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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Test Guideline 207

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
OECD GLP; UK GLP; EC Commission Directive 2004/10/EC

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
413-890-4
EC Name:
-
Cas Number:
82469-79-2
Molecular formula:
C28H50O8
IUPAC Name:
1,2,3-trihexyl 2-(butanoyloxy)propane-1,2,3-tricarboxylate
Constituent 2
Reference substance name:
1,2,3-propanetricarboxylic acid, 2-(butoxy), tri-n-hexyl est er
IUPAC Name:
1,2,3-propanetricarboxylic acid, 2-(butoxy), tri-n-hexyl est er
Details on test material:
Citroflex® B-6; n-Butyryltri-n-hexyl citrate (BTHC); CAS # 82469-79-2; Lot/Batch No. 0000085635; Purity 99.8%; Expiration Date 06 July 2009; Clear oily liquid; Storage conditions, room temperature in the dark.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Test concentrations were not corrected for purity of the test substance.

Sampling and analysis

Analytical monitoring:
no

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
Test soil was artificial OECD 207 soil (70% industrial quartz sand, 20% kaoline clay and 10% sphagnum peat). Calcium carbonate was added to adjust the pH to 6.0 ± 0.5. All concentrations tested refer to the material as supplied with no adjustment made for purity, and concentrations were prepared based on dry weight of soil. The concentrations used followed a standard range as recommended by the test guidelines. The test concentrations were formulated as stock solutions in acetone by direct dilution. Separate stock solutions were prepared for each treatment level. Stock solution concentrations were adjusted so that a constant volume (20 mL) was added to 200 g of bulk soil for each treatment level to prepare a premix which was subsequently added to an additional 2000 g of bulk soil for a total dry soil weight of 2200 g. Negative control soil was treated with acetone only. The acetone was evaporated overnight in a fume cupboard leaving the dry soil premixes ready to be mixed into the main soil bulk. On the day of treatment each premix was thoroughly mixed with the bulk soil by hand in a bowl prior to the addition of distilled water. Distilled water was gradually incorporated into the treated soil and the soil further mixed until a moisture content equivalent to approximately 35% of the dry weight was achieved. The soil in each test group was reweighed at the start of the study to ensure that the correct moisture content had been achieved.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Earthworms were obtained from Huntingdon Life Sciences stock. The earthworms were maintained in OECD 207 artificial soil in breeding boxes. Colonies were periodically fed with an oatmeal cereal as required and were kept moist with distilled water. Breeding boxes were stored in a temperature controlled laboratory (20°C ± 2°C) and were inspected daily.

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 body weight stratification procedure with the aid of a random number table. There were four replicates of ten earthworms per treatment and six treatment groups including the negative control group.

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Post exposure observation period:
None

Test conditions

Test temperature:
19.9°C to 20.6°C
pH:
6.0 ± 0.5
Moisture:
31% to 34% soil dry weight
Details on test conditions:
Exposures were conducted in appropriately labelled one-litre glass containers. The prepared soil (2200 g overall dry weight including 200 g premixes for each group) was divided approximately equally between four replicates per group. Earthworms in preweighed groups of ten were placed on the soil surface of each container. The containers were then covered with perforated polythene to retain moisture whilst providing a degree of ventilation. At termination of the study, the total bulk soil from each treatment groups was re-weighed to calculate the approximate moisture content.

Observations were conducted daily and any earthworms visible on the soil surface were evaluated for behavioural and pathological signs. 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 been no longer, 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 the front end. On Day 7, the medium was returned to the containers and surviving earthworms replaced on the surface.

Body weights of earthworms were recorded in all treatment and control replicates prior to treatment (Day 0) and on Day 14.

Nominal and measured concentrations:
0 mg/kg (control), 95, 171, 309, 556 and 1000 mg/kg dry soil weight (nominal)
Reference substance (positive control):
yes
Remarks:
Chloroacetamide

Results and discussion

Effect concentrationsopen allclose all
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
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:
A single mortality was observed in the control group at the Day 7 count. Body weights were unaffected by treatment.
Results with reference substance (positive control):
The LC50 values of Chloroacetamide to the earthworm, Eisenia foetida, were determined in a separate GLP compliant study and were as follows:

Day 7 LC50: 68.1 ppm (95% confidence limits 61.8 – 73.9 ppm)
Day 14 LC50: 64.9 ppm (95% confidence limits 58.4 – 72.5 ppm)

Results indicated that the laboratory system used for this type of study is valid.
Reported statistics and error estimates:
The lack of sufficient mortality precluded the calculation of an LC50. For body weight data Day 14 group means were adjusted using an analysis of covariance (ANCOVA) to normalise Day 14 means to Day 0 (baseline) data. Citroflex® B-6 treated groups were then compared to the negative control group using a two-tailed Williams’ test. There were no statistical differences in body weights between any of the treated groups and the control.

Any other information on results incl. tables

Cumulative Mortalities Following Treatment:

Group

Treatment

Nominal Treatment concentration (ppm)

No. of Earthworms

Day 7 Mortality  

Day 14 Mortality

Total Percent Mortality

1

Control

0

40

1

1

2.5

2

Citroflex® B-6

95

40

0

0

0

3

Citroflex® B-6

171

40

0

0

0

4

Citroflex® B-6

309

40

0

0

0

5

Citroflex® B-6

556

40

0

0

0

6

Citroflex® B-6

1000

40

0

0

0

Group Mean Body Weights (mg):

Group

Treatment

Nominal Treatment concentration (ppm)

Day 0  

Day 14

% Change

1

Control

0

460

450

-2.2

2

Citroflex® B-6

95

460

462

+0.4

3

Citroflex® B-6

171

460

449

-2.4

4

Citroflex® B-6

309

456

462

+1.3

5

Citroflex® B-6

556

460

470

+2.2

6

Citroflex® B-6

1000

459

450

-2.0

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
Under the conditions of this study, Citroflex® B-6 was of low acute toxicity to earthworms. The LC50 after 14 days was greater than 1000 ppm, the highest concentration tested.

The overall no observed effect concentration (NOEC) was considered to be ≥1000 ppm.