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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
THis study was conducted between 18 October 2016 and 27 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 222 and in compliance with GLP

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 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Alkyl (C12-C14) glycidyl ether
Label: Epoxide 8
51% C12 Glycidyl ether
21% C14 Glycidyl ether
5% C16 Glycidyl ether
13% N +1's
4% alpha addition Products
94% total active (label claim)
- Physical state: Clear colorless liquid
- Storage condition of test material: ambient
Specific details on test material used for the study:
Name: Oxirane, mono[(C12-14-alkyloxy)methyl] derivs
Physical state: Clear liquid
Batch Number: AAD1326100
Purity: 100% (Complex mixture used as product)
Storage: Refrigerated (2-8°C)
Expiry: 29 August 2019

Sampling and analysis

Analytical monitoring:
no

Test substrate

Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
Preparation of Test Item
Test Item Formulation
Eleven treatments were employed in this study.
i) Water control.
ii) Solvent control.
iii) Oxirane applied at 16.3 mg/kg dry soil.
iv) Oxirane applied at 29.4 mg/kg dry soil.
v) Oxirane applied at 52.9 mg/kg dry soil.
vi) Oxirane applied at 95.3 mg/kg dry soil.
vii) Oxirane applied at 171.5 mg/kg dry soil.
viii) Oxirane applied at 308.6 mg/kg dry soil.
ix) Oxirane applied at 555.6 mg/kg dry soil.
x) Oxirane applied at 1000 mg/kg dry soil.
xi) Mascot Systemic applied at 5 mg a.i./kg dry soil.

Method of Preparation
The treatments were prepared as follows:
Treatment Rate (mg/kg dry soil) Amount of test item used (µL) Final volume of acetone (mL) (nominal) mg/mL Solution code
Oxirane 1000 2473 10 220 A
555.6 1374 10 122.2 B
308.6 763 10 67.89 C
171.5 424 10 37.72 D
95.3 236 10 20.95 E
52.9 131 10 11.64 F
29.4 73 10 6.47 G
16.3 40 10 3.59 H

Treatment Rate (mg a.i./kg dry soil) Amount of test item used (µL) Final volume of RO water (mL) mg a.i./mL Sample code
Mascot Systemic - 416 100 2.1 I
5 10 mL of I 100 0.21 J

RO = reverse osmosis

Application of Test Item
Prior to treatment the initial moisture content of the soil was determined as 16.96% and the maximum water holding capacity (MWHC) as 65.55%. A moisture content equivalent to 50% of MWHC was selected as providing suitable conditions for earthworm development and the soil was pre-moistened to 25% MWHC. The volume required to achieve 50% of the maximum water holding capacity was calculated as 356.5 mL water/2200 g dry ISO earthworm soil.
The test item was dispersed in acetone, where 10 mLs from each treatment rate was placed over 50 g of dry sand and the solvent allowed allowed to evaporate before mixing into 2150 g dry soil equivalent. To bring the soil moisture content to 50% MWHC 356.5 mLs of reverse osmosis water was then mixed in using a hand held electric mixer.
For the water control and acetone controls, 95.5 g of untreated dry sand and 95.5 g of sand treated with 10 mL acetone respectively, were mixed into 4150 g dry soil equivalent of moist. Reverse osmosis water, 680.5 mL, was added to bring the soil to 50% of the maximum water holding capacity.
For the toxic reference 10 mL of Mascot Systemic treatment solution was poured over 95.5 g dry sand allowed to evaporate before being mixed into a 4150 g dry weight equivalent of moist soil. Reverse osmosis water, 680.5 mL, was added to bring the soil to 50% of the maximum water holding capacity.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Worm Selection
Earthworms were purchased from a reputable supplier; details are recorded only in the raw data to maintain supplier confidentiality.
Minimum and maximum ambient temperatures were recorded daily prior to the study start, with a range of 17.9 – 20.0C. The light intensity range was 422 – 505 lux controlled by a time switch set to provide a 16-hour light : 8-hour dark photoperiod.
Selection of adult worms for 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, the batch of worms was inspected for reproductive maturity by the supplier, 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 start 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

Test conditions

Test temperature:
18.0 - 22.5”C
pH:
5.9 - 6.4
Moisture:
32.25 - 37.90% equivalent to 49.17 - 57.77% Maximum Water Holding Capacity
Details on test conditions:
Medium (Test Soil)
An artificial soil (OECD 207) of the following composition was used as the test medium (Batch WS1602):
% w/w
Industrial quartz sand 70
Kaolin clay 20
Peat 10

pH was adjusted to 6.0 ± 0.5 using calcium carbonate
(approximately pH 6.5 prior to treatment)

Definitive Experimental Design and Study Conduct
Prior to application selected worms were rinsed in reverse osmosis water, blotted dry and individually weighed before being reweighed in replicates of ten.
Tests were conducted in plastic containers measuring approximately 11 x 17 x 5 cm, each covered with a fitted lid. After application, ca 663.9 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 chambers for the water control and solvent control, four replicate chambers for each treatment rate and eight for the positive control. Containers were labeled with the study director, study number, treatment, replicate number and date of application. The replicates of ten worms were then 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.

Test Conditions
The worms were maintained in a room which was designed to provide a satisfactory range of environmental conditions for the species. Minimum and maximum ambient temperatures were recorded daily throughout the study, with a range of 18.0 – 22.5”C and overall mean values of 19.4”C and 20.7”C respectively. The light intensity range was 403 - 755 lux controlled by a time switch set to provide a 16-hour light : 8-hour dark photoperiod.

Study Design

Assessments

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 and the adult worms humanely euthanized by freezing before being discarded.

Health
Adult worms were checked daily.

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 boxes 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 surface were removed with tweezers and counted. Test boxes 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 box, 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.

Moisture Content of Soil
Moisture content was recorded at Day 1 and Day 56. A container of soil was maintained under identical conditions to the test boxes and was used as a guide for maintaining the moisture content of the test boxes throughout the study. At termination, group replicate mean moisture content values were found to be approximately 32.25 to 37.90%, equivalent to 49.17 – 57.77% MWHC.

Test Soil pH
A sample was taken from the control and each treatment rate for pH sampling at the study start and completion.
Time (Days) Water control Solvent control Oxirane (mg /kg dry soil) Mascot Systemic
(5 mg a.i./kg dry soil)
16.3 29.4 52.9 95.3 171.5 308.6 555.6 1000
0 6.4 6.4 6.4 6.4 6.4 6.4 6.3 6.3 6.3 6.3 6.4
56 6.0 5.9 6.0 6.1 5.9 5.6 5.7 5.8 5.7 5.7 5.3

Pre-treatment soil pH was 6.5.

Termination
On Day 56 at termination of the test, the number of juvenile worms per container was determined. All test worms were then humanely euthanized by freezing before being discarded.

Data Evaluation

Validity Criteria
The data generated in this study met all of the validity criteria for the control. The criteria were:
• Each replicate (containing ten adults) to have produced ≥ 30 juveniles by the end of the test.
• The coefficient of variation of reproduction to be ≤ 30%.
• Adult mortality over the initial four weeks to be ≤ 10%

Statistics
Statistical analysis was carried out using SAS 9.1.3 (SAS Institute 2002).

Reference substance (positive control):
yes
Remarks:
Identity : Mascot Systemic Active ingredient: Carbendazim Appearance : White liquid Storage conditions: Room temperature Batch numbe r: 20150624 Expiry date : 28 June 2017 Purity : 505 g/L Date received : 29 September 2015

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Adult
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Adult
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
Juvenile
Key result
Duration:
56 d
Dose descriptor:
EC50
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
not determinable
Details on results:
Health and Mortality
No adult mortality was recorded at any treatment rate.
The LC50 for adult mortality at Day 28 was >1000mg/kg dry soil. A NOEC of 1000 mg/kg dry soil was achieved.

Bodyweights
The results are presented in Table 1.
There was no statistically significant adverse impact on adult bodyweight at any treatment rate.
The adjusted mean bodyweights on Day 28 at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil were 522, 522, 526, 534, 525, 519, 522 and 569 mg respectively compared to 520 mg in the solvent control.
The LC50 for mean bodyweight of the adult earthworm at Day 28 could not be estimated because there was not a dose-response relationship. A NOEC of 1000 mg/kg dry soil was achieved.
There was a statistically significant reduction (p<0.001***) in the bodyweight of the Mascot Systemic group at 5 mg a.i./kg dry soil.

Juvenile Worms
The numbers of juvenile worms counted at termination are shown in Table 2 .
Control group productivity was acceptable (mean of 146 and 156 juveniles per replicate in the water and solvent controls respectively). The co-efficient of variation of the number of juveniles in the solvent control group was 5.86% and in the water control 8.39%.
The mean number of juveniles produced at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil was 169, 146, 149, 146, 147, 153, 145 and 142 respectively compared to 156 in the solvent control. No juveniles were present in the Mascot systemic treatment group.
The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship. A NOEC of 1000 mg/kg dry soil was achieved.
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.
Results with reference substance (positive control):
See "details on results", above.

Any other information on results incl. tables

Table 1       % Adult Mortality and Treatment Mean Bodyweights (mg)

 Treatment  (mg/kg dry soil)   % Mortality  Time 0  Day 28(a)   Change (%)   p Value
 Water control  0  0  447  513  +148  0.696
 Solvent control  0  0  445  520  +16.9  -
 Oxirane  16.3  0  441  522  +18.4  1.000
   29.4  0  445  522  +17.3  1.000
   52.9  0  444  526  +18.5  1.000
   95.3  0  447  534  +19.5  1.000
   171.5  0  443  525  +18.5  1.000
   308.6  0  445  519  +16.6  1.000
   555.6  0  447  522

 +16.8

 1.000
   1000  0  446  569  +27.6  0.018*
 Mascot Systemic  5 mg a.i/kg dry soil  0  442  374  -15.4  0.001***

(a) Mean adjusted bodyweight

* p<0.05  *** p<0.001

      

Table 2       Mean Number of Juvenile Worms per Treatment Replicate

 Treatment  (mg/kg dry soil)  Day 56  p value
 Water control  0  146  0.185
 Solvent control  0  156  -
 Oxirane  16.3  169  0.566
   29.4  146  0.566
   52.9  149  0.566
   95.3  146  0.566
   171.5  147  0.566
   308.6  153  0.566
   555.6  145  0.308
   1000  142  0.155
 Mascot Systemic  5 mg a.i./kg dry soil  0  <0.001***

Co-efficient of variation solvent control = 5.86%

Co-efficient of variation water control = 8.39%

All rates were compared against the solvent control group using Dunnett’s test

*** p<0.001

                                

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
No adult mortality was recorded at any treatment rate.
The LC50 for adult mortality at Day 28 was >1000 mg/kg dry soil. The confidence intervals could not be obtained. A NOEC of 1000 mg/kg dry soil was achieved.
There was no negative statistically significant impact on adult bodyweight at any Oxirane, mono[(C12-14-alkyloxy)methyl] derivs treatment rate. The Mascot Systemic treated group had significantly lower body weights than the solvent control group (p<0.001***).
The adjusted mean bodyweights on Day 28 at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil were 522, 522, 526, 534, 525, 519, 522 and 569 mg respectively compared to 520 mg in the solvent control.
The LC50 for mean bodyweight of the adult earthworm at Day 28 could not be estimated because there was not a dose-response relationship. A NOEC of 1000 mg/kg dry soil was achieved.

There was no statistically significant reduction in the number of juveniles produced at any Oxirane, mono[(C12-14-alkyloxy)methyl] derivs treatment rate. The Mascot Systemic treatment group had significantly lower number of juveniles than the solvent control group (p<0.001***).
The mean number of juveniles produced at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil was 169, 146, 149, 146, 147, 153, 145 and 142 respectively compared to 156 in the solvent control. No Juveniles were present in the Mascot systemic treatment group.
The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship. A NOEC of 1000 mg/kg dry soil was achieved.
The study was considered valid as there was ≤ 10% adult mortality at four weeks and ≥30 juveniles had been produced in each solvent control replicate by the end of the test with the co-efficient 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 study was performed to determine the effects of Oxirane, mono[(C12-14-alkyloxy)methyl] derivs, (thereafter referred to as Oxirane or the test item in the text and tables) on the reproduction and growth of the earthworm, Eisenia fetida, in an artificial soil under laboratory conditions.  The method followed was that described in OECD 222 Guideline for the testing of Chemicals, Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 2016.

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

Findings

No adult mortality was recorded at any treatment rate.

The LC50 for adult mortality at Day 28 was >1000 mg/kg dry soil.  The confidence intervals could not be obtained.  A NOEC of 1000 mg/kg dry soil was achieved.

There was no negative statistically significant impact on adult bodyweight at any Oxirane, treatment rate.  The Mascot Systemic treated group had significantly lower body weights than the solvent control group (p<0.001***).

The adjusted mean bodyweights on Day 28 at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil were 522, 522, 526, 534, 525, 519, 522 and 569 mg respectively compared to 520 mg in the solvent control.

The LC50 for mean bodyweight of the adult earthworm at Day 28 could not be estimated because there was not a dose-response relationship.  A NOEC of 1000 mg/kg dry soil was achieved.

There was no statistically significant reduction in the number of juveniles produced at any Oxirane treatment rate.  The Mascot Systemic treatment group had significantly lower number of juveniles than the solvent control group (p<0.001***).

The mean number of juveniles produced at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil was 169, 146, 149, 146, 147, 153, 145 and 142 respectively compared to 156 in the solvent control.  No juveniles were present in the Mascot systemic treatment group.

The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship.  A NOEC of 1000 mg/kg dry soil was achieved.

The study was considered valid as there was ≤ 10% adult mortality at four weeks and ≥30 juveniles had been produced in each water and solvent 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