

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

sublethal responses in mysids

. Despite the well

- established use of mysid reproductive endpoints 42

such as fecundity, egg development time, and time to first brood release in st andard toxicity 43Invertebrate Two

- Generation ISR 16 May 2 8 , 2013

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

limited (Verslycke et al. 2004 a )

. For example

, w hile m uch attention has been given to 2

vitellogenin ( the precursor to the yolk protein vitellin in egg

- laying vertebrates and invertebrates ) 3

as an indicator of exposure to xeno

- estrogens in egg

- laying vertebrates, such studies were 4

lacking in mysids until recently (Ghekiere et al. 200 5 )

. In general, studies on the effects of 5

vertebrate

- type endocrine di sruptors on crustacean vitellogenesis and reproduction remain 6

fragmented and are often contradictory (Ghekiere et al. 2005)

. There is, however, a growing 7

weight

- of

- evidence that (xeno)estrogens (and potentially other vertebrate

- type endocrine 8

disruptors) a

re less effective in causing disruption of normal vitellogenesis in crustaceans than 9

they are in oviparous vertebrates

. Most likely, this is a result of the different hormonal control 10

strategies for vitellogenesis and reproduction in crustaceans compared w ith oviparous vertebrates 11

(Ghekiere et al

. 2005). 12

Time to maturation ( F 0 , F 1 ) 13

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

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

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

chemicals remains poorly understood

. Khan et al. (1992) asserted that sexual maturity, which 17

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

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

output

. 20

In the MTTT, time to maturation is determined for the F 0 and F 1 generation and typically 21

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

(McKenney 2005)

. 23

Time to first brood release

, Time to second brood release , and Interbrood d uration ( F 0 , F 1 ) 24

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

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

factors (McKenney 1996)

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

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

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

Appendix G). 30

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

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

(i.e., time between release of first and second brood) is determined for the F 0 and F 1 generation

. 33

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

surviv ed to produce a brood and produced some offspring. 35

Sex ratio in broods 1 and 2 ( F 0 , F 1 ) 36

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

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

. Intersexuality has also 38

been observed in natural populations of mysids (e.g., Mees et al

. 1 995) and other aquatic 39

arthropo ds (e.g., Moore and Stevenson 1991; 1994; Ford et al. 2004a

, b)

. T he origin of 40

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

attributable to EDCs 42

In the MTTT, sex ratio is determined for the F 0 and F 1 generation

. 43Invertebrate Two

- Generation ISR 17 May 2 8 , 2013

Fecundity (brood size ; F 0 , F 1 ) 1

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

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

. However, brood 3

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

and Greve, 2002 )

. Reduced mysid fecundity following contaminant expo sure is commonly 5

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

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

female in each of the first two broods for the F 0 and F 1 generation, and to tal number of young in 8

the first two broods in each replicate population for the F 0 and F 1 generation

. In addition, the 9

number of reproductively active females is determined for the F 0 and F 1 generation , total 10

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

in the first two broods

. 12

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

produced no offspring

. If a male member of a reproductive pair dies before production of the 14

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

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

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

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

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

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

3.1.3 Statistical Analyses 21

The MTTT collects quantitative data for more than 20 endpoints ( Annex 4

– A ttach

22

ment E ) that are typically collected in spreadsheets and then transferred to and analyzed with a 23

statistical software package

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

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

basket (i.e., replicate tank)

. Any endpoints that are significantly affected by the test chemical 26

should be report ed as such

. 27

Normality should be assessed using the Shap iro

- Wilk test (Shapiro and Wilk 1965)

. 28

Screened potential outliers are included in the tests

. Outliers can be tested in one generation 29

analyses using Tukey’s outlier rule on the untransformed data

. Outliers are assessed from 30

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

confounding outliers and treatment effects

. Any response more than 1.5 times the interquartile 32

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

considered an outlier by Tukey’s rule

. All statistical outliers should be screened to determine if 34

there is a non

- biological explanation for the outlier response

. Unless there is a valid reason for 35

excl usion, outliers should be included in data analyses

. If data deviate substantially from the 36

assumption of normality, data may be transformed (e.g.

, log transformation, square root 37

transformation, arc sin transformation) and retested

. If the normality assum ption is accepted for 38

either the untransformed or the transformed data

, heterogeneity of variances among the 39

concentration groups should be assessed with Levene’s te st with absolute residuals (Box 1953)

. 40

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

Levene’s tests

. If the normality assumption was invalid for both the untransformed and the 42

transformed data

, heterogeneity of variances among the concentration groups should not be 43Invertebrate Two

- Generation ISR 18 May 2 8 , 2013

assessed

, as the nonparametric proced ures are appropriate for data analyses

. If assumptions of 1

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

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

among treatmen t and control groups

. If one or both tests (i.e., normality, equal statistical 4

variance) fail, a nonparametric statistic such as a Kruskal

- Wallis test should be used to compare 5

treatment groups

. The appropriate multiple comparisons procedure should be sele cted to 6

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

parametric data, Dunn’s test for nonparametric data

. ). 8

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

each generation a s described above to compare the effects in the F 0 and F 1 generations

. 10

Comparisons of the F 0 and F 1 generations can be perform ed with a two

- way mixed effects 11

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

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

allowing unequal variances between the two generations

. It is strongly recommended that a 14

statistician be involved in both the design and analysis of the test resu lts

, since the test guideline 15

allows for some variation in experimental design

. 16



### 3.2 Summary of the Harpacticoid Copepod Development and Reproduction 17



### T est ( HCDRT ) P rotocol 18

ASTM published a full life cycle test using A. tenuiremis in 2004 (ASTM E2317

- 04; 19

ASTM 20 04)

. A first draft of an OECD copepod lifecycle guideline was written in 2003 as a co

20

project between UK, Denmar k , and Sweden

. The harpacticoids Nitocra spinipes and Tisbe 21

battagliai were included together with the calanoid Acartia tonsa

– T. battagliai wa s later 22

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

validation studies (OECD 2007)

. Revised draft guidelines were prepared in 2004, at which point 24

the harpacticoi d and calanoid guidelines were separated

. The guidelines were intended for 25

evaluation of adverse long

- term effects (incl uding endocrine disruptive effects) of various types 26

of chemicals (OECD 2007; OECD 2011)

. A first interlaboratory validation study was co nducted 27

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

. 28

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

second interlaboratory validation with A. tenuiremis (OECD 20 11)

. The HCDRT with 30

A. tenuiremis is described below

. The performance of this test is described in Section 4.2

. 31

3.2.1 Principle of the T est 32

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

estuarine/marine harpacticoid copepod species under semi

- static conditions

. The test is initiated 34

with newly hatched <24 hours old copepods ( F 0 generation), which are exposed individually in 35

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

added to seawater at a range of concentrations

. The test duration for the complete life

- cycle 37

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

any developmental

- rate changes induced by the test chemical of interest), which is sufficient 39

time for newly

- hatched copepods ( F 0 ) to develop to the adult stage, be mated, extrude two 40

clutches of eggs (broods) and produce offspring ( F 1 )

. Specifically, when the copepods reach 41

adulthood (around 15 to 18 days for A. tenuiremis at 25oC), the gender of each copepod is 42

determined by light microscopy

. Individual adult male:female mating pairs are then construc ted 43Invertebrate Two

- Generation ISR 19 May 2 8 , 2013

and allowed to mate for 7 to 14 days in new isolated microwell test chambers containing 1

seawater and the test substance added to seawater at the same range of concentrations as during 2

maturation/development

. The mating pairs are followed until the fema les have been fertilized 3

and have released at least two broods

. Hatching success of the first and second brood should be 4

determined, and the bioassay is usually terminated upon hatch of the second brood , or on day 36, 5

whichever occurs sooner

. 6

The biologic al endpoints measured include survival and development of the early 7

life

- stages, and multiple aspects of copepod reproduction (e.g., intersexuality, mating success, 8

brood size, abortion rates, number of necrotic eggs , and numbers of viable offspring produced)

. 9

Population growth rate and age:stage structure can also be calculated and predicted over multiple 10

generations using collected lifetable survival, sex

- ratio

, and fecundity data. 11

An overview of the methodology is provided in Table 3

- 2

. Details regarding test animals 12

and assay system, test substance concentration selection, test substance administration and 13

analytical determination, performance of the test and p erformance criteria, data reporting

, and 14

exposure are provided in Appendix F

. Biological endpoints of the HCDRT are described below. 15

Table 3

- 2

. Test Conditions for the Harpacticoid Copepod Development and Reproduction 16

Test (HCDRT) guideline

. 17

Recommended species Amphiascus tenuiremis

Test type Static renewal

Water temperature 25 ± 2oC

Salinity 18

- 4 0 o/oo

Photoperiod 1 2 h light, 1 2 h dark

Loading rate 20

- 4 0 animals per replicate microplate, each added to individual

microwells

three replicate test vessels (microplates) per concentrations and

control(s)

Microwell size 300 μ L total volume; 250 μ L 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., p rovide 9 0

- 95% water replacement in each test microwell

every 72

- h or less depending on stability of test substance)

. If

c oncentratio n 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 copepod s per test

concentration (start) 20

- 40/replicate; 3 replicates/treatment; 60

- 120 copepod s per test

concentration

No. of copepod mating pair s 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 μ l 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

terti olecta), a chrysophyte (e.g., Isochrysis galbana) and a

cryptophyte (e.g., Rhodomonas salina)

Cell density added to each test microwell at each feeding shouldInvertebrate Two

- Generation ISR 20 May 2 8 , 2013 target ~ 2 x 104 cells/microwell

. Aeration None , but dilution seawater should be aerated for 24 h, then filtered

at 0.2 μ m, 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 ( F 0

, F 1 – record by developmental stage )

Sex ratio ( F 0 )

Development rate (from nauplius to copepodite stage and adult

stage, respectively )

Fertilization success (number of mating pairs able to produce two

successive brood s)

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 brood s)

Optional endpoin ts 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 bro od

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 > 7 0% of saturation throughout exposure

temperature should vary by no more than ± 4 oC during the entire

test with a 36 day mean temperature of ± 2 °C

pH must not vary more than 0.5 units from the aver age 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., 3 0o/oo ± 3 o/oo )

sex

- ratio in F 0 - controls should on average range between 0.6 to 1.8

(± 0.2)

average number of viable off spring produced through two brood s 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 perio d or 25 days, whichever is less

Note: 1

Due to the limited amount of test solution volume ( approximately 250 μ L) in the HCDRT

, it may be necessary to 2

collect and pool individual microwell volumes within microplates within each treatment at the end of each renewal 3

period for water quality and test material measurements. 4

3.2.2 Endpoints 5

A detailed description of assay endpoints reflective of reproductive and developmental 6

impairment in aquatic arthropods, including copepods, is provided in a Detailed Review Paper 7

o n Aquatic Arthropods with an Emphasis on Developmental, Reproductive and Endocrine 8

Disruptive Effects (OECD 2006)

. L ifecycle endpoints for the recommended species, 9

A. tenuiremis

, are monitored daily in each microplate using light microscopy

. Growth 10Invertebrate Two

- Generation ISR 21 May 2 8 , 2013

(length /weight) is not recorded in the HCDRT

. A number of developmental and reproductive 1

endpoints are monitored and are discussed below

. 2

Survival 3

Survival of larvae (nauplii), juveniles (copepodites), and adults is checked daily up to and 4

including the last d ay of the test

. Mortali ty of mating pairs should be checked and recorded daily

. 5

The average mortality in the control(s) must not exceed 25% at the end of the exposure period 6

and no individual control replicate microplates should exceed 30% mortality

. Stage

- specific 7

survival data can be combined with sex ratio and fecundity data to estimate population growth 8

rate and age:stage structure ( Appendix F )

. 9

Rate of D evelopment 10

Various studies on copepods have demonstrated that larval development, which is tightly 11

linked to the molting process, is highly sensitive to a range of chemicals, including suspected 12

endocrine disruptors

. Adverse effect concentrations in these copepod studies were, in most cases, 13

lower than adverse effect concentrations for similar e ndpoints reported for other crustaceans

. 14

However, the mode of action of EDCs on crustaceans remains largely unknown, and very little is 15

known about the endocrine system of copepods (Kusk and Wollenberger 2007 and references 16

therein)

. The mean number of day s from brood extrusion to naupliar hatching has been used to 17

study egg development time in copepods following chemical exposure (Cary et al

. 2004)

. 18

Development rate can also be used in population growth rate and age:stage structure models 19

( Appendix F ). 20

Whi le growth is not measured in the HCDRT , the rate of development is measured as the 21

number of days it takes for development of each individually isolated copepod, from a first stage 22

nauplius to a juvenile copepodite to a reproductively mature adult

. 23

Reprod uction 24

Time to first brood release is one of the main endpoints often used in copepod life cycle 25

tests aimed at identifying effects of environmental pollutants with a suspected biological activity 26

(e.g., Breitholtz and Bengtsson . 2001; Chandler et al. 2004 a )

. Fecundity (brood size) has also 27

been evaluated in copepods following chemical exposure (e.g., Bejerano and Chandler 2003; 28

Hutchinson et al. 1999 a ; Breitholtz and Wollenberger 2003; Breitholtz et al. 2003)

. Because it is 29

an important measure of reproduc tive success, any reduction in fecundity can be interpreted as 30

an indication of reproductive toxicity (Khan et al. 1992)

. However, reductions in fecundity need 31

to be evaluated carefully to distinguish direct reproductive disruption from indirect effects on 32

fecundity due to growth effects (OECD 2006)

. 33

The HCDRT evaluates a number of reproductive performance measures, such as sex ratio, 34

fertilization success (number of mating pairs able to produce two successive broods ), time to 35

production of first brood and time interval between two brood s, offspring production through 36

two brood s

. Optional reproductive endpoints include number of necrotic or infertile eggs per 37

brood and number of aborted egg sacs

. Note that, r eproduction data in the HCDRT are expressed 38

on a p er female or per mating

- pair basis

. 39

3.2.3 Statistical Analyses 40

The HCRDT collects quantitative development and reproduction data that are typically 41

collected in spreadsheets and then transferred to and analyzed with a statistical software package 42Invertebrate Two

- Generation ISR 22 May 2 8 , 2013

( example of da ta entry sheets are provided in Annex 3 of HC DRT draft protocol

– Appendix F ). 1

OECD 2007; OECD 2011)

. 2

If desired, acute mortality data for selected time periods (e.g., ≤ 96 hours) can be analy z ed 3

based on the methods of Stephan ( 1977 ) to calculate median lethal (LC 50 ) concentrations of test 4

materials

. Statistically significant effects on survival of adult females are calculated using a 5

contingency table procedure, Fisher’s Exact Test ( Finney et al. 1963 ), which should be reported 6

as a separate endpoint. Th ose concentrations where mortality is significantly different from the 7

control are not included in the statistical analysis of the reproduction data

. 8

D evelopment rates, stage

- specific mortality, fertilization success, and sex ratios of the F 0 9

may all be a nalyzed statistically for significant differences relative to controls

. Reproductive 10

endpoints may be analy z ed as single end of test measurements or relative to different time 11

intervals during the exposure

. The mean number of F 1 offspring produced from the fertilized 12

females ( F 0 ) in each replicate is calculated

. Generally, the reproduction data are not adjusted for 13

mortality but are presented on a per female or per mating

- pair basis

. The mean number of 14

offspring produced by each control and treatment mating pair is calculated

. If an animal dies 15

after producing young, the total number of offspring produced up to the time of death is used

. On 16

occasions, it may be necessary to make an adjustment for mortality where those animals that died 17

during the exposure (i .e.

, before giving birth to any young) are not included in the statistical 18

analysis

. One

- way nested analysis of variance (ANOVA) is used to evaluate effects of the test 19

substance on the reproduction (and other chronic endpoints) of the copepods, and a mult iple 20

comparison procedure (e.g. Dunnett’s test [ Dunnet 1955 ] is used to define the treatment level 21

resulting in significantly different reproduction from the control (p=0.05)

. For ANOVA, 22

individual microwell mating pairs are nested within their particular microplate, which is then 23

replicated within a given treatment

. A nested model is necessary because microwells within a 24

microplate are not strictly independent of one another. 25

ANOVA is a parametric procedure based on the assumptions that the observations ar e 26

independent and normally distributed, and that the variance of the observations is homogeneous 27

across all toxicant concentrations and the control

. These assumptions should be checked prior to 28

using ANOVA, to determine if they have been met

. Non

- normal di stributed data may be 29

subjected to normalizing transformations such as log transforms, arc

- sine square root transforms, 30

etc., provided both treatment and control datasets are subjected to the same transformation. Tests 31

for validating these assumptions are Shapiro

- Wilks Test for normality and Bartlett’s Test for 32

homogeneity of variance ( US EPA 1988 )

. If the data do not meet the assumptions for ANOVA, 33

non

- parametric procedures such as Steel’s Many

- One

- Rank Test or Wilcoxon’s Rank Sum Test 34

( US EPA 1988 ) may be more appropriate. 35

If a statistically significant difference in survival, development rate, sex ratio, or 36

reproduction is detected between the control and solvent control, only the solvent control is used 37

as the basis for the calculation of results. If no significant differences exist between control and 38

solvent control data, these may be pooled for comparisons with test substance treatments. 39

40Invertebrate Two

- Generation ISR 23 May 2 8 , 2013



### 4 DEMONSTRATION AND OPTIMIZATION OF THE MYSID AND 1



### COPEPOD TEST METHODS 2

This section describes results of demonstrat ion and optimization studies that have been 3

performed in general accordance with the MTTT and HCDRT methods presented in Section 3 )

. 4



### 4.1 Mysid Two

- Generation Toxicity Test (MTTT) Guideline P erformance 5

McKenney (1998) synthesized the results of his earlier studies on the effects of chronic 6

exposure to various pesticides on A. bahia and concluded that life

- cycle endpoints (slowed 7

juvenile growth, inhibited reproductive c apacity, metabolic changes) were more sensitive than 8

survival

. Through these studies, McKenney also demonstrated that mysids are generally more 9

sensitive than most other aquatic species to chemical contaminants

. These early studies are not 10

summarized here and readers are referred to the original studies (McKenney 1982; McKenney 11

1986; McKenney and Matthews 1990; McKenney et al. 1991) and several reviews 12

(Verslycke et al. 2004 a ; Verslycke et al. 2007; OECD 2006)

. In a later transgenerational 13

exposure study wi th fenoxycarb , McKenney (2005) extend ed the life

- cycle test such that 14

juveniles, released by adults exposed since <24

- h our old juveniles, we re reared through 15

maturation without further exposure

. Neither maturation time, sex determination

, nor young 16

product ion was significantly altered during the life

- cycle exposure

. However, second

- generation 17

adults, exposed to fenoxycarb only as developing embryos and juveniles, produced fewer young 18

(at 6 μ g fenoxycarb/ L ) and contained significantly fewer males (at 1 μ g fenoxycarb/ L ). These 19

findings s uggest ed the need for, at least, two

- generational exposure protocols to adequately 20

predict the potential chronic impact of ED C s ( Verslycke et al. 2007; OECD 2006 ) and led to the 21

development of a proposed MTTT guideline

. A pro posed MTTT was submitted to OECD in 22

2006

. This guideline was initially proposed to OECD by US EPA in 2004 to estimate toxicity 23

associated with endocrine disruption and reproductive fitness for an identified potential EDC 24

(OECD 2004 a )

. The MTTT test is an e xtension of previous mysid life

- cycle test procedures 25

produced by ASTM (ASTM E1191) and US EPA ( OCSPP 850.1350)

. The MTTT has been used 26

in demonstration and optimization studies as described further below

. Collectively, these studies 27

form the basis for the recommended protocol described in Section 3.1

. 28

4.1.1 Demonstration S tud ies U sing the 2004 Draft MTTT Guideline 29

First set of demonstration studies 30

A first evaluation of the testing procedures and methods proposed by US EPA to OECD 31

in the draft guideline for a two

- generation mysid test to evaluate suspected endocrine

- disrupting 32

chemicals (OECD 2004 a ) was conducted through a demonstration study with three chemicals: 33

fenoxycarb

, 3,5

- dichlorophenol (3,5

- DCP ) , and fipronil (Putt and Cafarella 2006)

. These 34

chemicals each have a uniquely identified mode of action: fenoxycarb, a juvenile hormone 35

mimic designed to prevent target insects from molting to the adult stage; 3,5

- DCP, which inhibits 36

energy production through uncoupling of oxidative phosphorylati on; and fipronil, a central 37

nervous system disruptor that blocks the passage of chlorine ions through the GABA receptor

. 38

The endpoints examined in the F 0 and F 1 generations in this first evaluation included 39

survival, growth (total length

, dry weight , grow th rate between days 7 and 14 ), and reproductive 40

measures ( time to maturation, time to first and second brood release

, interbrood duration, number 41Invertebrate Two

- Generation ISR 24 May 2 8 , 2013

of young per female in each of the first two broods, total number of young in first two broods, 1

sex ratio , an d percentage of females that are reproductively active ). 2

Performance criteria for judging test data acceptability were: >70% survival of control 3

animals in all test phases over the duration of the chemical exposure; ≥75% of females in the F 0 4

and F 1 controls must produce young; and average number of young produced per control female 5

in the first two broods combined must be at least 8. 6

To check homogeneity of variance s in data obtained for each endpoint, Bartlett’s Test 7

(for normally distributed data) or Levene’s Test (when data were not normally distributed or 8

there was no variance in a treatment level) were used

. Shapiro

- Wilk’s test was used to evaluate 9

normality of the data distribution

. ANOVA was conducted on each data set to determine if a 10

treatmen t group in the data set was different from the other groups in that data set

. Treatment 11

level endpoints were compared with the performance of the solvent control data by using 12

William’s Test and Dunnett’s Test

. If necessary, mean values were transformed us ing square 13

root, arcsine square root, or log conversion procedures

. 14

Fenoxycarb 15

Detailed results of the demonstration study with fenoxycarb are presented in Putt and 16

Cafarella ( 2006 ) and are summarized here. 17

A 52

- day flow

- through t est was initiated at nomi nal concentrations of 0 ( solvent 18

control

- triethylene glycol ), 1.0, 2.5, 6.0, 16, and 40 μ g a ctive ingredient (a.i.) /L

. Analysis of the 19

test solutions demonstrated that the expected concentration

- gradient was generally maintained 20

during the exposure

. Mean measured concentrations of fenoxycarb ranged from 82 to 92% of 21

nominal

. Based on mean measured concentrations, the treatment levels were defined as 2.3, 4.9, 22

14, and 35 μg a.i./L

. Artificial seawater (commercially prepared salt formula added to labora tory 23

well water) was used as dilution water

. Newly prepared batches h ad a p H range of 7.6 to 8.2 and 24

a salinity of 20 ± 2‰

. 25

The test substance was introduced into 54

- L glass aquaria for a minimum of 48 hours 26

before the test began to allow the test solution to reach an equilibrium

. Three replicate aquaria 27

were used for each treatment level

. Exposure for the F 0 mysids began when <24h old individuals 28

were randomly distributed into the test aquaria from stock cultures

. Fifteen individuals were 29

added to each triplicate chamber within each single treatment exposure aquarium , such that each 30

treatment group received a minimum of 45 mysids

. T hree additional groups of 15 mysids were 31

randomly assigned to growth baskets within each replicate aquarium per treatme nt

. Exposures 32

were terminated 7 days after the mean day of release of the second brood of the F 1 mysids in the 33

solvent controls. 34

M ysids from both generations ( F 0 and F 1 ) for all treatments were fed twice daily, except 35

at test initiation and test termination

, where they were fed only once due to the time of test 36

initiation and termination (i.e., later in the day)

. For each generation, during the period prior to 37

pairing (e.g., the first 14 days), at least one of the two daily feedings was with brine shrimp 38

enriched with Selco ® , a supplemental substance high in saturated fatty acids

. During the period 39

subsequent to pairing (e.g., day 15 through test termination), the Selco ® enriched brine shrimp 40

were fed to the test organisms once every other day

. Rat ions for reproduction/growth baskets 41

(containing 15 mysids each) were approximately 1,800 nauplii/basket on days 1 to 3 ; 2,250 on 42

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 43Invertebrate Two

- Generation ISR 25 May 2 8 , 2013

cups (containing individually pa ired mysids) were fed approximately 900 nauplii/cup throughout 1

the test

. All treatments and controls received, as near as reasonably possible, the same ration of 2

food

. 3

M easured water quality parameters (temperature, pH, and salinity) stayed within the 4

spe cified limits ( Putt and Cafarella 2006 ). Dissolved oxygen levels dropped below 60% 5

saturation in both the controls and treatment levels at the same time , but this reduction is not 6

expected to have impacted study performance

, since all test performance crit eria were met

. 7

Results of the demonstration study are presented in Table 4

- 1

. F 0 generation mysid length 8

(day 14) was the most sensitive endpoint ( statistically significant effect in 2.5 to 40 μg/L 9

treatments )

. Length growth rate, time to first brood, number of offspr ing in the first brood

, and 10

total offspring from the F 0 generation were also statistically significantly different from controls 11

in 6, 16, and/or 40 μg/L treatments

. Compared to controls, significant increases were observed 12

for F 0 14

- day dry weight, dry w eight growth rate

, and F 1 total offspring production at the 13

2.5 μg/L treatment level , but similar results were not observed at any other treatment level . No 14

other significant F 1 generation effects were detected among any of the endpoints measured. 15

Table 4

- 1

. Results of the EPA demonstration study of the MTTT with fenoxycarb

. 16

Endpoint F 0 (Parental) F 1 (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

- -

[<LOQ] The limit of quantitation was 2 μg a.i./L, thus the lowest treatment level was not analytically verified. 17

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 18

[*] Statistical analysis for mean dry weight and length on day 0 was not performed since the nine subsamples 19

from the entire population of <24

- hour old mysids used to initiate the test were not related to dose levels 20

used in the study. 21

[N#] Sample size (n) was 3 for each treatment level or control, except as noted (e.g., for N8 , n = 8). 22

[↑] Indicates a statistically significant increase compared to control, based on Dunnett's, Williams' or 23

Bonferroni's test (α = 0.05). 24

[↓] Indicates a statistically significant decrease compared to control, based on Dunnett's, Williams' or 25

Bonferroni's test (α = 0.05). 26

27Invertebrate Two

- Generation ISR 26 May 2 8 , 2013

3,5

- dichlorophenol 1

Detailed results of the demonstration study wi th 3,5

- dichlorophenol (3,5

- DCP) are 2

presented in Putt and Cafarella ( 2006 ) and are summarized here. 3

A preliminary range finding test with 3,5

- DCP was conducted using an acute 4

flow

- through test exposing <24 hour mysids to nominal concentrations of 0.13, 0.3 2, 0.80, 2.0, 5

and 5.0 mg a.i./L and a solvent (acetone) control

. Two test vessels containing 10 mysids each 6

were established for each test concentration and the solvent control

. After 96 hours, 0, 5, 5, 25 7

and 100% mortality was observed among mysids expos ed to the 0.13, 0.32, 0.80, 2.0, and 5.0 mg 8

a.i./L treatment levels, respectively

. Mortality of 5% was observed among mysids exposed to the 9

solvent control

. 10

Based on the preliminary results, a 50

- day flow

- through test was initiated at nominal 11

concentratio ns of 0 (solvent control; triethylene glycol), 0.013, 0.032, 0.076, 0.20, and 12

0.50 mg a.i./L

. Analysis of the test solutions demonstrated that the expected concentration 13

gradient was generally maintained during the exposure

. Mean measured concentrations of 14

3,5

- DCP ranged from 56 to 98% of nominal, increasing with increasing concentration

. Based on 15

mean measured concentrations of 3,5

- DCP, the treatment levels tested were defined as 0.0072, 16

0.022, 0.072, 0.19, and 0.49 mg a.i./L

. Test procedures, feeding, and statistical data evaluation 17

procedures were the same as for the fenoxycarb experiment. 18

All measured water quality parameters (temperature, pH, and salinity) stayed within the 19

specified limits (Putt and Cafarella 2006)

. Dissolved oxygen levels dropped belo w 60% 20

saturation in both the controls and treatment levels at the same time, but this reduction is not 21

expected to have impacted study performance

, since all test performance criteria were met

. 22

Results from the 3,5

- DCP demonstration test are presented in Table 4

- 2

. Time to 23

maturation in the F 0 generation was the most sensitive endpoint in the mysid test ( statistically 24

significant effect in 0.0 32 to 0.5 m g/L treatments )

. Time to first brood, time to second brood, 25

interbrood duration, number of offspring in the first and second brood

, and total offspring 26

production in the F 0 generation were also sensitive endpoints

. Mysid F 0 day 7 dry weight did not 27

demonstrate a dose response ; however, the dry weights from the highest (0.5 mg/L) and second 28

lowes t (0.032 mg/L) treatment levels were significantly reduced

. Interbrood durations in the two 29

highest treatment levels ( 0. 20 and 0.5 0 m g/L) in the F 0 exposure were statistically s ignificant 30

compared to the solvent control

. In the F 1 generation, significant reductions in total number of 31

offspring per female were observed in the 0.0 13, 0.0 76

, and 0. 50 m g/L treatment levels

. The 32

percent of reproducing females was also a sensitive endp oint for adverse effects in the F 1 33

generation (0.5 mg/L)

. 34

35

36Invertebrate Two

- Generation ISR 27 May 2 8 , 2013

Table 4

- 2

. Results of the EPA demonstration study of the MTTT with 3,5

- DCP

. 1

Endpoint F 0 (Parental) F 1 (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

- - - ↓ ↓ ↓

- ↓

- ↓

% Reprodu cing Females

- - - - - - - - - ↓

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 2

[*] Statistical analysis for mean dry weight and length on day 0 was not performed since the nine subsamples from 3

the entire population of <24

- hour old mysids used to initiate the test were not related to dose levels used in the 4

study. 5

[N#] Sample size (n) was 3 for each treatment level or control, except as noted (e.g., for N8 , n = 8). 6

[↑] Indicates a statisticall y significant increase compared to control, based on Dunnett's or Williams' test (α = 0.05). 7

[↓] Indicates a statistically significant decrease compared to control, based on Dunnett's or Williams' or test (α = 8

0.05). 9

10

Fipronil 11

Detailed results of the dem onstration study with fipronil are presented in Putt and 12

Cafarella ( 2006 ) and are summarized here. 13

A 50

- day flow

- through test was initiated at nominal concentrations of 0 ( solvent control; 14

triethylene glycol ), 1.0

, 2.5

, 6.0

, 16 , and 40 n g a.i./L

. The two lowest nominal concentration 15

levels (1.0 and 2.5 ng/L) were not analytically measured during the study since they were below 16

the limit of quantitation (5 ng/L)

. Analysis of the remaining test solutions demonstrated that the 17

expected concentration gradient was generally maintained during the exposure

. Based on mean 18

measured concentrations of fipronil, these treatment levels were defined as 4.5, 16, and 19

44 ng a.i./L

. Test procedures, feeding, and statistical data evaluation procedures were the same as 20

for the fenoxycarb experiment. 21

All measured water quality parameters (temperature, pH, and salinity) stayed within the 22

specified limits (Putt and Cafarella 2006)

. Dissolved oxygen levels dropped below 60% 23

saturation in both the controls and treatment levels at th e same time, but this reduction is not 24

expected to have impacted study performance since all test performance criteria were met

. 25

The fipronil demonstration results are presented in Table 4

- 3

. F 0 generation day 7 length 26

and time to maturation were the most sensitive endpoints in the mysid two

- generation ex posure 27

to fipronil ( statistically significant effect in 1 to 40 ng/L treatments )

. Day 14 length, day 7 to 14 28Invertebrate Two

- Generation ISR 28 May 2 8 , 2013

length growth rate, and survival on day 14 in the F 0 generation were also sensitive endpoints

. In 1

the F 1 generation, day 14 survival was the most sensitive endpoint ( 40 ng/L)

. 2

Table 4

- 3

. Results of the EPA demonstrati on study of the MTTT with fipronil

. 3

Endpoint F 0 (Parental) F 1 (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

- - - - - - - - - -

[<LOQ] The limit of quantitation was 5 ng a.i./L, thus the two lowest treatment levels were not analytically verified. 4

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 5

[*] Statistical analysis for mean dry weight and length on day 0 was not performed since the nine subsamples 6

from the entire population of <24

- hour old mysids used to initiate the test were not related to dose levels 7

used in the study. 8

[N#] Sample size (n) was 3 for each treatment level or control, exce pt as noted (e.g., for N8 , n = 8). 9

[↑] Indicates a statistically significant increase compared to control, based on Dunnett's, Williams' or 10

Bonferroni's test (α = 0.05). 11

[↓] Indicates a statistically significant decrease compared to control, based on Du nnett's, Williams' or 12

Bonferroni's test (α = 0.05). 13

The following conclusions and recommendations based on the first set of demonstrations 14

studies were taken from Putt and Cafarella (2006): 15

 The F 0 and F 1 growth and reproduction endpoint data routinely pass ed tests for 16

normality and homogeneity, typically without requiring any data transformation 17

 Less than 24

- hour

- old mysid growth endpoints and sex ratio were the only two 18

general endpoints that did not elicit a statistically significant effect with at least one 19

chemical 20

 Growth rates were either less sensitive or equally as sensitive as the direct 21

measurement of either dry weight or length 22

 Mysid length was a more sensitive endpoint than dry weight ( i.e. , length was 23

significantly reduced with increasing tre atment level in two of three tests

– dry weight 24

was significantly impacted in these tests ) and easier to collect, especially with the 25

smallest mysids

. However, the sensitivity of the length endpoint varied for day 7 and 26

day 14 measurements

. Therefore, effects on length on day 7 do not necessarily 27

translate to effects on length on day 14 and vice versa

. Thus, measuring growth 28Invertebrate Two

- Generation ISR 29 May 2 8 , 2013 (particularly mysid length) at multiple time intervals over the first 14 days of 1

exposure is important if mysid length can be correlated to potential endocrine effects 2

 The proposed endpoints in the mysid two

- generation study design appear to be 3

relevant for identifying effects on growth and reproduction

. The 3,5

- DCP exposure 4

clearly demonstrated that the reproductive endpo ints are sensitive 5

 The study design for the two

- generation mysid study does not include any 6

biochemical, biomarker, or histopathology endpoints

. Therefore, a conclusive 7

determination of mode of action is not possible

. However, the growth and 8

reproductive e ndpoints evaluated in this demonstration are all relevant endpoints to 9

evaluate effects on mysid growth and reproduction

. This demonstration provided 10

strong evidence that multiple growth and reproduction endpoint monitoring is 11

necessary in the final study design to identify the wide range of potential effects from 12

an array of chemicals

. 13

Second set of demonstration studies 14

A second evaluation of the proposed draft guideline (OECD 2004 a ) was conducted 15

through a demonstration study with four chemicals: prochl oraz ( vertebrate aromatase inhibitor)

, 16

flutamide (vertebrate androgen receptor ant agonist) , ketoconazole (weak vertebrate androgen 17

receptor agonist) , and 4

- tert

- octylphenol ( vertebrate estrogen receptor agonist) ( Kropp and 18

Cullinan 2008)

. These chemicals h ad previously been used as reference chemicals in the 19

validation of several Tier 1 screening assays 20

(http://www.epa.gov/scipoly/oscpendo/pubs/assayvalidation/status.htm)

. The protocol used in 21

the demonstration study differed from the proposed 2004 guidelin e in four ways: (1) the mysids 22

for each study were fed with Artemia nauplii hatched from cysts that were not obtained from a 23

reference source (sensu Leger and Sorgeloos 1984), but were from a reliable source that has been 24

used successfully by the test labo ratory in many previous studies; (2) retention baskets used 25

during the testing were constructed from nylon mesh having a slightly larger mesh opening, 26

355 μm, than the 210

- to 330

- μm opening stipulated in the guideline and used in the first set of 27

demonstration studies; (3) mysid dry weights were measured to the nearest 1 μg instead of the 28

nearest 0.1 μg; and (4) the draft guideline specifies that females be paired with the largest 29

suspected males

― however, the laboratory used a dissecting microscop e to examine all mysids, 30

except for ovigerous females, to confirm gender and maturity before pairing

. None of these 31

modifications is likely to have had an adverse effect on the study. 32

Prochloraz 33

Detailed results of the demonstration study with prochloraz a re presented in 34

Aufderheide ( 2008 a ) and are summarized here. 35

A 21

- day flow

- through range

- finding test was initiated at nominal concentrations of 0 36

(dilution water control), 1.0, 10, 100, and 1,000 μg prochloraz/L

. Survival was the most 37

consistent indicator of a concentration

- dependent response during the range

- finding test and the 38

definitive test concentrations were set a t a nominal concentration range starting at approximately 39

one

- third the LC50 value (i.e., 300 μg prochloraz/L) after the initial 14 days of the range

- finding 40

test

. The definitive toxicity test was performed at nominal concentrations of 0 (control), 13.0, 41

21.6, 36.0, 60.0, and 100 μg prochloraz/L with three replicate test chambers for each control and 42

treatment level

. The mysids were initial ly fed according to the feeding regime specified by the 43

2004 OECD draft guideline

. However, due to poor reproductive activity during the first 27 days 44Invertebrate Two

- Generation ISR 30 May 2 8 , 2013

(i.e., increased time to first brood and low number of young in second brood) , feedings were 1

increased fr om 900 to 1,350 nauplii per brood cup by increasing the number of feedings from 2

two to three

. 3

All measured water quality parameters (temperature, dissolved oxygen pH, and salinity) 4

stayed within the specified limits (Aufderheide 2008 a )

. As a whole, more than 75% of control 5

pairs produced at least one brood and more than 70 % of control animals survived the first 28 6

days of testing, thus meeting the guidance

- specified acceptability criteria (Aufderheide 2008 a )

. 7

However, the numbers of F 1

- ge neration mysids were not sufficient to allow complete evaluation 8

of endpoints for that generation

. Analytical measurements of prochloraz made during the study 9

varied and showed occasional overlap of exposure concentrations during the study

. Reasons for 10

thi s variation in concentrations within dose levels were not identified

. The use of a self

- starting 11

siphon for water renewal was identified as a likely cause for nutritional issues by effectively 12

removing brine shrimp nauplii during draw down, and for loss of animals due to impingement 13

and mortality

. 14

Results from the prochloraz demonstration test are presented in Table 4

- 4

. All but one of 15

the variables (i.e., 7

- day weight) evaluated during the prochloraz study were nonresponsive as 16

indicated by signal

- to

- noise ratios <2 (Kropp and Cullinan 2008)

. Statistical analysis of the 17

endpoints based on the Kruskal

- Wallis and Dunn’s tests showed no significant differences 18

betwe en treatment mean responses and control mean responses

. 19

Table 4

- 4

. Results of the EPA demonstration study of the MTTT with prochloraz

. 20

Endpoint F 0 (Parental) F 1 (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 Offs p ring (1st and 2nd broods) ↓

- ↓ ↓ ↓ na na na na na

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 21

[na] Not assessed due to limited number of offspring. 22

[↑] Indicates a statistically significant increase compared to control, based on Dunnett's test (α = 0.05). 23

[↓] Indicates a statistically significant decrease compared to control, based on Dunnett's test (α = 0.05). 24

Dunnett’s test (conducted after removing the block effect), indicated that four endpoints 25

showed significant differences between the treatment and control responses (the significance of 26

these respons es may have been overestimated due to removal of the block effect, which reduced 27

the error for testing). Thus, responses of mysids to prochloraz generally were inconclusive 28

(Kropp and Cullinan 2008). This lack of response may have been partly attributable to the 29

variation in and overlap among doses to which the mysids were exposed. However, the poor 30

feeding regime and self

- starting siphon issues probably also had an important effect on the study

. 31Invertebrate Two

- Generation ISR 31 May 2 8 , 2013

Flutamide 1

Detailed results of the demonstration study with fl utamide are presented in Aufderheide 2

( 2008 b) and are summarized here

. The major modifications from the prochloraz study are an 3

increase of the feeding regime by about 50% more than the 2004 OECD draft guideline 4

specifications and the discontinuation of the self

- starting siphon when the F 0 adults were paired. 5

A 21

- day flow

- through range

- finding test was initiated at nominal concentrations of 6

0 (dilution water control), 0.00098, 0.0098, 0.097, and 0.98 mg flutamide/L

. None of the 7

biological parameters observe d for the range

- finding test demonstrated a concentration

8

dependent response to the flutamide exposure

. The definitive toxicity test was performed at 9

nominal concentrations of 0 (control), 0.125, 0.250, 0.500, 1.00, and 2.00 mg flutamide/L, as 10

determined f

rom the range

- finding test results, with three replicate test chambers for each control 11

and treatment level

. 12

All measured water quality parameters (temperature, dissolved oxygen pH, and salinity) 13

stayed within the specified limits (Aufderheide 2008 b )

. More than 70% of control animals 14

survived the first 28 days of testing, thus meeting the guidance

- specified acceptability criteria 15

(Aufderheide 2008 b )

. However , only 67% (instead of the guidance

- specified ≥75%) of control 16

pairs produced at least one brood

. Also, the numbers of F 1

- generation mysids were not sufficient 17

to allow evaluation of variables for that generation

. Analytical measurements of flutamide made 18

during the study were stable

, and there was no overlap among exposure concentrations during 19

the s tudy

. Average measured concentrations were ≥ 90% of the nominal values for each dose 20

level

. Three variables evaluated during the flutamide study, 7

- day length and 14

- day male and 21

female length, were determined to be responsive (signal

- to

- noise ratios >2)

. 22

Results from the flutamide demonstration test are presented in Table 4

- 5

. Statistical 23

analysis of the endpoints based on the Kruskal

- Wallis and Dunn’s tests showed no significant 24

differences between treatment mean responses and control mean responses

. Dunnett’s test 25

(conducted after removing the block effect) , indicated that four endpoints showed significant 26

differences between the treatment and control responses (the significance of these responses may 27

have been overestimated due to removal of the block effect

, which reduced the error for testing)

. 28

Three of the four treatment responses were at the highest dose level, which suggests a dose 29

response

. Thus, t he resp onses of mysids to flutamide probably should be considered as generally 30

inconclusive (Kropp and Cullinan 2008)

. The lack of response during the flutamide study may 31

have been partly attributable to inadequate diet. 32

Ketoconazole 33

Detailed results of the demon stration study with ketoconazole are presented in Hicks 34

(2008b) and are summarized here

. The major modifications from the prochloraz and flutamide 35

studies are a further increase of the feeding regime to three ad libitum feedings per day 36

(i.e., about 6,500 nauplii per day)

. F 1 mysids received a slightly larger ration

. Additionally, the 37

brine shrimp used each day were enhanced with a mixture of three commercially available 38

supplements: Lifeline Natural Health Enrichment HUFA, AlgaMac

- 3000, and AlgaMac

- Enhance

. 39

The self

- starting siphon was also no longer used

. 40

41Invertebrate Two

- Generation ISR 32 May 2 8 , 2013

Table 4

- 5

. Results of the EPA demonstration study of the MTTT with flutamide

. 1

Endpoint F 0 (Parental) F 1 (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 Offs p ring (1st and 2nd broods)

- - - - ↓ na na na na na

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 2

[na] Not assessed due to limited number of offspring. 3

[↑] Indicates a statistically significant increase compared to control, based on Dunnett's test (α = 0.05). 4

[↓] Indicates a statistically significa nt decrease compared to control, based on Dunnett's test (α = 0.05). 5

A 21

- day flow

- through range

- finding test was initiated at nominal concentrations of 6

0 (dilution water control), 1.0, 10, 100, and 1,000 μ g ketoconazole /L

. Survival was the most 7

consistent indicator of a concentration

- dependent response during the range

- finding test and the 8

definitive test concentrations were set at nominal concentration range starting at approximately 9

one

- third the LC 50 value (i.e., 150 μg ketoconazole/L) after 21 days of the range

- finding test

. 10

The definitive toxicity test was performed at nominal concentrations of 0 (control), 0 (vehicle 11

control; approximately 0.05 mL triethylene glycol/L), 3.3, 6.5, 13, 25, and 50 μg ketoconazole/L, 12

as determined from the range

- finding test results, with three replicate test chambers for the 13

control, vehicle

- control, and test

- substance treatments

. Mysids were fed ad libitum enriched 14

brine shrimp nauplii at least three times daily , as d etailed in Hicks ( 200 8 b)

. 15

All measured water quality parameters (temperature, dissolved oxygen pH, and salinity) 16

stayed within the specified limits ( Hicks 2008 b )

. In addition, F 0 and F 1 survival , percentage of F 0 17

and F 1 control and vehicle

- control female mysids that produced young, and average total young 18

per control and vehicle

- control female in the first two broods met the guidance

- specified 19

acceptability criteria , which are ≥ 75%, ≥ 70%, and 8, respectively (Hicks 2008 b )

. Analytical 20

measurements of ketoconazole made during the study were relatively variable, but there was no 21

overlap among exposure concentrations during the study

. Average concentrations were much 22

less than the nominal values for each dose level

. 23

The demonstration study results for ke toconazole are presented in Table 4

- 6

. Three F 0

- 24

g eneration variables evaluated during the ketoconazole study, 7

- day length and weight and total 25

number of young, were determined to be responsive (signal

- to

- noise ratios >2) . One 26

F 1

- generation variable, number of total young in the second brood, was determined to be 27

responsive

. Statistical analysis of the endpoints based on the Kruskal

- Wallis and Dunn’s tes ts 28

showed that the F 1

- generation number of young in the second brood for the level 3 dose 29

(8.64 μ g/L) was significantly different from that in the control treatment

. Dunnett’s test 30

(conducted after removing the block effect) , indicated that the F 1

- generation number of young in 31

t he second brood for the level 3, 4, and 5 doses were significantly different from that in the 32Invertebrate Two

- Generation ISR 33 May 2 8 , 2013

control treatment

. Dunnett’s test also indicated that six additional endpoints showed significant 1

differences between the treatmen t and control responses

. Three of these, F 0 number of young in 2

the first and second broods and total number of young, showed significant differences at the 3

highest dose level

. The fourth, F 0

- generation 7

- day weight, showed significant differe nces at the 4

do se levels 4 and 5

. The Dunnett’s test may have slightly over

- estimated the number of 5

significant responses ; thus the dose

- dependent response to ketoconazole may have occurred for 6

five endpoints, four of which measured reproductive responses ( i.e., total yo ung 1st brood F 0 , 7

total young 2nd brood F 0 , total young F 0 , and total young 2nd brood F 1 ) ( Kropp and 8

Cullinan 2008). 9

Table 4

- 6

. Results of the EPA demonstration study of the MTTT with ketoconazole

. 10

Endpoint F 0 (Parental) F 1 (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 Offs p ring (1st and 2nd broods)

- - - - ↓

- - ↓

- -

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 11

[↑] Indicates a statistically significant increase compared to control, based on Dunnett's test (α = 0.05). 12

[↓] Indicates a statistically significant decrease compared to control, based on Dunnett's test (α = 0.05). 13

4

- tert

- Octylphenol 14

Detailed results of the demonstration study with 4

- tert

- o ctylphenol are presented in Hicks 15

( 2008 a) and are summarized here

. The major modifications from the prochloraz and flutamide 16

studies are a further increase of the feeding regime to three ad libitum feedings per day 17

(i.e., about 5,200 nauplii per day)

. F 1 mysids received a slightly larger ration

. Additionally, the 18

brine shrimp used each day were enhanced with a mixture of three commercially available 19

supplements: Lifeline Natural Hea lth Enrichment HUFA, AlgaMac

- 3000, and AlgaMac

- Enhance

. 20

The self

- starting siphon was also no longer used

. 21

A 21

- day flow

- through range

- finding test was initiated at nominal concentrations of 22

0 (dilution

- water control), 1.0, 10, 100, and 1,000 μ g 4

- tert

- o ct ylphenol /L

. Survival was the most 23

consistent indicator of a concentration

- dependent response during the range

- finding test, and the 24

definitive test concentrations were set at nominal concentration range, starting at approximately 25

one

- third the 96

- hour LC50 for the range

- finding test (i.e., 502 μg 4

- tert

- octylphenol/L × 0.3, 26

where 0.3 is the correction factor used to account for approximately 30% test

- substance 27

recoveries during the range

- finding test)

. The definitive toxicity test was performed at nominal 28

c oncentrations of 0 (control), 0 (vehicle control; approximately 0.0 2 5 mL triethylene glycol/L), 29

3.3, 6.5, 13, 25, and 50 μg 4

- tert

- octylphenol /L, as determined from the range

- finding test results, 30

with three replicate test chambers for the control, vehicle

- control, and test

- substance treatments

. 31Invertebrate Two

- Generation ISR 34 May 2 8 , 2013

All measured water quality parameters (temperature, dissolved oxygen pH, and salinity) 1

stayed within the specified limits (Hicks 2008a )

. In addition, F 0 and F 1 survival, percentage of F 0 2

and F 1 control and vehicle

- control female mysids that produced young, and average total young 3

per control and vehicle

- control female in the first two broods met the guidance

- specified 4

acceptability criteria, which are ≥ 75%, ≥ 70%, and 8, respectively (Hicks 2008a )

. Analytical 5

measurements of 4

- tert

- octylphenol made during the study were highly variable

. There was some 6

overlap among exposure concentrations during the study, but this occurred only between the two 7

lowest dose levels

. Average concentrations were much l ess than the nominal values for each 8

dose level

. 9

The demonstration study results for 4

- tert

- octylphenol are presented in Table 4

- 7

. Four 10

F 0

- generation variables evaluated during the 4

- tert

- octylphenol study, 7

- day length and weight 11

and 14

- day female length and weight, were determined to be responsive

. Statistical analysis of 12

the endpoints based on the Kruskal

- Wallis and Dunn’s tests showed no significant differen ces 13

between treatment mean responses and control mean responses

. Dunnett’s test (conducted after 14

removing the block effect) indicated that nine endpoints showed significant differences between 15

the treatment and control responses (the significance of these responses may have been 16

overestimated due to removal of the block effect

, which reduced the error for testing)

. Thus, due 17

to the likely overestimation of significant responses, the results from the test with 18

4

- tert

- octylphenol generally should be interpret ed with caution (Kropp and Cullinan 2008)

. 19

A number of conclusions and recommendations based on the second set of 20

demonstrations studies are provided in Kropp and Cullinan (2008) and are discussed further in 21

Section 4.1.2

. 22

Table 4

- 7

. Results of the EPA demonstration study of the MTTT with 4

- tert

- Octylphenol

. 23

Endpoint F 0 (Parental) F 1 (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 Offs p ring (1st and 2nd broods)

- - - - ↓

- - - - na

Number reproducing females (1st brood)

- - - - ↓

- - - - na

Number reproducing females (2nd brood)

- - - - ↓

- - - - na

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 24

[na] Not assessed due to limited number of offspring. 25

[↑] Indicates a statistically significant increase compared to control, based on Dunnett's test (α = 0.05). 26

[↓] Indicates a statistically significant decrease compared to control, based on Dunnett's test (α = 0.05). 27Invertebrate Two

- Generation ISR 35 May 2 8 , 2013

4.1.2 Optimization S tud ies U sing the 2004 Draft G uideline 1

A third evaluation of the testing procedures an d methods proposed by USEPA to OECD 2

in the draft guideline for a two

- generation mysid test to evaluate suspected endocrine

- disrupting 3

chemicals (OECD 2004 a ) was conducted through an optimization study with four chemicals: 4

lindane (vertebrate aromatase inhi bitor) , prochloraz (vertebrate aromatase inhibitor) , atrazine 5

(weak vertebrate androgen and estrogen receptor agonist) , and perfluorodecanoic acid (interacts 6

with thyroid homeostasis) ( Kropp et al. 2009)

. The protocol used in the optimization study 7

differed from the 2004 proposed guideline in eight ways: 8

1. The Artemia nauplii were not from a reference source (sensu Léger and 9

Sorgeloos 1984), but were from a reliable source that has been successful in previous 10

studies 11

2. Mysids during the definitive test were fed ad libitum Artemia nauplii enriched with a 12

mixture of commercially available products (e.g., Salt Creek HUFA Enrich, 13

AlgaMac

- 3000, and AlgaMac

- Enhance) 14

3. Self

- starting siphons were not used as part of the exposure system design 15

4. The retention baskets were prepared with a slightly larger mesh, 355

- μm mesh 16

instead of the 210 to 330

- μm mesh specified by the guideline 17

5. The dry weights of the mysids were recorded to the nearest 1 μg instead of 0.1 μg 18

6. Instead of pairing females with the largest sus pected males, all mysids except for the 19

obviously ovigerous females were isolated and sex and maturity (i.e., male, 20

non

- ovigerous female, or immature) were confirmed with the aid of a dissecting 21

microscope 22

7. The F 0

- mysid exposure was initiated with at least 40 post

- larval mysids per replicate, 23

where each of the two retention baskets contained at least 20 post

- larval mysids 24

8. The number of F 0

- mysid brood pairs was targeted to be 10. 25

26

Items 1 through 6 were previously utilized in the demonstration studies with 27

ketoconazole and 4

- tert

- octylphenol and were shown to be test design improvements

. Items 7 28

and 8 were not used during the demonstration studies but were incorporated into the optimization 29

study to facilitate initiation of the F 1

- mysid exposure. 30

Lindane 31

Detailed results of the optimization study with lindane are presented in Hicks ( 2009a ) and 32

summarized here. 33

A 21

- day, flow

- through, range

- finding test was conducted at nominal concentrations of 34

0 (dilution

- water control), 0.19, 0.38, 0.75, 1.5, and 3.0 μg a.i ./L

. The estimated LC50 values for 35

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 36

indicator of a concentration

- dependent response during the range

- finding test

. The highest 37

definitive test concentration sel ected for the lindane definitive exposure was one fifth the 38

96

- hour LC50 for the range

- finding test (i.e ., 0.4 μ g/L)

. Nominal concentrations were 0 (control), 39

0.025, 0.05, 0.10, 0.20, and 0.4 μg a.i./L

. Analytical measurements of lindane made during the 40

st udy decreased during the course of the study (measured concentrations were 0.0182, 0.0375, 41

0.0808, 0.159, and 0.327 μg/L)

. Reasons for this decline in concentrations within dose levels 42

were not identified

. All test acceptability criteria were met

. 43Invertebrate Two

- Generation ISR 36 May 2 8 , 2013

The optimization study results for lindane are presented in Table 4

- 8

. All of the variables 1

evaluated during the lindane study were nonresponsive (signal

- to

- noise rati os <2)

. Statistical 2

analyses of the endpoints based on the Kruskal

- Wallis and Dunn’s tests showed that there w ere 3

few significant difference s between treatment mean responses and control mean responses : the 4

F 0 ( F 0 14

- day Male Length) was less than controls at 0.4 μg/L, the F 1 generation ( F 1 interbrood 5

duration) was longer th a n controls at 0.4 μg/L, and the total number of young per F 1 females was 6

greater than controls at 0.20 μg/L

. 7

Table 4

- 8

. Results of the EPA optimization study of the MTTT with lindane

. 8

Endpoint F 0 (Parental) F 1 (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 Offs p ring (1st and 2nd broods)

- - - - - - - - ↑

- Number reproducing females (1st brood)

- - - - - - - - - - Number reproducing females (2nd brood)

- - - - - - - - - -

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 9

[nr] Not reported for the F 0 generation. 10

[↑] Indicates a statistically significant increase compared to control, based on Kruskal

- Wallis/Dunn's test (α = 0.05). 11

[↓] Indicates a statistically significant decrease co mpared to control, based on Kruskal

- Wallis/Dunn's test (α = 0.05). 12

Prochloraz 13

Detailed results of the optimization study with prochloraz are presented in Hicks ( 2009 c ) 14

and are summarized here. 15

The highest definitive test concentration selected for the proc hloraz exposure was based 16

on the results of the range

- finding test and definitive exposure conducted as part of the 17

demonstration study ( see Section 4.1.1 )

. The highest prochloraz concentration tested during the 18

demonstration study was about one

- third the 14

- day LC50 (100 μg/L), but this was increased to 19

200 μg/L for the demonstration study

. An alytical measurements of prochloraz made during the 20

study generally were stable and there was no overlap among exposure concentrations during the 21

study

. Average measured concentrations were ≥90% of the nominal values for each dose level

. 22

All test acceptabi lity criteria were met

. 23

Results from the prochloraz optimization test are presented in Table 4

- 9

. Two 24

F 0

- generation variables evaluated during the prochloraz study, n umber of days to first and second 25

brood, were responsive (signal

- to

- noise ratios >2)

. Four F 1

- generation variables, number of days 26

to the first and second brood, interbrood duration, and total young in the second brood, were 27

determined to be responsive

. Statistical analyses of the endpoints was based on the 28

Kruskal

- Wallis and Dunn’s tests . 29Invertebrate Two

- Generation ISR 37 May 2 8 , 2013

Table 4

- 9

. Results of the EPA optimization study of the MTTT with prochloraz

. 1

Endpoint F 0 (Parental) F 1 (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 Offs p ring (1st and 2nd broods)

- - - - - - - - - - Number reproducing females (1st brood)

- - - - - - - - - - Number reproducing females (2nd brood)

- - - - - - - - - -

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 2

[nr] Not reported for the F 0 generation. 3

[↑] Indicates a statistically significant increase compared to control, based on Kruskal

- Wallis/Dunn's test (α = 0.05). 4

[↓] Indicates a statistically significant decrease compared to control, based on Kruskal

- Wallis/Dunn's test (α = 0.05). 5

Atrazine 6

Detailed results of th e optimization study with atrazine are presented in Hicks ( 2009b ) 7

and are summarized here. 8

The highest definitive test concentration selected for the atrazine definitive exposure was 9

one

- fifth the 96

- hour LC50 from a range

- finding test

. Survival was the mo st consistent indicator 10

of a concentration

- dependent response during the atrazine range

- finding test, but the number of 11

young per female also appeared to be a consistent indicator of a concentration

- dependent 12

response

. Analytical measurements of atrazine made during the study were stable and there was 13

no overlap among exposure c oncentrations during the study

. Average measured concentrations 14

were ≥90% of the nominal values for each dose level

. All test acceptability criteria were met

. 15

Results from the atra zine optimization test are presented in Table 4

- 10

. One variable 16

evaluated during the atrazine study, F 1 total number of young, was responsive (signal

- to

- noise 17

ratios >2)

. Statistical analyses of the endpoints based on the Kruskal

- Wallis and Dunn’s tests 18

showed that there were no significant differences between treatment mean responses and control 19

mean responses

. 20

Perfluorodecanoic acid (PFDA) 21

Detailed results of the op timization study with PFDA are presented in Hicks (2009 a ) and 22

are summarized here. 23

The highest concentration selected for the PFDA definitive exposure was based on these 24

range

- finding test results: 1) the effects observed on number of young per female wher e a 61% 25

reduction occurred at the 0.65 μg/L nominal treatment and a 36% reduction at the 0.10 μg/L 26

nominal treatment; and 2) the measured concentrations were 16% of nominal in the 0.10 μg/L 27

treatment and 51% of nominal in the 1.3 μg/L treatment . The highest PFDA definitive test 28Invertebrate Two

- Generation ISR 38 May 2 8 , 2013

nominal concentration selected by interpolating from the range

- finding test measured 1

concentrations was approximately one

- hundredth the 96

- hour LC50, or 50 μg/L, with four lower 2

concentrations arranged in a geometric seri es and a separation factor of approximately 2.5 to 3

bracket any biological effect. Analytical measurements of PFDA made during the study were 4

highly variable (CVs > 17%). There was no overlap among exposure concentrations during the 5

study, but the average c oncentrations of all dose levels were 56% to 67% of the nominal values. 6

All test acceptability criteria were met. 7

Table 4

- 10

. Results of the EPA optimization study of the MTTT with atrazine

. 8

Endpoint F 0 (Parental) F 1 (1st Gen eration)

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 Offs p ring (1st and 2nd broods)

- - - - - - - - - -

Number reproducing females (1st brood)

- - - - - - - - - - Number reproducing females (2nd brood)

- - - - - - - - - -

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 9

[nr] Not reported for the F 0 generation. 10

[↑] Indicates a statistically significant increase compared to control, based on Kruskal

- Wallis/Dunn's test (α = 0.05). 11

[↓] Indicates a statistically significant decrease compared to control, based on Kruskal

- Wallis/Dunn's test (α = 12

0.05). 13

Results from the PFDA optimization test are presented in Table 4

- 11

. All of the variables 14

evaluated during the PFDA study were nonresponsive (signal

- to

- noise ratios <2 )

. Statistical 15

analyses of the endpoints [conducted by Kropp and Cullinan (2009)] based on the 16

Kruskal

- Wallis and Dunn’s tests (of grouped treatment mean responses and control mean 17

responses) showed only two significant differences ( F 0 14

- day Female Weight and F 0 Days to 18

First Brood). No statistical differences were noted among individual treatment groups and 19

control groups for any endpoint. 20

4.1.3 Lessons Learned from Demonstration and Optimization Studies Using the 2004 21

Draft MTTT Guideline 22

A number of recommend ed modifications to the 2004 draft MTTT guideline were 23

suggested by Kropp and Cullinan (2008 ) and Kropp et al. ( 2009) based on the results of the 24

demonstration and optimization studies discussed in the previous sections, and they are repeated 25

below. These recommendations were considered when modifying the 2004 proposed OECD 26

protocol and draft the recommended MTTT guideline presented in Section 3.1

. 27Invertebrate Two

- Generation ISR 39 May 2 8 , 2013

Table 4

- 11

. Results of the EPA optimization study of the MTTT with perfluorodecanoic acid

. 1

Endpoint F 0 (Parental) F 1 (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 Offs p ring (1st and 2nd broods)

- - - - - - - - - - Number reproducing females (1st brood)

- - - - - - - - - - Number reproducing females (2nd brood)

- - - - - - - - - -

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 2

[nr] Not reported for the F 0 generation. 3

[*] Statistical analyses of these endpoints based on the Kruskal

- Wallis and Dunn’s tests (α = 0.05) showed that 4

there were significant differences between all grouped treatment mean responses and control mean responses 5

(F 0 14

- day Female Weight and F 0 Days to First Brood). 6

[↑] Indicates a statistically significant increase compared to control, based on Kruskal

- Wallis/Dunn's test (α = 0.05). 7

[↓] Indicates a statistically significant decrease compared to control, based on Kruskal

- Wallis/Dunn's test (α = 8

0.05). 9

10

Study design and statistical guidance 11

Statistical guidance provided by the 2004 draft MTTT guideline is minimal, essentially 12

only describing use of ANOVA or Kruskal

- Wallis tests and recommending that the laboratory 13

include a s tatistician in the process

. There is no guidance about the appropriate experimental 14

design (randomized versus blocked) th at should be used

. The correct statistical comparisons 15

when a vehicle control is required are also not addressed

. The lack of guidance could cause 16

confusion over the correct study design, the use of one

- tailed or two

- tailed tests, and whether the 17

treatment response should be compared with the dilution

- water or vehicle controls for tests that 18

employed both. 19

The design of the exposure experiment should be a completely randomized design unless 20

blocking is required

. In the case of the latter, blocks should be c haracterized by the 21

environmental qualities that make them different and relevant to the experiment

. Blocking makes 22

the analysis of the data more difficult, especially when a nonparametric analysis is required, and 23

provides a potentially underestimated var iance for testing when the blocks are artificial

. A 24

completely randomized design allows statistical comparisons to be run easily, whereas a block 25

design requires the fitting of a generalized linear model and more statistical expertise

. In all cases, 26

it is important to recognize that the replicates are the treatment tanks, not the individual retention 27

baskets within each tank

. Thus, the analyses should be conducted on the mean responses of the 28

treatment tanks. 29

Either the Kruskal

- Wallis, followed by the Dunn’ s test, or the Dunnett’s test is 30

appropriate to test the treatment responses under this proposed protocol

. One advantage of using 31

the Kruskal

- Wallis is that potential non

- monotonic effects can be evaluated

. When a vehicle 32Invertebrate Two

- Generation ISR 40 May 2 8 , 2013

control is used, it is appropriate to compare the vehicle

- control response to the dilution

- water 1

control response. However, the dilution

- water control is used to evaluate the acceptability of the 2

test and is not used to evaluate the treatment responses

. The treatment responses should be 3

ev aluated by comparing them to the vehicle

- control responses. 4

Self

- starting S iphon 5

All demonstration and optimization studies, for which the use of the self

- starting siphon 6

was discontinued, were completed successfully with no deterioration of water quality

. Thus, a 7

simple overflow drain or similar system that reduces the likelihood of impingement is 8

recommended as a modification of the proposed guideline

. If reference to a self

- starting siphon 9

remains in the guideline, a note of caution that the siphon like ly will increase impingement 10

should be added. 11

Feeding 12

Th e demonstration study comparisons of the control responses under the different feeding 13

regimes strongly suggested that mysids should be fed brine shrimp nauplii ad libitum (Kropp and 14

Cullinan 2008)

. T he comparisons further suggested that the brine shrimp should be enhanced by 15

exposing them to supplements that provide additional highly unsaturated fatty acids (HUFA), 16

especially docosahexaenoic acid (DHA), and eicosapentaenoic acid (EpA), both of which a re 17

relatively poor in natural strains of Artemia (Hanaee et al. 2005)

. Hanaee et al. (2005) found that 18

enriching Artemia cysts increased the HUFA content about 1.6 times and the DHA and EpA 19

content by 1.2 and 84 times, respectively

. The four demonstration studies described earlier 20

included a diet of Artemia nauplii that had been enriched with Lifeline Natural Health 21

Enrichment HUFA, AlgaMac

- 3000, and AlgaMac

- Enhance

. The addition of these or similar 22

supplements to the brine shrimp nauplii is a simple and co st

- effective way to help ensure that the 23

mysid diet is not a confounding factor during the evaluation of potential EDCs. 24

Initial T

est P opulation S ize 25

The size of the F 0

- mysid population used at test initiation during the optimization studies 26

was increased versus that specified in the proposed guideline and used during the demonstration 27

studies

. The number of F 0 mysids used to initiate the optimization study exposures was 20 per 28

retention basket, 5 more than the guideline

- specified number

. The targeted numbe r of F 0 mysid 29

pairs for the demonstration study exposures was 10 per replicate, three pairs more than the 30

guidelines

- specified number

. The larger initial F 0 population increased the population of the F 0 31

mysid generation available for pairing

. The increased number of F 0

- mysid pairs improves the 32

probability that a larger population of F 1 mysid would be available to initiate the F 1

- mysid 33

reproduction and growth retention baskets within a period of two days

. Therefore, F 1 mysids in 34

the growth retent ion baskets were available to supplement the F 1

- mysid pairing procedure, if 35

necessary, because the age of both sets of mysids was from 12

- to 16

- days old

. 36

It is recommended that the population size of the F 0 mysids be increased in each replicate 37

to 20 and the target number of F 0 mysid pairs to 10

. If the initial F 0 population is not increased, or 38

the increased population is not sufficient during some tests (i.e., reproduction is reduced in 39

response to the chemical being studied), the population of F 1 mysid s available for the pairing 40

procedure may not be sufficient to achieve the targeted number of seven F 1

- mysid pairs. 41Invertebrate Two

- Generation ISR 41 May 2 8 , 2013

Endpoint Clarification 1

Many endpoints are described in the 2004 draft MTTT guideline only by a simple 2

sentence or phrase and may require additional information to allow consistent determinations to 3

be made across testing laboratories

. A primary example concerns the treatment of mortality that 4

may arise during the testing, but that might not be exposure

- related

. For example, guidance could 5

b e included that directs a laboratory not to include mortality from impingement in the survival 6

endpoint

. Other examples are described in the following sub sections. 7

Dry Weight 8

The 2004 draft MTTT guideline provides considerable detail directing how the weig ht 9

endpoint is to be obtained, which is appropriate

. However, it is recommended that the 10

requirement to measure weight to the “nearest 0.1 μg” be changed to the “nearest 1 μg.” The 11

guideline instructions for dry weight measurements should be expanded to s pecify that weights 12

of males, females, and immature individuals should be measured and recorded

. Data analyses 13

should then be conducted for each gender. 14

Dead Young 15

Direction is needed to specify how dead young observed in broods produced by F 0 and F 1 16

mysids should be treated

. The guideline should specify that the occurrence and developmental 17

state of dead young could be determined and recorded, but should not be included in counts of 18

young per brood even if well

- developed. 19

Study Day 20

The Study Day selected to track ALL F 1

- brood pairs during the demonstration studies 21

was the age of the youngest mysid on the day following pairing, which may have been 1 to 2 22

days younger than the oldest

. However, during the optimization studies , the track ing of ALL 23

F 1

- brood pairs used a continuation of the day following initiation of the test, where study day 0 24

was test initiation (i.e., start of F 0

- mysid exposure)

. Criteria for the selection of the first study 25

day for F 1 mysids following pairing need to b e considered and defined in future guidance 26

documents

. The tracking of each F 1

- mysid brood cup individually would add adversely to the 27

complexity of the study and data evaluation/interpretation. 28

F

0 and F

1 Exposure Duration 29

The 2004 draft MTTT guideline does not clearly define the F 0 and F 1 exposure durations, 30

or state that a brood pair can be terminated at the time of second brood release

. If the intention of 31

the test design is to collect only data up to and including the day of second brood release by a 32

given brood pair, the guideline should clearly state that the collection of survival and 33

reproduction data beyond the day of second brood release is not required

. Therefore, an adult 34

pair could be t erminated at the time of second brood release or at 7 da ys after the median day of 35

second brood release in the control treatment

. The guideline also should define how control

36

treatment mysids without a mate (i.e., one of the two adults has died) are treated for calculation 37

of the median day of second brood rele ase and when treatment

- exposed mysids without a mate 38

and not producing a second brood are terminated. 39

Total Young Reproduction Data 40

The 2004 draft MTTT guideline describes the total young endpoint as the “Number of 41

young per female in each of the first two broods ( F 0 , F 1 )” but provides no further guidance about 42Invertebrate Two

- Generation ISR 42 May 2 8 , 2013

how this number is calculated

. Throughout the guidance document, number of young is 1

expressed as young per female, but in this case it is not

. During the optimization study, total 2

young was evaluated as number per female, but the total was calculated two ways

. For the first 3

calculation, the reproduction data for all females were treated equally (i.e., reproduction data for 4

females that die before producing a second brood or f emales not producing a second brood will 5

not be omitted)

. For the second, only the reproduction data for females that produced a second 6

brood were included

. Both sets of calculations were evaluated statistically and the results 7

occasionally differed

. The g uideline should clarify which approach meets the goals of the testing 8

so that the endpoint will be evaluated consistently by various testing laboratories. 9

Time to Maturation 10

The 2004 draft MTTT guideline lists Time to maturation, appearance of marsupial po uch 11

( F 0 , F 1 )” as one of the primary data types to be collected

. During the optimization studies , these 12

data were collected and reported, but they were not evaluated statistically

. This endpoint is 13

somewhat redundant with the time to first brood, which prob ably is a better indication of any 14

delay in reproduction caused by exposure to a test chemical

. It is recommend that Time to 15

maturation be dropped as an endpoint. 16

F

0 and F

1 Mysid Survival 17

Mysid survival was evaluated at multiple time

- points during the demonstration and 18

optimization studies

. The time

- points were days 14 (or the day of pairing) and 28 (or test 19

termination) for both generations

. However, the 2004 draft MTTT guideline does not clearly 20

identify how survival should be evaluated, which could c ause confusion about the point(s) at 21

which survival is to be evaluated. Several factors may complicate interpretation: 22

The number of mysids surviving at pairing day may be fewer than the number surviving 23

at termination because mysids from the growth basket have been used to achieve the desired 24

number of pairs (e.g., 11 mysids were present in a reproduction basket on day 13 but after 25

pairing there were 14 mysids present because three mysids from the growth basket were used to 26

obtain the targeted number of pa irs )

. In this case, evaluation of survival of all replicates and 27

treatments at the earliest day of pairing and again at termination of the mysid generation allows 28

survival to be evaluated at two time points. This method accounts for differences in mysid 29

po pulations at the start of each survival period. 30

Pairing of replicate(s) or treatment(s) mysids could occur on different days, particularly 31

for F 1 mysids

. For example, during a study, 7 groups may be paired 13 days after initiation, 32

whereas three groups may be paired 14 days after initiation

. During the demonstration and 33

optimization studies, survival on the pairing day achieved by most mysids, day 13 for the 34

example provided, was evaluated. 35

If the proposed guideline is revised so that pairs can be terminated as soon as they release 36

their 2nd brood, the guideline should specify whether survival in this case should be treated as 37

survival at termination or as survival at a particular time point (i.e., Day 28). 38

Reproduction Output as Number of Offspring /Female/Day 39

The 2004 draft MTTT guideline states that “Daily assessment of survival throughout the 40

test is necessary to provide a basis for expression and interpretation of reproductive output, that 41

is, number of offspring/female/d.” The value of expressi ng and interpreting reproductive output 42

as ” daily number of young per female” is not clear

. ”Daily” reproductive output does not truly 43Invertebrate Two

- Generation ISR 43 May 2 8 , 2013

reflect mysid reproduction cycles and therefore may not be appropriate

. An appropriate measure 1

of reproductive output is already obtained by scoring and evaluating the number of young per 2

female in each of the first two broods

. Therefore, the requirement for expression and 3

interpretation of reproductive output as number of offspring/female/day could be eliminated. 4

Dose Deter

mination and Power 5

The power of a test is defined as the probability of correctly rejecting the null hypothesis 6

(H 0 )

. Achieved statistical power is a function of the coefficient of variation (CV) of the 7

measured response, the number of replicates, and the percentage difference in the mean 8

responses observed. In general, sets of observations with large CVs have little power to detect 9

differences between means that differ by less than 20%

. An increase in the number of replicates 10

used during a test is required to either increase the power of the test for a given level of detection 11

or to decrease the difference between means that is to be detected for a given power 12

(Snedecor and Cochran 1980). The CVs observed during the optimization studies for most of the 13

cont rol data were less than 20% (Kropp et al. 2009)

. If a 10% difference between the control and 14

treatment means had occurred, the F 0 and F 1 measures of length and time to each brood would 15

have had a power greater than 80% for each chemical

. If a 20% differenc e between the control 16

and treatment means would have occurred, it would have provided at least 80% power for the 17

additional measures of the number of young for the F 0 and F 1 generations for each chemical

. 18

However, most of the percentage differences that were observed were less than 10% 19

(Kropp et al. 2009)

. The number of rep licates can be increased to detect smaller percentage 20

differences between the control and treatment means

. However, these smaller differences may 21

not be biologically relevant

. Also, an increase in the number of replicates increases the cost of 22

the laboratory element of the study

. The intent of the study should balance the power with the 23

ability to detect biologically relevant differences

. T he chemical dosing should be selected to 24

achieve relevant treatment

- related differences. 25

The general lack of treatment

- related differences during the optimization study could 26

have resulted from selected doses that were too low to bracket any biological effect that may 27

have been suggested by the range

- fi nding tests

. These results indicate the difficulties 28

encountered at selecting definitive test concentrations for a two

- generation test when the 29

objective is to establish a no

- observed and lowest

- observed effect concentration for both 30

generations

. The maxim um chemical concentrations used in these studies were calculated as 31

percentages of the LC50 (depending on the chemical, one

- hundredth to one

- third of the LC50) 32

estimated in the range

- finding test

. However, during the definitive exposures, the minimum 33

survival for both generations across all chemicals was 88%

. During the range

- finding tests for 34

the demonstration study , only the highest doses had an effect greater than 50% mortality

. Higher 35

chemical concentrations during this study could have created lar ger percentage differences with 36

only a slight increase in mortality

. The usefulness of a 96

- hour LC50 percentage (i.e., one

- fifth) 37

to select the highest definitive exposure concentration is questionable and may need to be further 38

supported by a longer

- term range

- finding test (e.g., 14 to 28 days)

. Setting a level of acceptable 39

mortality after 14 days of exposure at 20%, for example, and estimating the associated LC20 as 40

the maximum dose in the definitive test may be a better procedure for determining the ex posure 41

series

. This would not guarantee but should increase the likelihood of observing greater, and 42

biologically relevant, percentage differences between means in the two

- generation testing 43

scenarios. 44Invertebrate Two

- Generation ISR 44 May 2 8 , 2013



### 4.2 Proposed Harpacticoid Copepod Development and Reproduc tion Test 1



### Protocol P erformance 2

A copepod life

- cycle toxicity test protocol was previously established for Amphiascus 3

tenuiremis by the American Society for Testing and Materials (ASTM E2317

- 04)

. In addition, a 4

draft guideline w as proposed to OECD in 2004 t o evaluate effects of chemicals on A. tenuiremis 5

(OECD 2007)

. This draft guideline was used in two rounds of OECD validation studies as 6

described below

. In addition, numerous articles have been published based on the draft guideline 7

as described below

. Given that A. tenuiremis was the only species that was retained for the 8

second OECD interlaboratory validation, further details regarding the performance of lifecycle 9

tests with other calanoid and harpaticoid species are not summarized here, but have been 10

described in detail elsewhere ( e.g., Gourmelon and Ahtiainen 2007; Kusk and 11

Wollenberger 2007; OECD 2006; OECD 2007; OECD 2011 and references therein)

. 12

4.2.1 Demonstration and O ptimization S tudies U sing P roposed HCDRT P rotocol 13

Fipronil 14

A series of studies were c onducted using the A. tenuiremis life

- cycle test with the GABA

15

inhibitor insecticide fipronil (Cary et al. 2004; Chandler et al. 2004a,b)

. Chandler et al. (2004a) 16

reported on the development and demonstration of the microplate

- based life

- cycle developmenta l 17

and reproduction assay

. In the demonstration test, nominal concentrations of fipronil (0, 0.22, 18

0.36, and 0.60 μg/L) were prepared in seawater and copepods were exposed throughout the 19

embryo, nauplius, copepodite, and adult stages (16 to 17 days egg to e gg)

. Measured 20

concentrations (0.16, 0.22, and 0.42 μg/L) were within 68% of nominal and water quality 21

parameters met standard test guidelines

. 22

Results from the fipronil study are presented in Table 4

- 12

. Copepod survival was > 90% 23

during the test, and concentrations of ≥0.22 μg/L significantly delayed development from 24

copepodite to adult

. Reduced egg extrusion and reproductive failure were si gnificantly at 25

≥0. 22 μg/L fipronil

. A three

- generation population growth model also indicated a decline in 26

population size relative to controls at 0.16 μg/L (Chandler et al. 2004a)

. 27

Table 4

- 12

. R esults of the Chandler et al. demonstration study of the HCDRT with fipronil

. 28

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 ↓ ↓ ↓

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 29

[↑] Indicates a statistically significant increase compared to control. 30

[↓] Indicates a statistically significant decrease compared to control. 31

This study demonstrated that A. tenuiremis could be cultured and reared and development 32

and reproduction could be assessed using the life

- cycle assay

. Additional studies successfully 33

applied this life

- cycle test to evaluate population growth following exposure to fipronil and its 34Invertebrate Two

- Generation ISR 45 May 2 8 , 2013

degradation products (Chandler et al. 2004b) and to evaluate sex

- linked reproductive effects 1

(Cary et al. 2004)

. In another follow

- up study, this life

- cycle test was used to demonstrate the 2

effect of dissolved organic matter (DOM) on the toxicity of fipronil to A. tenuiremis (Bejar a no et 3

al. 2005)

. In this study, the presence of DOM enhanced the acute toxicity of fipronil and 4

mitigated the acute and chronic effects of chloro th a lonil and chlorpyrifos

. Thus, the authors 5

suggested that seawater

- only exposure (without DOM) may overestimate acute and chronic 6

pesticide effects under realistic conditions. 7

Atrazine 8

The A. tenuiremis life

- cycle test (2.5 generations) was conducted with nominal 9

concentrations of 0, 2.5, 25, and 250 μg/L of the triazine herbicide atrazine (Bejarano and 10

Chandler 2003)

. Measured conce ntrations were <0.1, 3.5, 30.3, and 246.6 μg/L and average 11

water quality parameters were pH = 8.25, salinity 30‰, and dissolved oxygen = 97%

. Survival 12

rates for all treatments was ≥ 95%

. 13

Results from the atrazine study are presented in Table 4

- 13

. No significant effects were 14

observed in the F 0 generation on development , sex ratios, hatching success, reproduction , and 15

malformation following atrazine exposure

. The number of viable offspring per female ( F 0 ) was 16

significantly reduced at 250 μg/L and estimated population production (using the Leslie matrix 17

model) was significantly reduced at 25 and 250 μg/L

. No significant effects were observed for F 1 18

survival, development, sex ratio, or hatching success

. However, the number of viable offspring, 19

malformations, and population production were affected at all test concentrations

. In addition, 20

reproductive failure ( F 1 ) was observed at 25 and 250 μg/L

. The results of the study pro vided a 21

successful demonstration of the copepod life

- cycle test and allowed for population growth 22

modeling. 23

Table 4

- 13

. R esults of the Bejarano and Chandler demonstration study of the HCDRT with 24

atrazine. 25

Endpoint F 0 (Parental) F 1 (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 ra t io

- - - - - - Developmental time

- - - - - - Hatching success

- - - - - - Viable offspring per female

- - ↓ ↓ ↓ ↓

Reproductive failure

- - - - ↑ ↑

Copepod malformations

- - - ↑ ↑ ↑

Estimated population production

- ↓ ↓ ↓ ↓ ↓

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 26

[↑] Indicates a statistically significant increase compared to control. 27

[↓] Indicates a statistically significant decrease compared to control. 28

3,5

- dichlorophenol (DCP) 29

The OECD (2 007) performed a prevalidation study following draft test guidelines using 30

two harpacticoid copepods ( N. spinipes and A. tenuiremis ) and one calanoid copepod ( A. tonsa )

. 31

Multiple laboratories conducted the developmental and reproduction test using 3,5

- DCP to 32

evaluate the performance of the test methods and sensitivity of the test specie s

. Nominal 33

3,5

- DCP concentrations were 0, 6.6, 20, 60, 180, and 540 μg/L

. 34Invertebrate Two

- Generation ISR 46 May 2 8 , 2013

Results from the DCP study are presented in Table 4

- 14

. In the developmental phase of 1

the harpacticoid life

- cycle test, mortality, development rate

, and sex ratio were assessed

. The 2

developmental data were consistent among the laboratories

, indicating lowest observed effect ive 3

concentrations (LOECs) between 6.6 and 20 μg/L

. The reproduction data were also consistent for 4

two of the three endpoints for the harpacti coid test with LOECs between 6.6 and 540 μg/L

. 5

Mortality, development, and reproduction were also m onitored for the calanoid copepod tests

. 6

Results for development and reproduction endpoints were variable (LOECs ranging from 6.6 to 7

540 μg/L) for the calanoid test and two labs found developmental endpoints to be most sensitive 8

while the third lab found r eproduction to be most sensitive

. One of the labs did not meet the 9

acceptability criteria for calanoid developmental data, limiting the comparability of the data. 10

Table 4

- 14

. R esults of the OECD demonstration study of the HCDR T with 3,5

- DCP. 11

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 ( F 0 ) DHI

- - - - ↓

DTU ↓

- - - ↓

OPUS ↓ ↓ ↓ ↓ ↓

Larval development ratio ( F 1 ) DHI

- - - ↓ ↓

DTU

- - - - ↓

OPUS

- ↓ ↓

- - Mean length* DHI

- - - ↑

- DTU

- - - - ↓

Mean egg production DHI ↑ ↑

- ↑

- DTU ↓ ↓ ↓ ↓ ↓

OPUS

- - - - ↓

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 12

[*] Endpoint not evaluated by one of the labs (IVL, BEI, or OPUS) 13

[↑] Indicates a statistically significant increase compared to control. 14

[↓] Indicates a statistically significant decrease compared to control. 15Invertebrate Two

- Generation ISR 47 May 2 8 , 2013

Following this series of tests, OECD (2007) provided several recommendations for future 1

development of copepod life

- cycle tests . The use of A. tenuiremis was recommended for the full 2

life

- cycle test due to its shorter testing time and better reproductive output and success, which 3

increases study power. The longer test duration necessary for N. spinipes and large 4

interlaboratory va riability for A. tonsa suggested that these species would be less practical 5

alternatives. In addition, the labs experienced difficulty with maintaining 3,5

- DCP 6

concentrations during the test, and thus recommended exploring other reference substances. 7

4.2.2 Lesso ns L earned from D emonstration and O ptimization S tudies U sing P roposed 8

HCDRT P rotocol 9

Several observations were provided following the demonstration and optimization studies 10

using multiple species of copepods (OECD 2007; Kusk and Wollenberger 2007)

. These 11

recommendations ultimately led to the decision to proceed with OECD inter

- laboratory 12

validation testing with only A. tenuiremis

. There were several key differences among the 13

copepod species tested that were recognized during the demonstration phases

. The test time to 14

run the harpacticoid full lifecycle test using N. spinipes was over 40 days, while the test time for 15

A. tenuiremis was approximately 25 days. This indicated that A. tenuiremis is more appropriate 16

to use for full life cycle testing to redu ce duration and costs (OECD 2007)

. The number of 17

replicates in the harpacticoid tests is high (at least 48 specimens per concentration)

, which allows 18

for strong statistical power (Kusk and Wollenberger 2007)

. In addition, the harpacticoid tests 19

focus on re production and development, while the calanoid test focuses more on development of 20

early life stages (Kusk and Wollenberger 2007)

. Demonstration tests also indicated that calanoid 21

tests were more variable than harpacticoid test s

. Finally, a standard ASTM m ethod was already 22

available to draw upon for the A. tenuiremis ( ASTM 2004 )

. Based on these factors, A. tenuiremis 23

was recommend for use in the development of a full life

- cycle test (OECD 2007)

. OECD (2011) 24

completed further interlaboratory studies with A. tenuiremis

, which are summarized in Section 5

. 25

26Invertebrate Two

- Generation ISR 48 May 2 8 , 2013



### 5 INTERLABORATORY EVAL UATION OF MYSID AND COPEPOD TE ST 1



### METHODS 2

Interlaboratory evaluation s of the MTTT and the H C D R T were conducted to : (1) evaluate 3

the value added of the MTTT over the exi sting mysid life cycle test; (2) compare the sensitivity 4

of the mysid and copepod multi

- gen methods; (3) evaluate the practical transferability of the 5

mysid and copepod multi

- gen methods; and (4) evaluate the quantitative reproducibility of the 6

mysid and c opepod multi

- gen methods

. 7

The following laboratories participat ed in the interlaboratory validation: 8

 Labs performing the MTTT: Smithers Viscient (SMV), University of North Texas 9

(UNT), and Wildlife International, Ltd

. (WLI). 10

 Labs performing the HCDRT: Block Environmental Services (BES), Brixham 11

Environmental Laboratory (BEL), and Centre for Environment, Fisheries & 12

Aquaculture Science (Cefas). 13

The following chemicals were used in the interlaboratory validation: lindane 14

( vertebrate aromatase inhibitor ) , vincloz ol in ( vertebrate androgen receptor antagonist) , and 15

4

- tert

- octylphenol (vertebrate estrogen receptor agonist)

. T he specifics for each study , as well as 16

the overall Quality Assurance Project Plan (QAPP) and Study Plans are described in the 17

individu al laboratory study report s , which are presented in Appendices C and D ( Block and 18

Bradbury 2012a,b; Hala and Garcia 2012 a,b ; Krueger et al. 2012; Le Page and Brown 2011; Lee 19

2012 ; Veauvy et al. 2012 )

. 20

Following completion of all studies, e ndpoint data were re

- analyzed by Battelle using a 21

common statistical analysis plan

. Results of this evaluation were reported in intra

- laboratory 22

analysis reports for each study ( Appendix G )

. The intra

- laboratory evaluation was composed of 23

two parts: (1) a comparison be tween the test chemical concentration groups and the concurrent 24

control groups; and (2) a comparison of the two generations to assess the benefits of a 25

two

- generation study as compared to a one

- generation life cycle study

. 26

In addition, an inter

- laboratory analysis Appendix C was conducted by Battelle and 27

consisted of three parts: (1) a comparison of the control group results to assess the range of 28

variation within studies, among studies within laboratories, and among laboratories; (2) a power 29

analyses base d on the control group results to potentially identify endpoints that can be detected 30

with high power; and (3) a comparison of the treatment group

- control group results for the key 31

parameters

. The inter

- laboratory analysis was carried out to answer a series of questions raised 32

by US EPA 33

1. I s the behavior of control values similar among labs? 34

2. A re dose

- response relationships similar among labs ? 35

3. F or the same chemical, is there value added by extending the test beyond one 36

generation ? 37

Results of the statistical re

- analysis are incorporated in to the following discussions of the 38

results of the MTTT test (i.e., results and statistical findings presented below are based on the 39

statistical re

- analysis of the data by Battelle [ Appendix C ] and differ, in some instances, from 40

statistical findings in the individual study reports)

. No statistical re

- analysis was conducted for 41Invertebrate Two

- Generation ISR 49 May 2 8 , 2013

the HCDRT studies

. As a result, t he HCDRT results are summarized from the original laborat ory 1

study reports

. 2



### 5.1 Lindane 3

Lindane, an organochlorine insecticide, was tested in three different laboratories using 4

the MTTT and the HCDRT with A. tenuiremis, for a total of six experiments

. The results of the 5

lindane inter

- validation study are described in individual laboratory study reports ( Appendices C 6

and D ) and are summarized below

. Results from the statistical re

- analysis by Battelle are 7

presented in Appendix C and are incorporated belo w

. 8

5.1.1 Exposure L evels 9

MTTT 10

Three laboratories participated in the lindane interlaboratory study of the MTTT : WLI, 11

SMV , and UNT

. Nominal l indane exposure concentrations for all laboratories were 0, 0.0 32, 12

0.0 8, 0.20, 0.5, a nd 1 .250 μg/L

. The mean measu red concentrations from each of the studies 13

ranged from 67 % to 147% of the nominal levels

. Measured concentrations were variable over the 14

test duration and generally greater than 70% of nominal concentrations

. Mean exposure 15

concentrations are presented in Table 5

- 1 and Table 5

- 2

. Temperature, dissolved oxygen 16

concentration, pH and salinity were measured daily in each experiment (each treatment and 17

control group) throughout the exposure period

. All water quality parameters were generally 18

within acceptable limits for mysid survival, growth, and reproduction, with minor exceptions for 19

water temperature at SMV and WLI

. These minor deviations from the guideline test conditions 20

did not negatively impact the results or interpretation of the studies

. 21

HCDRT 22

Three laboratories participated in the EPA

- led lindane interlaboratory study of the 23

HC DRT : BEL, Cefas, and BES

. Nominal l indane exposure concentrations were 0, 2.5, 5, 10, 15, 24

and 20 μg/L

. Time

- weighted mean measured concentrations were within acceptable limits and 25

ranged between 72 and 88% of nominal concentrations

. Mean exposure concentra tions are 26

presented in Table 5

- 3

. Water quality parameters were within the guideline acceptability criteria 27

(dissolved oxygen >70%, temperature 25 ± 2 o C., pH of 8.0 ± 0.4, and salinity of 30 ± 3‰) with 28

minor exceptions that were determined to not affect the test performance at any of the 29

laboratories. 30

In addition to the EPA

- led interlab oratory study, four laboratories participated in an 31

OECD interlaboratory evaluatio n of the HCDRT (OECD 2011)

. Since t he OECD method forms 32

the basis of the method evaluated by EPA, t he results from those experiments are presented here 33

as further validation of this protocol

. The four laboratories (USC, NOAA, Stockholm, and 34

Uppsala) e xamin ed the same nominal lindan e concentrations (0, 2.5, 5, 10, 15, and 20 μg/L)

. 35

Each of the laboratories experienced some difficulties with achieving the desired concentrations 36

due to l oss of lindane in solution (loss rates from 5 to 50%) or in the case of US C and Uppsala 37

exceeding the nominal concentrations by 50 to 150% for some treatments

. Measured 38

concentrations from each lab are presented in Table 5

- 4

. Most labs were able to maintain 39

physical parameters within guideline recommendations and requirements ; however, data were 40

not always recorded and reported consistently across labs. 41

42Invertebrate Two

- Generation ISR 50 May 2 8 , 2013

Table 5

- 1

. Survival and growth results of the EPA interlaboratory validation study of the 1

MTTT with lindane

. 2

Endpoint Laba F 0 (Parental) F 1 (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 ( F 1 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 ( F 1 only) SMV not applicable - - - - -

UNT

- - - - - Day 7 Length SMV

- - - - - - - - - ↓

UNT

- - - ↑ ↑

- - - ↗ ↗

Day 14 Length SMV ↑ ↑ ↑ ↑ ↑

- ↓ ↓

- -

UNT

- ↗ ↗ ↗ ↗

- ↓

- - - Termination Length SMV

- - - ↑

- - - ↓ ↑

-

UNT

- - - - - - - - - ↑

[a] Statistics were not performed (and data are not presented) for the results from WLI due to due to a lack of 3

organisms in the negative control group and due to insufficient numbers of offspring produced by the F 0 adults

. 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 re

8

analysis of test data (Battelle 2013). 9

[ ↗ ] Indicates a statistically significant increasing concentration trend (Linear trend test), bas ed on statistical re

10

analysis of test data (Battelle 2013). 11

[ ↘ ] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical re

12

analysis of test data (Battelle 2013). 13

14

15

16

17Invertebrate Two

- Generation ISR 51 May 2 8 , 2013

Table 5

- 2

. Reproduction results of the EPA interlaboratory validation study of the MTTT with 1

lindane

. 2

Endpoint Laba F 0 (Parental) F 1 (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

- - - - - - - - - -

[a] Statistics were not performed (and data are not presented) for the results from WLI due to due to a lack of 3

organisms in the negative control group and due to insufficient numbers of offspring produced by the F 0 adults. 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 re

8

analysis of test data (Battelle 2013). 9

[ ↗ ] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical re

10

analysis of test data (Battelle 2013). 11

[ ↘ ] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical re

12

analysis of test data (Battelle 2013). 13

14

15

16Invertebrate Two

- Generation ISR 52 May 2 8 , 2013

Table 5

- 3

. Results of the EPA interlaborat ory validation study of the HCDRT with lindane

. 1

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 ↓ ↓ ↓ ↓ ↓

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 2

[↑] Indicates a statistically significant increase compared to control. 3

[↓] Indicates a statistically significant decrease compared to control. 4

* The endpoint for dev elopment time to adult is considered unreliable because of the misidentification of 5

copepodites as adults as discussed in the report and therefore the overall NOEC and LOEC for this study has 6

been based on development to copepodite. 7

8

9Invertebrate Two

- Generation ISR 53 May 2 8 , 2013

Table 5

- 4

. Results of the OECD interlaboratory validation study of the HCDRT test with 1

lindane

. 2

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

-

[

- ] 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

[a] The mating success criterion of >70% was not met at the Uppsala lab. 7

[na] Not assessed

- population growth rate was only calculated for the 2.5, 10, and 20 μg/L treatments. 8

[nr] Not reported

– data for this parameter not reported in the OECD interlaboratory report. 9

5.1.2 Sensitive E

ndpoints 10

MTTT 11

All laboratories observed a number of sign ificant effects as indicated by the statistical 12

re

- analysis ( Appendix G )

, primarily in the F 0 generation (see Table 5

- 1 and Table 5

- 2 above )

. 13

C ontrol survival in the WLI study was below acceptability crite ria ; therefore these data were not 14

included in the statistical reanalysis by Battelle

. As a result, endpoint data as reported in the 15

original WLI study report are not summarized here

. 16

Survival endpoints w ere not significantly affected in the F 0 generation s

. A decreasing 17

trend (at the highest concentration) for r eproduction survival rate ( F 0 at termination ) was 18Invertebrate Two

- Generation ISR 54 May 2 8 , 2013

observed in the UNT test

. In the F 1 generation, significant decreases in reproduction survival rate 1

(day 7 and 14) and total survival rate (day 7 and 14) were found in the highest concentration 2

group in the UNT test

. Only terminal reproduction rate was found to be significant in the SMV 3

test with a decreasing trend for the highest concentration tested

. Several g rowth endpoints were 4

significantly increased as compared to the control in the F 0 generation but were variably 5

significantly increased or decreased as compared to the control in the F 1 generation ( Table 6

5

- 1 above )

. Fecundity endpoints (measured as the number of offspring) were variable among 7

UNT and SMV

, and consistent dose

- response relationships were not apparent

. Sex ratio was 8

unaffected at all test concentrations and in both genera tions

. A decreasing trend was observed for 9

the two highest concentration groups (UNT) for t ime to matur ity in the F 0 generation

. Time to 10

first and second brood were significantly reduced compared to the control in the F 0 generation in 11

several concentration groups at both SMV and UNT

. However, the F 1 time to first and second 12

brood increased compared to the control in several concentration groups at SMV and not at UNT

. 13

Overall, the most consistent endpoints in the l indane inter

- laboratory studies were the gro wth 14

endpoints (length)

. 15

A ll biological acceptability criteria were met during the tests at UNT and S M V

. At W LI

, 16

more than 75% of F 0 females produced young

. However, o nly 50% of the control F 1 females 17

produced young , even though more than 75% of F 1 females in two of the treatment groups 18

produced young

. Further, the F 1 generation in the controls did not produce an average combined 19

total number of young in th e first two broods ≥8 (i. e., seven were produced per female, and so 20

th e criterion was missed by 13% )

. Due to the lack of organisms in the WLI test, statistics were 21

not performed. 22

HCDRT 23

HCDRTs conducted by BES and Cefas met acceptability criteria ; however, the tests 24

conducted by BEL were considered unreliable

. BEL experienced practical difficulties carrying 25

out the copepod test due to issues with the identification of copepodite maturation and 26

determination of sex

. The poor reliability and problems regarding the quality of the data have 27

been acknowledged and for these reasons, particular care should be taken when drawing 28

conclusions from the BEL reproduction data

. 29

Copepod survival was significantly affected at 15 and 20 μg/L as reported by BEL and 30

BES, while no significant change in survival was noted by Cefas

. Lindane had a significant 31

inhibitory ef

fect on copepod development into both copepodite and adult life

- stages ( Table 5

- 3 32

above ). Specifically, lindane significantly increased the time to develop from the na upliar to the 33

copepodite life

- stages and also significantly increased the time necessary for exposed copepods 34

to mature into sexually mature adults (at concentrations as low as 2.5 μg/L)

. Sex ratio was not 35

significantly affected during any of the tests

. Li ndane was observed to have a significant effect 36

on copepod reproduction, with a reduction in the percentage of successful mating pairs and a 37

reduction in the total number of viable eggs and hatched nauplii

. These results were consistent in 38

the two labs wit h reliable results (BES and Cefas)

. The lowest concentration at which lindane 39

affected reproductions was 2.5 μg/L (nominal). 40

Biological results from the OECD interlab oratory study are presented in Table 5

- 4 above

. 41

Surv ival was significantly reduced at lindan e concentrations of 10, 15, and 20 μg/L at one or 42

more labs, respectively

. T hree of four participating laboratories reported consistent 43

concentration

- dependent delays in development rates, consistent naupliar

- specific mortality, 44Invertebrate Two

- Generation ISR 55 May 2 8 , 2013

consistent mating

- success depressions, and consistent reduction of fecundity (OECD, 2011)

. 1

Interlab oratory comparisons of lindane life table data using the Leslie matrix model 1 2

(Leslie 1945) produced consistent patterns of response at 10 and 20 μg/L

, indicating significant 3

reductions in population growth rates

. Results for time to first and second brood and number per 4

brood were not reported in the OECD interlaboratory study and thus are not comparable to the 5

EPA EDSP interlab oratory studies. 6

5.1.3 Conclusions 7

MTTT 8

The interlaboratory results for two of the three labs (UNT and SMV) were within 9

acceptable test method guidelines, while one lab experienced poor pe rformance in the F 1 10

generation (WLI)

. In general, labs demonstrated the ability to perform the test according to the 11

proposed method

. Lindane exposure significantly affected growth and reproduction in the mysid

, 12

primarily in the F 0 generation ; however, endpoint results were generally variable between 13

laboratories and between generations ( Table 5

- 1 and Table 5

- 2 above ). 14

HCDRT 15

The HCDRT interlaboratory validation study with lindane was conducted by seven 16

laboratories ( three in an EPA

- led study and four in an OECD

- led study )

. All laboratories 17

generally were able to perform the test within acceptable guidelines with some difficulties in 18

producing consistent lindane test concentrations, differentiating age differences for 19

deve lopmental endpoints, and achiev ing the desired mating success criterion

. Due to these issues, 20

OECD (2011) recommended that further work be conducted to address the proficiency

, 21

manpower, and t raining of laboratories performing the test, continue validation of the guideline 22

to identify preferred endpoints, and obtain additiona l information on the basic biology of the test 23

species

. However, despite some of the se difficulties, most labs detected delays in development 24

and reduced fecundity at the higher test concentrations ( Table 5

- 3 and Table 5

- 4 above ). 25

No consistent biological responses were observed between the MTTT and the HCDRT

. 26

T he test concentrations in the MTTT (highest test concentration of 1.25 μg /L) were all below 27

those tested in the HCDRT (lowest test concentration of 2.5 μg /L)

. 28



### 5.2 Atrazine 29

Atrazine was tested in three laboratories using only the HCDRT with A. tenuiremis , for a 30

total of three experiments

. The results of the atrazine inter laboratory validation study are 31

described i n OECD (2011) and summarized below. 32

Three laboratories participated in the OECD (2011) atrazine interlaboratory study of the 33

HCDRT : USC, NOAA, Stockholm

. Nominal atrazine exposure concentrations were 0, 26

, 43

, 72

, 34

120 , and 2 0 0 μg/L

. M easured concentrations were within 50 % of nominal concent rations

, and 35

atrazine was stable with relatively little loss from initial measured concentrations

. Mean 36

exposure concentrations are presented in Table 5

- 5

. Most labs were able to maintain physical 37

parameters within guideline recommendations and requirements ; however, physical and copepod 38

response data were not always recorded and reported consistently across labs. 39

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 2 8 , 2013 Table 5

- 5

. Results of the OECD interlaboratory validation study of the HCDRT with atrazine

. 1

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

- - - - -

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. Note that time 2

to 1st and 2nd brood and number of offspring in 1st and 2nd Brood were not reported. 3

[↑] Indicates a statistically significant increase compared to control. 4

[↓] Indicates a statistically significant decrease compared to control. 5

[nr] Not reported

– data for this parameter not reported in the OECD interlaboratory report. 6

5.2.1 Sensitive E

ndpoints 7

Control survi val was within the guideline criterion of >80% for all labs except the carrier 8

solution control from the Stockholm lab

. Stockholm attributed the normal

- control result to non

9

random selection of control nauplii at experimental set up, and low stock culture densities prior 10

to the beginning of the test

. Survival, development

, and reproduction results are presented in 11

Table 5

- 5

. No labs found concentration dependency or tr eatment

- specific significant differences 12

in mortality, development rate, sex ratios, or mating success at any test concentration or for any 13

life

- stage relative to controls (i.e., nauplius, copepodite, or adult)

. However, the number of viable 14

offspring prod uced through two clutches or broods at 120 and 200 ug/L atrazine were 15

significantly depressed rel ative to controls (USC and NOAA labs)

. All labs achieved >70% 16

mating pair success in controls except for Stockholm

, which achieved 44.2% success. Stockholm 17

nor mal control matings could not be completed because that set of microplates was accidentally 18

dropped prior to clutch extrusion. Solvent control matings that were successful by Stockholm 19

produced two

- brood clutch sizes of ~10 offspring

, which was close to th e mean response seen in 20

all atrazine treatments, and within the guideline two

- brood clutch

- size criterion of > 8 viable 21

offspring in controls

. 22

5.2.2 Conclusions 23

The HCDRT interlaboratory evaluation with atrazine was conducted by three laboratories

. 24

Two of three laboratories were able to perform the te st within acceptable guidelines

. T he 25DRAFT

Invertebrate Two

- Generation ISR 57 May 2 8 , 2013 biological results for most endpoints were consistent across labs and showed a 1

concentration

- dependent decrease in number of nauplii hatched over two clutches ( Table 5

- 5 ). 2



### 5.3 Vinclozolin 3

Vinclozolin was tested using the MTTT at UNT and the HCDRT at BES , for a total of 4

two experiments

. The results of the study are described in the laboratory test reports ( Hala and 5

Garcia 2012a; Block and Bradbury 2012b) and summarized below

. Results from the statistical 6

re

- analysis of the MTTT data produced at UNT by Battelle are presented in Appendix C and are 7

incorporated below. 8

5.3.1 Exposure L evels 9

MTTT 10

One laboratory (UNT) tested the effects of vinclozolin using the MTTT assay

. Nominal 11

test concentrations were 0, 25, 50

, 100, 200

, and 600 μg/L

. The time

- weighted average 12

concentrations of vinclozolin in the exposure tanks were < 0.00156 (control), 8.2, 24 .8, 37.3, 13

119. 5 and 402.5 μg /L ( Table 5

- 6 and Table 5

- 7 )

. The measured values were 33 to 67% of 14

nominal concentrations

. Water quality parameters were measured throughout the mysid test and 15

reported as: dissolved oxygen was ≥ 6.9 mg/L

, temperature rang ed from 25 to 27°C , pH ranged 16

from 7.8 to 8.5

, and light intensity ranged from 33 to 98 foot

- candles

. All water quality 17

parameters were within acceptable limits for mysid survival, growth and reproduction. 18

Table 5

- 6

. Survi val and growth results of the EPA validation study of the MTTT with 19

vinclozolin

. 20

Endpoint Lab F 0 (Parental) F 1 (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 (F 1 only) UNT not applicable

- - - - - Day 7 Dry Weight UNT

- - - - - - - - - - Day 14 Dry Weight UNT

- - - - ↓

- - - - ↓

Termination Dry Weight UNT

- - - - ↘

- - - - - Day 0 Length(F 1 only) UNT not applicable

- - - - ↗

Day 7 Length UNT

- - - - - - - - - - Day 14 Length UNT ↑

- ↑

- - - - - - - Termination Length UNT

- - - - - - ↗ ↗ ↗ ↗

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 21

[↑] Indicates a statistically significant increase compared to control (ANOVA F

- test), based on statistical re

22

analysis of test data (Battelle 2013). 23

[↓] Indicates a statistically significant decrease compared to control (ANOVA F

- test), based on statistical re

24

analysis of test data (Battelle 2013). 25

[ ↗ ] Indicates a statistically significant increasing concentration trend (Linear trend test), based on s tatistical re

26

analysis of test data (Battelle 2013). 27

[ ↘ ] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical re

28

analysis of test data (Battelle 2013). 29

30DRAFT

Invertebrate Two

- Generation ISR 58 May 2 8 , 2013 Table 5

- 7

. Rep roduction results of the EPA validation study of the MTTT with vinclozolin

. 1

Endpoint Lab F 0 (Parental) F 1 (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

- - - - ↑

- - - - -

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 2

[↑] Indicates a statistically significant increase compared to control (ANOVA F

- test), based on statistical re

- analysis 3

of test data (Battelle 2013). 4

[↓] Indicates a statistically significant decrease compared to control (ANOVA F

- test), based on statistical 5

re

- analysis of test data (Battelle 2013). 6

[ ↗ ] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical 7

re

- analysis of test data (Battelle 2013). 8

[ ↘ ] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical 9

re

- analysis of test data (Battelle 2013). 10

HCDRT 11

Vinclozolin concentrations of 12.96, 2 1.6, 36, 60, and 100 μg/L were tested in one 12

laboratory (BES)

. The chemical analysis of v inclozolin test concentrations showed that 13

concentrations of the test substance were all below the laboratory reporting limit ( Table 5

- 8 )

. A 14

review of the procedure for preparing the test solutions indicated that the solutions were prepared 15

from the primary stock solutions properly. Moreover, the primary stock solutions used to prepare 16

the test concentrations were measured to be within 10 percent of the expected concentration of 17

200 mg/L

. A review of the sample handling follow ing collection indicated th at samples were 18

submitted to the laboratory outside the recommended holding ti me

. These data suggest that test 19

chemical was likely lost through adsorption onto test chambers, sample storage bottles, as well as 20

through natural degradation processes prior to analysis. 21

5.3.2 Sensitive Endpoints 22

MTTT 23

The vinclozolin mysid two

- generation toxic ity test conducted by UNT met all control 24

acceptability criteria. Endpoint responses are summarized in Table 5

- 6 and Table 5

- 7 . Survival 25

endpoints for F 0 generation were generally not significantly different from the control (based on 26

ANOVA between group means), although some survival endpoints show ed a decreasing trend 27

for the highest concentration group (based on a linear trend test across treatment groups). 28

A significant reduction in survival was found at the second highest concentration for the F 1 29

generation for day 14 survival rate. Growth, meas ured as dry weight, was reduced compared to 30

the control in both generations. However , growth measured as length increased as compared to 31

the control in several exposure groups and in both generations ( Table 5

- 6 ). 32DRAFT

Invertebrate Two

- Generation ISR 59 May 2 8 , 2013 Table 5

- 8

. Results of the EPA validation study of the HCDRT with vinclozin

. 1

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

- - - - -

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 2

[↑] Indicates a statistically significant increase compared to control. 3

[↓] Indicates a statistically significant decrease compared to control. 4

The time to brood release showed an increasing trend in both generations in the highest 5

exposure groups

. Sex ratio (male/female) was significantly increased compared to the control in 6

the F 0 generation in the highest exposure group, but was unaffected in F 1

. A decreasing trend in 7

reproduction performance (% of females reproducing in the F 0 generation) was observed at the 8

highest concentration, but was unaffected in the F 1 generation

. Fecundity showed a decreasing 9

trend, which was most apparent for the highest concentration group in the F 1 generation ( Table 10

5

- 7 ). 11

HCDRT 12

All water quality and biological performance criteria were met for this study

. Vinclozolin 13

did not affect copepod survival at any exposure level by day 16 of the test, with the survival rate 14

not significantly reduced further by test termination on day 36

. Similarly, the developmental 15

endpoints tested were also not significantly affected, as vinclozolin did not significantly affect 16

the time for A. tenuiremis na uplii to mature into the copepodite stage and subsequent 17

transformation to the adult stage at test concentrations up to 100 μg/L

. Vinclozolin had no effect 18

on gender assignment for all concentrations tested

. Vinclozolin did, however, significantly delay 19

th e time for mated females to extrude their first brood at the 21.6 and 36 μg/L exposure levels, 20

although the time for females to extrude their second brood was not delayed at any exposure 21

level tested (see Table 5

- 8 )

. Vinclozolin did not significantly reduce or increase the number of 22

hatched offspring at any test concentration, although an appreciable decrease in hatched 23

offspring in the 100 μg/L treatment was noted

. La stly, with regards to reproductive endpoints, 24

fertilization success was not significantly affected for any test treatment when compared to the 25

carrier control

. An appreciable decrease (not significant) in the number of successful mating 26

pairs was noted in organisms exposed vinclozolin concentrations of 21.6, 36, and 60 μg/L. 27DRAFT

Invertebrate Two

- Generation ISR 60 May 2 8 , 2013 5.3.3 Conclusions 1

MTTT 2

The MTTT conducted by UNT was within acceptable test method guidelines

. Exposure 3

to vinclozolin significantly affected a number of growth (decreased weight, increased length) and 4

reproduction (increased sex ratio in F 0 , de creased fecundity in F 1 , decreased number of actively 5

reproducing females in F 0 , increased interbrood duration in F 1 ) parameters in both generations

, 6

whereas survival endpoints were generally not significantly affected (compared to controls)

. The 7

results indicate that UNT successfully conducted the MTTT test with minimal deviation from the 8

test guideline. 9

HCDRT 10

The HC DR T conducted by BES was within acceptable test method guidelines

. However, 11

since all measured vinclozo lin concentrations were below t h e laboratory reporting limit, results 12

were reported on a nominal basis

. Vinclozolin did not affect any of the copepod survival and 13

development endpoints at any exposure level

. However, since the true exp osure concentrations 14

are uncertain, these results should be interpreted with caution

. Of the measured reproduction 15

endpoints, only the time to first brood was significantly increased at 21.6 and 36 μg/L, but not at 16

higher exposure levels

. 17

Vinclozolin expo sure resulted in significant reproductive delay s (measured as time to 18

first brood release) at some treatment levels in both copepod and mysid tests ; however, 19

consistent concentration

- response relationships were not observed in either test

. 20



### 5.4 4

- tert

- octylphenol 21

4

- tert

- octylphenol was tested in two different laboratories using the MTTT method

. 22

Copepod interlaboratory studies were not conducted with this chemical

. The results of the 23

inter laboratory validation study are described in the individua l laboratory study reports 24

( Claude et al. 2012 ; Lee 2012a,c )

. Results from the statistical re

- analysis of the MTTT data 25

produced at SMV and WLI by Battelle are presented in Appendix C and are incorporated below. 26

5.4.1 Exposure L evels 27

Nominal 4

- tert

- octylphenol e xposure concentrations were 0.26, 0.64, 1.6, 4.0, and 28

10 μg/L for both laboratories (SMV and WLI)

. Analysis of the test solutions throughout the 29

exposure period demonstrated that the expected concentration

- gradient was generally maintained 30

during the exposure

. Mean measured concentrations of 4

- tert

- octylphenol ranged from 98 to 150% 31

(SMV) and 74 to 127% (WLI) of nominal

. The mean measured concentrations of 32

4

- tert

- octyl phenol (as active ingredient) for each lab are presented in Table 5

- 9 and Table 5

- 10

. 33

Based on the results, it was determined that the appropriate quality control was maintained 34

during the analyses of the 4

- tert

- octylphenol exposure solution samples in both laboratories. The 35

water quality parameters of both labs remained within tolerance limits as defined in the test 36

protocol. 37

38DRAFT

Invertebrate Two

- Generation ISR 61 May 2 8 , 2013 Table 5

- 9

. Re production re sults from the EPA validation study of the MTTT using 1

4

- tert

- Octylphenol. 2

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

[na] Not assessed due to insufficient production of live young to continue an F1 generation in all treatment groups. 3

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 4

[ ↑ ] Indicates a statistically significant increase compared to control (ANOVA F

- test), based on statistical re

- analysis of t est data 5

(Battelle 2013). 6

[ ↓ ] Indicates a statistically significant decrease compared to control (ANOVA F

- test), based on statistical re

- analysis of test data 7

(Battelle 2013). 8

[ ↗ ] Indicates a statistically significant increasing concentration trend (Line ar trend test), based on statistical re

- analysis of test 9

data (Battelle 2013). 10

[ ↘ ] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statisticalre

- analysis of test 11

data (Battelle 2013). 12

13

5.4.2 Sensitive Endpoints 14

4

- tert

- octylphenol exposure at SMV indicated that the F 0 generation appeared to be more 15

sensitive (ten endpoints affected) than the F 1 generation (four endpoints affected). A summary of 16

the responses for each endpoint in each generation is presented in Table 5

- 9 and Table 5

- 10

. The 17

most sensitive endpoints were those associated w ith fecundity (reduced number of offspring in 18

broods 1 and 2 and increased interbrood duration) and growth (increased weight and length), 19

whereas survival endpoints were not significantly affected in both generations. The results from 20

WLI similarly indicat ed that the most sensitive endpoints were reproduction (reduced number of 21

offspring in broods 1 and 2) and growth (reduced weight and increased length at day 14), 22

whereas survival endpoints were unaffected. The reproductive effects apparent in the F0 23

gener ation (WLI) resulted in insufficient production of live young to continue an F1 generation 24

in all treatment groups. Therefore, statistical analysis was not performed on the exposed groups 25

of the F1 generation (WLI), and only the F1 control group was evalua ted and found to perform 26

within test acceptability criteria. 27DRAFT

Invertebrate Two

- Generation ISR 62 May 2 8 , 2013 Table 5

- 10

. Survival and growth results from the EPA validation study of the MTTT using 1

4 tert

- Octylphenol

. 2

Endpoint Lab F 0 (Parental) F 1 (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 (F 1 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(F 1 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

[na] Not assessed due to insufficient production of live young to continue an F 1 generation in all treatment groups. 3

[

- ] Indicates an endpoint was measured, but was not statistically significantly different from control. 4

[↑] Indicates a statistically significant increase compared to control (ANOVA F

- test), based on statistical re

- analysis 5

of test data (Battelle 2013). 6

[↓] Indicates a statistically significant decrease compared to control (ANOVA F

- test), based on statistical 7

re

- analysis of test data (Battelle 2013). 8

[ ↗ ] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical 9

re

- analysis of test data (Battelle 2013). 10

[ ↘ ] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical 11

re

- analysis of test data (Battelle 2013). 12

5.4.3 Conclusions 13

The interlaboratory results for the SMV and WLI labs demonstrated that the test was 14

performed successfully and within acceptable test method guidelines, except for the F 1 15

generation from WLI

. At WLI, the reproductive effects apparent in the F 0 generation resulted in 16

insufficient production of live young to continue an F 1 generation in all treatment groups

. Several 17

g rowth and reproduction endpoints in the F 0 generation were found to be sensitive in both 18

laboratories, but the responses were not fully consistent between laboratories (i.e., weight and 19

length effects were inconsistent, whereas fecundity effects were consi stent)

. The reduction in 20

fecundity in the F 0 generation was the most consistent effect observed in both laboratories

. 21DRAFT

Invertebrate Two

- Generation ISR 63 May 2 8 , 2013 4

- tert

- oc t ylphenol appears to result in increas ed growth as compared to the control, although 1

growth endpoint variability was observed bet ween both studies. 2

3DRAFT

Invertebrate Two

- Generation ISR 64 May 2 8 , 2013 

### 6 DISCUSSION AND SUMMARY OF THE MTTT AND HCDRT METHODS 1



### 6.1 Reproducibility of the T est M ethod s 2

MTTT 3

McKenney f

irst demonstrated transgenerational effects in A. bahia using a 4

two

- generation exposure protocol ( Section 4.1 )

. These studies led to the development of a 5

proposed MTTT guideline (OECD , 2004 a )

. The MTTT guideline was used in demonstration and 6

optimization studies using a number of representative endocrine

- active chemicals in two 7

different laboratories ( Section 4.1 )

. Subsequently, the MTTT was used in an inter

- laborat ory 8

validation study consisting of six studies carried out at four laboratories with three representative 9

endocrine

- active chemicals (i.e., lindane, vinclozolin, and 4

- tert

- octylphenol ; Section 5 )

. D ata for 10

25 different endpoints (8 growth parameters, 11 reproduction parameters, and 6 survival 11

parameters) collected during the inter

- laboratory validation study were re

- analyzed to evaluate 12

the reproducibility of the recommended MTTT included as Appendix C

. Results of the MTTT 13

validation studies can be summarized as follows: 14

 Except for minor deviations from the guidelines test conditions, t he different 15

lab oratories were able to successfully execute the proposed MTTT

. I n one study

, one 16

laboratory was unable to achieve sufficient reproduction in the controls to complete 17

the second generation within the guidelines acceptability criteria (i.e., WLI in the 18

lindane study)

. 19

 Large laboratory

- to

- laboratory variability was observed in the reproduction and 20

growth endpoint data collected for the control groups

. Specifically, the coefficients of 21

variability (CVs) across laboratories and studies in the control groups in the F 0 22

generation exceeded 20 % for 7 out of 11 reproduction endpoints and five out of eight 23

growth endpoints ( Table 6

- 1 )

. Conversely, the CVs across laboratories and studies in 24

the control grou ps in the F 0 were below 20% for all six survival endpoints

. T here is 25

no clear consensus on what constitu tes an acceptable inter

- laboratory CV ; however, 26

desired maximum CV s of 20 and 30% have been used in the past (e.g., Moore 27

et al., 2000)

. While there is a positive association between the F 0 and F 1 CVs, 28

particularly for the reproduction and survival paramete rs, t he CVs in the F 1 29

generation tended to exceed those for the F 0 generation for the same parameter ( Table 30

6

- 1 ). 31

 Significant differences were observed in the technical proficiency (as estimated by 32

the variability of the endpoint measurements in the control groups) among labs that 33

participated in the i nter

- laboratory validation study , indicating difficulties in 34

transferability of the MTTT between these labs

. 35

 Significant variability was observed within laboratories between studies for the 36

reproductive and survival endpoints in the control groups, wherea s that variability 37

was smaller for the growth endpoints (length and weight ). 38

 Where the same chemical was tested in two laboratories (i.e., lindane and 39

4

- tert

- octylphenol), dose

- response relationships were generally not consistent (based 40

on comparisons of s ignificantly affected endpoints at each treatment level) among the 41

laboratories

. Day 14 F 0 length was the only growth parameter with consistently 42

detected significant increases at 0.08 μg lindane/L or above at two labs

. Number of 43

offspring, days to first a nd second brood, and length at day 14 provided the most 44DRAFT

Invertebrate Two

- Generation ISR 65 May 2 8 , 2013

consistent response in the inter

- laboratory studies (see summary tables in Section 5 )

. 1

Number of offspring i n brood 2 and total number of offspring in two broods were the 2

only two endpoints that showed a significant trend across 4

- tert

- octylphenol 3

concentration groups

. 4

 Growth endpoints were generally affected at lower concentrations than reproductive 5

endpoints i n the inter

- laboratory validation studies

. 6

 E ndpoint responses between the F 0 and F 1 generations were not consistent and were 7

significantly different in several instances

. 8

Table 6

- 1

. Ranges of coefficient of varia tion (CV) in the F 0 and F 1 generations based on six 9

studies carried out at four laboratories with three chemicals ( see Section 5 )

. 10

Category Parameter CV

– F 0 CV

– F 1

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

HCDRT 11

Chandler and colleagues conducted a range of studies with the harpacticoid copepod A

. 12

tenuiremis

, which served as initial development studies for the HCDRT ( Section 4.2 )

. A copepod 13

life

- cycle toxicity test protocol was established for A. tenuiremis by the American Society for 14

Testing and Materials in 2004 (ASTM E2317

- 04)

. In a ddition, a draft guideline was proposed to 15

OECD in 2004 to evaluate effects of chemicals on A. tenuiremis (OECD, 2007)

. This draft 16

guideline was used in two rounds of OECD validation studies (OECD 2007, 2011) and an EPA

17

led inter

- laboratory validation stud y ( Section 5 ) and results can be summarized as follows: 18

 Based on the prevalidation study (OECD 2007), in which two harpacticoid ( N. 19

spinipes and A. tenuiremis ) and one calanoid copepod ( A. tonsa ) were tested, the 20

A. tenuiremis assay was recommended for further validation due to its shorter test 21

time, bet t er reproductive output and success, its focus on reproductive and 22DRAFT

Invertebrate Two

- Generation ISR 66 May 2 8 , 2013

developmental endpoints, its greater statistical power due to high replication, and its 1

availability as an existing ASTM method

. 2

 Lindane was tested at the same concentration s in three laboratories as part of the 3

EPA

- led interlaboratory validation study and in four laboratories as part of the OECD 4

validation study

. Laboratories in the OECD validation study had difficult ies obtaining 5

the desired chemical concentrations

. While most of the seven test laboratories were 6

able to perform the test within guideline r ecommendations and requirements, data 7

w ere not always recorded and reported consistently among laboratories

. For example, 8

one of the labs in the OECD validation study experienced difficulties with the 9

identification of copepodite matura tion and determinati on of sex

. Lindane generally 10

caused reduced survival, developmental delay, and reduced fecundity, although 11

differences were observed in the concentrations at which these effects were reported 12

by different labs

. In addition, some labs did not find the s ame endpoints to be 13

significantly affected

. Due to these issues, OECD recommended that further work be 14

conducted to address the proficiency, manpower, and training of laboratories 15

performing the test, continue validation of the guideline to identify preferred 16

endpoints, and obtain additional information on the basic biology of the test species. 17

 Atrazine was tested in three laboratories as part of the OECD interlaboratory 18

validation study

. One of the laboratories was not able to achieve the required mating 19

succ ess in the controls ; however the other two labs were able to perform the test 20

within acceptable guidelines and reported consistent biological responses 21

(i.e., reduced number of nauplii hatched over two clutches in two highest test 22

concentrations with none of the other en dpoints significantly affected)

. 23

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

. While the 24

test was conducted within test method guidelines, all measured vinclozolin 25

concentrations were undetected

. 26



### 6.2 Data Interpretation 27

MTTT 28

The MTTT is proposed as a Tier 2 test to be used in EPA’s EDSP

. If Tier 2 testing is 29

indicated based on results of Tier 1 screening, EPA generally expects that the summary narrative 30

and characterization would not only explain the basis of the conclusions regarding potential 31

interactions with the estrogen (E), androgen (A), and/ or thyroid (T) hormonal pathways, but also 32

provide a discussion on why additional data are needed for assessing potential adverse outcomes 33

via potential endocrine disruption (US EPA 2011)

. 34

While vertebrate

- type steroids have been detected in several crustaceans, including 35

mysids (Verslycke et al., 2002), their endogenous origin is questioned in many cases (LaFont, 36

2000)

. Crustaceans are believed to lack E, A

, and T hormone axes

. In addition, little is known 37

about the potential of chemicals that may affect the E, A

, and T hormone axes through any 38

known modes of action (i.e., chemicals that are expected to be identified though Tier 1 screening) 39

to interfere with inver tebrate hormone axes or the endpoints measured in the MTTT

. While dose 40

response information can be obtained for the growth and reproductive endpoints evaluated in the 41

MTTT, the study design for the MTTT does not include any biochemical, biomarker, or 42

histo pathology endpoints

. Therefore, a specific mode of action determination is not possible, and 43DRAFT

Invertebrate Two

- Generation ISR 67 May 2 8 , 2013 would require additional endpoint measurements or be based on extrapolation of a documented 1

mode of action in other arthropods

. From a risk assessment perspective, the distinction between 2

endocrine or metabolic toxicity based on sound mechanistic evidence is not crucial . This 3

distinction is probably less crucial than identifying the most sensitive endpoints that can lead to 4

long

- term impacts at the population level (Verslycke et al. 2007). 5

Dose

- response information, such as LOEC, NOEC

, and ECx information for the 6

biological endpoints included in the MTTT, should be collected since such data will be required 7

to support chemical risk assessment

. In addition, common pop ulation modeling approaches can 8

be employed based on the data obtained in the MTTT to estimate population

- level effects 9

(Raimondo and McKenney 2005a)

. 10

HCDRT 11

The HCDRT is proposed as an option or alternative Tier 2 test to be used in EPA’s EDSP

. 12

Crustacean s, including copepods, are believed to lack E, A

, and T hormone axes

. In addition, 13

little is known about why chemicals that may affect the E, A

, and T hormone axes through any 14

known modes of action (i.e., chemicals that would be identified in Tier 1) may interfere with 15

invertebrate hormone axes or the endpoints measured in the HCDRT

. Nevertheless, it must be 16

assessed whether a vertebrate

- active endocrine active substance may affect an invertebrate to 17

complete a comprehensive assessment of potential risks

. 18

Dose

- response information, including LOEC, NOEC

, and ECx information for the 19

biological endpoints included in the HCDRT, will be required to support subsequent risk 20

assessment

. In addition, the survival, sex ratio, and fecundity data obtained in the HCDRT can be 21

used to calculate and predict population growth rate (e.g., intrinsic rate of natural increase 22

[r

m or λ ] and age:stage structure

. Such modeling may produce additional valuable data for 23

interpreting data collected in the HCDRT and potentially extrap olating results to 24

population

- level effects (OECD [158] 2011). 25



### 6.3 Strengths and Limitations of E ach T est 26

6.3.1 Strengths and Limitations of the MTTT 27

The recommended MTTT describes a two

- generation toxicity test with an estuarine 28

mysid that considers growth, surviva l, and reproductive endpoints in parents and offspring as an 29

integrated measure of toxicant exposure

. The test is intended to serve as a higher

- tier test with an 30

aquatic arthropod for collecti ng definitive concentration

- response information on adverse effe cts 31

suitable for ecological risk assessment

. As with any toxicological assay, strength and limitations 32

must be recognized and considered when evaluating data and drawing conclusions

. Significant 33

advantages

, limitations , and potential further refinements of the MTTT are summarized below. 34

Advant a ges of the MTTT 35

 Many environmental testing lab oratories have established experience with A. bahia 36

due to its long

- standing use as a standard test model for regulatory toxicity testing

. 37

Laboratories were generally able to successfully perform the MTTT within the 38

recommended acceptability criteria

. 39

 Mysids have been used as a test model for evaluating reproductive toxicity for several 40

decades and an increasing body of literature is available regarding their use for 41DRAFT

Invertebrate Two

- Generation ISR 68 May 2 8 , 2013

evalua ting endocrine toxicity, including the availability of mechanistic endpoints 1

(e.g., vitellin, ecdysteroid receptor gene expression ). 2

 The test can be conducted in continuous or intermittent flow

, which has significant 3

advantages with respect to ensuring co nsistent water quality and maintaining desired 4

chemical exposure concentrations

. 5

 The control variability of the survival and several of the reproduction (i.e., days to 6

brood release and offspring production) and growth (i.e., length) endpoints , especially 7

in the F 0 generation

, indicate that these should be able to detect significant adverse 8

effects with adequate statistical power

. 9

 Common population modeling approaches can be employed based on the data 10

obtained in the MTTT to estimate population

- level effects (Raimondo and 11

McKenney 2005a)

. 12

Limitations of the MTTT 13

 A large number of endpoints are included and there is some redundancy between 14

endpoints ( e.g.

, growth is evaluated by both weight and length )

. In addition, s everal 15

endpoints require ani mals to be sacrificed

, resulting in reduced replication for other 16

endpoints

. 17

 Several aspects of the MTTT, such as the collection of newly hatched offspring, 18

pairing of mature males/females, etc. are time

- consuming and resource

- demanding

. 19

Also, care and exp erience is required to minimize non

- treatment related mortality due 20

to handling or impingements

. 21

 Considerable variability was observed in endpoint responses between different 22

laboratories, resulting in reduced power to detect significant differences

. Some of the 23

variability between different laboratories may be attributable to intrinsic differences 24

in the laboratory animal populations , small differences among laboratories in feeding 25

and feed quality, etc. 26

 Some of the endpoints were consistently non

- responsive (e.g., sex ratio, time to 27

maturation)

, and their value may need to be evaluated further

. 28

 The addition of a second generation significantly adds to the time and resources 29

required to pe rform the MTTT

. The value of the additional information o btained from 30

the second generation was not obvious based on the results of the inte r

- laboratory 31

validation studies

. As discussed in the potential further refinement section, additional 32

chemicals may need to be tested before the value of the second generati on can be 33

adequately evaluated

. 34

 In cases where the test chemical causes reductions in fecundity, it may be difficult t o 35

complete the second generation of the MTTT due to insufficient young

. 36

 While the MTTT provides general guidance for conducting statistical analysis, more 37

specific guidance may be needed to ensure consistency

. Statistical evaluation of the 38

many endpoints and the potential for confounding factors (e.g., fecundity is related to 39

female body size in mysids) requires a qualified statist ician. 40DRAFT

Invertebrate Two

- Generation ISR 69 May 2 8 , 2013 Potential further refinements of the MTTT 1

The lack of treatment

- related differences could have resulted from selected doses that 2

were too low to bracket any biological effect that may have been suggested by an acute 3

range

- finding test

. It may be bet ter to select definitive exposure concentration s based on a 4

longer

- term range

- finding test

. For example, the guideline provides an alternative of conducting 5

a pared

- down life

- cycle range

- finding test ( e .g

., five breeding pairs at three dose levels + 6

controls carried through 21 days)

. The value of the latter alternative should be evaluated further

. 7

The lack of treatment

- related differences could also have resulted from the selection of 8

test chemicals used in the different validation stu dies

. The selected chemicals were 9

representative endocrine active chemicals in vertebrates, but may not exhibit endocrine toxicity 10

in arthropods

. Further validation with known arthropod endocrine active chemicals (e.g., insect 11

growth regulators, ecdysone a gonists, etc.) may provide better data for evaluating strengths and 12

weaknesses of the currently proposed MTTT

. Such chemicals are also potentially more likely to 13

result in transgenerational toxicity

. 14

It may be sufficient to use length measurements to evalu ate growth, given the difficulty 15

of accurately measuring changes in weight in mysids

. Furthermore, it may be sufficient to 16

calculate a growth rate based on length at Day 0 and termination

. This would reduce the amount 17

of animals n eeded for collecting growt h endpoint data, resulting in greater replication or 18

potentially lower loading rates if sufficient statistical pow er is maintained

. It may also be worth 19

exploring whether non

- lethal methods for obtaining length measurements are possible 20

(e.g., digital imag ing and processing). 21

6.3.2 Strengths and Limitations of HCDRT 22

Significant advantages and limitations of the HCDRT are summarized below

. 23

Advantages of HCDRT 24

 Relative to other harpacticoid copepod studies in the literature

, A. tenuiremis is more 25

chronically sensitive than all other species published to date where there is 26

comparative data (ASTM 2004 ). 27

 Laboratories were generally able to successfully culture A. tenuiremis , harvest gravid 28

copepods, and hatch healthy nauplii needed to conduct the HCDRT

. Labs als o 29

generally demonstrated the ability to execute the HCDRT within the stated 30

performance requirements

. 31

 Microplate test ing allows for easy (individual

- based) tracking of endpoints and high 32

replication resultin in strong statistical power

. 33

 Relatively consis tent concentration

- dependent delays in development rates, increased 34

naupliar

- specific mortality, mating

- success depressions, and reduction of fecundity 35

were observed during the lindane interlaboratory validation study

. 36

 Population modeling may be valuable but is not currently included in guideline

. 37

 A. tenuiremis has a rapid life cycle (16 to 17 d at 25  ̊C) and can be easily cultured in 38

water

- only and sediment:water matrices. 39

Limitations of the C opepod T est 40

 Commercial testing laboratories generally have no pr ior experience with this species

. 41

I ssues of proficiency in the successful application of the test method were identified 42DRAFT

Invertebrate Two

- Generation ISR 70 May 2 8 , 2013

as an issue with the HCDRT (OECD 2011)

. For example, some labs had initial 1

difficulties with the identification of copepodite maturatio n and determination of sex

. 2

 Microwell plate design results in difficulties for continuous monitoring of water 3

quality and difficulties producing consistently accurate and precise measured 4

concentrations

. Consequently, test results need to be reported in no minal 5

concentrations rather than measured concentrations (OECD 2011 )

. 6

 Absence of effects on most of the endpoints reported in the atrazine study 7

(OECD 2011)

. 8

 P ossible lack of sensitivity of the population of A. tenuiremis used in the study 9

(OECD 2011 ). 10

 Influence of mortality (males more sensitive than females) on the sex ratio 11

(OECD 2011 ). 12

 Manpower required in the study (50% time of a technician ). 13

 Limited knowle dge of the copepod endocrine system

. 14

 Small size limits biochemical studies

. 15

Potential F urther R efinements of the HCDRT 16

Some recommendations for further work were provided by OECD (2011) and are 17

repeated here: 18

 Performing the test method for controls alone to reassure that the test performance 19

and the validity criteria set out in the HCDRT can be met by the laboratories 20

 N eed for more information on the basic biology of this test species 21

 It would be very useful to have more specific information on how different feeding 22

levels affect the life history progression of this copepod. 23



### 6.4 Rationale for the U se of M ysid V ersus C opepod T est 24

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

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

survival: 27

 While both the MTTT and HCDRT require significant laboratory experience, t here is 28

significantly more experience with the use of mysids as a toxicity test model 29

compared to copepods 30

 There is a large amount of published toxicity data for A. bahia

, and available 31

evidence suggest that mysids are g enerally more sensitive than other standard toxicity 32

test species 33

 Our understanding of the basic biology of mysids (specifically A. bahia ) is 34

significantly greater than for the proposed harpacticoid copepod species 35

 Mysids have been used as a test model for evaluating reproductive toxicity for several 36

decades

, and an increasing body of literature is available regarding their use for 37

evaluating endocrine toxicity, including the availability of new mechanistic 38

endpoints (e.g., vitellin, ecdysteroid receptor g ene expression) 39

 Similar to copepods, mysids are of ecological importance, have a wide geographic 40

distribution , are available year

- round in the field, and can be easily transported and 41

cultured 42DRAFT

Invertebrate Two

- Generation ISR 71 May 2 8 , 2013  The MTTT test system allows for continuous or intermittent flo w exposures

, which 1

has significant advantages over the static renewal conditions employed in the 2

HCDRT with respect to ensuring consistent water quality and maintaining desired 3

chemical exposure concentrations 4

 A more robust inter

- laboratory validation stu dy has been conducted with the MTTT 5

than with the HCDRT where additional validation studies are still underway 6

 The greater body size of mysids makes them more amenable to additional 7

biochemical /molecular measurements 8

 The MTTT includes well

- established gro wth endpoints

, which appeared to be among 9

the most sensitive among the endpoints tested, whereas the HCDRT does not include 10

growth measurements 11

 Although b oth data obtained through the MTTT and HCDRT can be used for 12

population modeling , the MTTT has data th rough two generations

. 13

14

15DRAFT

Invertebrate Two

- Generation ISR 72 May 2 8 , 2013 

### 7 REFERENCES 1

2

[Includes works cited and works consulted] 3

4

Andersen, HR, Wollenberger, L, Halling

- Sorensen, B, and Kusk, KO. 2001. Development of 5

Copepod Nauplii to Copepodites

— A Parameter for Chronic Toxicity Including Endocrine 6

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– 2829. 7

8

ASTM (American Society for Testing and Materials). 1997. Standard Guide for Conducting 9

Life

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- 97. American Society for Testing and 10

Mater ials. West Conshohocken, Pennsylvania. 11

12

ASTM (American Society for Testing and Materials). 2004. Standard Guide for Conducting 13

Renewal Microplate

- Based Life

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ASTM Standard No. E2317

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Aufderheide, J

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Bechmann, RK. 1999. Effect of the Endocrine Disru pter Nonylphenol on the Marine Copepod 31

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33

Bejarano, AC, and Chandler, GT. 2003. Reproductive and Developmental Effects of Atrazine on 34

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Bejarano, A C, Chandler, GT, and Decho, AW. 2005

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42

Bejarano, AC, Chandler, GT, He, L, and Coull, BC. 2006

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8

Block, DS, and Bradbury , N. 2012 b

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Box, G EP. 1953

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Breitholtz, M. and Bengtsson, BE. 2001. Oestrogens Have No Hormonal Effect on the 17

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Breitholtz, M, Wollenberger, L, and Dinan, L. 2003. Effects of Four Synthetic Musks on the Life 21

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8DRAFT

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### 8 LIST OF APPENDICES 1

2



### APPENDIX A . VALIDATION OF SCRE ENING AND TESTING AS SAYS 3



### PROPOSED FOR THE EDS P ( DRAFT JULY 2007) 4



### APPENDIX B . DETAILED REVIEW PA PER ON AQUATIC ARTHROPOD S IN 5



### LIFE CYCLE TOXICITY TESTS (OECD NO. 55, JULY 2006) 6



### APPENDIX C

. REPORTS ON THE INTER

- LABORATORY VALIDATION 7



### TRIALS FOR THE MITT 8

C.1 Inter

- laboratory Statistical Analysis [Mysid Data Across All Studies], 2013 9

10

C.2 Mysid Study with Vin clozolin (100.02) , University of North Texas, 2012 11

12

C.3 DRAFT Mysid Study with Lindane (100.01) , University of North Texas, 2012 13

14

C.4 Mysid Studies with Lindane and 4

- tert

- Octylphenol (185A

- 102

- 1,

- 2A ) , Wildlife 15

International , 2012 16

17

C.5 Mysid Studies with Lindane and 4

- tert

- Octylphenol (13888.6109, 6111) , Smithers 18

Viscient, 2012 19

20



### APPENDIX D . REPORT S ON THE I NTER

- LABORATORY V ALIDATION 21



### T RIALS FOR THE HCDRT 22

D.1 Report of Progress on the Interlaboratory Validation of the OECD Harpacticoid Copepod 23

Development and Reproduction Test (OECD No. 158, August 2011) 24

25

D.2 Validation Report of the Full Life Cycle Test with the Harpacticoid Copepods Nitocra 26

spinipes and Amphiascus tenuiremis and the Calanoid Copepod Acartia tonsa (OECD 27

No. 79, August 2007) 28

29

D .3 Copepod Study with Lindane (27569) , Block Environmental Services, 2012 30

31

D.4 Copepod Study with Vinclozolin (27568) , Block Environmental Services, 2012 32

33

D.5 Copepod Study with Lindane (BR0476/B) , Brixham, 2011 34

35DRAFT

Invertebrate Two

- Generation ISR 89 May 2 8 , 2013 D.6 Copepod Study with Lindane (C5069) , Cef as, 2011 1

2



### APPENDIX E

. MYSID TWO

- GENERATION TEST GUID ELINE (DRAFT 3



### PROPOSAL FOR A STANDARDIZED PROTOCO L AS AN IN VIVO TIER 2 4



### TEST ) , APRIL 2013 5



### APPENDIX F

. HARPACTICOID COPEPOD DEVELOPMENT AND 6



### REPRODUCTION TEST WI TH AMPHIASCUS TENUIREMI S ( STANDARDIZED 7



### OECD DRAFT PROTOCOL FOR THE HCD RT AS A POTENTIAL 8



### ALTERNATIVE TO THE M TTT ), MAY 2009 9



### APPENDIX G . INTRA

- LABORATORY STATISTIC AL ANALYSIS REPORTS 10



### FOR TWO

- GENERATION INVERTEBR ATE STUDIES 11

G.1 Intra

- laboratory statistical analysis for Mysid Lindane Study at Smith ers Viscient, 2013 12

13

G.2 Intra

- laboratory statistical analysis for Mysid Octylphenol Study at Smithers Viscient, 14

2013 15

16

G.3 Intra

- laboratory statistical analysis for Mysid Lindane Study at University of North Texas, 17

2013 18

19

G.4 Intra

- laboratory statistical ana lysis for Mysid Vinclozolin Study at University of North 20

Texas, 2013 21

22

G.5 Intra

- laboratory statistical analysis for Mysid Octylphenol Study at Wildlife International, 23

2013 24