Registration Dossier

Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Remarks:
Effects on Reproduction and Growth of the Earthworm
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 30 November 2016 and 22 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear, colourless liquid
Details on test material:
Identification: Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide (CAS# 6731-36-8)
Description: Clear colorless liquid
Batch: 0905137109
Label: Trigonox 29 0905137109 2kg
Purity: 93.4%
Date Received: 18 December 2012
Expiry Date: 01 June 2019
Storage Conditions: Stored frozen at approximately -20 °C; used/formulated at ambient temperature <30 °C
Specific details on test material used for the study:
Purity/Concentration: 1,1-Di(tert-butylperoxy)-3,3,5-trimethylcyclohexane 93.4%
3,3,5-trimethylcyclohexane 2.56%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
On preparation 2 x 5 mL samples of each Trigonox 29 test solution and the water and solvent controls were taken and placed into scintillation vials for analysis of test substance concentration.

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
Range-finding test:
Each replicate was treated individually.
The test substance item (2 mL per replicate from each prepared solution) was placed over
10 g of dry sand and the treated dry sand was then left for the solvent to evaporate. The treated sand was then placed into 651.78 g of moist soil (equivalent to 540 g dry soil) and mixed for 2 minutes before 80.72 mL of RO water was added to bring the moisture content to 35%. The treated soil was then mixed for ten minutes using an electric mixer.
For the water and solvent controls 80.72 mL RO water was mixed into a 540 g soil dry weight equivalent of moist soil with 10 g of untreated dry sand for the water control and 10 g acetone treated sand for the solvent control to bring the soil to 35% moisture content per replicate. The mixing procedure was as for the Trigonox 29 treatments.

Definitive test:
Prior to treatment the initial moisture content of the soil was determined as 18.52% and the maximum water holding capacity (MWHC) as 66.57%. A moisture content equivalent to 50% of MWHC was selected as providing suitable conditions for earthworm development and the soil was not pre-moistened as the moisture content was greater than 25% of the MWHC. The volume required to achieve 50% of the maximum water holding capacity was calculated as 332.98 mL water/2200 g dry ISO earthworm soil.
The test item (8.8 mL from each prepared solution) was placed over 44 g of dry sand, the treated sand was then left to allow the solvent to evaporate. After evaporation of the solvent the treated sand was placed into 2555.29 g of moist soil (equivalent to 2156 g dry soil) and mixed for two minutes, 332.98 mL of RO water was then added to bring the soil moisture content to 50% MWHC. The treated soil was then mixed for ten minutes using an electric mixer.
For the water and solvent controls 635.69 mL RO water was mixed into a 4116 g soil dry weight equivalent of moist soil with 84 g of untreated dry sand for the water control and acetone (16.8 mL) treated sand for the solvent control to bring the soil to 50% of the maximum water holding capacity.
For the toxic reference 100 mL of Mascot Systemic treatment solution was mixed into a 4116 g dry weight equivalent of moist soil to which 84 g of untreated dry sand had been added. A further 535.69 mL of reverse osmosis water was added to bring the soil to 50% of the maximum water holding capacity.
The mixing procedure for the water and solvent controls and the toxic reference treatment was as for the Trigonox 29 treatments.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Earthworms (Eisenia fetida) and dried rabbit manure were purchased from a reputable supplier of laboratory livestock. Prior to study start the worms were maintained in multipurpose compost and fed regularly. Details of suppliers are recorded only in the raw data to maintain supplier confidentiality.
Selection of adult worms for both the acute range finder and the reproduction study was based primarily on individual weights falling within the specified weight range of 300 – 600 mg at the start of the treatment period. Prior to treatment for the definitive phase, the batch of worms was inspected for reproductive maturity, i.e. production of egg cocoons or presence of a clitellum, the presence of which confirmed suitability for use.
Approximately 24 hours prior to study starts adult worms were transferred to untreated artificial soil.

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Remarks:
Range finding test duration: 14 d. Definitive experiment duration: Adults: 28 d, Juveniles: 56 d
Post exposure observation period:
not applicable

Test conditions

Test temperature:
Range-finding test: 17.7 – 20.0°C
Definitive test: 19.3 - 21.6ºC
pH:
6.3-6.6
Moisture:
Moisture content was recorded at Day 1 and Day 56. A container of soil was maintained under identical conditions to the test containers and was used as a guide for maintaining the moisture content of the test containers throughout the study. At termination, group replicate mean moisture content values were found to be approximately 32.75 to 36.39%, equivalent to 49.17 – 55.09% MWHC.
Details on test conditions:
TEST SYSTEM
Range-finding test:
The test was conducted in labelled one-litre glass containers. The prepared soil (550 g dry weight) was placed in each container, two containers per rate. Earthworms in pre-weighed groups of ten were placed on the soil surface of each container. The containers were then covered with polythene to retain moisture. At termination of the study, the soil from each replicate of each treatment group was re-weighed to calculate the approximate moisture content. At the end of the study the earthworms were humanely euthanised by freezing before they were discarded with the soil.
Earthworms were rinsed in distilled water, blotted dry and individually weighed to ensure that all individual weights fell within the specified range (300 - 600 mg). Earthworms were randomly allocated to treatment using a bodyweight stratification procedure with the aid of a random number table.

Definitive test:
Prior to conducting the definitive study the potential use of glass test chambers as used in the acute range finder was evaluated. As the number of juveniles present on Day 56 did not meet the validity criteria of ≥30 per replicate, the definitive study was conducted in plastic containers measuring approximately 11 x 17 x 5 cm, each covered with a fitted lid.
Prior to application selected worms were rinsed in reverse osmosis water, blotted dry and individually weighed before being reweighed in replicates of ten.
After application, ca 666.4 g of moist soil (equivalent to 500 g dry soil at 50% of the maximum water holding capacity) was transferred into each of eight replicate containers for the water control, solvent control and toxic reference and four replicate containers for each Trigonox 29 treatment rate. Containers were labeled with the name of the Study Director, study number, treatment, replicate number and date of application. The replicates of ten worms were allocated randomly to each treatment using a bodyweight stratification procedure with the aid of a random number table.
Dried rabbit manure was used as food. One day after application of the test item, 5 g of dried manure was uniformly distributed onto the surface of the soil in each test container and wetted with 5 mL of reverse osmosis water. Worms at each treatment rate were fed weekly in a similar way if two or more replicates per rate were assessed as having consumed 50% or more of the food. The quantity of water given was based on the amount of water loss from the moisture control box each week.
After four weeks (after removal of adult worms), the juvenile worms were fed by carefully mixing 5 g of food (dried manure) into the substrate of each container. Juvenile worms were not fed further during the four-week rearing period.


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
An artificial soil (OECD 207) of the following composition was used as the test medium:

Industrial quartz sand 70 % w/w
Kaolin clay 20 % w/w
Peat 10 % w/w

pH was adjusted to 6.0 ± 0.5 using calcium carbonate
(Untreated soil prior to treatment pH 6.5 (definitive))
Soil batches: WS1602 (Rangefinder) and WS1701 (Definitive).

- Stability and homogeneity of test material in the medium: The mean concentrations of test formulations analysed for the study were within ± 10% of nominal concentrations, confirming acurate formulation.

OTHER TEST CONDITIONS
Range-finding test - Lighting was continuous at an intensity of 428 – 660 lux.
Definitive test: The light intensity range was 405 - 694 lux controlled by a time switch set to provide a 16-hour light: 8-hour dark photoperiod.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Range-finding test:
Assessment of health
Any earthworms visible on the soil surface were observed daily for behavioural and pathological signs.
Assessment of mortality
On Days 7 and 14, the medium was emptied from the test containers and the earthworms removed for counting. Mortalities, live earthworms and those unaccounted for were recorded (earthworms which died beneath the soil surface may have decomposed and no longer be detectable at the counts on Days 7 and 14). Mortality was assessed, if necessary, by testing the reaction of each earthworm to a mechanical stimulus at either end. On Day 7, the medium was returned to the containers and surviving earthworms replaced on the surface.
Assessment of bodyweights
Bodyweights of earthworms were recorded in both treatment and control replicates prior to treatment and on Day 14.

Definitive test:
Counts of Worms and Mortalities
On Day 28 of the study the soil was removed from the containers. The numbers of live adults were recorded. The soil was returned to the containers for the juvenile development phase and the adult worms were humanely euthanised by freezing before being discarded as clinical waste.
Health
Any worms visible on the soil surface were observed daily for behavioural and pathological signs.
Weights
Adult worms were weighed individually and then in replicates of ten worms prior to treatment and at the end of the four week adult exposure period in replicates of ten.
Juvenile Worms
The number of surviving juvenile worms in each replicate was determined on Day 56. Test containers were placed in a water bath set to 60°C to encourage juvenile worms to come to the soil surface. Worms visible on the soil were removed with tweezers and counted. Test containers were then removed from the water bath and emptied onto a tray. As soon as possible after removal from the water bath, a sample of soil was taken from each test container, checked to ensure it contained no worms and weighed accurately, before being dried and used to determine the moisture content at termination. The remaining soil from each container was searched carefully by hand and any remaining worms removed with tweezers and counted.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Range finding study: 0.1, 1.0, 10, 100 and 1000 a.i/kg dry soil
- Definitive study: 5.04, 9.07, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 a.i./kg dry soil

- OTHER INFORMATION
Termination
On Day 56 at termination of the test, the number of juvenile worms per container was determined. All test worms were then humanely euthanised by freezing prior to being discarded as clinical waste.
Nominal and measured concentrations:
Analysis of the concentrations of Trigonox 29 in the application treatment solutions confirmed nominal levels of the test substance were within applied limits ± 20% for all treatment groups, confirming accurate formulation. Therefore, nominal concentrations were used to calculate the results.
Reference substance (positive control):
yes
Remarks:
Mascot Systemic (active ingredient: Carbendazim)

Results and discussion

Effect concentrationsopen allclose all
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
224 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: There was not a sufficient dose-response relationship to provide a reliable estimate and this value should be used with caution.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
95.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: The 7.5% mortality (p 0.035*) recorded at 16.3 mg a.i./kg dry soil is not considered to be biologically significant as it is an outlying result.
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
141 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: bodyweight
Remarks on result:
other: The confidence interval was 96.0 – 207.1 mg a.i./kg dry soil.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
95.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: bodyweight
Duration:
56 d
Dose descriptor:
LC50
Effect conc.:
95.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks:
number of juveniles
Remarks on result:
other: There was not a sufficient dose-response relationship to provide a reliable estimate and this value should be used with caution.
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
52.9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks:
number of juveniles
Remarks on result:
other: The significant decrease observed at 16.3 mg a.i./kg dry soil is not considered of biological significance as it can be considered a result of the low level of adult mortality recorded at this rate.
Details on results:
Rangefinder
A range finding test was performed using rates of 0.1, 1, 10, 100 and 1000 mg a.i./kg dry soil. At seven days after treatment 15% mortality was observed at 1000 mg a.i./kg dry soil which increased to 100% at 14 days after treatment. No mortality was observed at 7 or 14 days in either control group or in the 0.1, 1, 10 and 100 mg a.i./kg dry soil Trigonox 29 treatment rates.
There was a reduction in the mean bodyweight of 24.3% at 100 mg a.i./kg dry soil 14 days after treatment.
The 14-day LC50 value was estimated to be between 100 and 1000 mg a.i./kg dry soil.

Definitive test:
Health and Mortality
Adult mortality of 0, 0, 7.5, 5, 2.5, 2.5, 10, 95, 100 and 100% was recorded at rates of 5.04, 9.07, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil respectively.
The 16.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil Trigonox 29 treated groups had significantly higher mortality than the solvent control group.

Bodyweights
The Trigonox 29 treated groups at 555.6 and 1000 mg a.i./kg dry soil were excluded from the analysis of body weight for adult earthworms as there were no surviving earthworms on Day 28.
The 171.5 and 308.6 mg a.i./kg dry soil Trigonox 29 treated groups had significantly lower adult bodyweight than the solvent control group (p<0.001).

Juvenile Worms
Solvent control group productivity was acceptable (mean of 172 juveniles per replicate). The coefficient of variation of the number of juveniles in the negative solvent control group was 4.39%.
The mean number of juveniles produced at rates of 5.04, 9.07, 16.3, 29.4, 52.9, 95.3 and 171.5, mg a.i./kg dry soil was 167, 173, 141, 212, 196, 96 and 2 respectively compared to 172 in the solvent control and 158 in the water control.
The Trigonox 29 treated groups at 308.5, 555.6 and 1000 mg a.i./kg dry soil and the positive control group were excluded from the analysis of number of juveniles on Day 56 as there were only two surviving earthworms at 308.5 mg a.i./kg dry soil and none at 555.6 and 1000 mg a.i./kg dry soil. No juveniles were produced in the Mascot Systemic treatment.
The 16.3, 95.3 and 171.5 mg a.i./kg dry soil Trigonox 29 treated groups had significantly fewer juveniles than the solvent control group (p=0.042). The 29.4 and 52.9 mg a.i./kg dry soil Trigonox 29 treated groups had significantly more juveniles than the solvent control group (p=0.042). The solvent control group had significantly more juveniles than the water control group (p=0.005).


Results with reference substance (positive control):
The 5 mg a.i./kg dry soil Mascot Systemic treated group had significantly higher mortality than the water control group (p<0.001).
The 5 mg a.i./kg dry soil Mascot Systemic treated group had significantly lower adult bodyweight than the water control group (p<0.001).
There was a statistically significant reduction (p<0.001***) in the mean number of juveniles of the Mascot Systemic group at 5 mg a.i./kg dry soil.

Any other information on results incl. tables

% Adult Mortality and Treatment Mean Bodyweights (mg)

Treatment

(mg a.i./kg dry soil)

% Mortality

Day of study

Change
(%)

p value

Time 0 (mg)

Day 28(a)(mg)

Water control

0

10

318

496

+56

0.586

Solvent control

0

0

316

522

+65.2

-

Trigonox 29

5.04

0

314

608

+93.6

1.000

9.07

0

316

646

+104.4

1.000

16.3

7.5

315

548

+74.0

1.000

29.4

5

317

529

+66.9

1.000

52.9

2.5

315

544

+72.7

1.000

95.3

2.5

314

546

+73.9

1.000

171.5

10

317

259

-18.3

<0.001

308.6

95

317

0.2

-100

<0.001

555.6

100

324

-

-

-

1000

100

314

-

-

-

Mascot Systemic

5 mg a.i./kg dry soil

5

315

353

+12.1

<0.001

 

p values are for comparison with solvent control using Williams’ test for the treatment rates and compared against the water control using t-test for the Mascot Systemic

 (a)Mean adjusted bodyweight

- Not applicable

 

 

Mean Number of Juvenile Worms per Treatment Replicate

Treatment

(mg a.i./kg dry soil)

Day 56

pvalue

Water control

0

158

0.005**

Solvent control

0

172

-

Trigonox 29

5.04

167

0.957

9.07

173

1.000

16.3

141

0.042*

29.4

212

0.042*

52.9

196

0.042*

95.3

96

0.042*

171.5

1.8

0.042*

308.6

0

-

555.6

-

-

1000

-

-

Mascot Systemic

5 mg a.i./kg dry soil

0

-

p values are for comparison with solvent control using Williams’ test for the treatment rates and t-test for the Mascot Systemic

*p< 0.05, **p< 0.01

-Not applicable

Chemistry Analysis (soil)

The homogeneity was assessed with respect to the level of concentration at nominal concentrations of 5 and 10 mg a.i./kg dry soil. Homogeneity, expressed as a mean, was 43% nominal at 5 mg a.i./kg dry soil and 90% nominal at 1000 mg a.i./kg dry soil.

Preliminary Investigation of Aerobic Transformation

Study VN47RM, Trigonox29:Preliminary Investigation of Aerobic Transformation in Surface Water and Aquatic Sediment Systems (non-GLP), was conducted in the department of Metabolism.

In the simulation study14C-test item was applied to samples of both aquatic sediment and surface water. Determination of radioactivity by liquid scintillation counting (LSC) was conducted at Time 0 and seven and 14 days after application. Total recovery, expressed as % applied radioactivity, was 19.8% in the aquatic sediment system and 23.5% from the surface water at Time 0. A lack of stability and reactivity/binding and the resulting chemical changes in the test substance meant that only the dosing solutions were analysed to demonstrate accurate addition of the test material to the test system.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Adult mortality of 0, 0, 7.5, 5, 2.5, 2.5, 10, 95, 100 and 100% was recorded at rates of 5.04, 9.07, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil respectively. No mortality was recorded in the water or solvent controls.
The 16.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil Trigonox 29 treated groups had significantly higher mortality than the solvent control group. The 5 mg a.i./kg dry soil Mascot Systemic treated group had significantly higher mortality than the water control group (p<0.001).
The LC50 for the mortality on Day 28 was 224 mg a.i./kg dry soil. However, there was not a sufficient dose-response relationship to provide a reliable estimate and this value should be used with caution.
The NOEC for mortality on Day 28 was 95.3 mg a.i./kg dry soil. The 7.5% mortality (p 0.035*) recorded at 16.3 mg a.i./kg dry soil is not considered to be biologically significant as it is an outlying result.
The Trigonox 29 treated groups at 555.6 and 1000 mg a.i./kg dry soil were excluded from the analysis of body weight for adult earthworms as there were no surviving earthworms on Day 28.
The 171.5 and 308.6 mg a.i./kg dry soil Trigonox 29 treated groups had significantly lower adult bodyweight than the solvent control group (p<0.001). The 5 mg a.i./kg dry soil Mascot Systemic treated group had significantly lower adult bodyweight than the water control group (p<0.001).
The LC50 for the mean body weight of the adult earthworms at Day 28 was 141.0 mg a.i/kg dry soil. The confidence interval was 96.0 – 207.1 mg a.i./kg dry soil.
The NOEC for the mean bodyweight at Day 28 was 95.3 mg a.i./kg dry soil.
The Trigonox 29 treated groups at 308.5, 555.6 and 1000 mg a.i./kg dry soil and the positive control group were excluded from the analysis of number of juveniles on Day 56 as there were only two surviving earthworms at 308.5 mg a.i./kg dry soil and none at 555.6 and 1000 mg a.i./kg dry soil. No juveniles were produced in the Mascot Systemic treatment.
The coefficient of variation of the number of juveniles in the solvent control group was 4.39%.
The 16.3, 95.3 and 171.5 mg a.i./kg dry soil Trigonox 29 treated groups had significantly fewer juveniles than the solvent control group (p=0.042). The 29.4 and 52.9 mg a.i./kg dry soil Trigonox 29 treated groups had significantly more juveniles than the solvent control group (p=0.042). The water control group had significantly fewer juveniles than the solvent control group (p=0.005).
The LC50 for the number of juveniles on Day 56 was 95.6 mg a.i./kg dry soil. However, there was not a sufficient dose-response relationship to provide a reliable estimate and this value should be used with caution.
The NOEC for the number of juvenile worms on Day 56 was 52.9 mg a.i./kg dry soil. The significant decrease observed at 16.3 mg a.i./kg dry soil is not considered of biological significance as it can be considered a result of the low level of adult mortality recorded at this rate.
The study was considered valid as there was ≤ 10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition application of the toxic reference Mascot Systemic at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.
Executive summary:

A preliminary range finding study was conducted at 0.1, 1, 10, 100 and 1000 mg a.i./kg dry soil to determine the acute toxicity of Trigonox 29 to the earthworm Eisenia fetida in an artificial soil under laboratory conditions(OECD 207) and to ensure selection of appropriate treatment rates for the reproduction study. Water and solvent controls were also included.

At seven days after treatment 15% mortality was observed at 1000 mg a.i./kg dry soil which increased to 100% at 14 days after treatment. No mortality was observed at 7 or 14 days in either control group or at 0.1, 1, 10 and 100 mg a.i./kg dry soil Trigonox 29 treatment rate. There was a reduction in the mean bodyweight of 24.3% at 100 mg a.i./kg dry soil 14 days after treatment. The 14-day LC50value was estimated to be between 100 and 1000 mg a.i./kg dry soil. 

A study was then performed to determine the effects of Trigonox 29 on the reproduction and growth of the earthworm, Eisenia fetida. The method followed was that described in OECD 222 Guideline for the Testing of Chemicals, Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 2004.

Thirteen groups of worms were allocated to the study. Ten groups of 40 worms were treated withthe test itemat rates of 5.04, 9.07, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i/kg dry soil. Water and solvent control groups of 80 worms were maintained in untreated soil to act as negative controls. A positive control group, 80 worms, was treated with Mascot Systemic (active ingredient carbendazim) at 5 mg a.i./kg dry soil. Adult worms were removed from the soil four weeks after treatment and the juvenile worms reared for a further four weeks.

Findings

In the reproduction study adult mortality of 0, 0, 7.5, 5, 2.5, 2.5, 10, 95, 100 and 100% was recorded at rates of 5.04, 9.07, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry mg a.i./kg dry soil respectively. No mortality was recorded in the water or solvent controls.

The 16.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil Trigonox 29 treated groups had significantly higher mortality than the solvent control group. The 5 mg a.i./kg dry soil Mascot Systemic treated group had significantly higher mortality than the water control group (p<0.001).

The LC50for the mortality on Day 28 was 224 mg a.i./kg dry soil. However, there was not a sufficient dose-response relationship to provide a reliable estimate and this value should be used with caution.

The NOEC for mortality on Day 28 was 95.3 mg a.i./kg dry soil. The 7.5% mortality (p 0.035*) recorded at 16.3 mg a.i./kg dry soil is not considered to be biologically significant as it is an outlying result.

The Trigonox 29 treated groups at 555.6 and 1000 mg a.i./kg dry soil were excluded from the analysis of body weight for adult earthworms as there were no surviving earthworms on Day 28.

The 171.5 and 308.6 mg a.i./kg dry soil Trigonox 29 treated groups had significantly lower adult bodyweight than the solvent control group (p<0.001). The 5 mg a.i./kg dry soil Mascot Systemic treated group had significantly lower adult bodyweight than the water control group (p<0.001).

The LC50for the mean body weight of the adult earthworms at Day 28 was 141.0 mg a.i/kg dry soil. The confidence interval was 96.0 – 207.1 mg a.i./kg dry soil.

The NOEC for the mean bodyweight at Day 28 was 95.3 mg a.i./kg dry soil.

The Trigonox 29 treated groups at 308.5, 555.6 and 1000 mg a.i./kg dry soil and the positive control group were excluded from the analysis of number of juveniles on Day 56 as there were no surviving earthworms.

The coefficient of variation of the number of juveniles in the solvent control group was 4.39%.

The 16.3, 95.3 and 171.5 mg a.i./kg dry soil Trigonox 29 treated groups had significantly fewer juveniles than the solvent control group (p=0.042). The 29.4 and 52.9 mg a.i./kg dry soil Trigonox 29 treated groups had significantly more juveniles than the solvent control group (p=0.042). The solvent control group had significantly more juveniles than the water control group (p=0.005).

The LC50for the number of juveniles on Day 56 was 95.6 mg a.i./kg dry soil. However, there was not a sufficient dose-response relationship to provide a reliable estimate and this value should be used with caution.

The NOEC for the number of juvenile worms on Day 56 was 52.9 mg a.i./kg dry soil. The significant decrease observed at 16.3 mg a.i./kg dry soil is not considered to be of biological significance as it can be considered a result of the low level of adult mortality recorded at this rate.

The study was considered valid as there was10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition application of the toxic reference Mascot Systemic at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.