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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
Study period:
19 Jul - 14 Sep 2017
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:
July 2016
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichteamt Hildesheim, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The respective test item amount was weighed, dispersed in demineralised water and added to the artificial soil (2400 g soil dry weight). Demineralised water (269 mL) was added to adjust the humidity of the artificial soil to a moisture of 54% of the maximum water holding capacity (WHC). 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 (bred in covered plastic vessels containing potting compost, fed with a litter of dried stinging nettle leaves and porridge oats).
- Age at test initiation (mean and range, SD): Earthworms of homogeneous age were used in the 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 - 12 months.
- Weight at test initiation (mean and range, SD): 0.35 - 0.54 g
- Other: The worms were washed with demineralised water and dried gently on a paper towel before introduced into each test vessel.
- Feeding: The earthworms were fed with cattle manure (Lehr- und Forschungsgut Ruthe, Sarstedt, Germany) weekly during the first 4 weeks (5 g on Days 0, 7, 14, 21, 28). 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.

ACCLIMATION
- Acclimation period and conditions: Earthworms were adapted to the artificial soil to be used for the test two days prior to test start.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Test temperature:
20 ± 2 °C
Moisture:
- 54% of the maximum water holding capacity (WHC).
- Throughout the test the replicates were weighed weekly. 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.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Round plastic boxes with an inner diameter of 15 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 (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Components of artificial soil:
- 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)
- 0.20 % calcium carbonate (CaCO3) to achieve a pH of 6.0 ± 0.5
- Pretreatment: Two days before test start the artificial soil (20000 g) was pre-moistened with 1920 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.

OTHER TEST CONDITIONS
- Photoperiod: 16 h
- Light intensity: 400 - 800 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The body weight of the adult earthworms was determined on Day 0 and 28 for each replicate. After the first four weeks, adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded. After a further four weeks, the number of offspring hatched from the cocoons was counted for each test container.

TEST CONCENTRATIONS
- Range finding study: yes, non-GLP
- Test concentrations: 1, 10, 100, 1000 mg/kg soil dry weight
- Results used to determine the conditions for the definitive study: After 14 days of incubaton, behavioural differences, mortalities as well as earthworm biomass were determined. 100 mortality was identified at the highest concentration while no mortalities were observed in the other concentrations.
Nominal and measured concentrations:
Nominal: 9.375, 18.75, 37.5, 75, 150 mg/kg dw soil
Reference substance (positive control):
yes
Remarks:
Carbendazim (the test is conducted once within 12 months. The most recent test was carried out from 09 Feb - 06 Apr 2017.
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
37.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality and reproduction
Details on results:
- Mortality at end of exposure period: No evident earthworm mortalities (< 10 %) of adult earthworms were observed in the controls and the test item concentrations 9.375, 18.75 and 37.5 mg/kg soil dry weight. At the test item concentrations 75 and 150 mg/k soil dry weight mortality statistically significantly were observed.
- Changes in body weight of live adults (% of initial weight) at end of exposure period: see field "any other information on results incl. tables"
- No. of offspring produced: see field "any other information on results incl. tables"
- Behaviour and Pathological Symptoms: see field "any other information on results incl. tables"

Results with reference substance (positive control):
- Results with reference substance valid?: yes
- Relevant effect levels: Significant effects on reproduction were observed between 1 and 2 mg reference substance/kg soil dry weight (in line with requirements of the guideline).

DETAILS ON BIOLOGICAL RESULTS.

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

Application rate Replicate
[mg test item/kg SDW] 1 2 3 4 5 6 7 8 Mean
Control 10 0 10 0 0 0 0 0 2.5
9.375 0 0 10 0  -   -   -   -  2.5
18.75 0 10 0 0  -   -   -   -  2.5
37.5 0 10 10 0  -   -   -   -  5
75 0 20 10 10  -   -   -   -  10
150 80 50 80 40  -   -   -   -  62.5

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

Application rate Observation parameter Replicate
[mg test item/kg SDW] 1 2 3 4 5 6 7 8
Control A
H
 9/10
1/10
 10/10
 -
 9/10
1/10
 10/10
 -
 10/10
 -
 10/10
 -
 10/10
 -
 10/10
 -
9.375 A
H
 10/10
 -
 10/10
 -
 9/10
1/10
 10/10
 -
 -   -   -   - 
18.75 A
H
 10/10
 -
 9/10
1/10
 10/10
 -
 10/10
 -
 -   -   -   - 
37.5 A
H
 10/10
 -
 9/10
1/10
 9/10
1/10
 10/10
 -
 -   -   -   - 
75 A
D
H
 10/10
 -
 -
 8/10
 -
 2/10
 9/10
 -
 1/10
 8/10
 1/10
 4/10
 -   -   -   - 
150 A
D
H
 2/10
 -
 8/10
 3/10
 2/10
 5/10
 2/10
 -
 8/10
 5/10
 1/10
 4/10
 -   -   -   - 

A = no obvious pathological symptoms

D = spontaneous segmentation and separation

H = earthworm dead

Table 3: Body Weight Changes of the Adult Earthworms

Application rate Replicate Mean body weight change of earthworms per replicate [%] Mean ± SD [%] Statistical Significance
[mg test item/kg SDW]
Control 1 34.1 23.3 ± 10.1  -
2 13.6
3 11.4
4 13.6
5 27.3
6 27.3
7 38.6
8 20.5
9.375 1 26.7 26.7 ± 1.84 No
2 24.4
3 26.7
4 28.9
18.75 1 31.1 27.4 ± 7.10 No
2 34.1
3 26.7
4 17.8
37.5 1 15.9 18.5 ± 4.66 No
2 22.2
3 22.7
4 13.3
75 1 24.4 24.2 ± 2.30 No
2 22.7
3 27.3
4 22.2
150 1 11.4 5.70 ± 19.3 Yes
2 -22.2
3 22.2
4 11.4

Table 4: Reproduction Rate after 8 weeks.

Application rate Mean ± SD of juveniles % of control Statistical Significance
[mg test item/kg SDW]
Control 143 ± 19.8  -  -
9.375 134 ±25.2 93.7 No
18.75 138 ±26.2 96.5 No
37.5 156 ±11.1 109 No
75 88 ± 12.0 61.5 Yes
150 10 ±5.06 6.99 Yes

Further calculated effect values:

- LOEC mortality, reproduction = 78 mg/kg SDW

- LOEC biomass = 150 mg/kg SDW

- NOEC biomass = 75 mg/kg SDW

- LC10 mortality (CI) = 42.9 mg/kg SDW (n.d.)

- LC20 mortality (CI) = 66 mg/kg SDW (n.d.)

- LC50 mortality (CI) = 150 mg/kg SDW (n.d.)

- EC10 biomass (CI) = 81.5 (62.8- 106) mg/kg SDW

- EC20 biomass (CI) = 94.9 (73.8- 122) mg/kg SDW

- EC50 biomass (CI) = 127 (94.2- 172) mg/kg SDW

- EC10 reproduction (CI) = 54.0 (32.0-90.9) mg/kg SDW

- EC20 reproduction (CI) = 62.8 (44.9 - 88.0) mg/kg SDW

- EC50 reproduction (CI) = 83.8 (53.0- 128) mg/kg SDW

VALIDITY CRITERIA.

Table 5: Validity criteria for OECD 222.

Criterion from the guideline to be satisfied in the controls

Outcome

Validity criterion fulfilled

Each replicate (containing 10 adults) to have produced ≥ 30 juveniles by the end of the test.

112 – 170 juveniles

yes

The coefficient of variation of reproduction is ≤ 30%.

13.9%

yes

Adult mortality over the initial 4 weeks of the test is ≤ 10%.

2.5% mortality in control

yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
The study conducted according to OECD 222 with Eisenia fetida as the test organism resulted in a NOEC concerning mortality and reproduction of 37.5 mg test item/kg soil dry weight.
Executive summary:

The toxicity of the substance on soil macroorganisms was performed according to the OECD guideline 222 and GLP. The test organism Eisenia fetida, was exposed to nominal test item concentrations of 9.375, 18.75, 37.5, 75, 150 mg/kg dw soil and a control for 8 weeks. Each concentration was mixed into artificial soil containing 5 % peat. The body weight of the adult earthworms was determined on Day 0 and 28. After the first four weeks, adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded. After a further four weeks, the number of offspring hatched from the cocoons was counted for each test container.

After 28 days of exposure in soil, neither evident earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and the test item concentrations 9.375, 18.75 and 37.5 mg test item/kg SDW. However, at the test item concentrations 75 and 150 mg test item/kg SDW statistically significant mortalities were observed. There were no statistically significant differences in earthworm body weights at the test item concentrations 9.375 to 75 mg test item/kg SDW compared to the control. At the concentration of 150 mg test item/kg SDW the body weight gain was statistically significantly reduced. After a further four weeks, the reproduction rate (average number of juveniles produced) was 143 in the control and ranged from 10 to 156 in the test item treatment rates. There was no statistically significant difference in earthworm reproduction in the treatment rates 9.375 to 37.5 mg test item/kg SDW compared to the control. However, at the test item concentrations 75 and 150 mg/kg SDW the earthworm reproduction was statistically significantly reduced. The study resulted into a NOEC (28 d) concerning mortality and reproduction of 37.5 mg test item/kg SDW.

Description of key information

NOEC (28 d) = 37.5 mg test item/kg dw (Eisenia fetida, OECD 222)

Key value for chemical safety assessment

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

Additional information

One study is available testing the toxicity of the substance on soil macroorganisms according to the OECD guideline 222 and GLP. The test organism Eisenia fetida, was exposed to nominal test item concentrations of 9.375, 18.75, 37.5, 75, 150 mg/kg dw soil and a control for 8 weeks. Each concentration was mixed into artificial soil containing 5 % peat. The body weight of the adult earthworms was determined on Day 0 and 28. After the first four weeks, adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded. After a further four weeks, the number of offspring hatched from the cocoons was counted for each test container.

After 28 days of exposure in soil, neither evident earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and the test item concentrations 9.375, 18.75 and 37.5 mg test item/kg SDW. However, at the test item concentrations 75 and 150 mg test item/kg SDW statistically significant mortalities were observed. There were no statistically significant differences in earthworm body weights at the test item concentrations 9.375 to 75 mg test item/kg SDW compared to the control. At the concentration of 150 mg test item/kg SDW the body weight gain was statistically significantly reduced. After a further four weeks, the reproduction rate (average number of juveniles produced) was 143 in the control and ranged from 10 to 156 in the test item treatment rates. There was no statistically significant difference in earthworm reproduction in the treatment rates 9.375 to 37.5 mg test item/kg SDW compared to the control. However, at the test item concentrations 75 and 150 mg/kg SDW the earthworm reproduction was statistically significantly reduced. The study resulted into a NOEC (28 d) concerning mortality and reproduction of 37.5 mg test item/kg SDW.