2-ethylhexyl benzoate

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2017 to 12 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil:
1 Pre-test: Prior to treatment the initial moisture content of the soil was determined as 20.47% and the volume required to achieve 35% moisture content was calculated as 162 mL reverse osmosis (RO) water/1100 g dry soil. The required water was added after the treated sand (10 g) had been mixed into a 1090 g dry weight equivalent of moist soil. The treated moist soil was mixed thoroughly using a hand held electric mixer.
2 Main study: Prior to treatment the initial moisture content of the soil was determined as 21.21% and the maximum water holding capacity (MWHC) as 59.33%. 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 190.25 mL water/2200 g dry ISO earthworm soil which was added after the test item in sand had been mixed into a 2180 g dry weight equivalent of moist soil. Treatment soil was mixed using a hand held electric mixer.

- Controls:
1 Pre-test: 162 mL RO water was mixed into a 1090 g dry weight equivalent of moist soil plus 10 g sand, to bring the soil to 35% moisture content.
2 Main study: 363.17 mL RO water was mixed into a 4162 g dry weight equivalent of moist soil to which 38 g dry sand had been added to bring the soil to 50% of the maximum water holding capacity.
Carbendazim solution (100 mL) was mixed into a 4162 g dry weight equivalent of moist soil to which 38 g dry sand had been added. An additional 263.17 mL RO water was also mixed in to achieve a soil concentration of 4.775 mg a.i./kg dry soil and 50% of the maximum water holding capacity.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earth worm
- Source: undisclosed supplier
- Age at test initiation; 2 months to 1 year (of reproductive maturity, i.e. production of egg cocoons or presence of a clitellum)
- Weight at test initiation : average per replicate 338-344 mg

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions (same as test or not): yes
- Health during acclimation (any mortality observed): no data

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

4 wk

Remarks:

adults during 4 weeks, followed by treatment of juveniles for 4 weeks

Test temperature:

18.1 – 22.0 ºC

pH:

day 1: 6.0-6.2
day 56: 6.5-6.9

Moisture:

day 1: adjusted to 50% of MWHC (59.33%)
day 56: group mean : 32.74 to 34.48% equivalent to 55.17 – 58.11% MWHC

Details on test conditions:

TEST SYSTEM
- Test container (material, size): plastic container 11 x 17 x 5 cm, each covered with a tight fitting lid
- Amount of soil or substrate: 500 g dry soil (648 g wet soil (50% MWHC)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per positive control (Carbendazim 4.77 mg/kg dw): 8

PROPERTIES OF ARTIFICIAL SOIL (OECD 207)
- % industrial quartz sand: 70
- % peat: 10
- % kaolin clay: 20
- Maximum water holding capacity (in % dry weigth): 59.33%
- pH: 6.4 --> adjusted to 6.0 with calcium carbonate

OTHER TEST CONDITIONS
- Photoperiod: 12/12 h
- Light intensity: 408 - 711 lux

EFFECT PARAMETERS MEASURED;
Adults: mortality on day 28; behavioural and pathological signs daily; body weight prior to treatment (individually) and on day 28 (per replicate)
Juveniles: survivors on day 56

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Range finding study: There was an adverse effect on survival at 14 days after treatment with 0% mortality at 62.5, 125 and 250 mg a.i./kg dry soil, 5% mortality at 500 mg a.i./kg dry soil and 30% mortality at 1000 mg a.i./kg dry soil. No mortality was observed in the water control group. There were no biologically significant treatment related differences in bodyweight of surviving earthworms between the treatment and control groups at 62.5, 125, 250 and 500 mg a.i./kg dry soil. A biologically significant treatment related effect was observed at 1000 mg a.i./kg dry soil

Nominal and measured concentrations:

16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil

Reference substance (positive control):

yes

Remarks:

Carbendazim 4.77 mg/kg dw

Duration:

4 wk

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

in adults

Duration:

4 wk

Dose descriptor:

NOEC

Effect conc.:

555.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

in adults

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

number of surviving juveniles

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

number of surviving juveniles

Details on results:

- Mortality at end of exposure period: Adult mortality of 0% was recorded at the 16.3, 29.4, 52.9, 95.3, 171.5, 308.6 and 555.6 mg a.i./kg dry soil treatment rates. Mortality of 32.5% was recorded at 1000 mg a.i./kg dry soil treatment rate (***p<0.001). This compared to 0% control mortality. Mortality of 10% was recorded in the positive control treatment.
- Total mass of adults at beginning of test: mean 338-344 mg
- Changes in body weight of live adults (% of initial weight) at end of exposure period: +94.7, +84.9, +95.3, +91.5, +102.1, +109.8, +106.1, +109.1 and +113.3% at 0, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil (positive control -33.3%)
- No. of offspring produced on day 56: 238, 256, 271, 256, 239, 259, 275, 239 and 185 at 0, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil (positive controll 0.25)

Results with reference substance (positive control):

Mortality: 10%
Bodyweight changes: - 33.3%
No of surviving juveniles: 0.25

Reported statistics and error estimates:

no statistically significant effects on adult mortality except at 1000 mg/kg dw (p<0.001 Fishers exact test)
statistically significant effects on adult bw at 171.5 (p = 0.047), 308.6 (p=0.047), 555.6 (p=0.027) and 1000 mg/kg dw (p=0.007, fishers exact test))
no statistically significant effects on number of juveniles

% Adult Mortality and Mean Bodyweights and Bodyweight Change (mg)

Treatment	(mg a.i./kg dry soil)	% Mortality	p-value	Day of study			(%) Change^(b)	p-value
				Day 0	Day 28	Day 28^(a)
Water control	0	0	-	341	664	664	+94.7	-
2-ethylhexyl benzoate	16.3	0	>0.999	344	636	632	+84.9	1.000
	29.4	0	>0.999	341	666	666	+95.3	1.000
	52.9	0	>0.999	341	653	652	+91.5	1.000
	95.3	0	>0.999	338	683	685	+102.1	0.399
	171.5	0	>0.999	338	709	711	+109.8	0.047*
	308.6	0	>0.999	343	707	705	+106.1	0.047*
	555.6	0	>0.999	341	713	713	+109.1	0.027*
	1000	32.5	<0.001***	339	723	724	+113.3	0.007**
Carbendazim	5	10	0.003**	339	226	227	-33.3	<0.001***

- Not applicable

(a) adjusted mean weights

(b) calculated from non-adjusted means

*p<0.05, **p<0.01, ***p<0.001 Fishers exact test

Mean Number of Juvenile Worms per Treatment Replicate

 

Treatment	(mg a.i./kg dry soil)	Day 56	pvalue
Water control	0	238	-
2-ethylhexyl benzoate	16.3	256	0.471
	29.4	271	0.060
	52.9	256	0.729
	95.3	239	1.000
	171.5	259	0.471
	308.6	275	0.212
	555.6	239	1.000
	1000	185	0.409
Carbendazim	5	0.25	<0.001***

Co-efficient of variation of the water control group = 5.85%

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

***p<0.001

Validity criteria fulfilled:

yes

Conclusions:

The adult LC50 was determined to be greater than 1000 mg a.i./kg dry soil, the highest rate tested. A treatment related NOEC of 555.6 mg a.i./kg dry soil was achieved.
The EC50 for the number of juveniles on Day 56 could not be determined because there was not a clear dose-response relationship. A NOEC of 1000 mg a.i./kg dry soil was achieved.

Executive summary:

In a test according to OECD 222 eight groups of four replicates (each 10 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 a.i./kg dry soil, mixed into the soil. A similar water control group with eight replicates was maintained in untreated soil to act as a negative control, and a positive control group, with eight replicates, was treated with carbendazim at ca.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.

Adult mortality of 0% was recorded at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6 and 555.6, mg a.i./kg dry soil treatment rates. Mortality of 32.5% was recorded at 1000 mg a.i./kg dry soil treatment rate (p<0.001), compared to 0% mortality in the water control group. Adult mortality of 10% was recorded in the positive control treatment.

The adult LC50 was determined to be greater than 1000 mg a.i./kg dry soil, the highest rate tested.The No Observed Effect Concentration (NOEC) for adult mortality was 555.6 mg a.i./kg dry soil.

There was no statistically significant adverse effect on biomass as a function of adult bodyweight at any test item treatment rate. There was however a statistically significant increase in bodyweights. The adjusted mean bodyweight in the positive control group was 227 mg, significantly lower than the water control group (p<0.001), with a reduction of 33.3% in adjusted mean bodyweights.

There was no statistically significant reduction in the number of juveniles produced at any test item treatment rate up to and including 1000 mg a.i./kg dry soil. The EC50 for the number of juveniles on Day 56 could not be determined because there was not a clear dose-response relationship. A NOEC (reproduction) of 1000 mg a.i./kg dry soil was achieved.

Description of key information

In a test according to OECD 222 eight groups of four replicates (each 10 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 a.i./kg dry soil, mixed into the soil. A similar water control group with eight replicates was maintained in untreated soil to act as a negative control, and a positive control group, with eight replicates, was treated with carbendazim at ca.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.

Adult mortality of 0% was recorded at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6 and 555.6, mg a.i./kg dry soil treatment rates. Mortality of 32.5% was recorded at 1000 mg a.i./kg dry soil treatment rate (p<0.001), compared to 0% mortality in the water control group. Adult mortality of 10% was recorded in the positive control treatment.

The adult LC50 was determined to be greater than 1000 mg a.i./kg dry soil, the highest rate tested.The No Observed Effect Concentration (NOEC) for adult mortality was 555.6 mg a.i./kg dry soil.

There was no statistically significant adverse effect on biomass as a function of adult bodyweight at any test item treatment rate. There was however a statistically significant increase in bodyweights.The adjusted mean bodyweight in the positive control group was 227 mg, significantly lower than the water control group (p<0.001), with a reduction of 33.3% in adjusted mean bodyweights.

There was non-statistically significant reduction in the number of juveniles produced at any test item treatment rate up to and including 1000 mg a.i./kg dry soil. The EC50 for the number of juveniles on Day 56 could not be determined because there was not a clear dose-response relationship. A NOEC (reproduction) of 1000 mg a.i./kg dry soil was achieved.

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:

555.6 mg/kg soil dw