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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:
19 Feb 2015 to 10 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study According to GLP
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
yes
Remarks:
In the control and two test item concentrations the initial pH value was 5.4 which is slightly lower than the range of pH 6.0 +/- 0.5 recommended by the guideline. This deviation is considered to have had no impact on the outcome of the study.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Vehicle:
yes
Details on preparation and application of test substrate:
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 content of the test item Benzyl Salicylate (purity 99.9%).

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 was 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 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.
Test organisms (species):
Eisenia fetida
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: kept at ECT since February 1994. Originally delivered by Co. Landenberger
- Age at test initiation: adult (with clitellum)
- Weight at test initiation: between 300 and 600 mg

ACCLIMATION
- Acclimation period: at least 24 hours
- Acclimation conditions (same as test or not): in untreated artificial soil
- Health during acclimation (any mortality observed): not reported
Study type:
laboratory study
Substrate type:
artificial soil
Total exposure duration:
14 d
Test temperature:
18.0 - 21.9°C (recommended 20 ± 2 °C)
pH:
Day 0: 5.4 - 5.6 (recommended 5.5 – 6.5)
Day 14: 5.3 - 5.5

In the control and at the concentrations of 178 and 562 mg test item/kg soil (dw) the initial pH value was slightly lower (5.4) than recommended by the guideline (5.5 – 6.5). Since the worms showed no effects on behaviour (e.g. avoidance of the substrate) this deviation is considered to have had no impact on the outcome of the study.
Moisture:
Soil moisture (day 0): 49.4 - 53.8% of WHCmax (recommended 40-60% of WHCmax)
Soil moisture (day 14): 46 - 48.3% of WHCmax

WHCmax = the maximum water capacity of the artificial soil
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1.7L preservation jars (Co. Bauer, Tamm) covered by perforated twist-off tops
- Amount of soil or substrate: 500g dw (dry weight) per test vessel
- No. of organisms per container (treatment): ten worms per replicate
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control (solvent treated): 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 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 Kow ≥ 2) 5% instead of 10% peat was used considering the influence on bioavailability (EPPO 2003).
- Maximum water holding capacity (in % dry weight): adjusted to 40 – 60% of WHCmax using deionised water
- Pretreatment of soil: 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.

OTHER TEST CONDITIONS
- Light cycle: continuous light
- Light intensity: 405 - 728 Lux (recommended 400 - 800 Lux)
- Feeding: none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality (%)
- Biomass (% of initial weight) determined after 14 days
- Morphology and behaviour

VEHICLE CONTROL PERFORMED: yes, solvent control

TEST CONCENTRATIONS
- Range finding study: 0.1, 1.0, 10.0, 100 and 1000 mg test item/kg artificial soil dry weight (dw).
- Definitive Test concentrations: 100, 178, 316, 562 and 1000 mg test item/kg artificial soil dry weight (dw)
- Results used to determine the conditions for the definitive study: Adult mortality after 14 days in range finder = 0% (0.1 to 100 mg test item/kg soil dw) and 70% (1000 mg test item/kg soil dw).
Nominal and measured concentrations:
Control
Solvent Control
Nominal: 100, 178, 316, 562 and 1000 mg test item/kg artificial soil dry weight
Reference substance (positive control):
yes
Remarks:
Chloroacetamide
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
562 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
5% mortality was observed in the control, the solvent control and at the concentration of 178 mg test item/kg soil dry weight (dw). At the concentrations of 100 and 562 mg test item/kg soil (dw) no mortality was observed. At the concentration of 316 and 1000 mg test item/kg soil (dw), 2.5% and 45% mortality was observed, respectively.

The percent of the initial weight of the earthworm biomass after 14 days was 106.7% for the control, 105.1% for the solvent control and 103.1% to 109.1% for all concentrations of the test item tested, respectively.

There were no effects on behaviour or morphology of the earthworms.
Results with reference substance (positive control):
The LC50 value for the toxic reference item, chloroacetamide, was determined as 74.9 mg/kg and is within the range, 20 - 80 mg Chloroacetamide/kg soil dry weight (dw), given in the ISO guideline 11268-1 (1997).
Reported statistics and error estimates:
Since mortality was < 50% the LC50 was not calculated and is regarded to be > 1000 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 highest test item concentration tested. Therefore, the NOEC(Mortality) was determined as 562 mg test item/kg soil (dw). Accordingly, the LOEC(Mortality) was determined as 1000 mg test item/kg soil (dw).

Table 1: Effects of Benzyl Salicylate on Mortality and Biomass of Eisenia fetida after 14 days of exposure

 Concentration

[mg test item/kg soil (dw)]

Mortality Day 7

[%]

Mortality Day 14

[%]

Biomass

[% of initial weight] 

 Control 5.0 5.0   106.7
 Solvent Control 5.0  5.0  105.1
 100 0.0 0.0  105.1
 178 5.0 5.0  103.1
 316 2.5 2.5  107.5
 562 0.0 0.0  106.2
 1000 45.0 45.0  109.1
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to earthworm Eisenia fetida in artificial soil has been investigated and gave a 14 day LC50 value of > 1000mg test item/kg soil dry weight (dw). The Lowest Observed Effect Concentration was considered to be 1000 mg test item/kg soil (dw). The No Observed Effect Concentration was 562 mg test item/kg soil (dw).

Description of key information

The acute toxicity of Benzyl Salicylate to the earthworm Eisenia fetida in artificial soil has been determined. The LC50 is regarded to be > 1000 mg test item/kg soil (dw).

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
1 000 mg/kg soil dw

Additional information

The acute toxicity of the test item to earthworm Eisenia fetida in artificial soil test has been investigated and gave a 14 day LC50 value of > 1000mg test item/kg soil dry weight (dw). The Lowest Observed Effect Concentration was considered to be 1000 mg test item/kg soil (dw). The No Observed Effect Concentration was 562 mg test item/kg soil (dw).