### DATA EVALUA TIO N RECORD

F I SH LIFE-CYCLE TOXICITY T E ST

§ 72-5



### I. 

### CHEM I C A L: Ethoprop PC Code No.: 041 1 01



### 2. TEST MATERIAL: E th opro ph os Tec hni ca l P ur i ty: 94.8 % 

## (w:w)

3. CITAT IO N:

A uth o r: Di o nn e, E

.

Titl e: E th o prop

- C hroni c T o xi c it y t o th e S h ee p s head Minn ow



## (Cy prin odo n v ari ega tu s) Durin g a F ull L i fe -C y cle

E xp os ur e.

S tu dy Co m p l et i o n D a t e: Jun e 29, 20 0 4

Labora t o ri es: S prin g b o rn S m i th ers Laborato ri es

790 M a in Street

W areham, MA 0257 1

- 1 075

Sponsor : B aye r CropScience

17745 South Metcalf Avenue

St il we ll

, K S 66085

Laboratory R eport I D: 1 3798.6 1 54**~** 

### M RlD No

. : 463 15401

DP Bar co d e: D35 92 01, D 3 0 6508



### 4. R E VIEWED BY: C hri st ie E. Pado v a

, St a ff Sc i e nti s t

, D y n a m ac Co rp o ration

Sig n at ur e : D ate : 11 / 23 / 04



### APPROV E D BY: Te ri Mye r s, Ph

. D.

, S t aff Sc i e n t i st, Dy n amac Corporat i on

Sig n ature:



## 5. APPROVED BY:

## ;::**p:ing~ PP** **~B-V**



## S i g n a tur e: 

# j**V'J'~** Date: 1 217 / 04

Date: 4

- 30

- 09

## DATA EVALUATION RECORD

FISH LIFE

- CYCLE TOXICITY TEST

§72

- 5

1. CHEMICAL : Ethoprop PC Code No. : 041101

2. TEST MATERIAL : Ethoprophos Technical Purity : 94.8% (w:w)

3. CITATION :

Author : Dionne, E.

Title : Ethoprop

- Chronic Toxicity to the Sheepshead Minnow

( Cyprinodon variegatus ) During a Full Life

- Cycle

Exposure.

Study Completion Date : June 29, 2004

Laboratories : Springborn Smithers Laboratories

790 Main Street

Wareham, MA 02571

- 1075

Sponsor : Bayer CropScience

17745 South Metcalf Avenue

Stilwell, KS 66085

Laboratory Report ID : 13798.6154

MRID No. : 46315401

DP Barcode : D359201

, D306508

4. REVIEWED BY : Christie E. Padova, Staff Scientist, Dynamac Corporation

Signature: Date: 11/23/04

APPROVED BY : Teri Myers, Ph.D., Staff Scientist, Dynamac Corporation

Signature: Date: 12/7/04

5. APPROVED BY : Keith Sappington , OPP/EFED/ERB

- V

Signature: Date: 4

- 30

- 09

### DP Barcode: D306508 MRID No: 46315401



## 2 6. STUDY PARAMETERS :

Scientific Name of Test Organism: Cyprinodon variegatus

Age of Test Organism: Em bryos, >24 to <48 hours old (F 0 

## generation)

Definitive Test Duration: 112 Days (Approx. 3.5 months)

Study Method: Flow

- through

Type of Concentrations: Mean

- measured

7. CONCLUSIONS :

The 3.5

- month chronic toxicity of Ethoprophos Technical (ethop rop) to the full life stage

of Sheepshead minnow ( Cyprinodon variegatus ) was studied under flow

- through

conditions. Fertilized eggs (280 embryos/treatment, >24 to <48 hours old) were exposed to

the test material at nominal concentrations of 0 (negative co ntrol), 1.0, 2.0, 4.0, 8.0, and 16

ppb a.i. Mean

- measured concentrations were <0.22 (control), 1.2, 2.0, 3.7, 7.4, and 15 ppb

a.i., respectively, and were generally stable during the study.

Following the completion of hatching on Day 6, larvae were red uced to 100 per treatment

level. At 4

- weeks post

- hatch, the juveniles were again reduced to 50 per treatment level.

Spawning was initiated 8 weeks following hatching: four groups of two male and five

female per test level were assigned to spawning aquar ia, and hatchability trials and early

life stage studies were performed for the F 1 

## generation. Following hatching, the F 1 

## generation was maintained for 4 weeks. The F 0 

## portion of test was terminated 16 weeks

after hatching (Day 118).

This study i s class ified as SUPPLEMENTAL because of several protocol deviations

involving measurement of embryo viability and poor statistical power for the F1 generation

endpoints.

Results Synopsis:

NO A EC: 2.0

LO A EC: 3.7

Endpoint(s) Affected: growth (weight) of 4

- week old F1 minnows (32% reduction at

LOAEC)

8. ADEQUACY OF THE STUDY :

A. Classification: SUPPLEMENTAL

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## 3 B. Rationale:

C. Repairability: This study is not considered upgradable

.

9. GUIDELINE DEVIATIONS :

1. The tempera ture ranged from 24

- 27 ° C throughout the study, which is less than the

recommended temperature of 30 ° C for this species.

2. It was not reported if aeration of the test aquaria was necessary during the study.

3. F 1



## - generation fish were maintained for only 4 we eks, instead of the required 8

weeks.

4. The reviewer

- calculated high

- low ratio of mean

- measured concentrations was 1.7

for the nominal 2.0 ppb treatment level, exceeding the 1.5 limit.

5. T his study design o nly included 2 true replicates. Although consistent with the

minimum number of replicates recommended in the study guideline, the small

number of replicates may have compromised the statistical power of the test,

particularly for F1 generation endpoints (see Table 1 below). Since the F1

endpoints are only addressed by full life cycle studies, these endpoints are of

particular importance in the study evaluation. For ethoprop, however, the general

lack of a dose

- response was observed for all but one F1 generation endpoint (i.e.,

modest % reductions in mean responses in the treatments relative to controls).

Thus, for those F1 endpoints where a dose

- response was not observed, the poor

statistical power of the F1 endpoint comparisons is moot. The one exception is

growth of F1 generation fish measured at 4 wee ks post hatch. For this endpoint, a

32% reduction in mean wet weight of 4

- week post hatch minnows relative to

negative controls occurred at 3.7, 7.4, 15 ppb but these were not found to be

statistically significant. Reductions in length were of up to 11% were also

observed in these treatments relative to controls. Since the variance in control

treatments for these endpoints is very small, the lack of statistical power appears

to be due to the small number of replicates (2) used in the study. Rather than

invalidating the study, this reviewer is re

- interpreting the study to consider the

32% reduction in wet weight of 4

- week post

- hatch F1 minnows at 3.7, 7.4, and 15

ppb to be biologically significant . T herefore, the NOAEC from this study is

now interpreted to be 2.0 ppb and the LOAEC is interpreted to be 3.7 ppb,

based on mean measured concentrations.

### DP Barcode: D306508 MRID No: 46315401



## 4 6. Embryo viability measurements were based on a single subsample of 100

embryos and appears to have introduced bias in the % hatching success

calculation ( see Figure 1 below). However, control % hatching success still

meets the guideline recommended 75% minimum for sheepshead minnow, when

Day 1 embryo viability measurements are used instead of the Day 0 subsample.

Table 1. Minimum Significant Differences (MSD) based on Dunnetts Statistical

Comparisons from the Ethoprop Fish Full Life Cycle Study (MRID MRID 46315401)

MSD (%) Endpoint Type Generation Comments

18.7 % hatch_F0 hatch F0 Reasonable MSD observed

17.4 % viability surv F0 Reasonable MSD obs erved

2.9 length_d28 growth F0 Reasonable MSD observed

11.6 weight_d28 growth F0 Reasonable MSD observed

52 % survival_F0 surv F0 Reasonable MSD observed

4.9 length_d28_male growth F0 Reasonable MSD observed

7.6 length_d28_fem growth F0 Reasonabl e MSD observed

9.4 length_fem growth F0 Reasonable MSD observed

16.6 weight_male growth F0 Reasonable MSD observed

52.9 # eggs_F1 repro F1 17% reduction at 15 ppb, no

consistent dose

- response < 15 ppb (1)



### 48.3 # eggs/fem_F1 repro F1 16% reduction at 15 ppb, no

consistent dose

- response < 15 ppb (1)



### 35.4 % hatch_F1 repro F1 25% reduction @ 15 ppb, no

consistent dose

- response < 15 ppb

27.2 length_F1 growth F1 12% reduction @ 15 ppb, 10% @ 7.4

& 3.7 ppb

62.6 # hatch_d7 hatch F1 2% reduction @ 15 ppb, no

consistent dose

- response

>32 weight_F1 growth F1 32% reduction in F1 weight @ 3.7,

7.4 and 15 ppb (Kruskal Walis)

(1) 

### a cursory analysis of data for this endpoint from 5 fish full life cycle studies suggest that it is typically

subject to high variabili ty in controls

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## 5 Figure 1. Comparison of Corrected Sheepshead Minnow % Hatch:

Mean Day 1 vs. Day 0 Subsample Embryo Viability

Relative Difference Between MEAN D0 and D1-Corrected

Percent Hatch for Sheepshead Minnow FLC

11%

2% 5% 14% 11% 12%

7%

0% 20% 40%

1 2 3 4 5 6 7

Compound% Difference Rel. to Day 1 

### SHM % Hatch Correction:

Day 0 Subsample vs. D1 Viability

60 70 80 90 100 110 120

60 80 100 120

Day 0 Subsample-CorrectedDay 1 ViabilityCorrected



### Data

1:1 line



## 10. SUBMISSION PURPOSE : This study was submitted to provide data on the toxici ty of

ethoprop to the full life

- cycle of s heeps head minnows for the purposes of chemical re

registration.

11. MATERIALS AND METHODS :

A. Test Organisms

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## 6 Guideline Criteria Reported Information

Species

Prefer Sheepshead minnow ( Cyprinodon

variegatus ) or Fathead minnow

( Pimephales promelas ). Sheepshead minnow ( Cyprinodon

variegatus )

Source and Acclimation Embryos were obtained from Aquatic

Biosystems, Fort Collins, CO. The

embryos were held in natural filtered

seawater at 22 ° C for approx. 1 hour, then

at 25 ° C until assignment.

Age at beginning of test

Embryos, 2 to 24 hours old Embryos, >24 to <48 hours old

Feeding

Fish should be fed at least twice daily and

should not be fed for at least 24 hours

prior to test termination. F 0 

## and F 1 

## larvae were fed live brine

shrimp nauplii thre e times daily during the

first 28 days post

- hatch. After 14 days

post

- hatch, small amounts of dry flaked

food was also offered daily. The juvenile

and adult fish were fed frozen brine

shrimp and Zeigler Prime Flakes twice

daily. Feedings were withheld 2 4 hours

before weight determinations.

Embryo Exposure (4 to 5 Days)

Embryos ( ≤ 24 hours old) from at least 3

separate spawns should be randomly

distributed to embryo cups.

A minimum of 50 embryos ( ≤ 24 hrs old)

per replicate cup, 4 cups per treatment

sho uld be used.

Parameters measured :

⋅ Survival of embryos

⋅ Time required to hatch

⋅ Hatching success Days 0

- 6

Embryos (> 24 to <48 hours old) were

randomly ass igned into embryo incubation

cups; the number of origin spawns was not

reported.

Each cup contained 70 embryos, with two

cups per replicate and two replicate

aquaria per treatment level (total of 280

embryos per treatment).

Parameters measured :

⋅ Hatching success

⋅ Time to hatch

⋅ Survival of fry at 4 weeks post

- hatch

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## 7 Guideline Criteria Reported Information

⋅ Survival of fry for 4 weeks

Dead and fungused embryos should be

counted and removed daily. Mortality was determined daily. Dead

embryos were removed.

Larval

- Juvenile Exposure (From Hatch

to 8 Weeks)

After hatching, each group of larvae is

randomly reduced to a minimum of 25 fish

an d released in replicate larval growth

chambers. The random selection must

include any fish that are lethargic or

deformed.

Parameters measured :

⋅ Fish survival (determined by

counting the number of live fish in

each replicate growth chamber

weekly).

⋅ Total lengths (mm) of all fish at 4

and 8 weeks after hatching. Hatch to 8 Weeks Post

- Hatch

When hatching was complete (on Day 6),

25 larvae were impartially selected from

each cup and transferred to the larval

growth chambers (two chambers within

each replicat e aquarium, 100 larvae per

treatment). At 4 weeks post

- hatch,

juvenile fish from the two growth

chambers were combined and impartially

reduced to 25 per replicate (50 per

treatment).

Parameters measured :

⋅ Survival of fry/juvenile fish at 8

weeks post

- ha tch

⋅ Total lengths (mm) of all surviving

fish at 4 and 8 weeks post

- hatch

(gender

- specific at 8 weeks).

⋅ Wet weights (mg) of fish

discontinued from exposure (at

thinning) at 4 weeks post

- hatch

Juvenile

- Adult Exposure (From 8 weeks

posthatch to the end of t he spawning

phase [32

- 40 weeks])

At 20

- 24 weeks after hatching, mature fish

are placed in a spawning tank of the same

concentration (4 males and 4 females

randomly chosen and assigned). The

spawning tank is divided into 4 individual

spawning chambers wit h appropriate

spawning substrates.

The substrates are examined daily and 8 to 16 Weeks Post

- Hatch

Two spawning groups (2 male and 5

female per group) were established for

each replicate aquarium. The first group

was established on Day 61 (55 days post

hatc h), and the second 14 days following

the first. Females killed by male

aggression were not replaced; however,

males were replaced in order to maximize

egg fertilization success.

The spawning substrates are examined

daily and embryos removed, counted, a nd

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## 8 Guideline Criteria Reported Information

embryos removed, counted, and recorded

separately for each pair.

For fathead minnow, adult exposure

should be terminated when no spawning

occurs for one week. For sheepshead

minnow , testing should be terminated after

spawning is observed for 2 weeks. examined for fertility.

Adult exposure was terminated on Day

118 (112 days post

- hatch).

Parameters measured :

⋅ Survival of adult fish

⋅ Fecundity (eggs/female/day)

⋅ Total lengths (mm) and wet weights

(g) of all surviving fish at Day 118

(gender

- specific )

Second Generation Embryo Exposure

(4 to 5 days)

50 embryos from each conc. level are

randomly selected and transferred to

incubation cups for hatch. Use the same

test procedures as those for parental

generation.

Embryos not selected are discarded. F 1 

## Embryo Exposure

50 embryos from spawns of ≥ 50 eggs were

incubated in each incubation cup as

previously described (200 embryos per

treatment level).

Second Generation Larval

- Juvenile

Exposure (From Hatch to 4

- 8 weeks)

After hatching, 25 larvae are rele ased in

each growth chambers (2 chambers per

treatment).

Each group of 2 nd 

## generation fish is

terminated 8 weeks after hatching.

Fish are blotted, weighed, and measured

before being discarded. F 1 

## Larval

- Juvenile Exposure

Groups of 25 newly

- hatched larv ae were

randomly released into each larval growth

chamber (100 larvae per treatment level).

Each group of F 1



## - generation fish was

terminated 28 days after hatching.

Fish were weighed (wet) and measured for

total length.

Comments : 850.1400 guideline r equirements specify that the hatching success of the controls for

sheepshead minnows should be >75%; however, the mean hatching success of the controls in this

study (57% ; page 27 of report ) does not meet guideline requirements. A sub

- sample of 100

embryo s was examined for viability, and based on visible developmental stages, it was estimated

that 68% of the eggs were viable (p. 18). Embryo hatching success values were adjusted by the

### DP Barcode: D306508 MRID No: 46315401



## 9 study authors using this estimated percent viability.

B. Test Syst em

Guideline Criteria Reported Information

Test Water

Sheepshead Minnow

1. Natural seawater (sterilized and

filtered) or a commercial mixture.

2. Natural seawater with a salinity of

> 15 parts per thousand (weekly range

of salinity <6% and monthly pH range

<0.8 pH units).

Fathead Minnow

1. Reconstituted water or water from

unpolluted well or spring (sterilized

and tested for pollutants).

2. Hardness of 40 to 48 mg/L as CaCO 3 

## and pH of 7.2 to 7.6. 1. Natural filtered seawater collected

from Cape Cod Canal, Bourne, MA.

2. Salinity of 31

- 33 ‰ and pH 7.6

- 8.0.

N/A

Test Temperature

Sheepshead : 30 ° C.

Fathead : 25 ° C and should not remain

outside the range of 24 to 26 ° C for more

than 48 hours. 24

- 27 ° C

N/A

Photo

- period

16

- hour light/8

- ho ur dark.

Light intensity of 10

- 100 lumens at water

surface. 16

- hour light/8

- hour dark cycle

Light intensity of 52

- 116 foot

- candles

(560

- 1200 lux)

Dosing Apparatus

1. Intermittent flow proportional

diluters or continuous flow serial

diluters.

2. A min imum of 5 toxicant 1. Intermittent

- flow proportional diluter.

2. Five toxicant concentrations with a

dilution factor of 0.5.

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## 10 Guideline Criteria Reported Information

concentrations with a dilution factor

< 0.5.

3. One control should be used. 3. A dilution water (negative) control was

used.

Toxicant Mixing

1. Mixing chamber recommended but

not required.

2. Test solution completely mixed

before introduction into the test system

(aeration should not be used for

mixing).

3. Flow splitting accuracy must be

within 10% and periodically checked. 1. The diluter system incorporated a

mixing chamber.

2. Yes

3. The flow

- splitting accuracy was <4%

(p. 19).

Exposure System/Test Vessels

Exposure tanks should be all glass or

glass with a plastic or stainless steel frame

(30.5 x 30.5 x 91.4 cm or 30.5 x 30.5 x 61

cm for fathead, and 45 x 90 x 26 cm for

sheepshead).

Larval chambers should have glass

bottoms and drains that allow water to be

drawn down to 3 cm.

Test water depth in adult tanks and larval

chambers should be a minimum of 15 cm. Gla ss exposure aquaria (60 x 30 x 30 cm)

were used, with a fill volume of 27 L and

depth of 15 cm.

The larval growth chambers were

constructed of glass and Nitex screening

and measured 30 x 13 x 25 cm; two larval

growth chambers were positioned within

each aquarium.

During spawning, designated aquaria each

contained two spawning baskets made of

8.5

- mesh nylon screening. Each basket

measured 30 cm 2 

## (depth of approx. 12

cm) and was placed over a removable egg

collection tray. The trays were

constructed wi th 3

- cm high glass sides

and 40

- mesh nylon screening bottom.

Embryo and Fry Chambers

120 mL glass jars with bottoms replaced

with 40 mesh stainless steel or nylon

screen. Chambers can be oscillated The embryo incubation cups were 5 cm

diameter glass jars with 40

- mesh Nitex

screen bottoms and stainless steel wire

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## 11 Guideline Criteria Reported Information

vertically using rocker arm apparatus (2

rpm motor) or placed in separate

chambers with self

- starting siphons. handles.

Flow Rate

Flow rates to adult tanks or larval

chambers should provide 90%

replacement in 8

- 12 hours, and maintain

DO at above 75% of saturation. The

toxicant level cannot drop below 20%

with fish in the tank. During the pre

- spawning phase, the flow

rate was 7.9 volume additions per day.

During the spawning phase, the fl ow rate

was 7.7 volume additions per day.

Aeration

Dilution water should be aerated to insure

dissolved oxygen concentrations at or

near 100% saturation. Test tanks and

embryo chambers should not be aerated. Not specified

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## 12 C. Chemical System

Guideline Criteria Reported Information

Nominal Concentrations

Minimum of 5 concentrations and a

control, all replicated; plus solvent control

if appropriate.

Toxicant conc. must be measured in one

tank at each toxicant level every week. 0 (negative control), 1.0, 2.0, 4.0, 8.0, and

16 ppb a.i.

Toxicant concentrations were measured

weekly from alternating replicate aquaria

in each test group.

Other Variables

1. DO must be measured at each conc.

at least once a week.

2. Test water temp. must be r ecorded

continuously.

3. Freshwater : A control and one conc.

must be analyzed weekly for pH,

alkalinity, hardness, and conductance.

Natural seawater : must maintain a

constant salinity and not fluctuate

more than 6% weekly; monthly pH

range <0.8 pH units. 1. DO was measured daily in each

replicate aquarium.

2. Temperature was measured daily in

each replicate aquarium, and was also

continuously monitored in one aquarium

on both levels of the diluter system.

3. pH and salinity were measured daily in

e ach replicate aquarium. The salinity did

not fluctuate.

Solvents

Should not exceed 0.1 ml/L in a flow

through system. Acceptable solvents are:

dimethylformamide, triethylene glycol,

methanol, acetone, ethanol. None used.

Comments : None.

12. REPO RTED RESULTS :

A. General Results

Guideline Criteria Reported Information

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## 13 Guideline Criteria Reported Information

Quality assurance and GLP compliance

statements were included in the report? Yes

Data Endpoints must include :

⋅ survival of P and F 1 

## embryos, time

required to hatch, and h atching

success;

⋅ survival and total length of P fish at 4

and 8 weeks after hatching;

⋅ weights and lengths of F 1 

## fish at 8

weeks;

⋅ incidence of pathological or

histological effects; and

⋅ observations of other effects or

clinical signs. Data Endpoint s included :

⋅ survival of F 0 

## and F 1 

## embryos, time

required to hatch, and hatching

success;

⋅ survival and total lengths of F 0 

## fish at

4 and 8 weeks after hatching;

⋅ wet weights of fish discontinued from

exposure at thinning 4 weeks after

hatching;

⋅ survival of F 0 

## fish at 16 weeks after

hatching (112 days post

- hatch; test

termination);

⋅ total lengths and weights (gender

specific) of surviving F 0 

## fish at 16

weeks after hatching;

⋅ F 0 

## fecundity (eggs/female/day)

⋅ total lengths and wet weights of F 1



## fish at 4 weeks a fter hatching

⋅ incidence of pathological or

histological effects;

⋅ observation of other effects or

clinical signs

Raw data included? Yes

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## 14 F 0 

## Results :

Nominal

Conc.

(ppb a.i.) Mean Measured

Conc.

(ppb a.i.) (SD) %

Hatch 1 

### 4

- Week Post

Hatch %

Survival 8

- Week Post

Hatch %

Survival 2 

### Terminal

% Survival 2, 3



### Negative

Control <0.22 82 98 98 86

1.0 1.2 ± 0.10 86 100 100 84

2.0 2.0 ± 0.25 82 98 98 76

4.0 3.7 ± 0.28 85 99 98 80

8.0 7.4 ± 0.64 90 99 100 82

16 1 5 ± 1.3 77 100 98 94

Data obtained from Tables 2, p. 34, and Tables 4

- 6, pp. 36

- 38.

1 

### Adjusted for a mean 68% viability of the eggs used to initiate the study.

2 

### Based on 50 fish/treatment, thinned at 4 weeks post

- hatch.

3 

### 16 weeks post

- hatch.

Mea n

Measured

Conc.

(ppb a.i.) Mean Total Length (mm) Wet Weight (g)

4 Weeks

Post

hatch 8 Weeks Post

- hatch Test Termination 4 Weeks

Post

hatch Test Termination

_ _ _ _ _ _

Negative

Control 28.9 45.2 42.5 53.1 47.5 0.387 3.1 2.1

1.2 28.6 44.7 41.6 53.3 46.9 0.382 3.1 1.9

2.0 28.5 45.1 42.0 53.2 47.3 0.382 3.0 2.1

3.7 28.5 45.6 40.8 53.2 46.8 0.382 3.0 2.1

7.4 28.4 45.5 42.4 54.1 47.4 0.373 3.1 2.0

15 28.0* 44.4 41.9 50.9* 46.1 0.371 2.6* 1.9

Data obtained from Tables 4 and 5, pp. 36

- 37.

* Significantly reduced compared to the control, based on Williams’ Test.

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## 15 Mean Measured Conc.

(ppb a.i.) Total Number of Eggs 1 

### Number of Eggs/

Female/Day

Negative Control 6888 25

1.2 6622 25

2.0 6770 25

3.7 8309 30

7.4 6876 25

15 5656 22

Data obtained from Table 6, p. 38.

1 

### Values represents mean of two replicates.



## Toxicity Observations : A slight (3%), but statistically

- significant reduction in mean total

length of lar val fish was observed at the 15 ppb a.i. group compared to the control (28.0

versus 28.9 mm) at 4 weeks post

- hatch (Table 4, p. 36). No differences in lengths were

observed at the 8

- week measurement; however, at study termination, the mean total length

an d wet weight of males at the 15 ppb a.i. level (50.9 mm and 2.6 g) were statistically less

than the controls (53.1 mm and 3.1 g). No other treatment

- related effects were observed on

any F 0 

## parameter. All embryo groups, at all exposure levels and the cont rol, hatched in 6

days (p. 27). No deformities or internal abnormalities were observed among the terminated

F 0 

## adult fish (p. 28).

F 1 

## Results:

Mean Measured

Concentration

(ppb a.i.) % Hatch 4 Week

Post

- Hatch

% Survival 4 Week

Post

- Hatch

Length (mm) 4 Week

Post

- Hatch

Wet Weight (g)

Negative Control 68 95 25.1 0.256

1.2 60* 95 21.1 0.160

2.0 71 98 25.0 0.251

3.7 74 93 22.6 0.173

7.4 66 100 22.5 0.175

15 52* 97 21.9 0.173

Data obtained from Table 7, p. 39.

* Signifi cantly reduced compared to the control, based on Fisher’s Exact Test.

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## 16 Mean Measured

Concentration

(ppb a.i.) Percent Hatched

5 Days 6 Days 7 Days 8 Days

Negative Control 0 0 94 6

1.2 0 6 92 2

2.0 4 25 71 0

3.7 0 0 94 6

7 .4 0 0 81 19

15 0 8 92 0

Data obtained from Table 8, p. 40.



## Toxicity Observations : A statistically

- significant reduction in hatching success was

observed at the 15 ppb a.i. test group compared to the control (52 versus 68%; Table 7, p.

39). T he reduced hatching success at the highest concentration was the result of the

performance of one spawning group in a total of four maintained during the study (p. 28).

The mean percent hatching success for the other three groups was 70%. Embryos exposed at

1.2 ppb a.i. had a statistically

- significant reduction in hatching success compared to the

control (60 versus 68%, respectively); however, this difference was not considered to be

treatment

- related as no concentration response was observed at the three higher levels. No

other statistical differences were observed on any parameter assessed.

B. Reported Statistical Results

Data obtained for the F 0 

## generation that were statistically analyzed included hatching

success; 28

- day post

- hatch survival, tot al length and wet weight; 55

- day post

- hatch survival,

male total length, and female total length; fecundity (eggs/female/day); 112

- day post

- hatch

survival, male total length, male wet weight, female total length, and female wet weight.

The time to hatch d ata were empirically estimated. Data obtained for the F 1 

## generation that

were statistically analyzed included hatching success; time

- to

- hatch; and 28

- day post

- hatch

survival, total length, and wet weight.

Continuous data (growth and reproduction endpoi nts) were analyzed for assumptions of

normality and homogeneity, followed by William’s Test using TOXSTAT (1996) statistical

software. Binomial data (survival and hatching success endpoints) were analyzed by

Fisher’s Exact Test using SYSTAT (1999) statist ical software. All statistical conclusions

were made at the 95% level of certainty.

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## 17 The no observed effect concentration (NOEC) is the highest test concentration causing no

adverse effects. The lowest observed effect concentration (LOEC) is the lowes t test

concentration causing adverse effects. The maximum acceptable toxicant concentration

(MATC) was calculated as the geometric mean of the NOEC and the LOEC.

Biological Endpoint NOEC

(ppb a.i.) LOEC

(ppb a.i.) MATC

(ppb a.i.)

F 0 

## Generation

Hatching success 15 >15 >15

Time to hatch 15 >15 >15

4

- week survival 15 >15 >15

4

- week length 7.4 15 11

4

- week wet weight 15 >15 >15

8

- week survival 15 >15 >15

8

- week length, male 15 >15 >15

8

- week length, female 15 >15 >15

16

- week survival 15 >15 >15

16

- week length, male 7.4 15 11

16

- week length, female 15 >15 >15

16

- week wet weight, male 7.4 15 11

16

- week wet weight, female 15 >15 >15

Fecundity (eggs/female/day) 15 >15 >15

F 1 

## Generatio n

Hatching success 7.4 15 11

Time to hatch 15 >15 >15

4

- week survival 15 >15 >15

4

- week length 15 >15 >15

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## 18 Biological Endpoint NOEC

(ppb a.i.) LOEC

(ppb a.i.) MATC

(ppb a.i.)

4

- week weight 15 >15 >15

8

- week survival Not

determined Not

determined Not

determined

8

- week length Not

determined N ot

determined Not

determined

8

- week weight Not

determined Not

determined Not

determined

NOEC: 7.4 ppb a.i.

LOEC: 15 ppb a.i.

MATC: 11 ppb a.i.

Endpoint(s) Affected: F 0 

## 4

- week post

- hatch length; F 0 

## 16

- week (terminal) post

- hatch length

and wet w eight of males; and F 1 

## hatching success (same conclusions)

13. REVIEWER'S STATISTICAL RESULTS :

Data obtained for the F 0 

## generation that were statistically analyzed included hatching success,

28

- day post

- hatch survival, total length and wet weight, 55

- day post

- hatch survival, male total

length, and female total length, fecundity (eggs/female/day), 112

- day post

- hatch survival, male

total length, male wet weight, female total length, and female wet weight. The time to hatch data

were also statistically a nalyzed by comparing the number of groups hatching following treatment

on days 6, 7, and 8. Data obtained for the F 1 

## generation that were statistically analyzed included

hatching success, 28

- day post

- hatch survival, total length, and wet weight.

All da ta were analyzed to determine if they satisfied the assumptions of ANOVA (i.e., normality

and homogeneity of variances). If they did, the NOEC and LOEC were determined using this

test via TOXSTAT statistical software. If data did not satisfy these assump tions, the NOEC and

LOEC values were determined using the non

- parametric Kruskal

- Wallis test. These analyses

were conducted using TOXSTAT statistical software.

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## 19 Biological Endpoint NOEC

(ppb a.i.) LOEC

(ppb a.i.)

F 0 

## Generation

Hatching success 1 5 >15

Time to hatch 15 >15

4

- week survival 15 >15

4

- week length 15 >15

4

- week wet weight 15 >15

8

- week survival 15 >15

8

- week length, male 15 >15

8

- week length, female 15 >15

16

- week survival 15 >15

16

- week length, male 15 >15

16

- week length, female 15 >15

16

- week wet weight, male 15 >15

16

- week wet weight, female 15 >15

Fecundity (eggs/female/day) 15 >15

F 1 

## Generation

Hatching success 15 >15

Time to hatch 15 >15

4

- week survival 15 >15

4

- week length 15 >15

4

- week weight 15 >15

The reviewer’s analyses revealed no significant effects of treatment on any parameter.

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## 20 14. REVIEWER'S COMMENTS :

Quality control samples were prepared at each sampling interval and remained with the set

of exposure solution samples throughout the analytical process; samples were prepared in

saltwater at a nominal concentrations of 0.500, 4.00, and 20.0 ppb a.i. (p. 24 and Table 3, p.

35). Recoveries ranged from 82.6

- 128% (n=51, includes two outliers >120%)

.

A method validation study conducted prior to initiation of the definitive test established a

mean recovery of 91.8 ± 7.47% for ethoprop from filtered seawater (Table 1B, p. 91). The

LOQ was 0.136 ppb a.i.

This study was performed according to U.S. E PA (FIFRA) Good Laboratory Practice

Standards (40 CFR 160, 1993) with the exception of the collection for the water and food

contaminant screening analyses. A Quality Assurance Statement was provided.

The reviewer’s conclusions differed from the study author’s, in that the reviewer’s analyses

revealed no significant effects of treatment on any endpoint, while the study author’s

analyses revealed significant reductions in F 0 

## 4

- week post

- hatch length, F 0 

## 16

- week

(terminal) post

- hatch length and wet weight of males, and F 1 

## hatching success. Differences

in these conclusions are attributed to the different statistical methods that these results are

based on. In the reviewer’s analysis, ANOVA did not reveal any significant differences

among the control and t reatment groups, so post

- hoc multiple comparison tests (i.e.,

William’s test) were not warranted.

However, the number of true replicates used in the study is small (2) but does comply with

the minimum specifications in the supporting documents of the 8 50.1500 guideline (Hansen

et al., 1978). Regardless of the number of replicates used in a test, the primary issue of

concern is the extent to which statistically significant differences can be detected relative to

the control. An analysis of the minimum significant difference (MSD) reported for the

ethoprop study indicates that the MSD is very large for some endpoints particularly from the

F1 generation fish (see Table 1 above). Since the F1 endpoints are only addressed by full

life cycle studies, these endpoints are of particular importance in the study evaluation. For

ethoprop, however, the general lack of a dose

- response was observed for all but one F1

generation endpoint (i.e., modest % reductions in mean responses in the treatments relative

to contr ols). Thus, for those F1 endpoints where a dose

- response was not observed, the poor

statistical power of the F1 endpoint comparisons is moot. The one exception is growth of F1

generation fish measured at 4 weeks post hatch. For this endpoint, a 32% redu ction in

mean wet weight of 4

- week post hatch minnows relative to negative controls occurred

at 3.7, 7.4, 15 ppb but these were not found to be statistically significant. Reductions in

length were of up to 11% were also observed in these treatments relati ve to controls. Since

the variance in control treatments for these endpoints is very small, the lack of statistical

power appears to be due to the small number of replicates (2) used in the study. Rather than

### DP Barcode: D306508 MRID No: 46315401



## 21 invalidating the study, this reviewer is re

- i nterpreting the study to consider the 32%

reduction in wet weight of 4

- week post

- hatch F1 minnows at 3.7, 7.4, and 15 ppb to be

biologically significant

. Therefore, the NOAEC from this study is now interpreted to

be 2.0 ppb and the LOAEC is interpreted to be 3.7 ppb, based on mean measured

concentrations.

15. REFERENCES :

APHA, AWWA, WPCF. 1995. Standard Methods for the Examination of Water and

Wastewater. 19 th 

## Edition, Washington, DC, 2

- 23

- 25.

ASTM. 2002. Standard practice for conducting acute t oxicity tests with fishes,

macroinvertebrates and amphibians. Standard E729

- 96. American Society for Testing and

Materials, 100 Barr Harbor Drive, West Conshohocken, PA 19428.

Hansen, D.L., et al. 1978. Life

- cycle toxicity test using sheepshead minno w ( Cyprinodon

variegatus ). In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA

- 600/9

-

78

- 010, March 1978.

Macek, K.J. and B.H. Sleight, III. 1977. Utility of toxicity tests with embryo and fry of fish in

evaluating hazards associated wi th chronic toxicity of chemicals to fishes. Symposium

Proceedings, ASTM, Memphis, Tennessee, October, 1976: 137

- 146.

McKim, J.N. and D.A. Benoit. 1971. Effect of long

- term exposure to copper on survival,

reproduction and growth of brook trout ( Salveli nus fontinalis ). J. Fish. Res. Bd. Can. 28 :

655

- 662.

McKim, J.M. 1977. Evaluation of tests with early

- life

- stage of fish for predicting long

- term

toxicity. J. Fish. Res. Board Can. 34: 1148

- 1154.

Mount, D.I. and W.A. Brungs. 1967. A simplified do sing apparatus for fish toxicological

studies. Water Res . 1:20

- 29.

Rexrode, M. and T.M. Armitage. 1986. Standard Evaluation Procedure; Fish Life

- Cycle

Toxicity Tests. Hazard Evaluation Division, Office of Pesticide Programs. Washington,

D.C. 20460. EPA 540/9

- 86

- 137.

Sprague, J.B. 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute

toxicity. Water Res . 1: 20

- 29.

SYSTAT®. 1999. SPSS, Inc.

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## 22 U.S. EPA. 1989. Federal Insecticide, Fungicide and Rodenticide Act (FIFRA); Good Laboratory

Practice Standards; Final Rule (40 CFR, Part 160).

U.S. Environmental Protection Agency. 1996. Ecological Effects Test Guidelines, OPPTS

850.1400: Fish Early

- Life Stage Toxicity Test , (Public Draft), Environmental Protection

Agency, Off ice of Pesticide Programs. Washington, D.C.

West, Inc., and D.D. Gulley. 1996. Toxstat, Release 3.5. West, Inc., Cheyenne, WY.

### DP Barcode: D306508 MRID No: 46315401



## 23 APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION :

hatching success

File: 5401hs Transform: NO TRANSFORMAT ION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 80.667 16.133 1.152

Within (Error) 6 84.000 14.000

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

Total 11 164.667

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

hatching success

File: 5401hs Transf orm: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 56.500 56.500

2 1.2 59.000 59.000

- 0.668

3 2.0 56.500 56.500 0.000

4 3.7 57.500 57.500

- 0.267

5 7.4 61.500 61.500

- 1.336

6 15 53.000 53.000 0.935

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

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## 24 hatching success

File: 5401hs Transform: NO T RANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIF ICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 10.589 18.7

- 2.500

3 2.0 2 10.589 18.7 0.000

4 3.7 2 10.589 18.7

- 1.000

5 7.4 2 10.589 18.7

- 5.000

6 15 2 10.589 18.7 3.500

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

hatching success

File: 5401hs Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 56.500 56.500 58.200

2 1.2 2 59.000 59.000 58.200

3 2.0 2 56.500 56.5 00 58.200

4 3.7 2 57.500 57.500 58.200

5 7.4 2 61.500 61.500 58.200

6 15 2 53.000 53.000 53.000

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

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## 25 hatching success

File: 5401hs Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 58.200

1.2 58.200 0.454 1.94 k= 1, v= 6

2.0 58.200 0.454 2.06 k= 2, v= 6

3.7 58.200 0.454 2.10 k= 3, v= 6

7.4 58.200 0.454 2.12 k= 4, v= 6

15 53.000 0.935 2.13 k= 5, v= 6

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

s = 3.742

Note: d f used for table values are approximate when v > 20.

hatching success (% viability)

File: 5401hsv Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 194.667 38.933 1.557

Within (Error) 6 150.000 25.000

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

Total 11 344.667

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

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## 26 hatching success (% viability)

File: 5401hsv Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 81.500 81.500

2 1.2 85.500 85.500

- 0.800

3 2.0 81.500 81.500 0.000

4 3.7 84.500 84.500

- 0.600

5 7.4 90.000 90.000

- 1.700

6 15 77.000 77.000 0.900

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

hatching success (% viability)

File: 5401hsv Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 14.150 17.4

- 4.000

3 2.0 2 14.150 17.4 0.000

4 3.7 2 14.150 17.4

- 3.000

5 7.4 2 14.150 17.4

- 8.500

6 15 2 14.150 17.4 4.500

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

hatching success (% viability)

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## 27 File: 5401hsv Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROU P ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 81.500 81.500 84.600

2 1.2 2 85.500 85.500 84.600

3 2.0 2 81.500 81.500 84.600

4 3.7 2 84.500 84.500 84.600

5 7.4 2 90.000 90.000 84.600

6 15 2 77.000 77.000 77.000

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

hatching s uccess (% viability)

File: 5401hsv Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC . SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 84.600

1.2 84.600 0.620 1.94 k= 1, v= 6

2.0 84.600 0.620 2.06 k= 2, v= 6

3.7 84.600 0.620 2.10 k= 3, v= 6

7.4 84.600 0.620 2.12 k= 4, v= 6

15 77.000 0.900 2.13 k= 5, v= 6

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

s = 5.000

Note: df used for table values are approximate when v > 20.

day 28 leng th

File: 5401l28 Transform: NO TRANSFORMATION

ANOVA TABLE

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## 28 ------------------------------------------------------------------------------

SOURCE DF SS MS F

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

Between 5 0.998 0.200 2.198

Within (Error) 6 0.545 0.091

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

Total 11 1.543

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

day 28 length

File: 5401l28 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

TRANS FORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 28.950 28.950

2 1.2 28.650 28.650 0.994

3 2.0 28.450 28.450 1.657

4 3.7 28.450 28.450 1.657

5 7.4 28.350 28.350 1.989

6 15 28.000 28.000 3.149 *

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

da y 28 length

File: 5401l28 Transform: NO TRANSFORMATION

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## 29 DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimu m Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 0.854 2.9 0.300

3 2.0 2 0.854 2.9 0.500

4 3.7 2 0.854 2.9 0.500

5 7.4 2 0.854 2.9 0.600

6 15 2 0.854 2.9 0.950

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

day 28 length

File: 5401l28 Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEA N MEAN

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

1 control 2 28.950 28.950 28.950

2 1.2 2 28.650 28.650 28.650

3 2.0 2 28.450 28.450 28.450

4 3.7 2 28.450 28.450 28.450

5 7.4 2 28.350 28.350 28.350

6 15 2 28.0 00 28.000 28.000

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

day 28 length

File: 5401l28 Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

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## 30 ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 28.950

1.2 28.650 0.995 1.94 k= 1, v= 6

2.0 28.450 1.659 2.06 k= 2, v= 6

3.7 28. 450 1.659 2.10 k= 3, v= 6

7.4 28.350 1.991 2.12 k= 4, v= 6

15 28.000 3.152 * 2.13 k= 5, v= 6

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

s = 0.301

Note: df used for table values are approximate when v > 20.

day 28 weight

File: 5401w28 Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 387.000 77.400 0.308

Within (Error) 6 1510.000 251.667

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

Total 11 1897.000

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

day 28 weight

File: 5401w28 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

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## 31 TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 387.500 387.500

2 1.2 381.500 381.500 0.378

3 2.0 382.000 382.000 0.347

4 3.7 382.000 382.000 0.347

5 7.4 372.500 372.500 0.946

6 15 371.500 371.500 1.009

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

day 28 weight

File: 5401w28 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 44.895 11.6 6.000

3 2.0 2 44.895 11.6 5.500

4 3.7 2 44.895 11.6 5.500

5 7.4 2 44.895 11.6 15.000

6 15 2 44.895 11.6 16.000

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

day 28 weight

File: 5401w28 Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

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## 32 IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 387.500 387 .500 387.500

2 1.2 2 381.500 381.500 381.833

3 2.0 2 382.000 382.000 381.833

4 3.7 2 382.000 382.000 381.833

5 7.4 2 372.500 372.500 372.500

6 15 2 371.500 371.500 371.500

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

day 28 weight

File: 5401w28 Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

I DENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 387.500

1.2 381.833 0.357 1.94 k= 1, v= 6

2.0 381.833 0.357 2.06 k= 2, v= 6

3.7 381.833 0.357 2.10 k= 3, v= 6

7.4 372.500 0.946 2.12 k= 4, v= 6

15 371.500 1.009 2.13 k= 5, v= 6

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

s = 15.864

Note: df used for table values are approximate when v > 20.

day 55 length males

File: 5401l55 Transform: NO TRANSFORM

KRUSKAL

- WALLIS ANOVA BY RANKS

- TABLE 1 OF 2

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

TRANSFORMED MEAN CALCULATED IN RANK

GROUP IDENTIFICATION MEAN ORIGINAL UNITS SUM

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

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## 33 1 control 45.250 45.250 13.000

2 1.2 44.750 44.750 10.500

3 2.0 45.100 45.100 9.500

4 3.7 45.600 45.600 18.500

5 7.4 45.500 45.500 21.000

6 15 44.450 44.450 5.500

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

Calculated H Value = 6.592 Critical H Value Table = 11.070

Since Calc H < Crit H FAIL TO REJECT Ho:All group s are equal.

day 55 length males

File: 5401l55 Transform: NO TRANSFORM

DUNNS MULTIPLE COMPARISON

- KRUSKAL

- WALLIS

- TABLE 2 OF 2

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

GROUP

TRANSFORMED ORIGINAL 0 0 0 0 0 0

GROUP IDENTIFICATION MEAN MEAN 6 2 3 1 5 4

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

6 15 44.450 44.450 \

2 1.2 44.750 44.750 . \

3 2.0 45.100 45.100 . . \

1 control 45.250 45.250 . . . \

5 7.4 45.500 45.500 . . . . \

4 3.7 45.600 45.60 0 . . . . . \

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

* = significant difference (p=0.05) . = no significant difference

Table q value (0.05,6) = 2.936 SE = 3.580

day 55 female length

F ile: 540155f Transform: NO TRANSFORMATION

ANOVA TABLE

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

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## 34 SOURCE DF SS MS F

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

Between 5 4.537 0.907 0.696

Within (Error) 6 7.820 1.303

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

Total 11 12.357

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

day 55 female length

File: 540155f Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

T RANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 42.550 42.55 0

2 1.2 41.600 41.600 0.832

3 2.0 42.000 42.000 0.482

4 3.7 40.750 40.750 1.577

5 7.4 42.550 42.550 0.000

6 15 41.850 41.850 0.613

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

d ay 55 female length

File: 540155f Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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## 35 ----------------------------------------------------------------------------

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 3.230 7.6 0.950

3 2.0 2 3.230 7.6 0.550

4 3.7 2 3.230 7.6 1.800

5 7.4 2 3.230 7. 6 0.000

6 15 2 3.230 7.6 0.700

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

day 55 female length

File: 540155f Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 42.550 42.550 42.550

2 1.2 2 41.600 41.600 41.8 00

3 2.0 2 42.000 42.000 41.800

4 3.7 2 40.750 40.750 41.717

5 7.4 2 42.550 42.550 41.717

6 15 2 41.850 41.850 41.717

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

day 55 female length

File: 540155f Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

### DP Barcode: D306508 MRID No: 46315401



## 36 IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 42.550

1.2 41.800 0.657 1.94 k= 1, v= 6

2.0 41.800 0.657 2.06 k= 2, v= 6

3.7 41.717 0.730 2.10 k= 3, v= 6

7.4 41.717 0.730 2.12 k= 4, v= 6

15 41.717 0.730 2.13 k= 5, v= 6

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

s = 1.142

Note: df used for table values are approximate when v > 20.

F0 survival

File: 5401s Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 374.667 74. 933 0.301

Within (Error) 6 1496.000 249.333

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

Total 11 1870.667

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

F0 survival

File: 5401s Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control <Treatment

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

TRANSFORMED MEAN CALCULATED IN

### DP Barcode: D306508 MRID No: 46315401



## 37 GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 86.000 86.000

2 1.2 84.000 84.000 0.127

3 2.0 76.000 76.000 0.633

4 3.7 80.000 80.000 0.380

5 7.4 82.000 82.000 0.253

6 15 94.000 94.000

- 0.507

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

F0 survival

File: 5401s Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 44.686 52.0 2.000

3 2.0 2 44.686 52.0 10.000

4 3.7 2 44.686 52.0 6.000

5 7.4 2 44.686 52.0 4.000

6 15 2 44.686 52.0

- 8.000

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

F0 survival

File: 5401s Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

### DP Barcode: D306508 MRID No: 46315401



## 38 ------ -------------------- --- ----------- ----------- -----------

1 control 2 86.000 86.000 81.500

2 1.2 2 84.000 84.000 81.500

3 2.0 2 76.000 76.000 81.500

4 3.7 2 80.000 80.000 81.500

5 7.4 2 82.000 82.000 82.000

6 15 2 94.000 94.000 94.000

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

F0 survival

File: 5401s Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 81.500

1.2 81.500 0.285 1.94 k= 1, v= 6

2.0 81.500 0.285 2.06 k= 2, v= 6

3.7 81.500 0.285 2.10 k= 3, v= 6

7.4 82.000 0.253 2.12 k= 4, v= 6

15 94.000 0.507 2.13 k= 5, v= 6

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

s = 15.790

Note: df used for table values are approximate when v > 20.

F0 male length

File: 5401ml Transform: NO T RANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 12.867 2.573 3.034

### DP Barcode: D306508 MRID No: 46315401



## 39 Within (Error) 6 5.090 0.848

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

Total 11 17.957

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

F0 male length

File: 5401ml Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 53.000 53.000

2 1.2 53.350 53.350

- 0.380

3 2.0 53.300 53.300

- 0.326

4 3.7 53.400 53.400

- 0.434

5 7.4 54.200 54.200

- 1.303

6 15 50.850 50.850 2.335

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

F0 male length

File: 5401ml Transfor m: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

### DP Barcode: D306508 MRID No: 46315401



## 40 1 control 2

2 1.2 2 2.606 4.9

- 0.350

3 2.0 2 2.606 4.9

- 0.300

4 3.7 2 2.606 4.9

- 0.400

5 7.4 2 2.606 4.9

- 1.200

6 15 2 2.606 4.9 2.150

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

F0 male length

File: 5401ml Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 53.000 53.000 53.450

2 1.2 2 53.350 53.350 53.450

3 2.0 2 53.300 53.300 53.450

4 3.7 2 53.400 53.400 53.450

5 7.4 2 54.200 54.200 53.450

6 15 2 50.850 50.850 50.850

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

F0 male length

File: 5401ml Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 53.450

1.2 53.450 0.489 1.94 k= 1, v= 6

### DP Barcode: D306508 MRID No: 46315401



## 41 2.0 53.450 0.489 2.06 k= 2, v= 6

3.7 53.450 0.489 2.10 k= 3, v= 6

7.4 53.450 0.489 2.12 k= 4, v= 6

15 50.850 2.334 * 2.13 k= 5, v= 6

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

s = 0.921

Note : df used for table values are approximate when v > 20.

F0 female length

File: 5401fl Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 2.907 0.581 0.236

Within (Error) 6 1 4.760 2.460

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

Total 11 17.667

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

Critical F value = 4.39 (0 .05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

F0 female length

File: 5401fl Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 47.400 47.400

2 1.2 47.000 47.000 0.255

### DP Barcode: D306508 MRID No: 46315401



## 42 3 2.0 47.100 47.100 0.191

4 3.7 46.300 46.300 0.701

5 7.4 47.300 47.300 0.064

6 15 46.100 46.100 0.829

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

Dunne tt table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

F0 female length

File: 5401fl Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 4.439 9.4 0.400

3 2.0 2 4.439 9.4 0.300

4 3.7 2 4.439 9.4 1.1 00

5 7.4 2 4.439 9.4 0.100

6 15 2 4.439 9.4 1.300

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

F0 female length

File: 5401fl Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 47.400 47.400 47.400

2 1.2 2 47.000 47.000 47.050

3 2.0 2 47.100 47.100 47.050

### DP Barcode: D306508 MRID No: 46315401



## 43 4 3.7 2 46.300 46.300 46.800

5 7.4 2 47.300 47.300 46.800

6 15 2 46.100 46.100 46.100

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

F0 female length

File: 5401fl Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN W ILLIAMS P=.05 WILLIAMS FREEDOM

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

control 47.400

1.2 47.050 0.223 1.94 k= 1, v= 6

2.0 47.05 0 0.223 2.06 k= 2, v= 6

3.7 46.800 0.383 2.10 k= 3, v= 6

7.4 46.800 0.383 2.12 k= 4, v= 6

15 46.100 0.829 2.13 k= 5, v= 6

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

s = 1.568

Note: df used for table values are approximate when v > 20.

F0 male weight

File: 5401mw Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 0.467 0.093 2.906

Within (Error) 6 0.190 0.032

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

Total 11 0.65 7

### DP Barcode: D306508 MRID No: 46315401



## 44 ------------------------------------------------------------------------------

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

F0 male weight

File: 5401mw Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 3.050 3.050

2 1.2 3.050 3.050 0.000

3 2.0 3.050 3.050 0.000

4 3.7 3.050 3.050 0.000

5 7.4 3.150 3.150

- 0.559

6 15 2.550 2.550 2.795

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

F0 male weight

File: 5401mw Transform: NO TRANSFORMATION

DUNN ETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN O RIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 0.506 16.6 0.000

3 2.0 2 0.506 16.6 0.000

4 3.7 2 0.506 16.6 0.000

### DP Barcode: D306508 MRID No: 46315401



## 45 5 7.4 2 0.506 16.6

- 0.100

6 15 2 0.506 16.6 0.500

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

F0 male weight

File: 5401mw Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 3.050 3.050 3.070

2 1.2 2 3.050 3.050 3.070

3 2.0 2 3.050 3.050 3.070

4 3.7 2 3.050 3.050 3.070

5 7.4 2 3.150 3.150 3.070

6 15 2 2.550 2.550 2.550

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

F0 male weight

File: 5401mw Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 3.070

1.2 3.070 0.112 1.94 k= 1, v= 6

2.0 3.070 0.112 2.06 k= 2, v= 6

3.7 3.070 0.112 2.10 k= 3, v= 6

7.4 3.070 0.112 2.12 k= 4, v= 6

15 2.550 2.810 * 2.13 k= 5, v= 6

### DP Barcode: D306508 MRID No: 46315401



## 46 ----------------------------------------------------------------------------

s = 0.178

Note: df used for table values are ap proximate when v > 20.

F0 female weight

File: 5401fw Transform: NO TRANSFORM

KRUSKAL

- WALLIS ANOVA BY RANKS

- TABLE 1 OF 2

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

T RANSFORMED MEAN CALCULATED IN RANK

GROUP IDENTIFICATION MEAN ORIGINAL UNITS SUM

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

1 control 2.100 2.100 17.500

2 1.2 2.000 2.000 13.000

3 2.0 2.050 2.050 14.500

4 3.7 2.000 2.000 13.000

5 7.4 2.000 2.000 14.000

6 15 1.850 1.850 6.000

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

Calculated H Value = 2.879 Critical H Val ue Table = 11.070

Since Calc H < Crit H FAIL TO REJECT Ho:All groups are equal.

F0 female weight

File: 5401fw Transform: NO TRANSFORM

DUNNS MULTIPLE COMPARISON

- KRUSKAL

- WALLIS

- TABLE 2 OF 2

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

GROUP

TRANSFORMED ORIGINAL 0 0 0 0 0 0

GROUP IDENTIFICATION MEAN MEAN 6 4 5 2 3 1

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

6 15 1.850 1.850 \

4 3.7 2.000 2.000 . \

5 7.4 2.000 2.000 . . \

2 1.2 2.000 2.000 . . . \

### DP Barcode: D306508 MRID No: 46315401



## 47 3 2.0 2.050 2.050 . . . . \

1 control 2.100 2.100 . . . . . \

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

* = significant difference (p=0.05) . = no significant difference

Table q value (0. 05,6) = 2.936 SE = 3.548

total eggs

File: 5401e Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 1807373.750 361474.750 0.874

Within (Error) 6 2482450.500 413741. 750

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

Total 11 4289824.250

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

total eggs

File: 5401e Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 3444.000 3444.000

2 1.2 3311.000 3311.000 0.207

3 2.0 3385.000 3385.000 0.092

### DP Barcode: D306508 MRID No: 46315401



## 48 4 3.7 4154.500 4154.500

- 1 .105

5 7.4 3438.000 3438.000 0.009

6 15 2828.000 2828.000 0.958

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

total eggs

File: 5401e Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 1820.334 52.9 133.000

3 2.0 2 1820.334 52.9 59.000

4 3.7 2 1820.334 52.9

- 710.500

5 7.4 2 1820.334 52.9 6.000

6 15 2 1820.334 52.9 616.000

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

total eggs

File: 5401e Transform: NO TRANSF ORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATIO N N MEAN MEAN MEAN

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

1 control 2 3444.000 3444.000 3573.625

2 1.2 2 3311.000 33 11.000 3573.625

3 2.0 2 3385.000 3385.000 3573.625

4 3.7 2 4154.500 4154.500 3573.625

### DP Barcode: D306508 MRID No: 46315401



## 49 5 7.4 2 3438.000 3438.000 3438.000

6 15 2 2828.000 2828.000 2828.000

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

total eggs

File: 5401e Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression m odel) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 3573.625

1.2 3573.625 0.202 1.94 k= 1, v= 6

2.0 3573.625 0.202 2.06 k= 2, v= 6

3.7 3573.625 0.202 2.10 k= 3, v= 6

7.4 3438.000 0.009 2.12 k= 4, v= 6

15 2828.000 0.958 2.13 k= 5, v= 6

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

s = 643.228

Note: df used for table values are approximate when v > 20.

eggs/female/day

File: 5401ed Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 74.750 14.950 0.790

Within (Error) 6 113.500 18.917

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

Total 11 188.250

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

### DP Barcode: D306508 MRID No: 46315401



## 50 Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

eggs/female/day

File: 5401ed Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT S IG

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

1 control 25.500 25.500

2 1.2 24.500 24.500 0.230

3 2.0 25.000 25.000 0.115

4 3.7 30.000 30.000

- 1.035

5 7.4 25.000 25.000 0.115

6 15 21.500 21.500 0.920

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

eggs/female/day

File: 5401ed Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho :Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 12.309 48.3 1.000

3 2.0 2 12.309 48.3 0.500

4 3.7 2 12.309 48.3

- 4.500

5 7.4 2 12.309 48.3 0.500

### DP Barcode: D306508 MRID No: 46315401



## 51 6 15 2 12.309 48.3 4.000

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

eggs/female/day

File: 5401ed Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 c ontrol 2 25.500 25.500 26.250

2 1.2 2 24.500 24.500 26.250

3 2.0 2 25.000 25.000 26.250

4 3.7 2 30.000 30.000 26.250

5 7.4 2 25.000 25.000 25.000

6 15 2 21.500 21.500 21.500

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

e ggs/female/day

File: 5401ed Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. S IG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 26.250

1.2 26.250 0.172 1.94 k= 1, v= 6

2.0 26.250 0.172 2.06 k= 2, v= 6

3.7 26.250 0.172 2.10 k= 3, v= 6

7.4 25.000 0.115 2.12 k = 4, v= 6

15 21.500 0.920 2.13 k= 5, v= 6

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

### DP Barcode: D306508 MRID No: 46315401



## 52 s = 4.349

Note: df used for table values are approximate when v > 20.

hatching success F 1

File: 5401h1 Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 616.417 123.283 1.702

Within (Error) 6 434.500 72.417

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

Total 11 1050.917

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

hatching success F1

File: 5401h1 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

T RANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 68.000 68.00 0

2 1.2 59.000 59.000 1.058

3 2.0 70.000 70.000

- 0.235

4 3.7 74.000 74.000

- 0.705

5 7.4 66.000 66.000 0.235

6 15 52.500 52.500 1.821

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

### DP Barcode: D306508 MRID No: 46315401



## 53 Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

h atching success F1

File: 5401h1 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 24.083 35.4 9.000

3 2.0 2 24.083 35.4

- 2.000

4 3.7 2 24.083 35.4

- 6.000

5 7.4 2 24.083 35.4 2.000

6 15 2 24.083 35.4 15.500

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

hatching success F1

File: 5401h1 Transform: NO TRANSFORMATION

WILL IAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 68.000 68.000 68.000

2 1.2 2 59.000 59.000 67.667

3 2.0 2 70.000 70.000 67.667

4 3.7 2 74.000 74.000 67.667

5 7.4 2 66.000 66.000 66.000

6 15 2 52.500 52.500 52.500

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

### DP Barcode: D306508 MRID No: 46315401



## 54 hatching success F1

File: 5401h1 Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABL E 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 68.000

1.2 67.667 0.039 1.94 k= 1, v= 6

2.0 67.667 0.039 2.06 k= 2, v= 6

3.7 67.667 0.039 2.10 k= 3, v= 6

7.4 66.000 0.235 2.12 k= 4, v= 6

15 52.500 1.821 2.13 k= 5, v= 6

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

s = 8.510

Note: df used for table values are approximate when v > 20.

survival F1

File: 5401s1 Transform: NO TRANSFORM

KRUSKAL

- WALLIS ANOVA BY RANKS

- TABLE 1 OF 2

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

TRANSFORMED MEAN CALCULATED IN RANK

GROUP IDENTIFICATION MEAN ORIGINAL UNITS SUM

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

1 control 95.000 95.000 12.000

2 1.2 95.000 95.000 10.500

3 2.0 98.000 98.000 15.500

4 3.7 93.000 93.000 6.500

5 7.4 100.000 100.000 21.000

6 15 97.000 97.000 12.500

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

Calculated H Value = 4.889 Critical H Value Table = 11.070

Since Calc H < Crit H FAIL TO REJECT Ho:All groups are equal.

### DP Barcode: D306508 MRID No: 46315401



## 55 survival F1

File: 5401s1 Transform: NO TRANSFORM

DUNNS MULTIPLE COMPARI SON

- KRUSKAL

- WALLIS

- TABLE 2 OF 2

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

GROUP

TRANSFORMED ORIGINAL 0 0 0 0 0 0

GROUP IDENTIFICATION MEAN MEAN 4 1 2 6 3 5

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

4 3.7 93.000 93.000 \

1 control 95.000 95.000 . \

2 1.2 95.000 95.000 . . \

6 15 97.000 97.000 . . . \

3 2.0 98.000 98.000 . . . . \

5 7.4 100.000 100.000 . . . . . \

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

* = significan t difference (p=0.05) . = no significant difference

Table q value (0.05,6) = 2.936 SE = 3.503

length F1

File: 5401l1 Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 27.064 5.413 0.935

Within (Error) 6 34.725 5.787

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

Total 11 61.789

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

### DP Barcode: D306508 MRID No: 46315401



## 56 Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups equal

length F1

File: 5401l1 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Con trol<Treatment

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

TRANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 25.050 25.050

2 1.2 21.000 21.000 1.684

3 2.0 24.950 24.950 0.042

4 3.7 22.650 22.650 0.998

5 7.4 22.450 22.450 1.081

6 15 21.950 21.950 1.289

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

length F1

File: 5401l1 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimum Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 6.808 27.2 4.050

3 2.0 2 6.808 27.2 0.100

4 3 .7 2 6.808 27.2 2.400

5 7.4 2 6.808 27.2 2.600

6 15 2 6.808 27.2 3.100

### DP Barcode: D306508 MRID No: 46315401



## 57 ------------------------------------------------------- -----------------------

length F1

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WILLIAMS TEST (Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 25.050 25.050 25.050

2 1.2 2 21.000 21.000 22.975

3 2.0 2 24.950 24.950 22.975

4 3.7 2 22.650 22.650 22.650

5 7.4 2 22.450 22.450 22.450

6 15 2 21.950 21.950 21.950

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

length F1

File: 5401l1 Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 25.050

1.2 22.975 0.863 1.94 k= 1, v = 6

2.0 22.975 0.863 2.06 k= 2, v= 6

3.7 22.650 0.998 2.10 k= 3, v= 6

7.4 22.450 1.081 2.12 k= 4, v= 6

15 21.950 1.289 2.13 k= 5, v= 6

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

s = 2.406

### DP Barcode: D306508 MRID No: 46315401



## 58 Note: df used for table values are approximate when v > 20.

weight F1

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KRUSKAL

- WALLIS ANOVA BY RANKS

- TABLE 1 OF 2

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

TRANSFORMED MEAN CALCULATED IN RANK

GROUP IDENTIFICATION MEAN ORIGINAL UNITS SUM

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

1 control 256.000 256.000 21.000

2 1.2 157.150 157.150 9.500

3 2.0 251.000 251.000 20.500

4 3.7 175.000 175.000 8.500

5 7.4 175.500 175.500 9.500

6 15 173.000 173.000 9.000

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

Calculated H Value = 7.035 Critical H Value Table = 11.070

Since Calc H < Crit H FAIL TO REJECT Ho:All groups ar e equal.

weight F1

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DUNNS MULTIPLE COMPARISON

- KRUSKAL

- WALLIS

- TABLE 2 OF 2

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

GROUP

TRANSFORMED ORIGINAL 0 0 0 0 0 0

GROUP IDENTIFICATION MEAN MEAN 2 6 4 5 3 1

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

2 1.2 157.150 157.150 \

6 15 173.000 173.000 . \

4 3.7 175.000 175.000 . . \

5 7.4 175.500 175.500 . . . \

3 2.0 251.000 251.000 . . . . \

1 control 256.000 256.000 . . .

. . \

### DP Barcode: D306508 MRID No: 46315401



## 59 ----------------------------------------------------------------------------

* = significant difference (p=0.05) . = no significant difference

Table q value (0.05,6) = 2.936 SE = 3.587

%hatch 6 days

File: 5401h6 Transform: NO TRANSFORMATION

KRUSKAL

- WALLIS ANOVA BY RANKS

- TABLE 1 OF 2

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

TRANSFORMED MEAN CALCULATED IN RANK

GROUP I DENTIFICATION MEAN ORIGINAL UNITS SUM

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

1 control 0.000 0.000 9.000

2 1.2 5.500 5.500 19.000

3 2.0 25.000 25.000 16.500

4 3.7 0.000 0.000 9.000

5 7.4 0.000 0.000 9.000

6 15 7.500 7.500 15.500

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

Calculated H Value = 5.582 Critical H Value Table = 11.070

Since Calc H < Crit H FAIL TO R EJECT Ho:All groups are equal.

%hatch 6 days

File: 5401h6 Transform: NO TRANSFORMATION

DUNNS MULTIPLE COMPARISON

- KRUSKAL

- WALLIS

- TABLE 2 OF 2

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

GROUP

TRANSFORMED ORIGINAL 0 0 0 0 0 0

GROUP IDENTIFICATION MEAN MEAN 1 4 5 2 6 3

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

1 control 0.000 0.000 \

4 3.7 0.000 0.000 . \

5 7.4 0.000 0.000 . . \

### DP Barcode: D306508 MRID No: 46315401



## 60 2 1.2 5.500 5.500 . . . \

6 15 7.500 7.500 . . . . \

3 2.0 25.000 25.000 . . . . . \

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

* = significant difference (p=0.05) . = no significant difference

Table q value (0.05,6) = 2.936 SE = 3.030

#hatch 7 days

File: 5401h7 Transform: NO TRANSFORMATION

ANOVA TABLE

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

SOURCE DF SS MS F

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

Between 5 889.417 177.883 0.407

Within (Error) 6 2619.500 436.583

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

Total 11 3508.917

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

Critical F value = 4.39 (0.05,5,6)

Since F < Critical F FAIL TO REJECT Ho:All groups e qual

#hatch 7 days

File: 5401h7 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 1 OF 2 Ho:Control<Treatment

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

T RANSFORMED MEAN CALCULATED IN

GROUP IDENTIFICATION MEAN ORIGINAL UNITS T STAT SIG

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

1 control 94.500 94.50 0

2 1.2 92.000 92.000 0.120

### DP Barcode: D306508 MRID No: 46315401



## 61 3 2.0 71.000 71.000 1.125

4 3.7 94.000 94.000 0.024

5 7.4 81.500 81.500 0.622

6 15 92.500 92.500 0.096

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

Dunnett table value = 2.83 (1 Tailed Value, P=0.05, df=6,5)

# hatch 7 days

File: 5401h7 Transform: NO TRANSFORMATION

DUNNETTS TEST

- TABLE 2 OF 2 Ho:Control<Treatment

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

NUM OF Minimu m Sig Diff % of DIFFERENCE

GROUP IDENTIFICATION REPS (IN ORIG. UNITS) CONTROL FROM CONTROL

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

1 control 2

2 1.2 2 59.132 62.6 2.500

3 2.0 2 59.132 62.6 23.500

4 3.7 2 59.132 62.6 0.500

5 7.4 2 59.132 62.6 13.000

6 15 2 59.132 62.6 2.000

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

#hatch 7 days

File: 5401h7 Transform: NO TRANSFORMATION

WILLIAMS TEST ( Isotonic regression model) TABLE 1 OF 2

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

GROUP ORIGINAL TRANSFORMED ISOTONIZED

IDENTIFICATION N MEAN MEAN MEAN

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

1 control 2 94.500 94.500 94.500

2 1.2 2 92.000 92.000 92.000

3 2.0 2 71.000 71.000 84.750

### DP Barcode: D306508 MRID No: 46315401



## 62 4 3.7 2 94.000 94.000 84.750

5 7.4 2 81.500 81.500 84.750

6 15 2 92.500 92.500 84.750

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

#hatch 7 days

File: 5401h7 Transform: NO TRANSFORMATION

WILLIAMS TEST (Isotonic regression model) TABLE 2 OF 2

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

ISOTONIZED CALC. SIG TABLE DEGREES OF

IDENTIFICATION MEAN WILLIAMS P=.05 WILLIAMS FREEDOM

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

control 94.500

1.2 92.000 0.120 1.94 k= 1, v= 6

2.0 84.750 0.467 2.06 k= 2, v= 6

3.7 84.75 0 0.467 2.10 k= 3, v= 6

7.4 84.750 0.467 2.12 k= 4, v= 6

15 84.750 0.467 2.13 k= 5, v= 6

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

s = 20.895

Note: df used for table values are approximate when v > 20.

%hatch 8 days

File: 5401h8 Transform: NO TRANSFORMATION

KRUSKAL

- WALLIS ANOVA BY RANKS

- TABLE 1 OF 2

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

TRANSFORMED MEAN CALCULATED IN RANK

GROUP IDENTIFICATION MEAN ORIGINAL UNITS SUM

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

1 control 5.500 5.500 14.500

2 1.2 2.500 2.500 13.500

3 2.0 0.000 0.000 9.000

4 3.7 6.500 6.500 15.500

5 7.4 19.000 19.000 16.500

### DP Barcode: D306508 MRID No: 46315401



## 63 6 15 0.000 0.000 9.000

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

Calculated H Value = 2.886 Critical H Value Table = 11.070

Since Calc H < Crit H FAIL TO REJECT Ho:All groups are equal.

%hatch 8 days

File: 5401h8 Transform: NO TRANSFORMATION

DUNNS MULTIPLE COMPARISON

- KRUSKAL

- WALLIS

- TABLE 2 OF 2

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

GROUP

TRANSFORMED ORIGINAL 0 0 0 0 0 0

GROUP IDENTIFICATION MEAN MEAN 3 6 2 1 4 5

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

3 2.0 0.000 0.000 \

6 15 0.000 0.000 . \

2 1.2 2.500 2.500 . . \

1 control 5.500 5.500 . . . \

4 3.7 6.500 6.500 . . . . \

5 7.4 19.000 19.000 . . . . . \

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

* = significant di fference (p=0.05) . = no significant difference

Table q value (0.05,6) = 2.936 SE = 3.030