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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 01, 2022 - April 29, 2022
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:
adopted April 4, 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 11268-1 (Effects of Pollutants on Earthworms. 1. Determination of Acute Toxicity Using Artificial Soil Substrate)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): Prior to weighing, the test item was ground with a tube mill. 2100.1 mg of the test substance was weighed for the limit concentration using an analytical balance and 10 g fine quartz sand was added. After mixing with a spoon to reach a homogeneous distribution of the test item within the sand the mixture was added to artificial soil equivalent to 2090 g dry weight, resulting in the following nominal concentration of the test item: 1000 mg test item/kg soil dry weight. There were no significant deviations from the target concentration (< 5%). The control was treated with the same amount of untreated sand per g substrate as the test item groups. While mixing the artificial soil in a laboratory mixer for approximately 5 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.
Test organisms (species):
Eisenia andrei
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworms Eisenia andrei
- Origin: Cultured under standardised conditions in ibacon laboratories in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature.
- Age at test initiation (mean and range, SD): Adult of approximately 10 to 11 months old, with clitellum.
- Weight at test initiation (mean and range, SD): Weight range 300 to 600 mg.
- Feeding: None.

ACCLIMATION
- Acclimation period: 1 day, in artificial soil and in the dark, under test conditions.
- Acclimation conditions (same as test or not): Same as test.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Remarks:
None
Post exposure observation period:
Not applicable
Test temperature:
Temperature maintained between 18°C and 22°C
pH:
pH was checked at the start and end of the main test in one sample per treatment group, according to ISO 10390 (CaCl2).
- pH range at experimental start: 5.8 to 5.9
- pH range at experimental end: 5.7 (see Table 6.3.1/1 in "Any other information on results incl. tables").
Moisture:
Water content was checked at the start and end of the experiment of one sample per treatment group, according to ISO 11465.
- At experimental start: 22.4% to 22.7% (53.2% to 53.9% of the maximum water holding capacity).
- At experimental end: 22.3% to 22.4% (53.0% to 53.3% of the maximum water holding capacity, see Table 6.3.1/1 in "Any other information on results incl. tables").
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Glass jars (1 L), loosely covered by glass-lids to enable exchange of air and to minimise evaporation, filled with 608.3 ± 1 g of the prepared soil (500 g dry weight plus deionised water).
- No. of organisms per container (treatment): 10 per test unit, i.e. 40 earthworms per treatment group
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: not applicable
- Introduction of Individuals: Washed, dried with a dry paper towel, weighed in groups of 10 earthworms and then placed on the surface of the treated or untreated (control) artificial soil.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Artificial soil: According to OECD 207.
• 10% Sphagnum-peat, as close to pH 5.5 to 6.0 as possible, air- dried and finely ground (<2 mm, with no visible plant remains) (Floragard, Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany)
• 20% Kaolin clay (Kaolinite content >30%) – (Erbslöh, 65558 Lohrheim, Germany)
• 69.6% fine quartz-sand (more than 50% by mass of particle size 0.05 mm to 0.2 mm) (Quarzwerke Frechen, Postfach 1780, 50207 Frechen, Germany)
• 0.4% Calcium carbonate (CaCO3) was added to adjust pH to 6.0 ± 0.5 (Merck, 64293 Darmstadt, Germany).
- Maximum water holding capacity (in % dry weigth): 42%

OTHER TEST CONDITIONS
- Test environment: Controlled environment room, with ventilated area
- Temperature: Maintained within the range of 18°C and 22°C
- Photoperiod: Continuous illumination
- Light intensity: Maintained within the range of 400 and 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Assessment of worm mortality: On day 7 and 14 after application the artificial soil was emptied from the jars onto a tray and the number of live and dead earthworms in each replicate was assessed. Missing earthworms and earthworms failing to respond to gentle stimulation were considered as dead. Living earthworms and the soil were placed back into the test units after the assessment on day 7.
- Behavioural abnormalities: Number of affected earthworms (e.g. not burrowing into the soil, lack of movement, rigidity, etc.) at day 0 (after introduction), day 7 and 14.
- Mean body weights: Per test container determined at experimental start (day 0) and 14 days after application, using the same washing and weighing procedures as at test initiation.

VEHICLE CONTROL PERFORMED: not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable (limit test)
- Range finding study: A non-GLP range finding test was performed with the following test concentrations: Control, 1.0, 10, 100, 500 and 1000 mg test item/kg soil dry weight.
- Results used to determine the conditions for the definitive study: No mortalities were observed at all treatment groups. Therefore, a limit test concentration at 1000 mg test item/kg soil dry weight was used for the definitive study. See table 7 of the attached study report for further details.
Nominal and measured concentrations:
Nominal concentration: 1000 mg test item/kg soil dry weight.
No analytical monitoring during the test.
Reference substance (positive control):
yes
Remarks:
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 January 2022 to February 2022.
Key result
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
Remarks on result:
other: Estimated value
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
Remarks:
and biomass
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
and biomass
Details on results:
- Mortality results: No mortality was observed at the single dose of 1000 mg test item/kg soil dry weight after 7 and 14 days exposure, which was not significantly different compared to the control (Fisher Exact Test, α = 0.05, one-sided greater). See Table 6.3.1/2 in "Any other information on results incl. tables".
- Changes in body weight of live adults (% of initial weight) at end of exposure period: At the single dose of 1000 mg test item/kg soil dry weight, the decrease of body weight of -11.5% was calculated to be statistically significantly different compared to the control (Student t-test, α = 0.05, two-sided). However, as this level of body weight changes is considered to be within the accepted biological variability of 20% (as the accepted validity criteria for body weight changes of the control is 20%, according to ISO 11268-1), the body weight changes in the test item treated group was not considered to be an adverse effect. See Table 6.3.1/3 in "Any other information on results incl. tables".
- Behavioural abnormalities: No behavioural effects were observed in any treatment group.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: In the most recent test with the reference item 2-Chloroacetamide (performed from January 2022 to February 2022 under ibacon study number 126166021) the LC50 after 14 days was determined to be 35.8 mg test item/kg soil (95% confidence limits: 34.5 to 37.1 mg test item/kg soil).
Reported statistics and error estimates:
A LC50 at days 7 and 14 was estimated, as no mortality was observed at the limit dose. Mortality data were analysed for significance by using Fisher’s Exact Binomial Test (two sample comparison, α = 0.05, one-sided greater).
Data on earthworm body weight changes were tested for normal distribution and homogeneity of variance using the Shapiro-Wilk ́s test and Levene ́s test (α = 0.01), respectively.
The data for body weight change were normally distributed and homogeneous, so the Student t-test (for homogeneous variances) was used for comparison of the treated and control group (two-sample comparison, two-sided, α = 0.05).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat® Solutions GmbH.

Table 6.3.1/1: pH and water content fo artificial soil

































































Treatment
Group



pH



% Water Content1



% of WHC1,2



Start



14 days



Start



14 days



Start



14 days



Control



5.9



5.7



22.7



22.4



53.9



53.3



1000



5.8



5.7



22.4



22.3



53.2



53.0



Minimum:   



5.8



5.7



22.4



22.3



53.2



53.0



Maximum:   



5.9



5.7



22.7



22.4



53.9



53.3



Test item dosage is  given as mg test item/kg artificial soil dry weight



1  The results represent rounded values calculated on the exact raw data



2 WHC = maximum water holding capacity; WHCmax was 42%



 


Table 6.3.1/2: Earthworm mortality





























































Treatment
Group



Mortality after 7 days



Mortality after 14 days



Mean Number of dead Earthworms



Mean
[%]


  

Mean Number of dead Earthworms



Mean
[%]


  

SD



Signifi-cance



SD



Signifi-cance



Control



0



0.0



0.0



-



0



0.0



0.0



-



1000



0



0.0



0.0



n.s.



0



0.0



0.0



n.s.



Test item dosage is given as mg test item/kg artificial soil dry weight



The results represent rounded values calculated from the exact raw data



 - Not applicable



n.s. = Not significantly different compared to the control, Fisher´s Exact Test, α = 0.05, one-sided greater



 


Table 6.3.1/3: Earthworm body weight changes


























































Treatment
Group



Test Start



After 14 days



 



Mean



Mean



Difference



Difference



Standard


 

[mg/worm]



[mg/worm]



[mg/worm]



%



Deviation



Significance



Control



446.1



437.5



8.7



-1.8



3.6



-



1000



420.8



372.3



48.5



-11.5



3.0



*



Test item dosage is given as mg test item/kg artificial soil dry weight



 - Not applicable



The results represent rounded values calculated from the exact raw data (% mean of 4 replicates, see Table 6 of the attached study report)



 * = significantly different compared to the control, Student t-test, α = 0.05, two-sided, but not considered to be an adverse effect


Validity criteria fulfilled:
yes
Remarks:
Control mortality was 0% at day 14 (validity criterion was met as should not exceed 10%); Control mean loss of biomass was 1.8% at day 14 (validity criterion was met as should not exceed 20%).
Conclusions:
In a 14-day acute toxicity study with the test substance to earthworms (Eisenia andrei) the 14-day LC50 was estimated to be >1000 mg test item/kg soil dry weight. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) related to mortality and biomass were determined to be ≥1000 mg test item/kg soil dry weight and >1000 mg test item/kg soil dry weight, respectively. The study is considered to represent worst case laboratory conditions.
Executive summary:

This study was performed to determine the acute toxicity of the test substance to the earthworm Eisenia andrei after 7 and 14 days of exposure in an artificial soil, according to OECD TG 207 and ISO 11268-1 with GLP compliance.


Following a preliminary non-GLP range finding test, earthworms were exposed to a control and a limit test concentration of 1000 mg test item/kg soil dry weight in groups of 40 (4 replicates per treatment group with 10 earthworms each) for a period of 14 days. The effect on mortality of earthworms after exposure over 14 days, behavioural effects and weight change were determined. 


A positive control study using 2-Chloroacetamide, performed from January 2022 to February 2022, was reported for reference purposes.





After 14 days of exposure, no mortality was observed in the control or the single test item concentration of 1000 mg test item/kg soil dry weight. At the single dose of 1000 mg test item/kg soil dry weight, the decrease of body weight of -11.5% was calculated to be statistically significantly different compared to the control. However, as this level of body weight changes is considered to be within the accepted biological variability of 20% (as the accepted validity criteria for body weight changes of the control is 20%, according to ISO 11268-1), the body weight changes in the test item treated group was not considered to be an adverse effect. In addition, no behavioural effects were observed in any treatment group.


In conclusion, the 14-day LC50 was estimated to be >1000 mg test item/kg soil dry weight. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) related to mortality and biomass were determined to be ≥1000 mg test item/kg soil dry weight and >1000 mg test item/kg soil dry weight, respectively. 




Description of key information

14d-LC50 (Eisenia andrei) >1000 mg test item/kg soil dry weight; OECD TG 207 and ISO 11268-1; Ibacon (2022).

Key value for chemical safety assessment

Additional information

No key value for chemical safety assessment is reported as no hazard was identified. The 14-day LC50 value was estimated to be greater than the highest tested concentration of 1000 mg test item/kg soil dry weight.