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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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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:
From 07 March 2022 to 15 August 2022
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))
Version / remarks:
2016
Deviations:
yes
Remarks:
Food was provided on day 0 instead of day 1. At test end, soil moisture deviated by more than 10 % from the initial value in all treatments. Temperature decreased to min. 17 °C for in total about 32 h. These deviations are considered to have no impact.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil: The respective test item amount was weighed out for each test item concentration and mixed thoroughly with quartz sand (10 g per replicate). The spiked quartz sand was given to the artificial soil (2360 g SDW). Demineralised water (300 g) was added to adjust the humidity of the artificial soil to a moisture of 54% of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 600 g soil dry weight was filled into each test vessel.
- Controls: Artificial soil moistened with demineralised water without test or reference item was used as control medium
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: Breeding stock culture maintained at the test facility
- Breeding: Organisms of the species Eisenia fetida are bred at the test facility in covered plastic vessels containing potting compost.
- Synchronisation of the earthworm population: Earthworms of homogeneous age and body weight were used in the definitive test. Synchronisation of the population was achieved by placing adult earthworms into breeding boxes and removing the adults after 4 weeks. Offspring from the remaining cocoons reached the adult age after 2 months at the earliest.
- Age at test initiation: Adult earthworms (with clitellum) aged 2 -12 months - Weight at test initiation: 0.38 to 0.57 g (wet mass)
- Feeding during breeding: Earthworms were fed with a litter of dried stinging nettle leaves and porridge oats. A sufficientamount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.
- Homogeneity of groups: The worms were washed with demineralised water and dried gently on a paper towel. Thehomogeneity of the population was checked by weighing the earthworms. After confirmation of homogeneity, groups of 10 earthworms (chosen by a randomised procedure) were weighed and introduced into each test vessel.

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions: Earthworms were acclimatised to the artificial soil to be used for the test (moisture content 54 % of the water holding capacity)
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Test temperature:
17 - 22 °C
pH:
6.14 - 6.61
Moisture:
23.9 - 31.4 % of dry weight
Details on test conditions:
TEST SYSTEM
- Test container: Round plastic boxes with an inner diameter of 15.0 cm corresponding to a bottom surface area of 177 cm2 and a height of 14 cm. Transparent and perforated lids enabled sufficient gas exchange, light input and prevented the test substrate from drying.
- Amount of soil or substrate: 600 g soil (dry weight)
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE
- Composition of artificial soil:
10 % peat, air-dried and finely ground
20 % kaolin, clay, kaolinite content > 30 %
69 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
0.30 % calcium carbonate (CaCO3) to achieve a pH of 6.0 ± 0.5
- Maximum water holding capacity: 45.0 (g/100 g dw)

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 529 ± 102 lx

EFFECT PARAMETERS MEASURED: After the first 4 weeks of the test, mortality and biomass of the adult worms were determined. After further 4 weeks the reproduction rate was determined by counting the hatched juveniles.

TEST CONCENTRATIONS
- Range finding study: A preliminary range finding test was performed with 4 test item concentrations each containing 2 replicates with 10 earthworms each.
- Test concentrations in range finding study: control, 1, 10, 100, 1000 mg/kg soil dry weight
- Results used to determine the conditions for the definitive study: No mortality was observed in neither test concentration in the range findings study. The highest change of live weight was observed in test concentration 1 mg/kg soil dry weight with -15 % (decrease of live weight).
Nominal and measured concentrations:
Nominal concentrations: 31.25, 62.5, 125, 250, 500, 1000 mg test item/kg soil dry weight (SDW).
Reference substance (positive control):
yes
Remarks:
Carbendazim; Test concentrations 0.5 - 1 - 2 - 4 mg a.i./kg SDW; According to guideline, significant effects should be between 1 and 5 mg a.i./kg SDW.
Key result
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
202 mg/kg soil dw
95% CI:
134 - 368
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
other: EC20
Effect conc.:
454 mg/kg soil dw
95% CI:
298 - 791
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
biomass
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
biomass
Duration:
8 wk
Dose descriptor:
LOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality at end of exposure period: Earthworm mortalities < 10 %
- Total mass of adults at beginning of test: 36.3 g in control, 18.1 - 18.2 g in each test vessel.
- Changes in body weight of live adults at end of exposure period: There were no statistically significant differences in earthworm biomass at any test item concentration compared to the control.
- No. of mean offspring produced: 84 - 122
- Morphological abnormalities: No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and all test item concentrations on day 28.
Results with reference substance (positive control):
- Results with reference substance valid? yes
According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg SDW.
- Significant effects levels: 0.5, 1, 2, 4 mg a.i./kg soil dry weight
Reported statistics and error estimates:
see "attachments"

Validity criteria:


- Adult mortality in the control group after the first four weeks of the definitive test should be ≤ 10 %; mortality in actual test: 0 %


- Number of juveniles per replicate in the control group should be ≥ 30; number of juvenils in actual test: 96 - 158


- Coefficient of variation calculated for the reproduction of the control group should be ≤ 30 %, coefficient in actual test: 15.5 %

Validity criteria fulfilled:
yes
Conclusions:
Following long-term toxicity effects were determined after 8 weeks:
LOEC (mortality, biomass) > 1000 mg/kg soil dry weight
NOEC (mortality, biomass) ≥ 1000 mg/kg soil dry weight
LOEC (reproduction) = 500 mg/kg soil dry weight
NOEC (reproduction) = 250 mg/kg soil dry weight
EC10, EC20, EC50 (reproduction): 202, 454 and > 1000 mg/kg soil dry weight, respectively.

Executive summary:

The long-term effects of Butyl methoxydibenzoyl methane on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016). The study was conducted under static conditions over 8 weeks with the test item concentrations 31.25, 62.5, 125, 250, 500, 1000 mg test item/kg soil dry weight (SDW). A control was tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control replicates and 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). The inserted earthworms had an individual body weight between 0.38 and 0.57 g at experimental starting. Neither evident earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and all test item concentrations after four weeks of exposure. There were no statistically significant differences in earthworm biomass at any test item concentration compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 124 in the control and ranged from 84 to 122 in the test item treatments. No statistically significant differences in earthworm reproduction were determined in the test item concentrations 31.25 to 250 mg/kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg/kg SDW the earthworm reproduction was statistically significantly reduced. The following effect levels were determined (mg/kg soil dry weight):


LOEC (mortality, biomass): > 1000


NOEC (mortality, biomass): ≥ 1000


LOEC (reproduction) = 500


NOEC (reproduction) = 250


EC10, EC20, EC50 (reproduction): 202, 454 and > 1000, respectively.

Description of key information

Following long-term toxicity effects to Eisenia fetida were determined after 8 weeks:
LOEC (mortality, biomass)> 1000 mg/kg soil dry weight
NOEC (mortality, biomass) ≥ 1000 mg/kg soil dry weight
LOEC (reproduction) = 500 mg/kg soil dry weight
NOEC (reproduction) = 250 mg/kg soil dry weight
EC10, EC20, EC50 (reproduction): 202, 454 and > 1000 mg/kg soil dry weight, respectively.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
202 mg/kg soil dw

Additional information

The long-term effects of the test item on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016). The study was conducted under static conditions over 8 weeks with the test item concentrations 31.25, 62.5, 125, 250, 500, 1000 mg test item/kg soil dry weight (SDW). A control was tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control replicates and 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). The inserted earthworms had an individual body weight between 0.38 and 0.57 g at experimental starting. Neither evident earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and all test item concentrations after four weeks of exposure. There were no statistically significant differences in earthworm biomass at any test item concentration compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 124 in the control and ranged from 84 to 122 in the test item treatments. No statistically significant differences in earthworm reproduction were determined in the test item concentrations 31.25 to 250 mg/kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg/kg SDW the earthworm reproduction was statistically significantly reduced. The following effect levels were determined (mg/kg soil dry weight):


LOEC (mortality, biomass): > 1000


NOEC (mortality, biomass): ≥ 1000


LOEC (reproduction) = 500


NOEC (reproduction) = 250


EC10, EC20, EC50 (reproduction): 202, 454 and > 1000, respectively.