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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
GLP compliance:
yes
Specific details on test material used for the study:
Identifier: EXP1200078
Appearance: Very dark brown (almost black) viscous liquid
Batch: E00275-350
Sample Expiration Date: end-2015
Purity:100%
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
The artificial soil was prepared in bulk by blending 70% sand, 20% kaolin clay, 10% sphagnum peat and 0.01% calcium carbonate (Appendix 3). The pH of the prepared bulk soil, stored dry at ambient temperature, was 5.7. The bulk artificial soil was
stored in a sealed container under ambient conditions until used to prepare the test soils.

Test soil was prepared by mixing the appropriate amount of test substance with solvent (THF), which was mixed with sand,
then dried to allow the solvent to evaporate.

The sand/test substance mixture was added to dry artificial soil as was sufficient reverse osmosis water to hydrate the soil.

Test soil components were mixed for a total of 20 minutes in order to achieve a homogeneous mixture.

Negative control soil was prepared in a similar manner as the treated soil but without the addition of test substance or
solvent.

Solvent control soil was prepared with THF but without test substance.

Seven-hundred-fifty grams of prepared soil were added to each of four test chambers for each of the treatment and control groups.

The test concentrations are reported as milligrams of test substance per kilogram of test soil on a dry weight basis (mg/kg).
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Earthworms (Eisenia fetida) for the test were from in-house cultures started with worms originally obtained from the
University of Maryland Wye Research and Education Center, Queenstown, Maryland.

Earthworms were cultured in a mixture of moist peat, fed saturated alfalfa, and held at a target temperature of 20 ± 5°C in
continuous light. Approximately 24 hours prior to the test, 320 adult worms (with clitellum) were selected and placed in a
container of prepared artificial soil substrate adjusted to a moisture content of approximately 34% by weight for the
acclimation period.

On the day of test initiation, the worms were rinsed briefly with water purified by reverse osmosis and indiscriminately
distributed by pairs into groups of ten worms each.

Each group of worms was weighed then placed on the soil surface in the appropriate test chamber. The worms were not
fed during testing.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Test temperature:
Soil temperature ranged from 20.7 to 21.5°C in the test groups at test initiation and ranged from 20.0 to 20.4°C at test
termination, within the desired range of 20 ± 2°C.
pH:
The soil pH ranged from 6.2 to 6.5 in the control and test soils at test initiation and ranged from 6.4 to 6.5 at test
termination.
Moisture:
Soil moisture content ranged from 32.8 to 33.3% at test initiation and from 31.3 to 32.3% at test termination, indicating that there was little change in soil moisture content during the test.
Details on test conditions:
Earthworms (Eisenia fetida) were exposed to a geometric series of five concentrations of EXP1200078 in soil. Nominal
concentrations of 62.5, 125, 250, 500 and 1000 mg/kg dry soil were selected in consultation with the sponsor.
A negative control group and a solvent control group were maintained concurrently in soil prepared without the addition of
EXP1200078. This route of administration was selected because it was representative of the natural exposure of
earthworms to chemicals.

Four replicate test chambers were maintained in each treatment and control group, with ten earthworms in each test chamber. Observations of mortality and clinical signs were conducted on Days 7 and 14.

The average weight of the live test organisms in each test chamber was determined at the beginning and end of the test.

The LC50 was determined by visual examination of the mortality data. The no observed-effect-concentration (NOEC) was determined by visual examination of the mortality and clinical observation data and statistical analysis of the body weight data.

At Wildlife International, reference toxicity tests with a reference toxicant, chloroacetamide, are periodically
conducted to assess the sensitivity of the test species and test procedures.
Nominal and measured concentrations:
Nominal test concentrations: Negative Control, Solvent Control, 62.5, 125, 250, 500 and 1000 mg
EXP1200078/kg dry soil

Reference substance (positive control):
yes
Remarks:
chloroacetamide
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Observations
The data from weekly observations of the earthworms for mortality and other signs of toxicity are presented in Table 2 (below). There were no mortalities in the negative control, solvent control or any of the treatment groups during the
14-day test. Because mortality was less than 50% in the highest concentration, the LC50 could not be statistically defined and was judged to be greater than the highest concentration tested. Earthworms in the negative and solvent control groups were normal in appearance and behavior throughout the test period. The 250 mg/kg dry soil treatment group contained one small
earthworm, the 500 mg/kg dry soil group had some earthworms with reduced reaction to mechanical stimulus on Day 7 and
one earthworm that was broken into two parts, presumably from an inadvertent injury that occurred during the removal of soil from the test chamber during observation procedures on Day 7. In the 1000 mg/kg dry soil group on Day 7, there were some thin earthworms and some worms with reduced reaction to mechanical stimulus. With the exception of the one small
earthworm in the 250 mg/kg group and the injured earthworm in the 500 mg/kg dry soil group, all earthworms appeared to be normal in appearance and behavior on Day 14. On Day 0, after earthworms were placed on the soil surface, some worms in
the 125, 500 and 1000 mg/kg dry soil groups were slow to burrow into the test soil, even after 1½ hours, indicating some
aversion to the test soils. On Day 7, after observations were completed and worms returned to the soil surface, all
earthworms burrowed, indicating that there was no aversion to the test soils after seven days.

Body Weights
Average individual body weights at test initiation and termination, and the change in body weight from test initiation to test termination, were calculated from the Day 0 and Day 14 replicate measurements (Table 3, below).
Statistical analyses were performed using SAS Version 8.2 statistical software (2). The negative and solvent control groups were compared statistically (T-Test of Means) and were not significantly different. Therefore the negative and solvent
groups were pooled for comparison with the test substance groups for loss in body weight using a Dunnett’s test (a = 0.05).
Data were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (a = 0.05). The change in body weight values (initial to final) were normally distributed and data analyzed had homogeneous variances.
The initial and final body weight values were normally distributed but not homogenous, however Dunnett’s test is robust with
respect to homogeneity therefore analyses were considered to be acceptable. No final body weight mean or change in body
weight (initial - final) mean in the treatment groups was statistically significant (p>0.05) when compared to the pooled control group mean. Therefore the NOEC was determined to be 1000 mg/kg dry soil, the highest concentration tested.

Results with reference substance (positive control):
The 14-day LC50 value for earthworms exposed to the reference substance, chloroacetamide, in an artificial soil substrate
was 28.7 mg a.i./kg dry soil with a 95% confidence interval of 15 and 60 mg a.i./kg dry soil.

The LC50 value of 28.7 mg a.i./kg dry soil falls within the range of 20 to 80 mg/kg specified in the ISO 11268-1 document.

Table 2

 

Cumulative Mortality and Observations of Earthworms

Exposed to EXP1200078 in an Artificial Soil Substrate

 

Concentration (mg/kg dry soil)

Rep.

Day 7

 

Day 14

Replicate Percent Mortality

Group Percent Mortality

Number Dead/Exposed

Effects1

Number Dead/Exposed

Effects1

Negative

Control

 

 

 

 

 

 

 

 

A

0/10

10 AN

0/10

10 AN

0

0

B

0/10

10 AN

0/10

10 AN

0

C

0/10

10 AN

0/10

10 AN

0

D

0/10

10 AN

0/10

10 AN

0

Solvent Control

A

0/10

10 AN

0/10

10 AN

0

0

B

0/10

10 AN

0/10

10 AN

0

C

0/10

10 AN

0/10

10 AN

0

D

0/10

10 AN

0/10

10 AN

0

62.5

A

0/10

10 AN

0/10

10 AN

0

0

B

0/10

10 AN

0/10

10 AN

0

C

0/10

10 AN

0/10

10 AN

0

D

0/10

10 AN

0/10

10 AN

0

125

A

0/10

10 AN

0/10

10 AN

0

0

B

0/10

10 AN

0/10

10 AN

0

C

0/10

10 AN

0/10

10 AN

0

D

0/10

10 AN

0/10

10 AN

0

250

A

0/10

9 AN, 1 S

0/10

9 AN, 1 S

0

0

B

0/10

10 AN

0/10

10 AN

0

C

0/10

10 AN

0/10

10 AN

0

D

0/10

10 AN

0/10

10 AN

0

500

A

0/10

10 AN

0/10

10 AN

0

0

B

0/10

10 AN

0/10

10 AN

0

C

0/10

10 AN

0/10

10 AN

0

D

0/10

5 AN, 4 R, 1 B2

0/10

9 AN, 1 B2

0

1000

A

0/10

7 AN, 3 T, 1 R

0/10

10 AN

0

0

B

0/10

8 AN, 2 R

0/10

10 AN

0

C

0/10

10 AN

0/10

10 AN

0

D

0/10

10 AN

0/10

10 AN

0

1Observed Effects:  AN = normal in appearance and behavior,  R = reduced reaction to mechanical stimulus, S = small, T = thin.

2B indicates broken worm, presumably damaged during removal of soil from test chamber during observation procedures on Day 7.

 


Table 3

 

Average Body Weights of Earthworms Exposed toEXP1200078 in an Artificial Soil Substrate

 

 

Concentration

(mg/kg dry soil)

 

 

Replicate

Average Earthworm Body Weights (g)1

Day 0

Day 142

Change in Body Weight2

Negative

Control

A

B

C

D

Mean ± Std. Dev.

0.50

0.47

0.45

0.50

0.48 ± 0.024

0.46

0.44

0.46

0.47

0.46 ± 0.013

-0.04

-0.03

+0.01

-0.03

-0.02 ± 0.022

Solvent

Control

A

B

C

D

Mean ± Std. Dev.

0.42

0.50

0.47

0.42

0.45 ± 0.039

0.42

0.45

0.44

0.40

0.43 ± 0.022

 0.00

-0.05

-0.03

-0.02

-0.03 ± 0.021

62.5

A

B

C

D

Mean ± Std. Dev.

0.49

0.43

0.46

0.48

0.47 ± 0.026

0.46

0.42

0.43

0.45

0.44 ± 0.018

-0.03

-0.01

-0.03

-0.03

-0.03 ± 0.010

125

A

B

C

D

Mean ± Std. Dev.

0.43

0.49

0.41

0.50

0.46 ± 0.044

0.41

0.45

0.39

0.47

0.43 ± 0.037

-0.02

-0.04

-0.02

-0.03

-0.03 ± 0.010

250

A

B

C

D

Mean ± Std. Dev.

0.44

0.45

0.42

0.43

0.44 ± 0.013

0.44

0.43

0.42

0.43

0.43 ± 0.008

 0.00

-0.02

 0.00

 0.00

-0.01 ± 0.010

500

A

B

C

D

Mean ± Std. Dev.

0.45

0.48

0.51

0.51

0.49 ± 0.029

0.45

0.44

0.49

0.47

0.46 ± 0.022

 0.00

-0.04

-0.02

-0.04

-0.03 ± 0.019

1000

A

B

C

D

Mean ± Std. Dev.

0.47

0.49

0.48

0.46

0.48 ± 0.013

0.43

0.47

0.46

0.45

0.45 ± 0.017

-0.04

-0.02

-0.02

-0.01

-0.02 ± 0.013

1Means and standard deviations were generated by SAS.  Manual calculations may differ slightly.

2No treatment group mean was statistically significantly different when compared to the respective pooled control group mean (Dunnett’s 2-tailed test,p>0.05).

 

 

 

Validity criteria fulfilled:
yes
Remarks:
The validity criterion for the test was met. Adult mortality of negative control earthworms was less than 10% at the end of the test. Performance of the positive controll (chloroacetamide) matched historical studies done in this laboratory.
Conclusions:
The 14-day LC50 estimation for earthworms exposed to EXP1200078 in an artificial soil substrate was determined to be
greater than 1000 mg/kg dry soil. The no-observed-effect-concentration was 1000 mg/kg dry soil, the highest concentration tested.
Executive summary:

The acute toxicity of the earthworm (Eisenia fetida) was determined in artificial soil containing weighed amounts of the test material. Total lenght of exposure was for 14 -days under controlled conditions. During that period of time, there was no effect on mortality or other measured parameters up to a maximum of 1000 mg/kg of dry soil.

Description of key information

The acute toxicity of the earthworm (Eisenia fetida) was determined in artificial soil containing weighed amounts of the test material. Total lenght of exposure was for 14 -days under controlled conditions. During that period of time, there was no effect on mortality or other measured parameters up to a maximum of 1000 mg/kg of dry soil. Study done according to OECD Guideline 207.

No long-term toxicity testing to soil macroorganisms is proposed, as in accordance to the Annex X, Paragraph 9.4, Column 2, exposure to soil is negligible and the CSA does not indicate the need to further investigate, with terrestrial RCRs <0.02 for all exposure scenarios.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
1 000 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
1 000 mg/kg soil dw

Additional information

The validity criterion for the test was met. Adult mortality of negative control earthworms was less than 10% at the end of the test. Performance of the positive control (chloroacetamide) matched historical studies done in this laboratory.