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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:
13 September 2016 through 06 January, 2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
NECTARYL :
Givaudan Item Ref. : 7446003
Lot No. : VE00441798
Purity : 95.3%
Date of Expiry : 27 May, 2017
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
The earthworms were tested in an artificial soil. The composition of this artificial soil was based on OECD Guideline No. 207 (1984) (the percentages refer to the dry weight):

- 5% Sphagnum peat (no visible plant remains; finely ground and air-dried)
- 20% Kaolin-Clay (kaolinite content > 30%)
- 74-75% Quartz sand (fine sand content with particles between 50 and 200 microns higher than 50%)
- 0.3-1% Calcium carbonate (CaCO3).

With respect to the properties of the test item (Log POW ≥ 2) 5% instead of 10% peat was used considering the influence on bioavailability (EPPO 2003). This is equivalent to a soil organic carbon content of approximately 2%.

The air dried peat was shredded in a chaff-cutter. Afterwards, all parts of the artificial soil were mixed homogeneously. The pH value was adjusted to 6 ± 0.5 using calcium carbonate. The moisture content was adjusted to 40 – 60% of WHCmax using deionised water. The water content of the artificial soil was determined by drying a small sample at 105 °C.


Pre-treatment of the Test Item :

The test item is not sufficiently soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone. All concentrations to be tested were based on the test item Nectaryl (purity 95.3%).

The test item was dissolved in an amount of acetone sufficient to prepare a stock solution. This stock solution was used to produce the various dosage solutions of the test item. An appropriate amount of the stock and the dosage solutions respectively were used to soak a portion of the quartz sand of the artificial soil by the solvent solution. The solvent control was prepared in the same way without the test item. After gentle evaporation of the solvent by placing under a fume hood, the quartz sand was coated by the test item.

The test item coated fraction of the quartz sand was then incorporated into the remainder of the artificial soil by thoroughly mixing it in for 5 minutes.

Finally, the treated artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g dw (dry weight) was used.


Chronological description of the test performance including methods used:

Preparation of the artificial soil:
- The dry constituents of the artificial soil were prepared as described, above. Calcium carbonate was used to adjust the pH-value to 6 ± 0.5. The maximum water capacity (WHCmax) of the artificial soil was determined.
- Four days before starting the test the artificial soil (per replicate 500 g dw) was pre-moistened with deionised water (40 – 60% of WHCmax).

Selection of worms:
- Adult worms were selected out of the culture in order to acclimatise them for at least 24 h in untreated artificial soil.

Test preparation (Day 0):
- Preparation of the test item solutions. The test item was dissolved in a sufficient volume of acetone to prepare a stock solution. This stock solution was dissolved with acetone to obtain one test item application solution (= dosage solution) for each test item concentration in such a way that an appropriate amount of these test item/acetone solutions were used to soak the 10% portion of the quartz sand which was separated before preparing the artificial soil. In the water control the same portion of quartz sand without solvent was added. In the solvent control same portions of quartz sand and acetone without test item were added.
- After gentle evaporating the solvent by placing the soaked quartz sand under a fume hood, the coated fractions of the quartz sand were incorporated into the remainder of the artificial soil to prepare the different test item soil concentrations. At the same time an appropriate volume of deionised water was added to adjust the final soil moisture (40 – 60% of WHCmax).
- The respective batches of artificial soil were mixed thoroughly until the test item solutions were homogeneously distributed.
- The respective artificial soil was filled into the appropriate test containers (corresponding to 500 g dw per container).
- The pH-value and the moisture of the artificial soil were determined once in each concentration and both controls (samples were taken from one test vessel).
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
The earthworms used in this test, Eisenia fetida (Lumbricidae, Earthworms) are kept at ECT Oekotoxikologie GmbH since February 1994. They were originally delivered by Co. Landenberger (D-72355 Schömberg).

Only adult worms (with clitellum) with a fresh weight between 300 and 600 mg were used. The selected test animals were acclimatised in untreated artificial soil at least 24 hours prior to the start of the test.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Remarks:
Observation made for mortality on Day 7 following application.
Test temperature:
20 +/- 2 °C
pH:
The pH value of the artificial soil substrate was adjusted to 6 +/- 0.5 using CaCO3.
Moisture:
Deionised water was employed to adjust the final soil moisture content during the study to between 40 and 60% maximum water holding capacity.
Details on test conditions:
Test Vessels : 1.7 L glass jars filled with 500 g dry weight equivalent of the artificial soil
Replicates : Four replicates per test level each containing 10 worms
Moisture Content : 40 - 60% Maximum WHC
Incubation Temperature : 20 +/- 2 °C
Light Cycle : Continuous light
Feeding : None

Introduction of worms (Day 0):
- The acclimatised worms were weighed individually and distributed into 32 units, each unit containing 10 individuals. These units were then organised in weight order, which is the heaviest unit to the lightest. These arranged units of 10 worms were separated into 4 groups consisting of eight units (=number of treatments (6) plus two controls). Within each group the units had approximately the same weight. Each of these groups was used for one replicate (e.g. replicate A of replicates A to D) of each treatment. Within the groups the units were assigned to the treatments.
- Afterwards the animals were put onto the soil surface of each test container.
- The test vessels were kept in a climate chamber with controlled abiotic conditions (especially temperature)

Day 7:
- The worms were removed carefully from their test containers. During this process, morphological and behavioural changes and the number of surviving worms in each test container were recorded. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. Afterwards the living worms were put back onto the soil surface in the containers.

Day 14:
- The worms were removed carefully from the test containers. During this process, morphological and behavioural changes and the number of surviving worms in each test container were recorded. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. The combined weight of the surviving worms from each replicate was determined.
- The pH-value and the moisture of the artificial soil were determined once in each concentration (samples were taken from one test vessel)
Nominal and measured concentrations:
The test item was tested in six concentrations. The concentrations of the test item (determined by a previous non-GLP Range-Finding Test were as follows :

132 mg test item/kg soil (dw)
198 mg test item/kg soil (dw)
296 mg test item/kg soil (dw)
444 mg test item/kg soil (dw)
667 mg test item/kg soil (dw)
1000 mg test item/kg soil (dw)

A Blank Control and a Solvent Control were run in parallel.
Reference substance (positive control):
yes
Remarks:
Chloroacetamide : Tested at 10, 20, 40 and 80 mg/kg tested frequently to assure the constant sensitivity of the test system (July 2016 - ECT Study No. IRA1606)
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
246.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI : 224.8 - 269.4 mg/kg dry weight
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
132 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
198 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Effects of NECTARYL on Mortality and Biomass of Esienia fetida :

Exposure Concn. Mortality Biomass
(mg test item/kg soil (dwt)) (%) (% of initial weight)

Day 7 Day 14 Day 14

Water / Blank Control 7.5 17.5 84.8
Solvent Control 0.0 0.0 94.5
132 0.0 0.0 76.9
198 7.5 35.0 63.7
296 27.5 70.0 57.9
444 45.0 92.5 62.0
667 92.5 100.0 -
1000 100.0 100.0 -
Results with reference substance (positive control):
Mortality: 5% mortality was observed in the control and at a concentration of 10 mg Chloroacetamide/kg soil (dw). 7.5% mortality occurred at concentrations of 20.0 Chloroacetamide/kg soil (dw). At a concentration of 40 mg Chloroacetamide/ kg soil (dw) 25% mortality was observed. At a concentration of 80.0 mg Chloroacetamide/kg soil (dw) 100% mortality was determined.
Comments: The LC50 value was calculated by probit analysis as 42.2 mg Chloroacetamide/kg soil (dw).This value is within the range (20 - 80 mg Chloroacetamide/kg soil dry weight (dw)) given in the ISO guideline 11268-1 (1997). In addition, the LC50 value is within the order of magnitude published in scientific literature (Kula 1998).
Validity criteria fulfilled:
yes
Conclusions:
Findings:
A clear dose-response effect was observed in the study. Although a mortality of 17.5% was observed in the water control, 0% mortality occurred in the solvent control and, as such, the study is considered valid. At the concentration of 132 mg test item/kg soil dry weight (dw) 0% mortality was also observed. At the concentrations of 198, 296 and 444 mg test item/kg soil (dw) mortality between 35.0 and 92.5% was observed. At the concentrations of 667 and 1000 mg test item/kg artificial soil (dw) 100% mortality was observed, respectively.
The LC50 value was calculated by Probit analysis using Linear Max. Likelihood Regression as 246.3 mg test item/kg soil (dw) (95% confidence interval: 224.8 – 269.4 mg test item/kg soil (dw)).
Fisher’s Exact Test (1-sided, p ≤ 0.05) showed a significant difference concerning mortality between the solvent control and the concentrations of 198, 296, 444, 667 and 1000 mg test item/kg (dw). Therefore, the NOECMortality was determined as 132 mg test item/kg soil (dw). Accordingly, the LOECMortality was determined as 198 mg test item/kg soil (dw).
The percent of the initial weight of the earthworm biomass after 14 days was 84.8% for the water control, 94.5% for the solvent control and 57.9% to 76.9% for all concentrations of the test item tested showing less than 100% mortality with no evident dose-response effect related to this end-point.
There were no effects on behaviour or morphology of the earthworms.
Executive summary:

Findings:

A clear dose-response effect was observed in the study. Although a mortality of 17.5% was observed in the water control, 0% mortality occurred in the solvent control and, as such, the study is considered valid. At the concentration of 132 mg test item/kg soil dry weight (dw) 0% mortality was also observed. At the concentrations of 198, 296 and 444 mg test item/kg soil (dw) mortality between 35.0 and 92.5% was observed. At the concentrations of 667 and 1000 mg test item/kg artificial soil (dw) 100% mortality was observed, respectively.

The LC50 value was calculated by Probit analysis using Linear Max. Likelihood Regression as 246.3 mg test item/kg soil (dw) (95% confidence interval: 224.8 – 269.4 mg test item/kg soil (dw)).

Fisher’s Exact Test (1-sided, p ≤ 0.05) showed a significant difference concerning mortality between the solvent control and the concentrations of 198, 296, 444, 667 and 1000 mg test item/kg (dw). Therefore, the NOECMortality was determined as 132 mg test item/kg soil (dw). Accordingly, the LOECMortality was determined as 198 mg test item/kg soil (dw).

The percent of the initial weight of the earthworm biomass after 14 days was 84.8% for the water control, 94.5% for the solvent control and 57.9% to 76.9% for all concentrations of the test item tested showing less than 100% mortality with no evident dose-response effect related to this end-point.

There were no effects on behaviour or morphology of the earthworms.

Description of key information

A clear dose-response effect was observed in the study. At the concentration of 132 mg test item/kg soil dry weight (dw) 0% mortality was also observed. At the concentrations of 198, 296 and 444 mg test item/kg soil (dw) mortality between 35.0 and 92.5% was observed. At the concentrations of 667 and 1000 mg test item/kg artificial soil (dw) 100% mortality was observed, respectively.

The LC50 value was calculated by Probit analysis using Linear Max. Likelihood Regression as 246.3 mg test item/kg soil (dw) (95% confidence interval: 224.8 – 269.4 mg test item/kg soil (dw)).

Fisher’s Exact Test (1-sided, p ≤ 0.05) showed a significant difference concerning mortality between the solvent control and the concentrations of 198, 296, 444, 667 and 1000 mg test item/kg (dw). Therefore, the NOECMortality was determined as 132 mg test item/kg soil (dw). Accordingly, the LOECMortality was determined as 198 mg test item/kg soil (dw).

The percent of the initial weight of the earthworm biomass after 14 days was 84.8% for the water control, 94.5% for the solvent control and 57.9% to 76.9% for all concentrations of the test item tested showing less than 100% mortality with no evident dose-response effect related to this end-point.

There were no effects on behaviour or morphology of the earthworms.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
246.3 mg/kg soil dw

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