# 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 e ach 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 ch emistry

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 timelineGen 

# 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 s ystem. 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 firs t 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 facilita te 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 t he 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 pool ed 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 replicat e 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

vitell ogenin 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 fertil ity 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

his topathology 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 individual s 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 F 1 and F2

The fecundity and fertility of reproductive pairs was assessed from the 12 th 

# through the 16 th 

# 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 populat e 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 hatc hlings within treatments were pooled before distribution during the 4 th 

# 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 be ing 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

th e 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., te stis, 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 proc edures 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 M MT using 4

- t

- octylphenol as the test agent.2

- G e n p r otoco l patte r ne d f r o m O ct y l P heno l (11 / 02 / 06)

E v

en t ( r o ws c r i t i ca l e v ent s on l y ) D a y D at e C a l en d D S td y W k S td y d F 0 W k F 0 da y F 0 R ep r d F 1 W k F 1 da y F 1 R ep r d F 2 W k F 2 da y C o l o r K e y

S e t

- u p i n c uba t or s w ith

~ 30 0 e m br y os N o te st ar t da y f or

e m br y o c o ll e cti o n is

T ue s da y

, no t M onday

S t ud y st ar t da te M o n 27

- N o v

- 06 331 H o li da y s

P u t 24 0 ( t o t a l) l ar v a e i n to 2

, 2 L t an k s T ue 28

- N o v

- 06 332 F0 / F 1 adu lt t er m i na ti on.

D a y 10 5 f o r ea ch

genera ti on

S e l e ct an d red ist r i bu te to 15

- 1 8 t an k s (1 0 in ea c h) T ue 29

- N o v

- 06 333 F0 / F 1 Fe c und it y /f er tility

P u t breed i n g pa i r s i n to 4 2 t an k s

S t ar t c he m ist r y in e x po s ur e s y st em M o n 26

- N o v

- 06 330 F0

, F1

, F 2

C u lli ng /s a m p li ng /s e t

- up

breed i n g pa i r s. F i r st c u ll

da y 22

, s e c on d c u ll da y s

43

- 44

A ss e ss pre

- e x po s ur e f er tilit y

- f e c und ity ( d 50

- 63) M o n 26

- N o v

- 06 330

E n d pre

- e x po s ur e f er tilit y

- f e c und ity 26

- N o v

- 06 330

M o ve breed i n g pa i r s i n to e x po s ur e s y st em

S t ar t e x po s ur e M o n 26

- N o v

- 06 330

S t ar t f er tilit y

- f e c und ity a ss e ss m en t ( d 65

- 85) T ues 26

- N o v

- 06 330

S t ar t F 1 ge n ( t w o i n c uba t or s pe r t an k ) T ues 26

- N o v

- 06 330

E n d f er tilit y

- f e c und ity a ss e ss m ent Fr i 26

- N o v

- 06 330

T er m i na te F 0 M o n 26

- N o v

- 06 330

R ed ist r i bu te F1 s 10 /t ank T ues 26

- N o v

- 06 330

T er m i na te F1 s if c on t ro ls ar e reprodu ci ng T ue s 26

- N o v

- 0 6 330

E v

en t ( 1 r o w fo r eac h da y o f stud y ) D a y D at e C a l en d D S td y W k S td y d F 0 W k F 0 da y F 0 R ep r d F 1 W k F 1 da y F 1 R ep r d F 2 W k F 2 da y D a y 06

- 0 7 ho li da y s S ta r t date

M o n 27

- N o v

- 06 331 M o n 25

- D e c

- 06 27

- N o v

- 06

S t ud y st ar t da te (F 0 e m br y o s c o ll e ct ed) T ue 28

- N o v

- 06 332 1 1 1 1 M o n 1

- J an

- 07

W ed 29

- N o v

- 06 333 2 2 M o n 15

- J an

- 07

T hu 30

- N o v

- 06 334 3 3 M o n 19

- Feb

- 07

Fr i 1

- D e c

- 06 335 4 4 M o n 28

- M a y

- 07

S at 2

- D e c

- 06 336 5 5 W ed 4

- J u l

- 07

S un 3

- D e c

- 06 337 6 6 M o n 3

- S ep

- 07

M o n 4

- D e c

- 06 338 7 7 M o n 8

- Oct

- 0 7

P u t 24 0 ( t o t a l) l ar v a e i n to 2

, 2 L t an k s T ue 5

- D e c

- 06 339 2 8 2 8 M o n 12

- N o v

- 07

W ed 6

- D e c

- 06 340 9 9 T hu 22

- N o v

- 07

T hu 7

- D e c

- 06 341 10 10 T ue 25

- D e c

- 07

Fr i 8

- D e c

- 06 342 11 11

S at 9

- D e c

- 06 343 12 12

S un 10

- D e c

- 06 344 13 13

M o n 11

- D e c

- 06 345 14 14

T ue 12

- D e c

- 06 346 3 15 3 15

W ed 13

- D e c

- 06 347 16 16

T hu 14

- D e c

- 06 348 17 17

Fr i 15

- D e c

- 06 349 18 18

S at 16

- D e c

- 06 350 19 19

S un 17

- D e c

- 06 351 20 20

M o n 18

- D e c

- 06 352 21 21

S e l e ct an d red ist r i bu te to 15

- 1 8 t an k s (1 0 in ea c h) T ue 19

- D e c

- 06 353 4 22 4 22

W ed 20

- D e c

- 06 354 23 23T hu 21

- D e c

- 06 355 24 24

Fr i 22

- D e c

- 06 356 25 25

S at 23

- D e c

- 06 357 26 26

S un 24

- D e c

- 06 358 27 27

M o n 25

- D e c

- 06 359 28 28

T ue 26

- D e c

- 06 360 5 29 5 29

W ed 27

- D e c

- 06 361 30 30

T hu 28

- D e c

- 06 362 31 31

Fr i 29

- D e c

- 06 363 32 32

S at 30

- D e c

- 06 364 33 33

S un 31

- D e c

- 06 365 34 34

M o n 1

- J an

- 07 1 35 35

T ue 2

- J an

- 07 2 6 36 6 36

W ed 3

- J an

- 07 3 37 37

T hu 4

- J an

- 07 4 38 38

Fr i 5

- J an

- 07 5 39 39

S at 6

- J an

- 07 6 40 40

S un 7

- J an

- 07 7 41 41

M o n 8

- J an

- 07 8 42 42

S e t u p 5 6 breed i n g pa i r s

S t ar t c he m ist r y in e x po s ur e s y st em T ue 9

- J an

- 07 9 7 43 7 43

W ed 10

- J an

- 07 10 44 44

T hu 11

- J an

- 07 11 45 45

Fr i 12

- J an

- 07 12 46 46

S at 13

- J an

- 07 13 47 47

S un 14

- J an

- 07 14 48 48

M o n 15

- J an

- 07 15 49 49

T ue 16

- J an

- 07 16 8 50 8 50

W ed 17

- J an

- 07 17 51 51

T hu 18

- J an

- 07 18 52 52

Fr i 19

- J an

- 07 19 53 53

S at 20

- J an

- 07 20 54 54

S un 21

- J an

- 07 21 55 55

M o n 22

- J an

- 07 22 56 56

T ue 23

- J an

- 07 23 9 57 9 57

W ed 24

- J an

- 07 24 58 58

T hu 25

- J an

- 07 25 59 59

Fr i 26

- J an

- 07 26 60 60

S at 27

- J an

- 07 27 61 61

S un 28

- J an

- 07 28 62 62

M o n 29

- J an

- 07 29 63 63

B eg in pre

- e x po s ur e f er tilit y

- f e c und ity T ue 30

- J an

- 07 30 10 64 10 64

W ed 31

- J an

- 07 31 65 65

T hu 1

- Feb

- 07 32 66 66

Fr i 2

- Feb

- 07 33 67 67

S at 3

- Feb

- 07 34 68 68

E n d pre

- e x po s ur e f er tilit y

- f e c und ity S un 4

- Feb

- 07 35 69 69

M o ve breed i n g pa i r s i n to e x po s ur e s y st em

S t ar t e x po s ur e M o n 5

- Feb

- 07 36 70 70 N o t e : t h is is a ct ua lly da y 6 9 dp f, c on sist en t w ith ou r o t he r t e sts. D. L.

S t ar t F 0 f er tilit y

- f e c und ity a ss e ss m ent T ue 6

- Feb

- 07 37 11 71 11 71 1

W ed 7

- Feb

- 07 38 72 72 2

T hu 8

- Feb

- 07 39 73 73 3

Fr i 9

- Feb

- 07 40 74 74 4

S at 10

- Feb

- 07 41 75 75 5S un 11

- Feb

- 07 42 76 76 6

M o n 12

- Feb

- 07 43 77 77 7

Fer tilit y

- f e c und ity a ss e ss m ent T ue 13

- Feb

- 07 44 12 78 12 78 8

W ed 14

- Feb

- 07 45 79 79 9

T hu 15

- Feb

- 07 46 80 80 10

Disi n f e ct s y st e m Fr i 16

- Feb

- 07 47 81 81 11

S at 17

- Feb

- 07 48 82 82 12

S un 18

- Feb

- 07 49 83 83 13

S w a p in cl ea n t an k s M o n 19

- Feb

- 07 50 84 84 14

S t ar t F 1 ge n (F ill l e ft i n c uba t or s) T ue 20

- Feb

- 07 51 13 85 13 85 15 1 1

S e c on d da y o f F 1 eg g c o ll e cti o n (F ill r i gh t i n c uba t or s) W ed 21

- Feb

- 07 52 86 86 16 2

T hu 22

- Feb

- 07 53 87 87 17 3

Fr i 23

- Feb

- 07 54 88 88 18 4

S at 24

- Feb

- 07 55 89 89 19 5

S un 25

- Feb

- 07 56 90 90 20 6

M o n 26

- Feb

- 07 57 91 91 21 7

T ue 27

- Feb

- 07 58 14 92 14 92 22 2 8

W ed 28

- Feb

- 07 59 93 93 23 9

T hu 1

- M ar

- 0 7 60 94 94 24 10

Fr i 2

- M ar

- 0 7 61 95 95 25 11

S at 3

- M ar

- 0 7 62 96 96 26 12

S un 4

- M ar

- 0 7 63 97 97 27 13

M o n 5

- M ar

- 0 7 64 98 98 28 14

La st w ee k o f f er tilit y /f e c und ity a ss e ss m ent T ue 6

- M ar

- 0 7 65 15 99 15 99 29 3 15

W ed 7

- M ar

- 0 7 66 100 100 30 16

T hu 8

- M ar

- 0 7 67 101 101 31 17

Fr i 9

- M ar

- 0 7 68 102 102 32 18

S at 10

- M ar

- 07 69 103 103 19

S un 11

- M ar

- 07 70 104 104 20

T er m i na te F 0 M o n 12

- M ar

- 07 71 105 105 21

R ed ist r i bu te F1 s 10 /t an k (F i r st c u ll) T ue 13

- M ar

- 07 72 16 106 4 22

W ed 14

- M ar

- 07 73 107 23

T hu 15

- M ar

- 07 74 108 24

Fr i 16

- M ar

- 07 75 109 25

S at 17

- M ar

- 07 76 110 26

S un 18

- M ar

- 07 77 111 27

T a k edo w n o f e x t r a F1? M o n 19

- M ar

- 07 78 112 28

T ue 20

- M ar

- 07 79 17 113 5 29

W ed 21

- M ar

- 07 80 114 30

T hu 22

- M ar

- 07 81 115 31

Fr i 23

- M ar

- 07 82 116 32

S at 24

- M ar

- 07 83 117 33

S un 25

- M ar

- 07 84 118 34

M o n 26

- M ar

- 07 85 119 35

T ue 27

- M ar

- 07 86 18 120 6 36

W ed 28

- M ar

- 07 87 121 37

T hu 29

- M ar

- 07 88 122 38

Fr i 30

- M ar

- 07 89 123 39

S at 31

- M ar

- 07 90 124 40

S un 1

- A pr

- 0 7 91 125 41

F in Clip M o n 2

- A pr

- 0 7 92 126 42

T ue 3

- A pr

- 0 7 93 19 127 7 43

F 1 C u ll to pa i r s ( s e c on d c u ll) W ed 4

- A pr

- 0 7 94 128 44

F 1 C u ll to pa i r s ( s e c on d c u ll) T hu 5

- A pr

- 0 7 95 129 45Fr i 6

- A pr

- 0 7 96 130 46

S at 7

- A pr

- 0 7 97 131 47

S un 8

- A pr

- 0 7 98 132 48

M o n 9

- A pr

- 0 7 99 133 49

T ue 10

- A pr

- 07 100 20 134 8 50

W ed 11

- A pr

- 07 101 135 51

T hu 12

- A pr

- 07 102 136 52

Fr i 13

- A pr

- 07 103 137 53

S at 14

- A pr

- 07 104 138 54

S un 15

- A pr

- 07 105 139 55

M o n 16

- A pr

- 07 106 140 56

T ue 17

- A pr

- 07 107 21 141 9 57

W ed 18

- A pr

- 07 108 142 58

T hu 19

- A pr

- 07 109 143 59

Fr i 20

- A pr

- 07 110 144 60

S at 21

- A pr

- 07 111 145 61

S un 22

- A pr

- 07 112 146 62

M o n 23

- A pr

- 07 113 147 63

T ue 24

- A pr

- 07 114 22 148 10 64

W ed 25

- A pr

- 07 115 149 65

T hu 26

- A pr

- 07 116 150 66

Fr i 27

- A pr

- 07 117 151 67

S at 28

- A pr

- 07 118 152 68

S un 29

- A pr

- 07 119 153 69

M o n 30

- A pr

- 07 120 154 70

S t ar t F 1 f er tilit y

- f e c und ity a ss e ss m ent T ue 1

- M a y

- 07 121 23 155 11 71 1

W ed 2

- M a y

- 07 122 156 72 2

T hu 3

- M a y

- 07 123 157 73 3

Fr i 4

- M a y

- 07 124 158 74 4

S at 5

- M a y

- 07 125 159 75 5

S un 6

- M a y

- 07 126 160 76 6

M o n 7

- M a y

- 07 127 161 77 7

Fer tilit y

- f e c und ity a ss e ss m ent T ue 8

- M a y

- 07 128 24 162 12 78 8

W ed 9

- M a y

- 07 129 163 79 9

T hu 10

- M a y

- 07 130 164 80 10

Fr i 11

- M a y

- 07 131 165 81 11

S at 12

- M a y

- 07 132 166 82 12

S un 13

- M a y

- 07 133 167 83 13

M o n 14

- M a y

- 07 134 168 84 14

S t ar t F 2 ge n (F ill l e ft i n c uba t or s) T ue 15

- M a y

- 07 135 25 169 13 85 15 1 1

S e c on d da y o f F 2 eg g c o ll e cti o n (F ill r i gh t i n c uba t or s) W ed 16

- M a y

- 07 136 170 86 16 2

T hu 17

- M a y

- 07 137 171 87 17 3

Fr i 18

- M a y

- 07 138 172 88 18 4

S at 19

- M a y

- 07 139 173 89 19 5

S un 20

- M a y

- 07 140 174 90 20 6

M o n 21

- M a y

- 07 141 175 91 21 7

T ue 22

- M a y

- 07 142 26 176 14 92 22 2 8

W ed 23

- M a y

- 07 143 177 93 23 9

T hu 24

- M a y

- 07 144 178 94 24 10

Fr i 25

- M a y

- 07 145 179 95 25 11

S at 26

- M a y

- 07 146 180 96 26 12

S un 27

- M a y

- 07 147 181 97 27 13

M o n 28

- M a y

- 07 148 182 98 28 14

La st w ee k o f f er tilit y /f e c und ity a ss e ss m ent T ue 29

- M a y

- 07 149 27 183 15 99 29 3 15W ed 30

- M a y

- 07 150 184 100 30 16

T hu 31

- M a y

- 07 151 185 101 31 17

Fr i 1

- J un

- 07 152 186 102 32 18

S at 2

- J un

- 07 153 187 103 19

S un 3

- J un

- 07 154 188 104 20

T er m i na te F 1 M o n 4

- J un

- 07 155 189 105 21

R ed ist r i bu te F2 s 10 /t ank T ue 5

- J un

- 07 156 28 190 16 4 22

W ed 6

- J un

- 07 157 191 23

T hu 7

- J un

- 07 158 192 24

Fr i 8

- J un

- 07 159 193 25

S at 9

- J un

- 07 160 194 26

S un 10

- J un

- 07 161 195 27

M o n 11

- J un

- 07 162 196 28

T ue 12

- J un

- 07 163 29 197 5 29

W ed 13

- J un

- 07 164 198 30

T hu 14

- J un

- 07 165 199 31

Fr i 15

- J un

- 07 166 200 32

S at 16

- J un

- 07 167 201 33

S un 17

- J un

- 07 168 202 34

M o n 18

- J un

- 07 169 203 35

T ue 19

- J un

- 07 170 30 204 6 36

W ed 20

- J un

- 07 171 205 37

T hu 21

- J un

- 07 172 206 38

Fr i 22

- J un

- 07 173 207 39

S at 23

- J un

- 07 174 208 40

S un 24

- J un

- 07 175 209 41

F in Clip M o n 25

- J un

- 07 176 210 42

T ue 26

- J un

- 07 177 31 211 7 43

F 2 C u ll to pa i r s ( s e c on d c u ll) W ed 27

- J un

- 07 178 212 44

F 2 C u ll to pa i r s ( s e c on d c u ll) T hu 28

- J un

- 07 179 213 45

Fr i 29

- J un

- 07 180 214 46

S at 30

- J un

- 07 181 215 47

S un 1

- J u l

- 07 182 216 48

M o n 2

- J u l

- 07 183 217 49

T ue 3

- J u l

- 07 184 32 218 8 50

W ed 4

- J u l

- 07 185 219 51

T hu 5

- J u l

- 07 186 220 52

Fr i 6

- J u l

- 07 187 221 53

S at 7

- J u l

- 07 188 222 54

S un 8

- J u l

- 07 189 223 55

M o n 9

- J u l

- 07 190 224 56

T ue 10

- J u l

- 07 191 33 225 9 57

W ed 11

- J u l

- 07 192 226 58

T hu 12

- J u l

- 07 193 227 59

Fr i 13

- J u l

- 07 194 228 60

S at 14

- J u l

- 07 195 229 61

S un 15

- J u l

- 07 196 230 62

M o n 16

- J u l

- 07 197 231 63

T ue 17

- J u l

- 07 198 34 232 10 64

W ed 18

- J u l

- 07 199 233 65

T hu 19

- J u l

- 07 200 234 66

Fr i 20

- J u l

- 07 201 235 67

S at 21

- J u l

- 07 202 236 68

S un 22

- J u l

- 07 203 237 69M o n 23

- J u l

- 07 204 238 70

S t ar t F 2 f er tilit y

- f e c und ity a ss e ss m ent T ue 24

- J u l

- 07 205 35 239 11 71

W ed 25

- J u l

- 07 206 240 72

T hu 26

- J u l

- 07 207 241 73

Fr i 27

- J u l

- 07 208 242 74

S at 28

- J u l

- 07 209 243 75

S un 29

- J u l

- 07 210 244 76

M o n 30

- J u l

- 07 211 245 77

Fer tilit y

- f e c und ity a ss e ss m ent T ue 31

- J u l

- 07 212 36 246 12 78

W ed 1

- A ug

- 07 213 247 79

T hu 2

- A ug

- 07 214 248 80

Fr i 3

- A ug

- 07 215 249 81

S at 4

- A ug

- 07 216 250 82

S un 5

- A ug

- 07 217 251 83

M o n 6

- A ug

- 07 218 252 84

S t ar t F 3 ge n (F ill l e ft i n c uba t or s) T ue 7

- A ug

- 07 219 37 253 13 85

S e c on d da y o f F 3 eg g c o ll e cti o n (F ill r i gh t i n c uba t or s) W ed 8

- A ug

- 07 220 254 86

T hu 9

- A ug

- 07 221 255 87

Fr i 10

- A ug

- 07 222 256 88

S at 11

- A ug

- 07 223 257 89

S un 12

- A ug

- 07 224 258 90

M o n 13

- A ug

- 07 225 259 91

T ue 14

- A ug

- 07 226 38 260 14 92

W ed 15

- A ug

- 07 227 261 93

T hu 16

- A ug

- 07 228 262 94

Fr i 17

- A ug

- 07 229 263 95

S at 18

- A ug

- 07 230 264 96

S un 19

- A ug

- 07 231 265 97

M o n 20

- A ug

- 07 232 266 98

La st w ee k o f f er tilit y /f e c und ity a ss e ss m ent T ue 21

- A ug

- 07 233 39 267 15 99

W ed 22

- A ug

- 07 234 268 100

T hu 23

- A ug

- 07 235 269 101

Fr i 24

- A ug

- 07 236 270 102

S at 25

- A ug

- 07 237 271 103

S un 26

- A ug

- 07 238 272 104

T er m i na te F 2 M o n 27

- A ug

- 07 239 273 105