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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From December 02, 1998 to February 26, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
yes
Remarks:
analyses were not performed because the substance was readily biodegradable and has a halflife of 40d in soil. It can not be ascertained whether or not the test substance was stable during the test.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
- Species/strain: Eisenia fetida fetida
- Source of the initial stock: Aquatic Research Organisms INC, Hampton, USA
- Culturing techniques: They were kept in covered plastic vessels (volume 60 L) containing potting compost.
- Age/weight: The weight of the adult earthworms was 0.30 to 0.60 g.
- Pre-treatment: The worms had been acclimatised to artificial soil one d prior to the test.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Test temperature:
18-21°C
pH:
5.56 - 6.06
Moisture:
32.9-35.8%
Details on test conditions:
- Adjustment of pH: No
- Light intensity / photoperiod: Continuous light
- Relevant degradation products: Not determined

Details on test system:
- Artificial soil test substrate: 10% peat (air dried and finely ground), 20% kaolin (kaolinite content 84-87%), 70% quartz sand (sand with particle
size of 100-400 µm) and 0.5% calcium carbonate. PH was 5.73 and moisture content was 20.7%.
- Test mixture: A total weight of 750 g of test substrate was put in each vessel.
- Size, volume and material of test container: Glass dishes of 1.0 l.
- Amount of artificial soil (kg)/ container: 0.750 kg w wt/container
- Nominal levels of test concentrations: 100, 180, 320, 580 and 1000 mg/kg dry weight artificial soil, based product with 51.7% in water
- Active ingredient levels of test concentrations: 52, 93, 165, 300 and 517 mg/kg dry weight artificial soil
- Number of replicates/concentration: 4
- Number of earthworms/test concentration: 40
- Number of earthworms/container: 10
- Light source: 400 to 800 lux
- Test performed in closed vessels due to significant volatility of test substrate: Yes, with perforated plastic film, but not because of volatilisation
but to prevent the test substrate from drying.

The test method was static. The test substance was applied once at test start. The individual live weights of the worms were reported after 14d of exposure. Other effects (pathological symptoms, changes in behaviour of the worms) of the test substance were reported after 7 and 14d of exposure.

Nominal and measured concentrations:
Nominal: 100, 180, 320, 580 and 1000 mg/kg dw of soil
Reference substance (positive control):
yes
Remarks:
2-Chloracetamide (99.5%)
Key result
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:
other: weight
Remarks on result:
other: corresponding to >517 mg a.i./kg soil dw
Key result
Duration:
14 d
Dose descriptor:
LC0
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight
Remarks on result:
other: corresponding to >517 mg a.i./kg soil dw
Details on results:
LC0 >1000 mg/kg dw
LC50 >1000 mg/kg dw
LC100 >1000 mg/kg dw
No significant mortality and effects were seen in any of the tested concentrations after 14d of exposure. There was no statistically significant difference at the test concentrations 100 - 1000 mglkg compared to the control group. The earthworms in the concentrations 100 to 1000 mg/kg dw showed normal behaviaur and no pathological symptoms. The highest tested concentration without mortality and any other effects was 1000 mg/kg dw.
Results with reference substance (positive control):
Results of the reference test with 2-Chloracetamide showed that the method is sensitive and valid.

For result tables, kindly refer to the attached background material section of the IUCLID.

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 14d NOEC in earthworm (Eisenia foetida) was determined at 1000 mg/kg dw (or 517 mg/kg dw based on a.i.) (nominal).
Executive summary:

A study was conducted to determine the toxicity to soil macroorganisms of the read across substance, C12 -16 ADBAC (51.7% active) according to OECD Guideline 207, in compliance with GLP. Earthworms (Eisenia foetida) were exposed to a single dose of the read across substance at nominal concentrations of 100, 180, 320, 580 or 1,000 mg/kg dw of artificial soil. No analytical dose verification was performed. The individual live weights of the worms were reported after 14 d of exposure. Other effects (pathological symptoms, behaviour of the worms) were reported after 7 and 14 d of exposure. Results of the reference test with 2 -chloracetamide show that the method was sensitive and valid. The substance did not cause a change in behaviour, weight and mortality of the earthworm at any of the tested concentrations after 14 d of exposure. This was probably due to adsorption onto soil. The highest tested concentration without mortality and any other effects was 1000 mg/kg dw. Under the study conditions, the 14 d NOEC in earthworm was 1000 mg/kg dw (or 517 mg a.i./kg dw) and the 14 d LC0 was > 1000 mg/kg dw (or > 517 mg a.i./kg dw) (Noack, 1999). Based on the results of the read across study, similar NOEC value is expected for the test substance.

Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From October 13, 2003 to November 11, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
Four groups of ten worms were treated with each concentration of test substance. Doses were applied as aqueous solutions and the same volume for all dose levels was used. The control was treated with water only. Worms were rinsed in dechlorinated water, blotted dry and individually weighed at the beginning of the study. At the end of the study they were cleaned in the same manner and weighed in groups of ten.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Test organisms:
- Common name: Earthworm (Eisenia foetida foetida)
- Source: Huntingdon Life Sciences
- Age at test initiation (mean and range, SD): More than 2 months
- Weight at test initiation (mean and range, SD): 300 - 600 mg
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Test temperature:
21.1 - 21.7°C
pH:
5.86
Moisture:
Moisture content was 35% of the dry weight at the beginning and 32-34% at the end of the study.
Details on test conditions:
Test system:
- Test container (material, size): One litre glass container covered with perforated plastic to retain moisture.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

Source and properties of substrate (if soil):
- Artificial OECD 207 soil, Batch 13/03, (Industrial quartz sand, kaolin clay and sphagnum peat) was used for the main test (pH approximately 5.86)

Other test conditions:
- Photoperiod: continuous light
- Light intensity: 684 lux

Effect parameters measured: Mortality, behavioural and pathological signs

Vehicle control: yes

NB. On Days 7 and 14 the medium was emptied from the containers and worms were removed and counted. On Day 7, the medium was returned to the containers and surviving worms placed on the soil surface.

Nominal and measured concentrations:
Nominal concentrations: 0, 953, 1715, 3086, 5556 or 10000 mg a.i./kg dw of soil
Reference substance (positive control):
yes
Remarks:
Chloroacetamide
Key result
Duration:
7 d
Dose descriptor:
LC50
Effect conc.:
7 160 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits 5560 - 7590 mg a.i./kg dw of soil
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
7 070 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits 5560 - 7400 mg a.i./kg dw of soil
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
953 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: body weight
Details on results:
- Mortality at end of exposure period: After 7d, all worms at 10000 and 2 worms at 5556 mg a.i./kg dw of soil were dead. By Day 14, one additional worm died at 5556 mg a.i./kg dw of soil.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: Group mean body weights were affected by treatment with test substance at 1,715 mg a.i./kg dw of soil and above.
- Morphological abnormalities: No
- Behavioural abnormalities: No
- Other biological observations: At 5556 mg a.i./kg soil dw several live worms were visible at the sides of the containers on Days 1 to 3 then again on Days 8 and 9. At the highest dose level, live worms were visible at the sides and on the surface of the soil in some containers during Days 1 to 6.
Results with reference substance (positive control):
The LC50 values of chloroacetamide to the earthworm, determined in a separate GLP compliant study, were 41 (95% confidence limits 37-43) and 39 (95% confidence limits 35-41) mg a.i./kg dw of soil at Day 7 and 14, respectively. The results obtained in the positive control test were considered acceptable.
Reported statistics and error estimates:
The LC50 values were calculated using a logistic model (Berkson, 1944). 95% confidence limits were estimated by the likelihood ration method (Williams, 1986).

Table 1. Mortality rate (%)

Group No.

Treatment

Initial

Mortality

Total worms

 

(mg a.i./kg dw of soil

No. worms

Day 7

Day 14

as %

1

0

40

0

0

0

2

953

40

0

0

0

3

1715

40

0

0

0

4

3086

40

0

0

0

5

5556

40

2

3

7.5

6

10000

40

40

40

100

 

Table 2. Mean bodyweights (mg)

Group

Treatment

(mg a.i./kg dw of soil

Day 0

Day 14

% decrease

1

0

388

334

14

2

953

389

340

13

3

1715

390

323

17

4

3086

386

304

21

5

5556

389

250

36

6

10000

392

n/d

n/d

 

n/d: no data (as all worms in this group had died)

For further details refere to the attachment under 'attached background materials'.

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of the read across study, the 14d LC50 and NOEC values for earthworm (Eisenia foetida) were 7070 and 953 mg a.i./kg soil dw (nominal) respectively.

Executive summary:

A study was conducted to determine the toxicity to soil macroorganisms of the read across substance C12-16 ADBAC (49.5% active) according to OECD Guideline 207, in compliance with GLP. Six groups of forty earthworm (Eisenia foetida) were allocated to an artificial soil containing 0, 953, 1715, 3086, 5556 or 10000 mg a.i./kg soil dw (nominal concentrations). No analytical dose verification was performed. Mortality was recorded on Days 7 and 14. Worms were weighed at the beginning and end of the study. After 7 days, all worms at 10000 and 2 worms at 5556 mg a.i./kg soil dw were dead. By Day 14, one additional worm died at 5556 mg a.i./kg soil dw. A treatment-related reduction in body weight was observed. Group mean body weights were affected by treatment with read across substance at 1715 mg a.i./kg soil dw and above. Under the study conditions, the 7 and 14 d LC50 values were 7160 and 7070 mg a.i./kg soil dw, respectively and the NOEC was 953 mg a.i./kg soil dw (nominal) (Rodgers, 2004). Based on the results of the read across study, similar EC50 and NOEC values is expected for the test substance.

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 13 September 2018 to 08 February 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
yes
Remarks:
These deviations are considered to have no impact on quality and integrity of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
On Day 0, after 28 days and on Day 55, the amount of the test substance in soil was analytically verified via LC-MS/MS for the control and all test substance concentrations using pooled samples of all respective replicates.
Vehicle:
yes
Remarks:
demineralised water
Details on preparation and application of test substrate:
The respective test substance amount for each test substance concentration was weighed out, dispersed in demineralised water (200 mL) and added to the artificial soil (2440 g SDW). Additional demineralised water (78 mL) was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 610 g SDW were filled into each test vessel.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
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.

Feeding
During breeding, the earthworms are fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Test temperature:
20 +/- 2 °C
pH:
The initial pH-value of the artificial soil was 6.12 (measured on Day -21 after preparation of the soil). The range of pH during test was 5.89 to 6.32.
Moisture:
Throughout the test the replicates were weighed weekly to determine water loss via evaporation. Evaporated water was replenished by adding demineralised water to maintain the soil moisture which should not deviate by more than 10% of the initial value at the end of the test. Additional moistening was performed in case the soil appeared too dry.
Moisture [%] of dry weight: 17.5 - 25.7
Details on test conditions:
Reference substance: Carbendazim
Time schedule : The test is conducted once within twelve months. The most recent test was carried out from 2019-01-24 to 2019-03-21 (Study ID 71901IRS / RBN70070).
Kind of application: Application into the artificial soil by mixing
Test concentration: 0.5 - 1 - 2 - 4 mg a.i./kg SDW
According to the guideline, earthworms are expected to show significant effects between 1 and 5 mg a.i./kg SDW.


Test Method
Test duration: 8 weeks
Number of replicates: 8 replicates per control and 4 replicates per test substance concentration
Number and age of the earthworms: 10 adult earthworms (with clitellum) aged 2-12 months were used per replicate. The difference of age did not deviate by more than 1 month.
Body (or live) weights of test organisms: 0.30 - 0.60 g (nominal), 0.36 to 0.55 g (actual) at experimental starting
Acclimation: Earthworms to be used for the test were acclimatised to the artificial soil two days prior to test start.
Homogeneity of groups: The worms were washed with demineralised water and dried gently on a paper towel. The homogeneity 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.
Test medium: Artificial soil consisted of the following components:
- 5 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.18 % calcium carbonate (CaCO3) to achieve a pH of 6.0 +/- 0.5
The WHCmax, the moisture content and the pH-value of the artificial soil were determined and adjusted. Two days before test start the artificial soil (21000 g) was pre-moistened with 1576 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

Test container
Test units containing about 610 g SDW were 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 enable sufficient gas exchange, light input and prevent the test substrate from drying.

Application
The respective test substance amount for each test substance concentration was weighed out, dispersed in demineralised water (200 mL) and added to the artificial soil (2440 g SDW). Additional demineralised water (78 mL) was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 610 g SDW were filled into each test vessel.

Temperature: 20 +/- 2 °C
Photoperiod:16 h light and 8 h dark
Light intensity: 400 - 800 lux
Feeding: The earthworms were fed with air-dried cattle manure weekly during the first 4 weeks. At test start and at Day 28 the food was mixed carefully into the soil, on the other days the food was placed onto the soil surface. No further feeding took place during the remaining 4 weeks of the test. The cattle manure was delivered by LEHR- UND FORSCHUNGSGUT RUTHE, Schäferberg 1, 31157 Sarstedt, Germany. Manure was obtained from cattle that were not subject to medication or treatment with substances, such as growth promoters, nematicides or similar veterinary products that could adversely affect the worms during the test. The manure was air-dried and finely ground before use.

Moistening: Throughout the test the replicates were weighed weekly to determine water loss via evaporation. Evaporated water was replenished by adding demineralised water to maintain the soil moisture which should not deviate by more than 10% of the initial value at the end of the test. Additional moistening was performed in case the soil appeared too dry.

Type and Frequency of Measurements and Observation

Measurements and observation
At experimental starting and experimental completion, the pH- value and moisture content of the test medium were determined in each treatment and the control from pooled samples of all replicates. The water content of each test container was checked weekly by weighing. The weight loss was replenished with the appropriate amount of demineralised water. The body weight of the adult earthworms was determined individually on Day 0 and Day 28 for each replicate. After the first four weeks, the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each replicate. After an additional 4 weeks, the number of offspring (juveniles) hatched from the cocoons was counted for each test container.

Determination of the test substance
On Day 0, after 28 days and on Day 55, the amount of the test substance in soil was analytically verified via LC-MS/MS for the control and all test substance concentrations using pooled samples of all respective replicates.

Observation parameters
A) no obvious pathological symptoms
B) no reaction to touching
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow excretion from the oral aperture
G) ulcer and skin bleedings
H) dead earthworms

Equipment
pH-Meter, pH3110, WTW
Hygrothermograph, LUFFT, LAMBRECHT
Drying cabinet, BINDER GmbH
Analytical balance, SARTORIUS
Top pan balances, KERN
Waterbath, MEMMERT
Automatic Timer
Mixer, Dynamic model FT97, THIEL GROßKÜCHENBEDARF
Mixer, SEVERIN
Luxmeter, TESTO

Chronological Test Description
Day -24 Artificial soil was produced.
Day -22 to -2 The pH-value, the WHCmax and the moisture content of the artificial soil were determined and adjusted.
Day -2 Earthworms were adapted to artificial soil moistened to 54 % of the maximum water holding capacity. Artificial soil for the study was moistened to 27 % of the maximum water holding capacity.

Experimental Starting (Day -0)
Earthworms were washed in demineralised water, dabbed dry on paper towels and weighed.
Application and moistening as described above.
Moisture content and pH-value were determined using pooled samples of each treatment and each control.
Feeding.
Earthworms were placed on the surface of the test medium. Sampling for analytical verification of the test substance in the controls and all test substance concentrations.

Day 7, 14, 21
Feeding and moistening as described above

Day 28
The test medium was searched carefully for adult earthworms, which were removed, counted and weighed. Morphological and behavioural changes were documented. The same test medium, including cocoons laid during the first 4 weeks of the test period, was incubated under the same conditions for an additional 4 weeks.
Feeding and moistening as described above
Sampling for analytical verification of the test substance in the control and all test substance concentrations

Day 35, 42, 49
Moistening as described above

Day 55
The number of offspring was determined by placing the containers into a water bath at 45 - 60 °C and removing all worms, which accumulated on the soil surface. Subsequently, the remaining soil was searched for juvenile earthworms. The total number of juveniles was assessed by counting.
Sampling for analytical verification of the test substance in the control and all test substance concentrations

Day 55 to 56
The moisture content and pH-value of the treatment rates 125, 250, 1000 and 2000 mg test substance/kg SDW and of the controls were analysed from pooled samples of all respective replicates. However, due to a technician error only three of four replicates could be used for analysing moisture content and pH-value at the test substance concentration 500 mg test substance/kg SDW (experimental completion).

Statistics and Calculations

Adult Mortality The arithmetic mean and NOEC were determined. Since no evident mortality occurred the LCx – values were not determined.

Reproduction
- Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend Analysis on Contrasts (Monotonicity of Concentration/Response)
- William’s Multiple Sequential t-test Procedure

The α-value (acceptable probability of incorrectly concluding that there is a difference) was  = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC50 - value for reproduction was calculated using 3-param. normal CDF (cumulative distribution function).

Biomass
- Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend Analysis on Contrasts (Monotonicity of Concentration/Response)
- Dunnett’s Multiple t-test Procedure

The α value (acceptable probability of incorrectly concluding that there is a difference) was  = 0.05. For the endpoint of biomass, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated.

Software
The data presented in the tables of the report were computer-generated and have been rounded for presentation. Thus, manual re-calculation of the data based on the results presented in this report may result in minor deviations from these figures. Calculations were carried out using the following software:
- Excel, MICROSOFT
- ToxRat Professional Version 3.3.0 , TOXRAT SOLUTIONS GMBH
- Sigma Plot, SPSS INC.
Nominal and measured concentrations:
62.5, 125, 250, 500, 1000 mg a.i. BKC/ADBAC/kg SDW corresponding to 125, 250, 500, 1000, 2000 mg test substance/kg SDW
Artificial soil moistened with demineralised water without test or reference item was used as control medium.
Reference substance (positive control):
yes
Remarks:
Carbendazim 0.5 - 1 - 2 - 4 mg a.i./kg SDW
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 2 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: equivalent to >1000 mg a.i. test substance/kg SDW
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 2 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: equivalent to ≥1000 mg a.i. test substance/kg SDW
Key result
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
ca. 500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: equivalent to 250 mg a.i. test substance/kg SDW
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
ca. 250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: equivalent to 125 mg a.i. test substance/kg SDW
Key result
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
ca. 589 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CI, 171 - >2000
Remarks:
equivalent to 295 mg a.i. test substance/kg SDW (95% CI, 85.5 - >1000)
Results with reference substance (positive control):
According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg SDW.

Results

Adult Mortality

No evident earthworm mortality (<10%) was observed in the controls and all test substance concentrations.

Table 1 Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate Replicate
[mg test substance/kg SDW] 1 2 3 4 5 6 7 8 Mean
Control 0 20 0 0 0 0 0 0 2.5
125 0 0 0 0 - 0
250 0 0 0 0 0
500 0 10 0 0 2.5
1000 0 0 0 0 0
2000 0 0 0 0 0

Pathological Symptoms and Changes in Behaviour

No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test substance concentrations.

Table 2 Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate

Replicate

[mgtest substance/kg SDW]

Observation parameter

1

2

3

4

5

6

7

8

Control

A

H

10/10

-

8/10

2/10

10/10

-

10/10

-

10/10

-

10/10

-

10/10

-

10/10

-

125

A

10/10

10/10

10/10

10/10

-

250

A

10/10

10/10

10/10

10/10

500

A

H

10/10

-

9/10

1/10

10/10

-

10/10

-

1000

A

G

9/10

1/10

10/10

-

10/10

-

10/10

-

2000

A

10/10

10/10

10/10

10/10

A = no obvious pathological symptoms, G = ulcer and skin bleedings, H = earthworm dead

Live Weight

At test start, the earthworms had individual weights of 0.36 - 0.55 g. There were no statistically significant differences in earthworm body weights in all treatment rates compared to the control.

Table 3 Body Weight Changes of the Adult Earthworms

Application rate

[mg test substance/kg SDW]

Replicate

Mean body weights per replicate

Mean body weight change of earthworms

Test start

28 days

Per replicate

Mean ± SD

Sig.

[g]

[g]

[g]

[%]

[g]

[%]

Control

1

0.45

0.45

0.00

0.0

0.01 ± 0.02

1.13 ± 3.96

2

0.45

0.46

0.01

2.2

3

0.45

0.44

-0.01

-2.2

4

0.44

0.46

0.02

4.5

5

0.45

0.44

-0.01

-2.2

6

0.45

0.48

0.03

6.7

7

0.44

0.46

0.02

4.5

8

0.44

0.42

-0.02

-4.5

125

1

0.44

0.44

0.00

0.0

0.02 ± 0.02

4.45 ± 4.80

No

2

0.45

0.46

0.01

2.2

3

0.44

0.46

0.02

4.5

4

0.45

0.50

0.05

11.1

250

1

0.44

0.47

0.03

6.8

0.01 ± 0.02

2.25 ± 3.72

No

2

0.44

0.43

-0.01

-2.3

3

0.45

0.46

0.01

2.2

4

0.44

0.45

0.01

2.3

500

1

0.44

0.40

-0.04

-9.1

0.00 ± 0.03

-0.02 ± 7.58

No

2

0.45

0.49

0.04

8.9

3

0.44

0.45

0.01

2.3

4

0.45

0.44

-0.01

-2.2

1000

1

0.44

0.45

0.01

2.3

0.02 ± 0.01

4.50 ± 2.60

No

2

0.44

0.47

0.03

6.8

3

0.45

0.48

0.03

6.7

4

0.45

0.46

0.01

2.2

2000

1

0.44

0.47

0.03

6.8

0.02 ± 0.01

3.93 ± 2.20

No

2

0.45

0.46

0.01

2.2

3

0.44

0.46

0.02

4.5

4

0.45

0.46

0.01

2.2

SD = Standard deviation, Sig. = Statistical Significance (Dunnett’s Multiple t-test Procedure,α= 0.05) compared to control

Reproduction Rate

The reproduction rate (average number of juveniles produced) was 83 juveniles after 8 weeks in the control and ranged from 18 to 74 juveniles in the test substance treatment rates. There were no statistically significant differences in earthworm reproduction at the treatment rates 125 and 250 mg test substance/kg SDW compared to the control. However, at the test substance concentrations 500 to 2000 mg test substance/kg SDW the earthworm reproduction was statistically significantly reduced. The coefficient of variation calculated for the reproduction of the control was 16.5 % and thus lower than 30 % meeting a validity criterion for this study.

Table 4 Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate

[mg test substance/kg SDW]

Replicate

Number of juveniles

Mean ± SD

CV              [%]

[%]

of control

Sig.

Control

1

67

83 ± 13.7

16.5

-

-

2

95

3

91

4

63

5

78

6

92

7

101

8

78

125

1

82

74 ± 26.7

36.0

89.2

No

2

86

3

34

4

92

250

1

68

67 ± 24.6

36.8

80.7

No

2

95

3

70

4

35

500

1

22

37 ± 13.7

36.9

44.6

Yes

2

55

3

38

4

34

1000

1

60

32 ± 21.3

66.7

38.6

Yes

2

8

3

30

4

31

2000

1

14

18 ± 2.83

15.7

21.7

Yes

2

20

3

20

4

18

SD = Standard deviation, CV = Coefficient of variation, Sig. = Statistical Significance (William’s Multiple Sequential t-test Procedure,α= 0.05) compared to the control

Measured Exposure Concentrations during the Definitive Test

Analytical evaluation of two main components of the test substance BKC/ADBAC (C12-BKC and C14-BKC) was carried out via LC/MS-MS for the various concentrations of 125 to 2000 mg/kg SDW and the control. For details of the analytical method, kindly refer to the attached background material section of the IUCLID. The recovery rates of the two components were in the ranges from 83% to 101% at Day 0, from 68% to 92% at Day 28 and at Day 55 from 25 to 56% of the nominal concentrations.

Table 5 Measured Concentrations and Percent of Nominal Concentration of the Component C12-BKC of BKC/ADBAC

Sampling date

Day 0

Day 28

Day55

Nominal concentration

C12-BKC

Test substance

[mg/kg SDW]

C12-BKC

[mg/kg SDW]

Meas. conc.
[mg/kg SDW]


[%]

Meas. conc.
[mg/kg SDW]


[%]

Meas. conc.
[mg/kg SDW]


[%]

2000

718

662

92

543

76

177

25

1000

359

361

101

331

92

103

29

500

180

174

97

161

90

80.5

45

250

89.9

84.3

94

71.5

80

30.4

34

125

44.9

43.0

96

37.3

83

20.5

45

Control

< LOQ

< LOQ

< LOQ

Meas. conc.= measured concentration of the component C12-BKC

% = Percent of the nominal concentration of C12-BKC

LOQ = Limit of quantification of the analytical method (0.100 mg test substance/kg SDW corresponding to 0.0359 mg C12-BKC/L

Table 6 Measured Concentrations and Percent of Nominal Concentration of the Component C14-BKC of BKC/ADBAC

Sampling date

Day 0

Day 28

Day55

Nominal concentration

C14-BKC

Test substance [mg/kg SDW]

C14-BKC

[mg/kg SDW]

Meas. conc.
[mg/kg SDW]


[%]

Meas. conc.
[mg/kg SDW]


[%]

Meas. conc.
[mg/kg SDW]


[%]

2000

281

234

83

191

68

99.6

35

1000

141

133

95

127

90

59.9

43

500

70.3

62.0

88

62.3

89

38.7

55

250

35.1

30.0

85

26.8

76

16.1

46

125

17.6

15.3

87

13.5

77

9.82

56

Control

< LOQ

< LOQ

< LOQ

Meas. conc.= measured concentration of the component C14-BKC

%= Percent of the nominal concentration of C14-BKC

LOQ =Limit of quantification of the analytical method (0.100 mg test substance/kg SDW corresponding to 0.0141 mg C14-BKC/L)

Validity Criteria

This study on the effects of BKC/ADBAC on the reproduction of earthworms of the species Eisenia fetida was performed according to OECD Guidelines 222 (July 2016). All validity criteria were fulfilled:

Table 7 Validity Criteria

Validity Criteria

Required

Test Results

Adult mortality in the control group after the first four weeks of the definitive test

≤10 %

2.5 %

Average number of juveniles in the control group

≥ 30

83

Coefficient of variation calculated for the reproduction of the control group

< 30 %

16.5 %

Reference substance data

Table 8 Reference substance - Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate
[mg a.i./kg

SDW]

Rep.

Number of juveniles

Mean±SD

CV

% of control

Significance*

Control

1

137

105± 18.8

17.9

-

-

2

96

3

131

4

105

5

98

6

87

7

96

8

90

0.5

1

109

90 ± 16.0

17.7

85.7

No

2

86

3

71

4

95

1

1

57

70 ± 12.2

17.4

66.7

Yes

2

79

3

63

4

82

2

1

87

78± 6.99

8.96

74.3

Yes

2

71

3

79

4

74

4

1

21

14± 8.19

58.5

13.3

Yes

2

20

3

8

4

5

Rep. = Replicate, a.i. = active ingredient, SD = Standard deviation, CV = Coefficient of variation

*) = Statistically significantly reduced compared tocontrol (Williams Multiple Sequential t-test Procedure)

According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg SDW.

Discussion and Conclusions

Under the conditions of this study, neither evident earthworm mortalities (<10%) nor evident pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or all test substance concentrations. There were no statistically significant differences in earthworm body weights in all test substance concentrations compared to the control. After an additional 4 weeks, the reproduction rate (average number of juveniles produced) was 83 juveniles in the control and ranged from 18 to 74 juveniles in the test substance treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rate 125 and 250 mg test substance/kg SDW compared to the control. However, at the test substance concentrations 500 to 2000 mg test substance/kg SDW the earthworm reproduction was statistically significantly reduced. All validity criteria recommended by the test guidelines were fulfilled.

For detailed results and figures, kindly refer to the attached bakground material section of the IUCLID.

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the LOEC (mortality, biomass), NOEC (mortality, biomass), LOEC (reproduction), NOEC (reproduction) and EC50 (reproduction) values for read across substance were reported to be >2000, ≥2000, 500, 250 and 589 mg test substance/kg SDW, respectively (equivalent to >1000, ≥1000, 250, 125 and 295 mg a.i./kg SDW, respectively).
Executive summary:

A study was conducted to determine the effects of read across substance (50% active in water) on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae), according to the OECD TG 222, in compliance with GLP. The study was conducted under static conditions over 8 weeks with the read across substance concentrations 125, 250, 500, 1000, 2000 mg//kg solid dry weight (SDW) corresponding to 62.5, 125, 250, 500, 1000 mg a.i./kg SDW. Each application rate was mixed into artificial soil containing 5% peat. A control including untreated artificial soil was tested under the same conditions as the read across substance treatments. A total of 80 test organisms were divided equally into 8 control replicates adnd another total of 40 test organisms were divided equally into 4 replicates for each read across substance treatment (i.e., 10 earthworms per replicate). They had an individual body weight between 0.36 and 0.55 g at the experimental starting. Each concentration level and control were analysed via LC-MS/MS analysis on Day 0, Day 28 and Day 55 using pooled samples of all replicates. The measured concentrations of the pooled samples of replicates were within the range of 83 to 101 % of the nominal values on Day 0, demonstrating the right preparation of the tested concentrations. After 28 days of exposure in soil, no read across substance-related earthworm mortalities (<10%), pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or all read across substance concentrations. There were no statistically significant differences in earthworm body weights in all read across substance concentrations compared to the control. After an additional 4 weeks, the reproduction rate (average number of juveniles produced) was 83 juveniles in the control and ranged from 18 to 74 juveniles in the read across substance treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 125 and 250 mg read across substance/kg SDW compared to the control. However, at the read across substance concentrations 500 to 2000 mg read across substance/kg SDW the earthworm reproduction was statistically significantly reduced. All validity criteria recommended by the test guidelines were fulfilled. Under the study conditions, the LOEC (mortality, biomass), NOEC (mortality, biomass), LOEC (reproduction), NOEC (reproduction) and EC50 (reproduction) values for read across substance were reported to be >2000, ≥2000, 500, 250 and 589 mg read across substance/kg SDW, respectively (equivalent to >1000, ≥1000, 250, 125 and 295 mg a.i./kg SDW, respectively). Based on the results of the read across study, similar effect levels can be expected for the test substance.

Description of key information

Based on the above information and in line with the biocides assessment report on the read across substance C12 -16 ADBAC, the 14 d LC50 of 7070 has been selected to express the acute toxicity of the test substance. Further, based on the chronic toxicity study with the read across substance, the 28-d NOEC of 125 mg/kg bw/day has been considered further for hazard/risk assessment.    

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
7 070 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
125 mg/kg soil dw

Additional information

Short-term toxicity study:  

Study 1.  A study was conducted to determine the toxicity to soil macroorganisms of the read across substance C12-16 ADBAC (49.5% active) according to OECD Guideline 207, in compliance with GLP. Six groups of forty earthworms (Eisenia foetida) were allocated to an artificial soil containing 0, 953, 1715, 3086, 5556 or 10000 mg a.i./kg soil dw (nominal concentrations). No analytical dose verification was performed. Mortality was recorded on Days 7 and 14. Worms were weighed at the beginning and end of the study. After 7 days, all worms at 10000 and 2 worms at 5556 mg a.i./kg soil dw were dead. By Day 14, one additional worm died at 5556 mg a.i./kg soil dw. A treatment-related reduction in body weight was observed. Group mean body weights were affected by treatment with read across substance at 1715 mg a.i./kg soil dw and above. Under the study conditions, the 7 and 14 d LC50 values were 7160 and 7070 mg a.i./kg soil dw, respectively and the NOEC was 953 mg a.i./kg soil dw (nominal) (Rodgers, 2004).

Study 2. A study was conducted to determine the toxicity to soil macroorganisms of the read across substance, C12 -16 ADBAC (51.7% active) according to OECD Guideline 207, in compliance with GLP. Earthworms (Eisenia foetida) were exposed to a single dose of the read across substance at nominal concentrations of 100, 180, 320, 580 or 1,000 mg/kg dw of artificial soil. No analytical dose verification was performed. The individual live weights of the worms were reported after 14 d of exposure. Other effects (pathological symptoms, behaviour of the worms) were reported after 7 and 14 d of exposure. Results of the reference test with 2 -chloracetamide show that the method was sensitive and valid. The substance did not cause a change in behaviour, weight and mortality of the earthworm at any of the tested concentrations after 14 d of exposure. This was probably due to adsorption onto soil. The highest tested concentration without mortality and any other effects was 1000 mg/kg dw. Under the study conditions, the 14 d NOEC in earthworm was 1000 mg/kg dw (or 517 mg a.i./kg dw) and the 14 d LC0 was > 1000 mg/kg dw (or > 517 mg a.i./kg dw) (Noack, 1999).

 Based on the above two studies, the same effect levels were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. They further stated that: “The findings of the two tests, although different in absolute values, are not in contrast. Since the second test provides a “higher than” value corresponding to a complete lack of lethal or sublethal effects, the 14d LC50 = 7070 mg/kg dry soil (US ISC) is selected to express the acute toxicity of Alkyl (C12-16) dimethylbenzyl ammonium chloride to soil dwelling invertebrates.” 

 

Long-term toxicity study:  

A study was conducted to determine the effects of read across substance (50% active in water) on mortality, biomass and the reproductive potential of the earthworm speciesEisenia fetida(Annelida, Lumbricidae), according to the OECD TG 222, in compliance with GLP. The study was conducted under static conditions over 8 weeks with the read across substance concentrations 125, 250, 500, 1000, 2000 mg//kg solid dry weight (SDW) corresponding to 62.5, 125, 250, 500, 1000 mg a.i./kg SDW. Each application rate was mixed into artificial soil containing 5% peat. A control including untreated artificial soil was tested under the same conditions as the read across substance treatments. A total of 80 test organisms were divided equally into 8 control replicates adnd another total of 40 test organisms were divided equally into 4 replicates for each read across substance treatment (i.e., 10 earthworms per replicate). They had an individual body weight between 0.36 and 0.55 g at the experimental starting. Each concentration level and control were analysed via LC-MS/MS analysis on Day 0, Day 28 and Day 55 using pooled samples of all replicates. The measured concentrations of the pooled samples of replicates were within the range of 83 to 101 % of the nominal values on Day 0, demonstrating the right preparation of the tested concentrations. After 28 days of exposure in soil, no read across substance-related earthworm mortalities (<10%), pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or all read across substance concentrations. There were no statistically significant differences in earthworm body weights in all read across substance concentrations compared to the control. After an additional 4 weeks, the reproduction rate (average number of juveniles produced) was 83 juveniles in the control and ranged from 18 to 74 juveniles in the read across substance treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 125 and 250 mg read across substance/kg SDW compared to the control. However, at the read across substance concentrations, 500 to 2000 mg read across substance/kg SDW the earthworm reproduction was statistically significantly reduced. All validity criteria recommended by the test guidelines were fulfilled. Under the study conditions, the LOEC (mortality, biomass), NOEC (mortality, biomass), LOEC (reproduction), NOEC (reproduction) and EC50 (reproduction) values for read across substance were reported to be >2000, ≥2000, 500, 250 and 589 mg read across substance/kg SDW, respectively (equivalent to >1000, ≥1000, 250, 125 and 295 mg a.i./kg SDW, respectively). Based on the results of the read across study, similar effect levels can be considered for the test substance.

Therefore, based on the above information and in line with the biocides assessment report on the read across substance C12 -16 ADBAC, the 14 d LC50 of 7070 has been selected to express the acute toxicity of the test substance. Further, based on the chronic toxicity study with the read across substance, the 28-d NOEC of 125 mg/kg bw/day has been considered further for hazard/risk assessment.