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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:
experimental study
Adequacy of study:
supporting study
Study period:
01 Aug 2013 - 15 Aug 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
ISO 11268-1 (Effects of Pollutants on Earthworms. 1. Determination of Acute Toxicity Using Artificial Soil Substrate)
Version / remarks:
1993
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatsministerium für Umwelt und Landwirtschaft, Freistaat Sachsen, Dresden, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil: weighing of test item followed by addition of quartz sand; mixture of test item and quartz sand is added to prepared amount of artificial soil resulting in desired concentrations.
- Controls: Blank control
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: reared under ambient laboratory conditions in the test facility (original breeding of animals was purchased from “W. Neudorff GmbH KG”, Emmerthal, Germany)
- Age at test initiation: approximately 3 months old with clitellum
- Weight at test initiation: 300 – 462 mg/worm

ACCLIMATION
- Acclimation period: Earthworms were acclimatised in a separate batch of the untreated artificial soil for approximately 24 hours before test start.
- Health during acclimation (any mortality observed): no mortality
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Test temperature:
18 - 22 °C
pH:
test start: 6.08 - 6.13
test end: 5.79 - 5.82
Moisture:
test start: 35.0 - 35.1% (equivalent to 56.1 – 56.3 % of WHC)
test end: 34.5 - 34.8% (equivalent to 55.3 – 55.8 % of WHC)
Details on test conditions:
TEST SYSTEM
- Test container: glass container (volume: 1 L) with a glass lid which allowed gaseous exchange
- Amount of soil or substrate: 500 g dry weight
- 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
Composition:
- 10 % sphagnum peat; origin: Torfwerk Moorkultur Ramsloh, Saterland, Germany, classified according to DIN 11540 (as close to pH 5.5-6.0 as possible, no visible plant remained, finely ground, dried to measured moisture content)
- 20 % kaolin clay (kaolinite content > 30 %); type: Kaolin W, origin: ERBSLÖH Lohrheim GmbH, 65558 Lohrheim, Germany - 0.5% calcium carbonate; origin: MERCK KGaA, 64271 Darmstadt, Germany
- 69.5 % industrial quartz sand; type: Millisil W3, origin: Quarzwerke GmbH, 50207 Frechen, Germany (fine sand is dominant with more than 50% of the particles between 50 and 200 μm)
- deionised water
- Maximum water holding capacity: 62.4 g/100 g soil dw
- Stability of test material in the medium: stable

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity: 550 lx (source: artificial light, Lumilux L58W)

EFFECT PARAMETERS MEASURED: mortality; behavioural and pathological symptoms at 7 and 14 d after start of exposure

Nominal and measured concentrations:
nominal: control, 100, 178, 316, 562 and 1000 mg test item/kg soil dw / control, 89.6, 159.49, 283.14, 503.56, 869.0 mg a.s./kg soil dw
Reference substance (positive control):
yes
Remarks:
2-chloroacetamide
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
>= 869 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 869 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
- Mean changes in body weight of live adults at end of exposure period in %: -6.8 (control), -7.2 (100 mg test item/kg d.w.), -6.6 (178 mg test item/kg d.w.), -6.9 (316 mg test item/kg d.w.), -7.0 (562 mg test item/kg d.w.), -7.1 (1000 mg test item/kg d.w.)
- Morphological abnormalities: none
- Behavioural abnormalities: none
- Other biological observations: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: LC50 = 24.9 mg a.i./kg soil d.w. (95% confidence limits ranging from 23.6 to 26.1 mg a.i./kg soil d.w.)
Reported statistics and error estimates:
No statistically significant mortality compared to the control was observed at any test item concentration (Fisher’s Exact Binomial Test with Bonferroni Correction, alpha = 0.05, one-sided greater). No abnormal behaviour or toxic symptoms of the worms were observed in the test item treatment groups or control group during the test. The test item caused no statistically significant (Williams-t-test, alpha = 0.05, one-sided greater) change in biomass (difference in fresh weight of surviving worms between test start and day 14 of exposure) compared to the control (6.8 % biomass decrease) at concentrations of 100, 178, 316, 562 and 1000 mg test item/kg soil d.w.

With the exception of 2.5% mortality at a concentration of 178 mg test item/kg soil dry weight no mortality was observed in any other test item treatment group and in the control group.

Validity criteria fulfilled:
yes
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Nov 2013 - 14 Jan 2014
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 13, 2004
Deviations:
no
Qualifier:
according to
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Version / remarks:
July 1998
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): All application solutions were prepared freshly on the day of application. Prior to the start of the test, the application solution of the highest concentration of the test item was prepared. The test item was mixed into deionised water as homogeneously as possible. The application solution of the highest test concentration was further diluted with deionised water to obtain the desired test concentrations. Uniform volumes of the application solutions were used for all replicates of the test concentrations. They were thoroughly mixed into the artificial soil of each replicate using a laboratory mixer in the order lowest test concentration to highest test concentration.
- Controls: The control group was treated first in the same way as described above but with deionised water only.
- See also Table 1 under "Any other information on material and methods incl. tables" below.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm (Eisenia fetida andrei)
- Source: strain of Prof. Graff, Forschungsanstalt für Landwirtschaft, Braunschweig, Germany. Strain has been held in the laboratory of the testing facility for several years at 22 ± 2°C
- Age at test initiation (mean and range, SD): Approx. 4 months old; Adult worms, at least 2 months old, but not older than 1 year, with a well-developed clitellum were used for the study. The age of the worms from the synchronised culture differed not more than 4 weeks.
- Weight at test initiation (mean and range, SD): mean body wet weight: 0.30 to 0.50 g per worm

ACCLIMATION
- Acclimation period: One day prior to the test start, the test organisms were acclimated to the artificial soil and test temperature

CULTIVATION
- During cultivation, the earthworms were fed with an animal manure / lucerne meal mixture in 14 day intervals. In order to obtain a synchronised culture of earthworms, 50 adult worms were kept in a closed rearing vessel with substrate and food for a period of 4 weeks. After this time, the adult worms were selected out of the substrate and the offspring hatched from the cocoons. The substrate consisted of about 43.8 % animal manure, 43.8 % peat, 5.84 % lucerne meal, 5.84 % silicon dioxide and 0.73 % calcium carbonate (dry weight in each case).
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Remarks:
Study consisted of 2 parts. Adults were exposed to the test item for a period of 4 weeks (1st part): thereafter, adults were removed from test vessels and cocoons and juveniles remained in test vessels for additional 4 weeks (2nd part).
Test temperature:
20 ± 2 °C
pH:
start: 6.15 - 6.22
end: 5.84 - 6.23
Moisture:
start: 30.0 - 31.9% water content (51.64 - 56.45% of WHCmax)
end: 32.1 - 34.6% water content (57.06 - 63.75% of WHCmax)
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Non-re-usable plastic boxes (length x width x height ca. 16.5 cm x 12 cm x 6 cm, area approximately 200 cm2); covered with transparent lids to prevent earthworms from escaping and to reduce evaporation during the test period. The lids were perforated to allow air exchange.
- Amount of soil or substrate: 500 g artificial soil (dry weight); depth of approximately 5 cm soil in the test vessels
- 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)
- Composition (percentage distribution on dry weight basis): Sphagnum peat (shredded): 10%, Kaolinite clay (content of Kaolinite = 30.2 %) 20%, Industrial quartz sand (Particle size: 0.20 mm – 0.05 mm = 91.35%): 69%, Food (dried ground cow manure): 1%
- The artificial soil was prepared by mixing the dry components intensely in a laboratory mixer. Then, the soil was pre-moistened with deionised water.
- Maximum water holding capacity (in % dry weigth): 45.35% (82.99 g water/100 g dry artificial soil)
- pH: Calcium carbonate for the adjustment was added

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour darkness
- Light intensity: 400 - 800 Lux (mean light intensity was 562 Lux at day 0, 552 Lux at day 28 and 518 Lux at day 56 of the study)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality and adverse effects of adults after 4 weeks of exposure; Growth of adults at the beginning and after 4 weeks; Reproduction in terms of surviving juveniles after 8 weeks


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
Nominal and measured concentrations:
control, 100, 178, 316, 562, and 1000 mg/kg dw (nominal)
control, 18.2, 32.4, 57.51, 102 and 182 mg a.i./kg dw (calculated)
Reference substance (positive control):
yes
Remarks:
Carbendazim EC 360 G
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:
other: reproduction and growth
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 182 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: reproduction and growth
Details on results:
- Mortality at end of exposure period: none
- Total mass of adults at beginning of test: 103,71 g
- Changes in body weight of live adults (% of initial weight) at end of exposure period: The mean body weight of the adult earthworms in the control group had increased during the 4 weeks of exposure. The mean body weight was 0.48 g per worm (+31.33% of the mean initial weight). The mean changes in body weight of the test item concentrations of 100, 178, 316, 562, and 1000 mg test item/kg dry weight artificial soil were +25.35, +20.76, +30.09, 36.38, and 41.56 % respectively.
- No. of offspring produced: In the control group, on average 181.9 juvenile earthworms per test vessel were found (corresponding to a mean reproduction rate of 18.2 juveniles per surviving adult). The mean reproduction of the test item concentrations of 100, 178, 316, 526 and 1000 mg test item/kg dry weight artificial soil were 175.0, 191.5, 195.3, 173.8 and 171.8 juveniles respectively. This corresponds to 96.2, 105.3, 107.4, 95.5 and 94.4 % of the reproduction of the control.
- Morphological abnormalities: none
- Behavioural abnormalities: none
Results with reference substance (positive control):
The toxic standard reference test, with the reference test item mixed into the artificial soil, was performed last from September 21 to November 28, 2012 (Study No.: Rg-R-Ref 19/12; Report No. kra-Rg-R-Ref 19/12; NON-GLP)
- Results with reference substance valid? yes
- Relevant effect levels: EC10, EC20 and EC50 for reproduction were calculated to be 3.06, 3.22, and 3.54 mg a.s./kg dry weight artificial soil, respectively. Confidence limits (95 %) could not be calculated. The results of the reference test indicated that the test system was sensitive to the reference test item.
Reported statistics and error estimates:
Growth: The homogeneity of variances of the data was checked by Cochran’s test. The homogeneity hypothesis was even after transformation rejected. The normal distribution of the data was tested by Kolmogorov-Smirnov test. The normality hypothesis of the data was accepted. The data were statistically evaluated by means of a U-test after Williams multiple sequential t-test. The statistical software package ToxRatPro Version 2.10@ was used for the calculation.
Reproduction: The homogeneity of variances of the data was checked by Cochran’s test. The homogeneity hypothesis was accepted. The normal distribution of the data was tested by Kolmogorov-Smirnov test. The normality hypothesis of the data was accepted. The data were statistically evaluated by means of a Williams multiple sequential t-test. EC10 and EC20 could not be calculated. The statistical software package ToxRatPro Version 2.10@ was used for the calculation.

Table 1: Validity criteria for OECD 207

Criterion from the guideline

Outcome

Validity criterion fulfilled

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

194, 206, 192, 184, 167, 198, 155, 159

yes

the coefficient of variation of reproduction to be ≤ 30 %

10.5%

yes

adult mortality over the initial 4 weeks of the test to be ≤ 10 %

0%

yes

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (56 d, reproduction) ≥ 1000 mg a.i./kg dw nominal (Eisenia fetida, OECD 222)

LC50 (14 d, mortality) > 869 mg/kg soil dw nominal (Eisenia fetida, OECD 207)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
869 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
1 000 mg/kg soil dw

Additional information

Two GLP guideline studies are available on the toxicty of the substance towards earthworms (Eisenia fetida) (M-489574-01-1, M-469589-01-1). One study invetigates the long-term toxicity according to OECD guideline 222 and the second study tested the short-term effects to E. fetida according to OECD guideline 207.

 

In the key study (M-489574-01-1) the long-term toxicity of the test item to earthworms was investigated. The study was performed according to OECD 222 and GLP. Adult earthworms (at least 2 months old, four replicates of 10) were exposed in an artifical soil system containing 69.5% quartz sand, 20% kaolin clay, and 10% sphagnum peat. The test organisms were exposed to concentrations of 100, 178, 316, 562, and 1000 mg/kg dry weight (corresponding to 18.2 – 182 mg a.i./kg dry weight). The study consisted of two parts. Adults were exposed to the test item for a period of 4 weeks (1st part). Thereafter, adults were removed from test vessels and cocoons and juveniles remained in test vessels for additional 4 weeks (2nd part). In addition, an untreated water control was tested. A toxic reference (Carbendazim EC 360 G) was tested in a separate study. Temperature ranged from 18 to 22 °C and the photoperiod was 16-hour light to 8-hour darkness at 400 - 800 lux. Mortality and adverse effects of adults after 4 weeks of exposure, growth of adults at the beginning and after 4 weeks, and reproduction in terms of surviving juveniles after 8 weeks were used to determine the long-term toxicity. No effects were observed up to the highest tested concentration of 1000 mg/kg dry weight. Therefore, NOEC (28 d, growth) and NOEC (56 d, reproduction) were ≥ 1000 mg/kg dry weight soil (corresponding to 182 mg a.i./kg dry weight soil). All validity criteria were met and the study is valid.

 

One further study is available investigating the short-term toxicity of the substances to the earthworm Eisenia fetida (M-469589-01-1). The study was performed according to OECD 207 and GLP with an artificial soil containing 69.5% quartz sand, 20% kaolin clay, 10% sphagnum peat and 0.5% CaCO3. Adult earthworms (about 3 months old, four replicates of 10) were exposed over a period of 14 d to concentrations of 89.6, 159.49, 283.14, 503.56 and 869 mg a.i./kg dry weight. In addition, an untreated water control was tested. A toxic reference (2-chloroacetamide) was tested in a separate study. Temperature ranged from 18 to 22 °C and the photoperiod was continuous light at 550 lux. Mortality and biomass change were determined after 14 d and were used to determine the effect concentrations. The LC50 (14 d) was > 869 mg a.i./kg dry weight soil (the highest concentration tested), the NOEC (14 d) was determined to be ≥869 mg a.i./kg dry weight soil. All validity criteria were met and the study is valid.