### (

A REP RODUCTION STUDY WITH THE EARTHWORM

IN AN ARTIFICIAL SOIL SUBSTRATE

FINAL REPORT

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER:

DOW STUDY NUMBER:

OECD Guideline 222 for the Testing o f Chemicals

Earthworm Reproduction Test ( Eisenia fetida/Eisenia andrei )

and

ISO 11268-2

AUTHORS:

ST UDY INITIATION DATE: 13 April 2012

STUDY COMPLETION DATE: 31 August 2012

SUBMI TTED TO:

The Dow Chemical Company

Midland, Michigan 48640

USA



# Wildlife International, Ltd.

8598 Commerce Drive

Easton, Maryland 21601 USA

(410) 822-8600

Page 1 of 41

# Wildlife International, Ltd.

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### Project Number

Dow Study Number

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT



### SPONSOR: The Dow Chemical Company

TITLE: ( A Reproduction Study with the

Earthwonn in an Artificial Soil Substrate

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER:

DOW STUDY NUMBER:

STUDY COMPLETION DATE: 31 August 2012

This study was conducted in compliance with Good Laboratory Practice Standards as

published by the U.S. Enviromnental Protection Agency in 40 CFR Part 792, 17 August 1989; OECD

Principles of Good LaboratOlY Practice (ENVIMCICHEM 

### (98) 17), Paris, 1998; and Japan MAFF,

(II 

### NohSan, Notification No. 6283, Agricultural Production Bureau, I October 1999), with the

following exceptions:

The stability under storage conditions at the test site detennined in accordance with Good Laboratory

Practice Standards.

Verification, stability and homogeneity of the test substance in the soil were not determined in

accordance with Good Laboratory Practice Standards.

Periodic analyses of soil, feed and water for potential contaminants were not performed according to

Good Laboratory Practice Standards but were performed using a certified laboratory and standard

U.S. EPA analytical methods.

Date

Senior Biologist

# Wildlife International, Ltd.

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### Project Number

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QUALITY ASSURANCE STATEMENT



### This study was examined for compliance with Good Laboratory Practice Standards as

published by the U.S. Enviromuental Protection Agency, 40 CFR Part 792,17 August 1989; OECD

Principles of Good Laboratory Practice (ENVIMC/CHEM (98) 17), Paris, 1998; and Japan MAFF

(II NohSan, Notification No. 6283, Agricultural Production Bureau, I October 1999).

The dates of all inspections and audits, and the dates that any findings were reported to the

Study Director and Laboratory Management were as follows:

ACTIVITY: DATE CONDUCTED:



### Protocol 17 February 2012



### Test Substance Preparation 16 April 2012

Day 28 Adult Observations 14 May 2012



### pH Measurements II June 2012

Data and Draft Report 10 August 2012

Final Report 31 August 2012



### All inspections were study-based unless otherwise noted.

Director of Regulatory and Ecotox Operations DATE REPORTED TO:

STUDY MANAGEMENT: DIRECTOR:

17 February 2012 02 April 2012

16 April 2012 17 April 2012

14 May 2012 15 May 2012

11 June 2012 12 June 2012

II August 2012 14 August 2012

31 August 2012 31 August 2012



### .3/ 

### /llJ61JSr .).0/2-



### Date

# Wildlife International, Ltd.

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### REPORT APPROVAL



### SPONSOR: The Dow Cheruical Company Project Number

Dow Study Number

TITLE: ( A Reproduction Study with the

Earthworm in an Artificial Soil Substrate

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER:

DOW STUDY NUMBER:

STUDY DIRECTOR:

Date •

Senior Biologist

WILDLIFE INTERNATIONAL. LTD. MANAGEMENT:



# 3 l 

# A**'J,,~t** L /) / 'Z.--



### Date

Mager of Plant and Invertebrate Toxicology

:--.**:~**

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TABLE OF CONTENTS

Title Page

................................................................................................ ....................................... 1

Good Laboratory Practice Compliance Statement ........................................................................ 2

Quality Assurance Statement ........................................................................................................ 3

Report Approval ............................................................................................................................ 4

Table of Contents .......................................................................................................................... 5

Summary

................................................................................................ ........................................ 7

Introduction ................................................................................................................................... 8

Objective ....................................................................................................................................... 8

Experimental Design ..................................................................................................................... 8

Materials and Methods .................................................................................................................. 9

Test Substance .................................................................................................................... 9

Test Organism .................................................................................................................... 9

Test Soil Preparation ........................................................................................................ 10

Test Chambers .................................................................................................................. 11

Physical Properties of Test Soil ....................................................................................... 11

Environmental Conditions ................................................................................................ 11

Observations ..................................................................................................................... 12

Body Weights ................................................................................................................... 12

Collection and Enumeration of Juveniles ......................................................................... 12

Data Analyses ................................................................................................................... 12

Results and Discussion ................................................................................................................ 13

Environmental Conditions and Physical Properties of Test Soil ..................................... 13

Mortality and Clinical Signs ............................................................................................ 14

Body Weights ................................................................................................................... 14

Reproductive Output ........................................................................................................ 14

Reference Toxicity Test .................................................................................................. 15

Validity Criteria ................................................................................................................ 15

Conclusions ................................................................................................................................. 15

References ................................................................................................................................... 16

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TABLE OF CONTENTS

- Continued

-

TABLES

Ta ble 1. Moisture, pH, and Temperature of the Test Soil ........................................ 17

Table 2. Mortality and Observations of Adult Earthworms Exposed to

i n an Artificial Soil Substrate for 28 Days ............. 18

Table 3. Average Body Weights of Adult Earthworms Exposed to

in an Artificial Soil Substrate for 28 Days ............. 19

Table 4. Number of Juveniles Produced by Earthworms Exposed to

in an Artificial Soil Substrate ................................. 20

APPENDICES

Appendix 1. Protocol and Deviation ............................................................................... 21

Appendix 2. Certificate of Analysis ................................................................................ 34

Appendix 3. Artificial Soil and Test Soil Preparation .................................................... 35

Appendix 4. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd.

Earthworm Bulk Soil with Dry Manure ....................................................... 37

Appendix 5. Analyses o f P esticides, O rganics and Metals in Wildlife International,

Ltd.

Municipal Water ......................................................................................... 39

Appendix 6. Personnel Involved in the Study ................................................................. 41

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SUMMARY

SPONSOR: The Dow Chemical Company

TITLE: ( A Reproduction Study with the

E arthworm in an Artificial Soil Substrate

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER:

DOW STUDY NUMBER:

TEST SUBSTANCE:

( Lot Number

TEST DATES: Study Initiation: April 13, 2012

Experimental Start (EPA): April 16, 2012

Experimental Start (OECD): April 15, 2012

Experimental Termination: June 13, 2012

TEST ORGANISM: Earthworm ( Eisenia fetida )

AGE OF TEST ORGANISM: Adult (with a clitellum)

SOURCE OF TEST ORGANISM: In -house culture started with earthworms obtained from

University of Maryland

Wye Research & Education Center

P.O. Box 169

Queenstown, MD 21658

NOMINAL TEST CONCENTRATIONS:

Negative Control, 62.5, 125, 250, 500 and 1000 mg/kg dry soil

RESULTS: EC50 for juvenile production: >1000 mg/kg dry soil

95% confidence limits: not calculable

NOEC for juvenile production: 1000 mg/kg dry soil

LOEC for juvenile production: >1000 mg/kg dry soil

LC50 for adult mortality: >1000 mg/kg dry soil

95% confidence limits: not calculable

NOEC for body weight: 1000 mg/kg dry soil

LOEC for body weight: >1000 mg/kg dry soil

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INTRODUCTION

The study was conducted by Wildlife International, Ltd. at t he Wildlife International, Ltd.

toxicology facility in Easton, Maryland. The in

- life portion of the test was conducted from 16 April

to 11 June 2012 (final soil moisture measurements were completed on June 13, 2012). A copy of the

raw da ta g enerated a t W ildlife I nternational, L td. a nd a c opy of t he f inal r eport are filed under

Project Number in the archives located at Wildlife International, Ltd. site. T he original

raw data and final report are archived at The Dow Chemical Company.

OBJECTIVE

The o bjective o f t his st udy w as t o d etermine the effects of on the

earthworm, Eisenia fetida , during an 8

- week exposure period in an artificial soil substrate. A dults

were exposed for 28 days and then removed to evaluate mortality and growth. The cocoons and the

soil were returned to test chambers for anoth er 28 days to evaluate effects upon reproductive output

(number of juveniles at test termination).

EXPERIMENTAL DESIGN

Adult ear thworms w ere ex posed t o a g eometric ser ies o f f ive t est co ncentrations of

in artificial soil. A n egati ve control group w as exposed to soil prepared

without the addition of the t est s ubstance a nd w as m aintained c oncurrently. F our r eplicate t est

chambers were maintained in each treatment group and there were eight replicate test chambers in

the control grou p. Ten earthworms were housed in each of the replicate test chambers. On the day

of test in itiation, th e e arthworms w ere r insed b riefly w ith w ell w ater, g ently b lotted o n p aper,

weighed in groups of 10, and then placed on the soil surface of a test chamber. The adult earthworms

were fed cow manure weekly during the first 28 days of the test, re moved from the test chambers on

Day 28, and observed for mortality and signs of toxicity. Test soil and cocoons were returned to test

chambers for an additional 2 8 days in order to determine the numbers of juveniles produced in each

replicate by the end of the test on D ay 56. C ow m anure w as al so ad ded t o t he so il o nce at t he

beginning of the cocoon exposure period to serve as a source of food for juvenile worms em erging

from c ocoons. The number o f j uveniles p resent i n each r eplicate at t est t ermination w as u sed t o

determine the NOEC for reproduction.

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The nominal test concentrations to which the earthworms were exposed were 62.5, 125, 250,

500 and 1000 milligrams o f per kilogram of dry soil (mg/kg). Selection of the

nominal test concentrations was made in consultation with the Sponsor.

MATERIALS AND METHODS

The study was conducted according to the procedures outlined in the protocol, “

A Reproduction Study with the Earthworm in an Artificial Soil Substrate” (Appendix 1).

The protocol was based upon procedures outlined in the Organization for Economic Cooperation and

Development ( OECD) G uideline N o. 222: “ Earthworm Reproduction Test (Eisenia fetida/andrei) ”

( 1 ) and ISO/FDIS 11268-2 ( 2 ).

Test Substance

The test substance used w as r eceived f rom T he D ow C hemical C ompany o n 1 3 F ebruary

2012 and assigned Wildlife International, Ltd. identification number 10404. The test substance was

a id entified a s: L ot CAS N o.

Information received from t he Sponsor indicated purity of the test substance to be

and the recertification date to be 01 F ebruary 2014 ( Appendix 2 ). T he test substance was

stored under ambient conditions.

Test Organism

Adult earthworms ( Eisenia fetida ) of mixed age were obtained from a Wi ldlife Internatio nal,

Ltd. culture that was established with earthworms obtained from the University of Maryland Wye

Research and Education Center in Queenstown, Maryland. T he identity of the original culture was

verified by the supplier. Earthworms were from synchronous cultures (individuals not differing in

age by more than four weeks) maintained in moist peat moss and fed saturated alfalfa and/or cow

manure.

Adult earthworms were transferred to the test room and held in two glass aquaria for

acclimation to test con ditions for 7 days prior to test initiation. One day prior to the test the worms

(380 with clitellum) were removed from t he c hamber a nd di vided i nto t en one

- liter g lass b eakers

containing prepared artificial soil substrate adjusted to a moisture content o f approximately 35% by

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weight, for t he soil acclimation period. E arthworms w ere f ed c ow manure t hroughout t he

acclimation period.

On th e d ay o f te st in itiation, th e e arthworms w ere r insed b riefly w ith w ell w ater, gently

blotted on paper, weighed in groups of 10, and then placed on the soil surface of a test chamber. The

test chambers were ordered randomly in order to minimize bias, which might arise from the selection

process. T he earthworms were fed cow manure during testing. On Day 1 of the test, 5 grams of

finely ground cow manure for food, and water to moisten the food, were added to the test chambers.

Food w as pr ovided a pproximately w eekly dur ing t he ne xt t hree weeks by adding food in a small

depression in the soil surface and covering it with a thin layer of soil. The amount of food supplied

was reduced i f uneaten food remained from t he previous feeding interval. On Day 28, after adult

earthworms were removed, approximately 5 a dditional grams of manure was gently mixed into the

test soil before it was returned to the test chambers.

Test Soil Preparation

The artificial soil was prepared in bulk by blending approximately 70% sand, 20% kaolin clay

and 10% sphagnum peat ( Appendix 3 ). T he pH of the bulk soil prior to h ydration was adjusted to

5.9 using calcium carbonate. The bulk artificial soil was stored in a sealed container under ambient

conditions until used to prepare the test soils.

Test soil was pr epared by pr emixing t he a ppropriate a mount of t he t est s ubst ance w ith an

aliquot o f d ry a rtificial s oil ( Appendix 3 ). Sufficient reverse osmosis water was added to the dry

artificial soil to achieve a moisture content of approximately 35% by weight. T est soil components

were mixed for a total of 25 minutes in order to achieve a homogeneous mixture. N egative control

soil was prepared in the same manner as the treated soil but with only the addition of water and with

a mixing time of 20 m inutes. S even hundred fifty grams of prepared soil were added to each test

chamber. Test concentrations are reported as milligrams per kilogram of soil on a d ry weight basis

(mg/kg).

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Test Chambers

The test chambers were one

- liter glass beakers covered with plastic wrap that was perforated

for air exc hange. All test chambers were identified with the project number, test concentration and

replicate. Test chambers were arranged in a randomized block design to minimize bias that might

result from a placement effect in the study room.

Physical Properties of Test Soil

Soil temperature was measured i n o ne r eplicate o f each t reatment an d co ntrol group at t est

initiation and termination (Day 56) us ing a ha nd

- held t hermometer. M oisture c ontent a nd pH

measurements were made on soil samples collected from each batch of soil prepared for treatment

and control groups at the time of test soil mixing. At test termination, samples for moisture content

and p H m easurements w ere co llected f rom o ne r eplicate o f each co ntrol an d t reatment g roup.

Measurements of pH were made using a Thermo Orion 525 Aplus pH meter. Soil moisture content

was determined by measuring the initial weight of the soil sample, and then weighing the soil sample

after drying for 2 or more days at approximately 105°C. The percent moisture was calculated using

the following formula:

% Moisture = [(wet weight - dry weight) ÷ wet weight] * 100.

Test ch ambers were weighed p eriodically t o monitor so il moisture l oss. Lost soil moisture

was replaced by adding water to the soil surface until weights approximated those at the start of the

test (Day 1) or those weights collected on Day 28 of the test (after adult earthworms were removed).

Environmental Conditions

During the test, the earthworms were maintained in an environmental room set to maint ain a

temperature of approximately 20 ± 2°C. A ir temperature was recorded daily in the environmental

room. The photoperiod during acclimation and testing was 16 hours of light and 8 hours of dark per

day and was provided by overhead fluorescent bulbs. T he target light intensity during the test was

approximately 400 to 800 lux, and was verified on Day 35 of the test at the top of each test chamber

using a Sper Scientific INC model 840006 light meter.

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Observations

At test initiation, the earthworms we re placed on the surface of the soil in each test chamber

and were observed for burrowing behavior. On Day 28 of t est, t he contents of each test chamber

were removed to determine the number of s urviving e arthworms. A ll s urviving e arthworms w ere

observed for gross physical an d b ehavioral ab normalities. A fter t he ad ults w ere r emoved,

approximately 5 grams of food were gently mixed by hand i nto t he t est s oil. T he s oil a nd a ny

cocoons a nd y oung w orms w ere r eturned t o t he t est c hambers. F ollowing obs ervatio ns a nd body

weight determinations, surviving earthworms were euthanized by freezing and holding for disposal

by incineration.

Body Weights

In order to ensure that t he w orms u sed i n t he t est w ere o f si milar si ze, 2 0 w orms w ere

collected indiscriminately f rom the worms acclimated for the test and weighed individually and the

mean weight and standard deviation calculated. Group weights for all earthworms in each replicate

were collected prior to being placed i n t he t est c hambers on D ay 0. O n D ay 28, a ll s u rviving

earthworms were removed from each replicate test chamber, rinsed with deionized water and blotted

on dr y pa per. G roup body w eights w ere m easured f or w orms i n e ach t est chamber, and average

individual body weights were calculated.

Collection and Enumeration of Juveniles

On Day 56, juveniles were removed from the test soil and counted. A ll replicates for each

treatment group were placed in a hot water bath (temperature of approximately 60 ̊C) for at least

approximately 20 minutes until the juvenile worms moved into the top layer, or were on t op, of the

soil. The juveniles were removed from each replicate, along with some soil, and placed in holding

containers prior to counting. The soil from each replicate was searched by hand to be sure that all

juveniles were collected with the heating procedure. The juveniles removed from each replicate test

chamber were counted and examined for physical abnormalities.

Data Analyses

Differences between the treatment groups an d the control group were evaluated to assess

potential effects on fecundity, body weight, and changes in body weight. Initial and final adult body

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weight a nd the n umber o f j uvenile w orms w ere d etermined f or each r eplicate. T reatment group

means were calculated and compared t o t he control group means using Dunnett’s Test in order to

determine w hich g roup(s), i f a ny, di ffered s ignificantly from the control at th e 0.05 level of

significance. P rior to conducting Dunnett’s test, the data were tested for homogeneity of variance

and normal distribution. Results of the statistical tests were used to help determine the no

- observed

effect

- concentration (NOEC) and lowes t- observed

- effect

- concentration (LOEC) for body weight and

production of juveniles. T he NOEC was defined as the maximum test concentration at which there

were no treatment

- related reductions in adult body weight or a reduction in the number of offspring.

The LOEC was defined as the lowest test concentration that showed effects for the same categories.

Statistical calculations were made using a p ersonal computer and commercially available software

( 3 ). T he E C/LC50 i s de fined a s t he co ncentration o f t est su bstance t hat cau sed a 5 0% r eduction

relative to the control group for a variable of concern. No EC/LC50 calculations were warranted for

body weights, adult mortality or reproduction due to the lack of a 50% effect at any test le vel.

RESULTS AND DISCUSSION

Environmental Conditions and Physical Properties of Test Soil

Average light intensity during the 16

- hour light period was 533 ± 86.1 l ux, with a range of

405 to 726 l ux. A ir t emperature i n the environmental room was wi thin t he de sired r ange of

approximately 20 ± 2°C with a mean air temperature of 21.6 ± 0.51°C (mean ± standard deviation)

during the test. T he m ean da ily a ir t emperatures i n t he r oom r anged f rom 21 t o 22° C, w ith t he

exception o f a d aily m ean t emperature o f 23°C on the last d ay o f t he t est ( the sl ightly el evated

temperature on the last day was a protocol deviation but there was no adverse impact on the test).

Soil temperature ranged from 19.9 to 20.1°C in each of the groups at test initiation and from 20.0 to

21.1°C at test termination, and was therefore within the desired range of 20 ± 2°C. S oil pH ranged

from 7.0 t o 7.3 a t t est i nitiation, a nd f rom 7.2 t o 7.3 a t t est t ermination. Soil moisture content

measured dur ing t he t est r anged f rom 34 t o 35% a t t e st i nitiation a nd f rom 41 t o 46% a t t est

termination. M easurements of soil pH, soil temperature, and soil moisture content at test initiation

and test termination are presented in Table 1

.

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Mortality and Clinical Signs

There were no m ortalities in the control group and treatment groups during the 28

- day adult

exposure period. All surviving earthworms in the control and the treatment groups were normal in

appearance an d b ehavior ( Table 2 ). E arthworms s howed no a version to s oils t reated w ith

S ince there was no mortality of adult earthworms in the study an LC50 value

for mortality was not calculated.

Body Weights

The body weights of the adult worms available for use in the test r anged from 325 to 576 mg

with a m ean of 422 ± 69.9 ( SD) m illigrams ba sed on 20 individually weighed worms. The

earthworms used in the study had a wet mass meeting the 250 t o 600 m g range specified in OECD

222. Average adult body weights at initiation and termination of the adult exposure period, and the

change in body weight from initiation to termination, were calculated from the Day 0 a nd Day 28

measurements ( Table 3 ). W hen c ompared w ith t he c ontrol g roup, t here w ere no a ppare nt ef fects

upon earthworm weight in the t reatment g roups dur ing t he 28

- day a dult e xposure pe riod a nd no

statistically significant differences between mean weights in treatment groups when compared to the

control group means, with the exception of the 500 m g/kg final body weight. The earthworms in the

500 mg/kg treatment g roup g ained w eight a nd t he c hange i n w eight m ean w as not s ignificantly

different from the control therefore the difference i n f inal body w eight w as not c onsidered t o be

related t o t reatmen t. T herefore, ba sed on body w eight da ta, t he L OEC w as determined to be

>1000 mg/kg and the NOEC 1000 mg/kg.

Reproductive Output

The mean number of juveniles in the negative control group on D ay 56 w as 232.8 ± 31.33

(mean ± standard deviation), with a coefficient of variation of 13.5%. In the 62.5, 125, 250, 500 and

1000 mg/kg dry soil treatment groups the mean number of juveniles was 185.3, 184.8, 190.0, 179.3

and 176.5, r espectively ( Table 4 ). T he j uveniles co llected f ro m t he c ontrol a nd t reatment g roups

were normal i n appearance and behavior. The mean number of juveniles produced in each of the

treatment groups was statistically significantly different from the mean number of j uveniles in the

control group with D unnett’ s Test ( p< 0.05). While m ean num ber of juveniles produced i n the

control group was higher than all of the treatment groups, all groups had at least one replicate with

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over 200 j uveniles pr oduced by 10 a dult e arthworms. G iven t hat the overall amount of juv enile

production in the study was quite high compared to historical data, and that there was no obv ious

dose- response pattern, the statistically significant differences were not considered to be biologically

meaningful. The g uideline v alidity c riteria f or numbers of j uveniles is 30 or more per negative

control replicate, a nd a c oefficient of v ariation of 30% or l ess f or t he c ontrol g roup, w hich ha s

8 replicates. T hat g ives a n i ndication of bot h t he t endency f or t his t est t o ha ve m uch l ower

reproduction a nd a hi gh a mount of v ariability. Therefore, the L OEC f or r eproduction w as

determined to be >1000 mg/kg, and the NOEC to be 1000 mg/kg, the highest test concentration.

Reference Toxicity Test

Wildlife International, Ltd. conducted a reference toxicity test with carbendazim in 2006 ( 4 ) to

document that the earthworms being cultured were sensitive to a known toxicant. The LC50 value

for the mortality of the adult earthworms exposed to carbendazim for 28 da ys was 7.149 mg a.i./kg

dr y soil, with a 95% confidence interval of 6.338 a nd 8.273 mg a.i./kg dry soil. T here were effects

upon a dult e arthworm w eight a t c oncentrations of 0.3 m g a .i./kg a nd i n t he 2, 4 a nd 8 m g a.i./kg

groups. The EC50 value for reproduction was 0.8914 mg a.i./kg dry soil, with a 95% confidence

interval of 0.8416 a nd 0.9718 m g a.i./kg dry soil. T he NOEC was 0.5 mg a.i./kg dry soil, and the

LOEC was 1 mg a.i./kg dry soil, based on the numbers of juveniles produced.

Validity Criteria

The test was considered to be acceptable based on the validity criteria. A dult mortality of

control e arthworms w as l ess t han 10% w ith no m ortalities oc curring i n t he c ontrol g roup. T here

were more than 200 juveniles produced in each of the eight replicates for the control group, t hereby

meeting the criterion of 30 or more juveniles per container. The coefficient of variation of

reproduction in the control group was 13.5, meeting the less than the 30% criterion.

CONCLUSIONS

There was no mortality of adult earthworms exposed to at 62.5, 125,

250, 500 a nd 1000 m g/kg dr y s oil f or 28 da ys. B ased on body w eight data, the NOEC was

determined to be 1000 mg/kg, the highest test concentration. T he NOEC for reproduction was also

determined to be 1000 mg/kg.

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REFERENCES

1. Organization for Economic Cooperation and Development . 2004. OECD Guideline for the

Testing of Chemicals; Guideline 222: “Earthworm Reproduction Test ( Eisenia fetida/andrei )”.

2. International Organization for Standardization . 1997. I SO/FDIS 112 68

- 2. Soil Quality

– Effect of P ollutants on E arthworms ( Eisenia fe tida )

– Part 2: D etermination of E ffects on

Reproduction.

3. SAS I nstitute, I nc. 1999. SAS/STAT User’s Guide, V ersion 8. Cary, NC, SAS Institute,

Inc.

4. Wildlife International, Ltd . 2007. Carbendazim: A Reproduction Study with the

Earthworm in an Artificial Soil Substrate

. Project No. 100R-114A.

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Table 1

Moisture, pH, and Temperature of the Test Soil

Test Initiation Test Termination

Nominal Concentration

(mg/kg dry soil) M oisture Content 1 (%) pH 1 Temperature 2 (°C) Moisture Content 3 (%) pH 3 Temperature 3 (°C)

Negative Control 34.3 7.0 19.9 40.8 7.3 21.1

62.5 34.2 7.1 20.1 44.6 7.3 20.0

125 34.5 7.0 20.0 43.6 7.2 21.0

250 34.5 7.3 20.0 43.2 7.3 21.0

500 34.3 7.0 19.9 46.1 7.2 20.9

1000 33.9 7.2 20.0 43.8 7.3 20.9

1 Measurements taken from prepared soil on Day 0 prior to allocation to test chambers.

2 Measurements taken from one replicate test chamber at each concentration on Day 0.

3 Measurements taken from one replicate test chamber at each concentration at test termination.

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Table 2

Mortality and Observations of Adult Earthworms Exposed to in an Artificial

Soil Substrate for 28 Days

Nominal Concentration

(mg/kg dry soil) Replicate NumberDead 1 Effect s 2 Replicate

Percent

Mortality Group Percent

Mortality

Negative Control

(0) A

B

C

D

E

F

G

H 0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10 10 AN

10 AN

10 AN

10 AN

10 AN

10 AN

10 AN

10 AN 0

0

0

0

0

0

0

0 0

62.5 A

B

C

D 0/10

0/10

0/10

0/10 10 AN

10 AN

10 AN

10 AN 0

0

0

0 0

125 A

B

C

D 0/10

0/10

0/10

0/10 10 AN

10 AN

10 AN

10 AN 0

0

0

0 0

250 A

B

C

D 0/10

0/10

0/10

0/10 10 AN

10 AN

10 AN

10 AN 0

0

0

0 0

500 A

B

C

D 0/10

0/10

0/10

0/10 10 AN

10 AN

10 AN

10 AN 0

0

0

0 0

1 000 A

B

C

D 0/10

0/10

0/10

0/10 10 AN

10 AN

10 AN

10 AN 0

0

0

0 0

1 Mortality data are presented as the number found dead per number exposed.

2 Observed Effects: AN = normal in appearance and behavior.

# W ildlife International, Ltd.

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Dow Study Number

Table 3

Average Body Weights of Adult Eart hworms Exposed to

in an Artificial Soil Substrate for 28 Days

Nominal

Concentration

(mg/kg dry soil) Replicate Average Earthworm Body Weights (g)

Day 0 1 Day 28 Change in Body

Weight 2

Negative Control

(0) A

B

C

D

E

F

G

H

M ean ± Std. Dev. 0.54

0.51

0.43

0.50

0.55

0.46

0.53

0.54

0.51 ± 0.043 0.63

0.62

0.63

0.63

0.67

0.62

0.64

0.63

0.63 ± 0.016 +0.09

+0.11

+0.20

+0.13

+0.12

+0.16

+0.11

+0.09

0.13 ± 0.038

62.5 A

B

C

D

Mean ± Std. Dev. 0.45

0.54

0.47

0.46

0.48 ± 0.041 0.6 4

0.64

0.58

0.62

0.62 ± 0.028 +0.19

+0.10

+0.11

+0.16

0.14 ± 0.042

125 A

B

C

D

Mean ± Std. Dev. 0.55

0.48

0.42

0.56

0.50 ± 0.066 0.66

0.55

0.62

0.66

0.62 ± 0.052 +0.11

+0.07

+0.20

+0.10

0.12 ± 0.056

250 A

B

C

D

Mean ± Std. Dev. 0.43

0.44

0.54

0.49

0.48 ± 0.051 0.58

0.57

0.60

0.60

0.59 ± 0.015 +0.15

+0.13

+0.06

+0.11

0.11 ± 0.039

500 A

B

C

D

Mean ± Std. Dev. 0.46

0.41

0.45

0.49

0.45 ± 0.033 0.59

0.61

0.60

0.53

0.58 ± 0.036* +0.13

+0.20

+0.15

+0.04

0.13 ± 0.067

1000 A

B

C

D

Mea n ± Std. Dev. 0.48

0.41

0.51

0.47

0.47 ± 0.042 0.58

0.58

0.59

0.62

0.59 ± 0.019 +0.10

+0.17

+0.08

+0.15

0.13 ± 0.042

1 Initial body weight means were not statistically significantly different ( p> 0.05) for any group when compared to the

control group mean using Dunnett’s Test.

2 Change in body weight (initial to final) values were not statistically significantly different ( p> 0.05) for any group

when compared to the control group mean using Dunnett’s 2

- Tailed Test.

* Final body weight means was statistically significantly different ( p< 0.05) for group when compared to the control

group mean using Dunnett’s Test.

# W ildlife International, Ltd.

- 20 - Project Number

Dow Study Number

Table 4

Number of Juveniles Produced by Earthworms Exposed to

in an Artificial Soil Substrate

Nominal Concentration

(mg/kg dry soil) Replicate Number of Juveniles 1

(Day 56) Mean Number of Juveniles 2

(Mean ± Standard Deviation)

Negative Control

(0) A

B

C

D

E

F

G

H 282

276

206

203

228

231

234

202 232.8 ± 31.33 3

62.5 A

B

C

D 168

207

189

177 185.3 ± 16.86

125 A

B

C

D 223

157

158

201 184.8 ± 32.72

250 A

B

C

D 203

195

196

166 190.0 ± 16.39

500 A

B

C

D 161

187

153

216 179.3 ± 28.48

1000 A

B

C

D 183

205

171

147 176.5 ± 24.19

1 All juveniles were normal in appearance and behavior.

2 All treatment groups were statistically different ( p< 0.05) from the control group with Dunnett’s Test.

3 Coefficient of Variation = S.D./mean *100 = 13.5%.

# W ildlife International, Ltd.

- 21 - Project Number

Dow Study Number

Appendix 1

Protocol and Deviation

# W ildlife International, Ltd.

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Dow Study Number

# W ildlife International, Ltd.

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

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Dow Study Number

Appendix 2

Certificate of Analysis

# W ildlife International, Ltd.

- 35 - Project Number

Dow Study Number

Appendix 3

Artificial Soil and Test Soil Preparation

Artificial Soil Preparation

The artificial soil was prepared in bulk by mixing the following constituents in a soil mixer

for approximately 30 minutes:

quartz sand 63.0 kg

kaolin clay 18.0 kg

sphagnu m peat 9.0 kg

calcium carbonate 0.9 kg

Test Soil Preparation

A total of 8 kg (wet weight) of test soil with calculated soil moisture content of approximately

35% was prepared for the control group and 4 kg (wet weight) was prepared for each of the t reatment

groups. Test substance concentrations were based on t he dr y w eight por tion of t he s oil. T he

calculated dry weight of 4 k g of prepared soil, with a moisture content of 35%, was 2.60 k g. To

account for the estimated 1.6% moisture content present in the bulk soil stored in ambient conditions,

1358 mL of water was added to 2642 g of bulk soil. In addition dry cow manure (10 g/kg soil) was

added for food. For the control group, 2716 mL of water was added to 5284 g of bulk soil and 53 g

of cow manur e. Components of the test soils were as follows:

Weights and Volumes Used in Preparation of Test Soils

Treatment Group

(mg/kg dry soil) Amount of

Bulk Soil

(g) Amount of

Cow Manure

(g) Amount of

(g) Amount of Reverse

Osmosis Wat er

(mL)

Negative Control 5284 53 0 2716

62.5 2642 26 0.1625 1358

125 2642 26 0.3250 1358

250 2641 26 0.6500 1358

500 2641 26 1.3000 1358

1000 2639 26 2.6000 1358

# W ildlife International, Ltd.

- 36 - Project Number

Dow Study Number

Appendix 3 (continued)

Bulk artificial soil was weighed i nto a t ared H obart ® 

### mixing bowl a nd t he a ppropriate

amount of manure was added. The test substance was weighed into a tared 100 mL glass beaker then

combined, by stirring with a metal spoon, with an aliquot of the dry soil. The test substance and soil

mixture was added to the Hoba rt ® 

### bowl and the soil mixed dry for approximately 5 minutes. Reverse

osmosis water, measured with 1 liter, 50 mL and 25 mL graduated cylinders, was added slowly while

the mixer was running and the soil mixed approximately 5 minutes. The sides, bottom and blade of

the Hobart ® 

### mixer were scraped before the soil was mixed an additional approximately 15 minutes.

Negative control soil was prepared in a similar manner, but without the addition of test substance and

without the initial dry mix. P repared soil, 7 50 grams, was weighed into labeled, 1 L glass beakers,

covered with plastic wrap, and placed in a randomized block in the test room.

# W ildlife International, Ltd.

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Dow Study Number

Appendix 4

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd.

Earthworm Bulk Soil with Dry Manure 1

Pesticides and Organics

Component Measured Concentration

(μg/kg) Component Measured Concentration

(μg/kg)

Aldrin < 0.84 Gamma BHC

– Lindane < 0.84

Alpha BHC < 0.84 Gamma Chlordane < 0.84

Alpha Chlordane < 0.84 Guthion (Azinphos

- methyl) < 68

Beta BHC < 1.9 HCB < 0.84

Bolstar < 68 Heptachlor < 0.84

Chlordane < 17 Heptachlor Epoxide < 0.84

Coumaphos < 68 Kepone < 7.1

Delta BHC < 0.84 Malathion < 68

Demeton

- O < 68 Merphos < 68

Demeton

- S < 68 Methoxychlor < 8.4

Diazinon < 68 Methyl Parath ion < 68

Dichlorvos < 68 Mevinphos < 68

Dieldrin < 1.7 Mirex < 1.7

Disulfoton < 76 Naled < 68

Dursban (Chlorpyrifos) < 68 o,p

- DDD < 1.7

Endosulfan I < 0.84 o,p

- DDE < 1.7

Endosulfan II < 1.7 o,p

- DDT < 1.7

Endosulfan Sulfate < 1.7 p,p

- DDD < 1.7

Endri n < 1.7 p,p

- DDE < 1.7

Endrin Aldehyde < 1.7 p,p

- DDT < 1.7

Endrin Ketone < 1.8 Phorate < 68

EPN < 68 Ronnel < 68

Ethion < 68 Stirophos < 68

Ethoprop < 68 Telodrin < 1.2

Ethyl Parathion < 68 Tokuthion < 68

Famphur < 68 Toxaphene < 33

Fensulfothion < 200 Trichloronate < 68

Fenthion < 68 Trithion < 68

1 Analyses performed by Lancaster Laboratories on samples collected on December 19, 2011.

# W ildlife International, Ltd.

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Dow Study Number

Appendix 4 (continued)

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd.

Earthwo rm Bulk Soil with Dry Manure 1

Metals

Component Measured Concentration

(mg/kg) Component Measured Concentration

(mg/kg)

Aluminum 9860 Magnesium 262

Antimony < 1.95 Manganese 7.60

Arsenic < 1.95 Mercury < 0.479

Barium 80.3 Nickel 1.35

Beryllium < 0.488 Nitrate by IC (solid) 2.3

Bromide by IC (solid) < 5.0 Nitrite by IC (solid) < 1.0

Cadmium < 0.488 Potassium 194

Calcium 5960 Selenium < 1.95

Chloride by IC (solid) 63.1 Silver < 0.488

Chromium 8.51 Sodium < 97.5

Cobalt < 0.488 Sulfate by IC ( solid) 107

Copper 1.43 Thallium < 2.93

Fluoride by IC (solid) 1.6 Vanadium 4.94

Iron 621 Zinc 5.00

Lead 10.8

1 Analyses performed by Lancaster Laboratories on samples collected on December 19, 2011.

# W ildlife International, Ltd.

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Appendix 5

Analyses of Pesticides, Organics and Metals in

Wildlife International, Ltd. Municipal Water 1

Pesticides and Organics

Component Measured Concentration

(μg/L) Component Measured Concentration

(μg/L)

Aldrin < 0.0098 Gamma BHC

– Lindane < 0.0098

Alpha BHC < 0.0098 Gamma Chlordane < 0.0 20

Alpha Chlordane < 0.0098 Guthion (Azinphos

- methyl) < 5.8

Beta BHC < 0.0098 HCB < 0.0098

Bolstar < 1.9 Heptachlor < 0.0098

Chlordane < 0.49 Heptachlor Epoxide < 0.0098

Coumaphos < 2.9 Kepone < 0.20

Delta BHC < 0.0098 Malathion < 2.9

Demeton

- O < 1. 9 Merphos < 1.9

Demeton

- S < 2.9 Methoxychlor < 0.098

Diazinon < 5.8 Methyl Parathion < 1.9

Dichlorvos < 2.9 Mevinphos < 4.8

Dieldrin < 0.020 Mirex < 0.25

Disulfoton < 1.9 Naled < 5.8

Dursban (Chlorpyrifos) < 2.9 o,p

- DDD < 0.020

Endosulfan I < 0.0098 o,p

- DDE < 0.74

Endosulfan II < 0.020 o,p

- DDT < 0.020

Endosulfan Sulfate < 0.020 p,p

- DDD < 0.020

Endrin < 0.020 p,p

- DDE < 0.020

Endrin Aldehyde < 0.098 p,p

- DDT < 0.020

Endrin Ketone < 0.020 Phorate < 1.9

EPN < 1.9 Ronnel < 1.9

Ethion < 1.9 Stirophos < 1.9

Ethoprop < 2.9 Telodrin < 0.0098

Ethyl Parathion < 1.9 Tokuthion < 1.9

Famphur < 2.9 Toxaphene < 3.0

Fensulfothion < 8.7 Trichloronate < 1.9

Fenthion < 1.9 Trithion < 1.9

1 Analyses performed by Lancaster Laboratories on samples collected on December 28, 2011.

# W ildlife International, Ltd.

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Dow Study Number

Appendix 5 (continued)

Analyses of Pesticides, Organics and Metals in

Wildlife International, Ltd. Municipal Water 1

Metals

Component Measured Concentration

(mg/L) Component Measured Concentration

(mg/L)

Aluminum < 0.200 Ma gnesium 1.76

Antimony < 0.0200 Manganese < 0.0050

Arsenic < 0.0200 Mercury < 0.00020

Barium 0.0638 Nickel < 0.0100

Beryllium < 0.0050 Nitrate Nitrogen < 0.50

Bromide < 2.5 Nitrite Nitrogen < 0.50

Cadmium < 0.0050 Potassium 6.92

Calcium 7.53 Selenium < 0.0200

Chloride 11.3 Silver < 0.0050

Chromium < 0.0150 Sodium 30.9

Cobalt < 0.0050 Sulfate 17.2

Copper < 0.0100 Thallium < 0.0300

Fluoride 0.70 Vanadium < 0.0050

Iron < 0.200 Zinc < 0.0200

Lead < 0.0150

1 Analyses performed by Lancaster Labor atories on samples collected on December 28, 2011.

# W ildlife International, Ltd.

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Appendix 6

Personnel Involved in the Study

The following key Wildlife International, Ltd. personnel were involved in the conduct or

management of this study:

1. Henry O. Krueger, Ph.D., Director of Aquatic, Plant and Insect Toxicology

2. John R. Porch, Manager of Plant and Invertebrate Toxicology

3. Anne B. Sindermann, Senior Biologist, Nontarget Plants and Invertebrates