Appendix D: MMT Protocol

Appendix D, Section 1: Protocol Summary

Briefly, the test consisted of continuously exposing three generations of fish starting with young adults 

in the first generation, designated F0. There were 6 treatments; five chemical concentration groups and a 

control group. Within each treatment incorporated six replicates. The measured endpoints included embryo 

hatch, growth, survival, fecundity and fertility, liver vitellogenin mRNA levels, the number of anal fin 

papillae (a sexual phenotype marker), histological evaluation of the gonad phenotype, and histopathology 

evaluation of kidney, liver and gonad. All of these endpoints are evaluated in the context of the genetic sex 

of each fish. Chemical concentrations are held constant in each treatment throughout the bioassay, to allow 

endpoint sensitivity comparisons across and within generations. Thus, although not discussed here in detail, 

precise dependable flow-through exposure and dilution systems as well as high-quality analytical chemistry 

is an integral part of the protocol.

The exposure in F0 began by delivering chemical to to breeding pairs of young adults (11 wpf). 

Fecundity and fertility were measured over five weeks and the F0 fish were terminated. Embryos were 

collected in the third week of F0 reproduction for starting F1. The exposure continued throughout F1 and 

F2; from hatching through reproductive development, and through the reproductive assessment (see figure 

2). Fish were sampled at various life stages in each generation to measure the effect endpoints listed above. 

The total in-life exposure spanned 29 weeks.

MMT Exposure and endpoint timeline

Gen

F0 1 2 3 4 5

F1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

F2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Endpoints

Reproducti

on F0 F1 F2

Hatch F

1

F

2

Survival F

0

F

1

F

1

F

1

F

2

F

2

F

2

Growth F

0

F

1

F

1

F

2

F

2

Vitellogenin F

0

F

1

F

1

F

2

F

2

Secondary 

sex

F

0

F

1

F

1

F

2

F

2

Histopathol

ogy

F

0

F

1

F

1

F

2

F

2
Table of the time-line and endpoints in the 4-tert-octylphenol MMT. The MMT was 29 weeks long and 

spanned 2 1⁄2 generations; F0, F1, and F2. The cells below study-week row indicate when various endpoints 

were measured in each generation. The life-stages of the test animals are color-coded as follows.

Appendix D, Section 2: Details of the MMT

The temperature throughout the test was 26 C. The photoperiod was 16h light and 8h dark. Water 

flow to each 2 L aquarium used throughout was 20 ml/min. The 6 treatments including controls, designated 

T1 through T6 (control dilution water only T1, through the highest treatment concentration T6). The 

proportionate chemical concentration difference between treatments was 0.5. The concentrations of toxicant 

were measured weekly in two replicates in each treatment over the course of the study. A brief description 

of intergenerational succession and of fish numbers employed in each replicate at various life-stages 

follows. The F0 generation started as a pooled cohort of eggs from at least 36 individual pairs of medaka in 

the fish culture system. After hatch they were raised unexposed in test aquaria until they were young adults 

(11 wpf). Each replicate of each treatment in the next two generations, F1 and F2, was started on two 

consecutive days in the third week of reproduction of F0 and F1 respectively. On each of the two days the 

eggs from each replicate pair were collected and counted. Up to 20 fertile eggs were randomly selected and 

placed in an incubator, also one for each day. All 6 replicate incubators for each treatment for each day were 

placed in an “incubator” aquarium, which was identical to all the other exposure tanks except that it held 

only incubators. Thus there were two incubator aquaria for each treatment; one which held all the replicate 

spawns for a treatment from the first day and the other from the second day of two consecutive spawning 

days. The chemical treatment was delivered to each 2 L incubator tank at the same rate as was delivered to 

the breeding pairs, i.e. 20 mL/min. Each incubator was aerated gently to facilitate chemical exchange 

between the incubator and the incubator tank and to minimize disease transmission between eggs. Dead eggs 

were removed from each incubator daily. Upon hatching each fish was moved from the incubator into the 

treatment tank which held the incubator. Thus during the incubation period, 8 tanks were employed for each 

treatment; 6 pair-breeding tanks and 2 incubator tanks.

The F1 and F2 hatchlings were held in the two incubator tanks per replicate until they were 3 wpf. 

Then, they were pooled and randomly distributed, 10 fish tank, to the 6 replicate tanks for each treatment. 

Extra fish were culled. For the remainder of each generation (F1 and F2) each treatment consisted of 6 

replicate 2 liter aquaria. At 8 wpf all the fish in each replicate are genetically sexed. One XX and one XY 

fish were selected to become the breeding pair for each replicate. The remaining fish were terminated and 

sampled for measurement of sub adult life-stage endpoints. Endpoints measured included growth, liver 

vitellogenin mRNA, gonad sex (histological), and secondary sex (anal fin papillae).

The 6 breeding pairs in each treatment were reared for an additional 4 weeks, during which time 

spawning usually begins. The time of first spawn for each pair was recorded. Reproductive assessment of 

each fish pair began in the 12 wpf. Fecundity and fertility was recorded for 4 days each week (Tue-Fri) for 5 

consecutive weeks. Beginning 16 wpf, the spawning pairs were terminated and adult endpoint data was 

collected. The measured endpoints included growth, liver Vtg levels, anal fin papillae number, and 

histopathology of gonad, kidney, and liver tissues.

Genotypic Sex

The sex of medaka was determined by identifying the presence of the sex-determining gene (DMY) 

in each fish. In normal conditions XX individuals express the female phenotype and XY individuals the 

male phenotype. Then, the genotypic sex was determined by non-destructively sampling a small piece of the 

caudal fin of each fish, its DNA, and then using PCR evaluating it for the presence of DMY.

Embryo Larva Juvenile Subadult Adult
Reproduction in F0, F1, F2; Hatch and Survival in F1 and F2

The fecundity and fertility of reproductive pairs was assessed from the 12th through the 16th week in 

each generation (F0,F1, and F2). On the morning of each day during evaluation (Tuesday through Friday of 

each week) egg clumps were removed from females and from the bottom of each tank and counted for 

fecundity and evaluated under the dissecting microscope for fertility. In the third week of the reproduction 

assessment, after the egg collection, viable embryos were placed in incubators to populate the subsequent 

generation. Each incubator was inspected daily and dead eggs were tallied and removed. Upon hatch, the 

larval fish were counted daily, removed from the incubator and placed in one of two tanks per treatment. In 

this fashion, replicate hatchlings within treatments were pooled before distribution during the 4th wpf of each 

generation, to the 6 replicate tanks per treatment. The difference between number of hatched individuals and 

the number tallied at 4 wpf was used to calculate the survival values for each of the two replicates per 

treatment.

Subadult survival, growth, vitellogenin, anal fin papillae, and gonad phenotype (F1, F2)

Beginning 8 wpf in F1 and F2, the genetic sex of each fish was determined as described above. Each 

fish was netted, anaesthetized (0.2 mg/L buffered MS-222), and fin clipped (either dorsally or ventrally). To 

retain the identity of each fish, while the genetic sex was being determined, one pair (up to 10 fish total, 

each with either a dorsally or ventrally clipped fin) were held in up to 5 small chambers within each 

replicate tank. After genetic sex was determined, one randomly selected XX, XY pair was placed back into 

the tank for subsequent spawning and fecundity/fertility assessment, and the remaining genetically-sexed 

fish were euthanized for evaluation of growth, liver vitellogenin mRNA, anal fin papillae number, and 

histologically-determined gonad phenotype (i.e., testis, ovary, or gonadal intersex).

Adult survival, growth, vitellogenin, anal fin papillae, and histopathology (F0, F1, F2)

At the end of the 12-16 wpf evaluation of fecundity and fertility (see above), all spawning pairs were 

euthanized with MS-222 and evaluated for effects on growth, liver Vtg mRNA, and anal fin papillae. Each 

fish was fixed in Bouin’s fluid for histological evaluation of the gonad phenotype as well as for gonad, 

kidney and liver pathology.

Statistics

The recommended statistical procedures can be found in detail in the statistics appendix and 

summarized in the body of the ISR. 

Appendix D, Section 3: Daily Protocol

Below is an example of the daily protocol followed by laboratory personnel at NHEERL/MED 

during the performance of a MMT using 4-t-octylphenol as the test agent. 

2-Gen protocol patterned from OctylPhenol (11/02/06)

Event (rows critical events only) Day Date Calend D Stdy Wk Stdy d F0 Wk F0 day F0 Repr d F1 Wk F1 day F1 Repr d F2 Wk F2 day Color Key

Set-up incubators with ~ 300 embryos

Note start day for

embryo collection is

Tuesday, not Monday

Study start date Mon 27-Nov-06 331 Holidays

Put 240 (total) larvae into 2, 2L tanks Tue 28-Nov-06 332

F0/F1 adult termination. 

Day 105 for each 

generation

Select and redistribute to 15-18 tanks (10 in each) Tue 29-Nov-06 333 F0/F1 Fecundity/fertility

Put breeding pairs into 42 tanks

Start chemistry in exposure system Mon 26-Nov-06 330

F0,F1,F2

Culling/sampling/set-up 

breeding pairs. First cull 

day 22, second cull days

43-44

Assess pre-exposure fertility-fecundity (d 50-63) Mon 26-Nov-06 330

End pre-exposure fertility-fecundity 26-Nov-06 330

Move breeding pairs into exposure system

Start exposure Mon 26-Nov-06 330

Start fertility-fecundity assessment (d 65-85) Tues 26-Nov-06 330

Start F1 gen (two incubators per tank) Tues 26-Nov-06 330

End fertility-fecundity assessment Fri 26-Nov-06 330

Terminate F0 Mon 26-Nov-06 330

Redistribute F1s 10/tank Tues 26-Nov-06 330

Terminate F1s if controls are reproducing Tues 26-Nov-06 330

Event (1 row for each day of study) Day Date Calend D Stdy Wk Stdy d F0 Wk F0 day F0 Repr d F1 Wk F1 day F1 Repr d F2 Wk F2 day Day 06-07 holidays Start date

Mon 27-Nov-06 331 Mon 25-Dec-06 27-Nov-06

Study start date (F0 embryos collected) Tue 28-Nov-06 332 1 1 1 1 Mon 1-Jan-07

Wed 29-Nov-06 333 2 2 Mon 15-Jan-07

Thu 30-Nov-06 334 3 3 Mon 19-Feb-07

Fri 1-Dec-06 335 4 4 Mon 28-May-07

Sat 2-Dec-06 336 5 5 Wed 4-Jul-07

Sun 3-Dec-06 337 6 6 Mon 3-Sep-07

Mon 4-Dec-06 338 7 7 Mon 8-Oct-07

Put 240 (total) larvae into 2, 2L tanks Tue 5-Dec-06 339 2 8 2 8 Mon 12-Nov-07

Wed 6-Dec-06 340 9 9 Thu 22-Nov-07

Thu 7-Dec-06 341 10 10 Tue 25-Dec-07

Fri 8-Dec-06 342 11 11

Sat 9-Dec-06 343 12 12

Sun 10-Dec-06 344 13 13

Mon 11-Dec-06 345 14 14

Tue 12-Dec-06 346 3 15 3 15

Wed 13-Dec-06 347 16 16

Thu 14-Dec-06 348 17 17

Fri 15-Dec-06 349 18 18

Sat 16-Dec-06 350 19 19

Sun 17-Dec-06 351 20 20

Mon 18-Dec-06 352 21 21

Select and redistribute to 15-18 tanks (10 in each) Tue 19-Dec-06 353 4 22 4 22

Wed 20-Dec-06 354 23 23
Thu 21-Dec-06 355 24 24

Fri 22-Dec-06 356 25 25

Sat 23-Dec-06 357 26 26

Sun 24-Dec-06 358 27 27

Mon 25-Dec-06 359 28 28

Tue 26-Dec-06 360 5 29 5 29

Wed 27-Dec-06 361 30 30

Thu 28-Dec-06 362 31 31

Fri 29-Dec-06 363 32 32

Sat 30-Dec-06 364 33 33

Sun 31-Dec-06 365 34 34

Mon 1-Jan-07 1 35 35

Tue 2-Jan-07 2 6 36 6 36

Wed 3-Jan-07 3 37 37

Thu 4-Jan-07 4 38 38

Fri 5-Jan-07 5 39 39

Sat 6-Jan-07 6 40 40

Sun 7-Jan-07 7 41 41

Mon 8-Jan-07 8 42 42

Set up 56 breeding pairs

Start chemistry in exposure system Tue 9-Jan-07 9 7 43 7 43

Wed 10-Jan-07 10 44 44

Thu 11-Jan-07 11 45 45

Fri 12-Jan-07 12 46 46

Sat 13-Jan-07 13 47 47

Sun 14-Jan-07 14 48 48

Mon 15-Jan-07 15 49 49

Tue 16-Jan-07 16 8 50 8 50

Wed 17-Jan-07 17 51 51

Thu 18-Jan-07 18 52 52

Fri 19-Jan-07 19 53 53

Sat 20-Jan-07 20 54 54

Sun 21-Jan-07 21 55 55

Mon 22-Jan-07 22 56 56

Tue 23-Jan-07 23 9 57 9 57

Wed 24-Jan-07 24 58 58

Thu 25-Jan-07 25 59 59

Fri 26-Jan-07 26 60 60

Sat 27-Jan-07 27 61 61

Sun 28-Jan-07 28 62 62

Mon 29-Jan-07 29 63 63

Begin pre-exposure fertility-fecundity Tue 30-Jan-07 30 10 64 10 64

Wed 31-Jan-07 31 65 65

Thu 1-Feb-07 32 66 66

Fri 2-Feb-07 33 67 67

Sat 3-Feb-07 34 68 68

End pre-exposure fertility-fecundity Sun 4-Feb-07 35 69 69

Move breeding pairs into exposure system

Start exposure Mon 5-Feb-07 36 70 70 Note: this is actually day 69 dpf, consistent with our other tests. D.L.

Start F0 fertility-fecundity assessment Tue 6-Feb-07 37 11 71 11 71 1

Wed 7-Feb-07 38 72 72 2

Thu 8-Feb-07 39 73 73 3

Fri 9-Feb-07 40 74 74 4

Sat 10-Feb-07 41 75 75 5
Sun 11

-Feb-07 42 76 766

Mon 12-Feb-07 43 77 777

Fertility-fecundityassessmentTue 13-Feb-07 44 12 78 12 788

Wed 14-Feb-07 45 79 799

Thu 15-Feb-07 46 80 80 10

Disinfectsystem Fr

i 16-Feb-07 47 81 81 11

Sat 17-Feb-07 48 82 82 12

Sun 18-Feb-07 49 83 83 13

Swap in cleantanks

Mon 19-Feb-07 50 84 84 14

StartF1 gen (Fillleftincubators)

Tue 20-Feb-07 51 13 85 13 85 1511

Second dayofF1 eggcollection (Fillrightincubators)

Wed 21-Feb-07 52 86 86 162

Thu 22-Feb-07 53 87 87 173

Fri 23-Feb-07 54 88 88 184

Sat 24-Feb-07 55 89 89 195

Sun 25-Feb-07 56 90 90 206

Mon 26-Feb-07 57 91 91 217

Tue 27-Feb-07 58 14 92 14 92 2228

Wed 28-Feb-07 59 93 93 239

Thu1-Mar-07 60 94 94 24 10

Fri2-Mar-07 61 95 95 25 11

Sat3-Mar-07 62 96 96 26 12

Sun4-Mar-07 63 97 97 27 13

Mon5-Mar-07 64 98 98 28 14

Lastweekoffertility/fecundityassessment

Tue6-Mar-07 65 15 99 15 99 293 15

Wed7-Mar-07 66 100 100 30 16

Thu8-Mar-07 67 101 101 31 17

Fri9-Mar-07 68 102 102 32 18

Sat 10-Mar-07 69 103 103 19

Sun 11-Mar-07 70 104 104 20

TerminateF0

Mon 12-Mar-07 71 105 105 21

Redistribute F1s 10/tank (Firstcull)

Tue 13-Mar-07 72 16 1064 22

Wed 14-Mar-07 73 107 23

Thu 15-Mar-07 74 108 24

Fri 16-Mar-07 75 109 25

Sat 17-Mar-07 76 110 26

Sun 18-Mar-07 77 111 27

Takedownofextra F1?Mon 19-Mar-07 78 112 28

Tue 20-Mar-07 79 17 1135 29

Wed 21-Mar-07 80 114 30

Thu 22-Mar-07 81 115 31

Fri 23-Mar-07 82 116 32

Sat 24-Mar-07 83 117 33

Sun 25-Mar-07 84 118 34

Mon 26-Mar-07 85 119 35

Tue 27-Mar-07 86 18 1206 36

Wed 28-Mar-07 87 121 37

Thu 29-Mar-07 88 122 38

Fri 30-Mar-07 89 123 39

Sat 31-Mar-07 90 124 40

Sun1-Apr-07 91 125 41

Fin ClipMon2-Apr-07 92 126 42

Tue3-Apr-07 93 19 1277 43

F1Cull to pairs(secondcull)

Wed4-Apr-07 94 128 44

F1Cull to pairs(secondcull)

Thu5-Apr-07 95 129 45
Fr

i6-Apr-07 96 130 46

Sat7-Apr-07 97 131 47

Sun8-Apr-07 98 132 48

Mon9-Apr-07 99 133 49

Tue 10-Apr-07 100 20 1348 50

Wed 11-Apr-07 101 135 51

Thu 12-Apr-07 102 136 52

Fri 13-Apr-07 103 137 53

Sat 14-Apr-07 104 138 54

Sun 15-Apr-07 105 139 55

Mon 16-Apr-07 106 140 56

Tue 17-Apr-07 107 21 1419 57

Wed 18-Apr-07 108 142 58

Thu 19-Apr-07 109 143 59

Fri 20-Apr-07 110 144 60

Sat 21-Apr-07 111 145 61

Sun 22-Apr-07 112 146 62

Mon 23-Apr-07 113 147 63

Tue 24-Apr-07 114 22 148 10 64

Wed 25-Apr-07 115 149 65

Thu 26-Apr-07 116 150 66

Fri 27-Apr-07 117 151 67

Sat 28-Apr-07 118 152 68

Sun 29-Apr-07 119 153 69

Mon 30-Apr-07 120 154 70

StartF1fertility-fecundityassessment

Tue1-May-07 121 23 155 11 711

Wed2-May-07 122 156 722

Thu3-May-07 123 157 733

Fri4-May-07 124 158 744

Sat5-May-07 125 159 755

Sun6-May-07 126 160 766

Mon7-May-07 127 161 777

Fertility-fecundityassessmentTue8-May-07 128 24 162 12 788

Wed9-May-07 129 163 799

Thu 10-May-07 130 164 80 10

Fri 11-May-07 131 165 81 11

Sat 12-May-07 132 166 82 12

Sun 13-May-07 133 167 83 13

Mon 14-May-07 134 168 84 14

StartF2 gen (Fillleftincubators)

Tue 15-May-07 135 25 169 13 85 1511

Second dayofF2 eggcollection (Fillrightincubators)

Wed 16-May-07 136 170 86 162

Thu 17-May-07 137 171 87 173

Fri 18-May-07 138 172 88 184

Sat 19-May-07 139 173 89 195

Sun 20-May-07 140 174 90 206

Mon 21-May-07 141 175 91 217

Tue 22-May-07 142 26 176 14 92 2228

Wed 23-May-07 143 177 93 239

Thu 24-May-07 144 178 94 24 10

Fri 25-May-07 145 179 95 25 11

Sat 26-May-07 146 180 96 26 12

Sun 27-May-07 147 181 97 27 13

Mon 28-May-07 148 182 98 28 14

Lastweekoffertility/fecundityassessment

Tue 29-May-07 149 27 183 15 99 293 15
Wed 30

-May-07 150 184 100 30 16

Thu 31-May-07 151 185 101 31 17

Fri1-Jun-07 152 186 102 32 18

Sat2-Jun-07 153 187 103 19

Sun3-Jun-07 154 188 104 20

TerminateF1

Mon4-Jun-07 155 189 105 21

Redistribute F2s 10/tank

Tue5-Jun-07 156 28 190 164 22

Wed6-Jun-07 157 191 23

Thu7-Jun-07 158 192 24

Fri8-Jun-07 159 193 25

Sat9-Jun-07 160 194 26

Sun 10-Jun-07 161 195 27

Mon 11-Jun-07 162 196 28

Tue 12-Jun-07 163 29 1975 29

Wed 13-Jun-07 164 198 30

Thu 14-Jun-07 165 199 31

Fri 15-Jun-07 166 200 32

Sat 16-Jun-07 167 201 33

Sun 17-Jun-07 168 202 34

Mon 18-Jun-07 169 203 35

Tue 19-Jun-07 170 30 2046 36

Wed 20-Jun-07 171 205 37

Thu 21-Jun-07 172 206 38

Fri 22-Jun-07 173 207 39

Sat 23-Jun-07 174 208 40

Sun 24-Jun-07 175 209 41

Fin Clip

Mon 25-Jun-07 176 210 42

Tue 26-Jun-07 177 31 2117 43

F2Cull to pairs(secondcull)

Wed 27-Jun-07 178 212 44

F2Cull to pairs(secondcull)

Thu 28-Jun-07 179 213 45

Fri 29-Jun-07 180 214 46

Sat 30-Jun-07 181 215 47

Sun1-Jul-07 182 216 48

Mon2-Jul-07 183 217 49

Tue3-Jul-07 184 32 2188 50

Wed4-Jul-07 185 219 51

Thu5-Jul-07 186 220 52

Fri6-Jul-07 187 221 53

Sat7-Jul-07 188 222 54

Sun8-Jul-07 189 223 55

Mon9-Jul-07 190 224 56

Tue 10-Jul-07 191 33 2259 57

Wed 11-Jul-07 192 226 58

Thu 12-Jul-07 193 227 59

Fri 13-Jul-07 194 228 60

Sat 14-Jul-07 195 229 61

Sun 15-Jul-07 196 230 62

Mon 16-Jul-07 197 231 63

Tue 17-Jul-07 198 34 232 10 64

Wed 18-Jul-07 199 233 65

Thu 19-Jul-07 200 234 66

Fri 20-Jul-07 201 235 67

Sat 21-Jul-07 202 236 68

Sun 22-Jul-07 203 237 69
M

on 23-Jul-07 204 238 70

StartF2fertility-fecundityassessment

Tue 24-Jul-07 205 35 239 11 71

Wed 25-Jul-07 206 240 72

Thu 26-Jul-07 207 241 73

Fri 27-Jul-07 208 242 74

Sat 28-Jul-07 209 243 75

Sun 29-Jul-07 210 244 76

Mon 30-Jul-07 211 245 77

Fertility-fecundityassessmentTue 31-Jul-07 212 36 246 12 78

Wed1-Aug-07 213 247 79

Thu2-Aug-07 214 248 80

Fri3-Aug-07 215 249 81

Sat4-Aug-07 216 250 82

Sun5-Aug-07 217 251 83

Mon6-Aug-07 218 252 84

StartF3 gen (Fillleftincubators)

Tue7-Aug-07 219 37 253 13 85

Second dayofF3 eggcollection (Fillrightincubators)

Wed8-Aug-07 220 254 86

Thu9-Aug-07 221 255 87

Fri 10-Aug-07 222 256 88

Sat 11-Aug-07 223 257 89

Sun 12-Aug-07 224 258 90

Mon 13-Aug-07 225 259 91

Tue 14-Aug-07 226 38 260 14 92

Wed 15-Aug-07 227 261 93

Thu 16-Aug-07 228 262 94

Fri 17-Aug-07 229 263 95

Sat 18-Aug-07 230 264 96

Sun 19-Aug-07 231 265 97

Mon 20-Aug-07 232 266 98

Lastweekoffertility/fecundityassessment

Tue 21-Aug-07 233 39 267 15 99

Wed 22-Aug-07 234 268 100

Thu 23-Aug-07 235 269 101

Fri 24-Aug-07 236 270 102

Sat 25-Aug-07 237 271 103

Sun 26-Aug-07 238 272 104

TerminateF2

Mon 27-Aug-07 239 273 105