5-bromo-1,3-dichloro-2-fluorobenzene

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Remarks:

The study was conducted to meet the national regulatory requirements in a non-EEA country.

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

A stock solution was prepared by dissolving 5250.4 mg of test substance in 25 mL acetone. A sequential 1:2 dilution series was prepared, starting with the 210.0 mg/mL stock solution by adding 12 mL of acetone to 12 mL of the stock solution or dilution. Then 10 mL of the corresponding dilutions were added to 20 g fine quartz sand in two steps of 5 mL. After the first half of the volume was added the treated sand was left for 90 minutes in a fume hood until the solvent had evaporated. Then, the second volume of 5 mL was added and the treated sand was left for an additional 90 minutes in a fume hood until the solvent had evaporated and then was mixed with a spoon. Then artificial soil equivalent to 2080 g dry weight was added to test item blended sand, to get to the nominal test concentrations.
The control was treated with the same amount of sand as the groups exposed to the test substance. The solvent control group was treated with the same amount of acetone and sand as the groups exposed to the test substance.
While mixing the artificial soil in a laboratory mixer for five minutes the soil of each treatment group was moistened with deionised water to achieve the required water content. Each group was treated in one batch and then split into 4 replicates.
The application order was 1. control, 2. solvent control, 3. test substance groups from low to high concentrations.

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

Life stage: adults
Age: 6 to 7 months, with clitellum
Body weight: 300 to 600 mg
Origin: cultured under standardised conditions at the testing facility in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature
Acclimatisation: 1 day, in artificial soil, under test conditions

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Post exposure observation period:

Not applicable

Test temperature:

18 to 22 °C

pH:

Start of test: 5.8 to 5.9
End of test: 5.7 to 6.0

Moisture:

Start of test: 24.6 to 27.8% (47.2 to 53.5% of the maximum water holding capacity)
End of test: 26.8 to 30.0% (51.6 to 57.7% of the maximum water holding capacity)

Details on test conditions:

Replicates: 4 per treatment group
Ten individuals were used per test unit, i.e. 40 earthworms per treatment group. The individuals were washed and then dried with a dry paper towel, weighed in groups of 10 earthworms and then placed on the surface of the artificial soil.

Test environment: controlled environment room in a ventilated area
Light regime: continuous illumination with intensity in the range from 400 to 800 lux
Test units: glass jars (1 L), loosely covered by glass lids, filled with 635.1 g of artificial soil
Artificial soil (prepared according to OECD TG 207): 10% sphagnum peat, air-dried and finely ground, with no visible plant remains (< 2 mm), 20% Kaolin clay (kaolinite content >30%), 69.7% fine quartz sand (>50% by mass with particle size in the range from 0.05 to 0.2 mm), 0.3% calcium carbonate
The artificial soil had a maximum water holding capacity of 52% of the dry weight.

Nominal and measured concentrations:

0 (control, solvent control groups), 62.5, 125, 250, 500 and 1000 mg/kg soil

Reference substance (positive control):

yes

Remarks:

2-chloroacetamide

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

245.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

No significant mortality was observed at test concentrations up to and including 125 mg/kg soil dw at day 7 or 14 when compared to the pooled control. The lowest observed effect concentration (LOEC) was 250 mg/kg soil dw. The mean mortality at 250 mg/kg soil dw was 52.5% after 7 days and 55.0% after 14 days. All earthworms were found dead after 7 days at test concentrations of 500 and 1000 mg/kg soil dw.
The body weight changes of the earthworms were not statistically significantly different compared to the pooled control up to and including the concentration of 125 mg/kg soil dw. At the concentration of 250 mg/kg soil dw body weight changes were statistically significant.
At 500 and 1000 mg/kg soil dw worms did not burry into the soil on the first day of the experiment after 1 hour. At the concentration of 250 mg/kg soil dw worms showed less signs of agitation and were very thin on days 7 and 14.

Results with reference substance (positive control):

The LC50 of 2-Chloroacetamide is determined at least once a year as a means of assuring that the laboratory test conditions are adequate and have not changed significantly over time. The most recent test was performed in August/September 2018. The test resulted in a LC50 of 30.1 mg/kg soil dw over 14 days, and the NOEC for body weight changes was 32.0 mg/kg soil dw.

Table 1: pH and water content of the artificial soil

Treatment group	pH at start	pH after 14 days	% water at start	% water after 14 days	% of WHC	% of WHC
Control	5.8	5.8	27.0	27.3	52.0	52.4
Solvent control	5.9	5.7	27.3	28.0	52.6	53.9
62.5 mg/kg	5.9	5.8	27.8	26.8	53.5	51.6
125 mg/kg	5.9	5.8	27.6	27.7	53.1	53.2
250 mg/kg	5.9	5.9	24.6	30.0	47.2	57.7
500 mg/kg	5.8	5.9	26.1	28.2	50.2	54.3
1000 mg/kg	5.8	6.0	25.7	28.6	49.4	54.9
Min	5.8	5.7	24.6	26.8	47.2	51.6
Max	5.9	6.0	27.8	30.0	53.5	57.7

WHC: maximum water holding capacity of artificial soil, which was 52% of dry weight

Table 2: Mortality of earthworms after 14 days

Treatment group	Mean mortality after 7 days	Standard deviation	Statistical significance	Mean mortality after 14 days	Standard deviation	Statistical significance
Control	0.0	0.0	-	0.0	0.0	-
Solvent control	0.0	0.0	Not significant	0.0	0.0	Not significant
Pooled control	0.0	0.0	-	0.0	0.0	-
62.5 mg/kg	0.0	0.0	Not significant	0.0	0.0	Not significant
125 mg/kg	0.0	0.0	Not significant	0.0	0.0	Not significant
250 mg/kg	52.5	49.9	*	55.0	52.6	*
500 mg/kg	100	0.0	*	100	0.0	*
1000 mg/kg	100	0.0	*	100	0.0	*

* significantly different compared to pooled control, Fisher’s Exact Test, α = 0.05, one-sided greater

Table 3: Body weight changes after 14 days

Treatment group	Mean at test start (mg/worm)	Mean after 14 days (mg/worm)	Difference in %	Standard deviation	Statistical significance
Control	411.7	387.0	-5.9	6.6	-
Solvent control	413.2	.93.4	-4.5	7.5	Not significant
Pooled control	412.5	390.2	-5.2	6.6	-
62.5 mg/kg	399.6	383.8	-3.7	4.4	Not significant
125 mg/kg	349.9	399.0	1.1	2.9	Not significant
250 mg/kg	395.1	297.7	-25.8	10.6	*
500 mg/kg	386.3	-	-	-	-
1000 mg/kg	388.5	-	-	-	-

* significantly different compared to the pooled control, Williams t-test, α = 0.05, one-sided smaller

Validity criteria fulfilled:

yes

Conclusions:

In this 14-day study on the short-term toxicity of the substance to earthworms, the LC50 was found to be 245.4 mg/kg soil dw.

Executive summary:

The short-term effects of the substance on the mortality, behaviour and body weight of earthworms (Eisenia andrei) were investigated over a period of 14 days under GLP in a laboratory study performed to OECD TG 207. The nominal test concentrations were 62.5, 125, 250, 500 and 1000 mg/kg soil dw. The artificial soil was prepared in accordance with the OECD guidance. The test material was dissolved in acetone and then incorporated into the soil. An untreated control (test with artificial soil under same conditions) and a solvent control (test with artificial soil and solvent) were included. All test groups were done with four replicates including ten adult earthworms. Assessments were done to study the effects of the substance on mortality, behaviour and body weight over the course of the study. The earthworms in the groups treated with 500 and 1000 mg/kg soil dw showed atypical behaviour, e.g. they did not burry into the soil in the first hour after placement on the treated soil. At the concentration of 250 mg/kg soil dw worms showed less signs of agitation on days 7 and 14. Statistically significant mortality was seen in the test groups exposed to 250, 500 and 1000 mg/kg soil dw, and all earthworms were dead after seven days at the two highest doses. The 14-day LC50 value in this study was 245.4 mg/kg soil dw. The body weight of worms in the group treated with 250 mg/kg soil dw was significantly lower than that of the controls after 14 days. Overall, the NOEC for adverse effects in the study on short-term toxicity to earthworms was 125 mg/kg soil dw over the study period of 14 days.

Description of key information

14-day LC50 = 245.4 mg/kg soil dw in earthworm - Eisenia andrei (GLP, OECD TG 207)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

245.4 mg/kg soil dw