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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
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:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
April 2004
Deviations:
yes
Remarks:
The weight of one worm and the test temperature (once) were slightly above the recommended values. These deviations had no impact on the integrity and quality of the study.
Qualifier:
according to
Guideline:
other: EU Method C.33 (Earthworm Reproduction Test (Eisenia fetida/ Eisenia andrei))
Version / remarks:
Commission Regulation (EC) No. 440/2008, amended by Commission Regulation (EC) No 2016/266
Deviations:
yes
Remarks:
The weight of one worm and the test temperature (once) were slightly above the recommended values. These deviations had no impact on the integrity and quality of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: not applicable (UVCB substance)
Analytical monitoring:
not required
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of mixing into soil: Application solutions for the different test concentrations were prepared by dispersing the test material in dichloromethane. For each test replicate, 10 mL of the respective application solution was applied on the surface of 50 g of quartz sand. After evaporating the solvent for 4 hours, the test material - quartz sand mixture was thoroughly mixed with an adequate amount of test soil.
- Controls: solvent control (without test material); untreated control (without solvent and test material)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dichloromethane (CH2Cl2)
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: in-house culture
- Age at test initiation (mean and range, SD): 9 months (difference between the worms maximum four weeks); synchronized adults with a visible clitellum.
- Weight at test initiation: mean 426 mg per earthworm, range 300-605 mg (one individual worm in the 320 mg/kg treatment group was slightly above the weight range required by the guideline. However, this had no impact on the integrity and quality of the study).

All earthworms came from the laboratory in-house culture (started with worms from Blades Biological Ltd, United Kingdom). The earthworms were not treated against any diseases before and after study initiation. Care was taken to minimise stress during holding and handling. Earthworms were kept in a 1:1 mixture of garden soil and horse manure before testing.

ACCLIMATION
- Acclimation period: five days before study initiation
- Acclimation conditions (same as test or not): same. During acclimatization the worms were fed with moistened and finely ground horse manure ad libitum to avoid starvation and to minimize weight loss and stress.

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Remarks:
adults worms were removed after 28 days and the juvenile worms were exposed for another 4 weeks
Test temperature:
19.4 – 23.2 °C with a mean temperature of 20.5 °C (continuous recording of the room temperature using a data logger). For one short period, the temperature was above the upper limit of the recommended range of 18 – 22 °C. This deviation is not considered to have had an impact on the outcome of the study since the performance of the control test organisms was normal.
pH:
Day 0: 5.5 – 5.7
Day 56: 5.9 – 6.0
Moisture:
Test initiation: 34 – 35%, corresponding to 58 – 60% of the MWHC.
Test termination: 34 – 37%, corresponding to 59 – 63% of the MWHC.

The water content of the test soils at test end did not vary by more than 10% from that at the start of the test, as recommended by the guidelines.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): plastic containers measuring 17 x 12 x 11.8 cm covered with perforated lids to enable air exchange and minimize evaporation from the artificial soil
- Amount of soil or substrate: approx. 500 g (dry weight)
- No. of organisms per container (treatment): 10 (allocated randomly)
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate):
Fine quartz sand: 70%
Kaolin clay: 20% (kaolinite content >30%)
Sphagnum peat: 10% (air dried and finely ground, with no visible plant remains)
- Maximum water holding capacity (in % dry weigth): 58%
- Storage (condition, duration): in a cool area in plastic container

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light: 8-hour dark
- Light intensity: 530 – 640 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality and growth (body weight) of adult worms after 4 weeks of exposure
- Number of offspring (juveniles and cocoons) after a further 4 weeks (effects on reproduction)

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 1, 10, 100 and 1000 mg/kg dry soil and control
- Results used to determine the conditions for the definitive study: no effects on adult earthworm survival and bodyweight; 11 – 25% inhibition of reproduction.
Nominal and measured concentrations:
nominal: 1000, 320, 100, 32, and 10 mg/kg soil (dry weight)
Reference substance (positive control):
yes
Remarks:
carbendazim at 0.5, 1.0, 1.9, 3.8, and 7.7 mg carbendazim/kg soil (dry weight)
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
adult body weight
Duration:
28 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:
Mortality at end of exposure period: no mortalities or sub-lethal effects were observed
- Total mass of adults at beginning of test: see Table 1
- Changes in body weight of live adults (% of initial weight) at end of exposure period: see Table 1
- No. of offspring produced: see Table 2
- No. of unhatched cocoons: see Table 3
- Morphological abnormalities: none
- Behavioural abnormalities: none
- Other biological observations: food consumption at 32 mg/kg dry soil was slightly less than in the other test item treatments and the controls. However, this did not result in statistically significantly reduced body weight change (Table 1) or number of juveniles (Table 2).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels:
Endpoint: reproduction
NOEC: 1.0 mg a.i./kg dry soil
LOEC: 1.9 mg a.i./kg dry soil
EC10: 0.6 mg a.i./kg dry soil (95% confidence limits 0.32-0.71)
EC20: 0.7 mg a.i./kg dry soil (95% confidence limits 0.49-0.87)
EC50: 1.2 mg a.i./kg dry soil (95% confidence limits 0.98-1.4)
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional, Version 3.2, ® ToxRat Solutions GmbH. The LC10, LC20 and LC50 could not be calculated due to no mortality up to and including the highest test concentration and were derived directly from the raw data. For reproduction, differences between the solvent control and the control were tested using the Student t-test (one-sided smaller, α = 0.05). Since mean number of juveniles in the control and the solvent control did not differ statistically significantly, differences between the test item treatments and the solvent control were tested using the Dunnett t-test (one-sided smaller, α = 0.05). The EC10, EC20 and EC50 could not be calculated due to missing statistically significant differences between the solvent control and the test item treatments up to and including the highest test concentration and were derived directly from the raw data.
Validity criteria fulfilled:
yes
Remarks:
Mean adult mortality over the initial four weeks was ≤ 10 %. The mean number of juveniles per test unit was ≥ 30 at test termination. The coefficient of variation calculated for the number of juveniles was ≤ 30 % at test termination.
Conclusions:
In a valid, reliable and conclusive study, the effects of the test item BYK-LP N 06649 on the earthworm species Eisenia fetida (Annelida: Lumbricidae) were tested according to the OECD testing guideline No. 222 (2016) and Commission Regulation (EU) No 2016/266 (2015). The tested concentrations were 10, 32, 100, 320 and 1000 mg/kg dry soil, endpoints were mortality and growth after 28 days of exposure of the adults and reproduction after additional 28 days. The test item BYK-LP N 06649 had no detrimental effects on any of the endpoints up to and including the highest tested concentration of 1000 mg/kg dry soil under the conditions of this test.

Description of key information

Toxicity to soil macroorganisms: not toxic to Eisenia fetida: NOEC (reproduction and growth) ≥1000 mg/kg soil dry weight (OECD 222, EU C.33)

Key value for chemical safety assessment

Additional information

No effects were observed on adult mortality, growth and reproduction up to the highest tested concentration (1000 mg/kg dw). As it could be concluded that the test material does not have adverse effects towards soil organisms at the highest recommended concentration in laboratory tests, no NOEC and thus no PNEC could be derived.