2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.03,19.05,17.06,14.07,12.023,25.024,32.025,30]hexatriaconta-1(36),2,5,7,9,11,14,16,18,20,23,25,27,29,32,34-hexadecaene

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD 207

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

DIN ISO 11268-1, 1997

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The test item was weighed out and dissolved in acetone. This solution was mixed thoroughly with quartz sand (10 g quartz sand per replicate) and the quartz sand evaporated to dryness. The treated quartz sand was given to the artificial soil. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 54 % of the maximum water holding capacity. Thereafter, the test medium was mixed with a long handled blender for at least three minutes and filled into the test vessels (555 g dry weight each).
- Controls: Artificial soil without test item was used as control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):Acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 2.5 mL per replicate
- Evaporation of vehicle before use: Yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida Savigny 1826 (Annelida, Lumbricidae)
- Source: Own breeding
- Age at test initiation (mean and range, SD): adult (with clitellum)
- Weight at test initiation (mean and range, SD): 0.50 g/worm


ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not):same as test
- Health during acclimation (any mortality observed): No mortality or abnormal behaviour observed

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

19-22 °C

pH:

Nominal test item
concentration
[mg/kg soil dry weight]
pH-value

day 0 day 14
Control 5.62 5.64
Solvent Control 5.65 5.42
1000 5.64 5.36

Moisture:

Nominal test item
concentration
[mg/kg soil dry weight] Moisture [%] of dry weight
day 0 day 14
Control 31.9 31.4
Solvent Control 31.6 30.9
1000 32.3 31.4

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass dishes (volume 1.5 L), covered with perforated plastic film to prevent the test medium from drying up
- Amount of soil or substrate: 555 g
- No. of organisms per container (treatment): 10 per container (40 per treatment)
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)

- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % quartz sand, 0.37 % calcium carbonate
- Organic carbon (%): Not measured
- Maximum water holding capacity (in % dry weigth): 64
- Pretreatment of soil: No pretreatment except pre-moistening two days before test start
- Storage (condition, duration): room temperature, protected from light, 16 days



OTHER TEST CONDITIONS
- Photoperiod: 16
- Light intensity: 573 +/- 39 lx


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The live weight of the earthworms was measured individually at the day of application (day 0) and at day 14.
Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 days after application.

Observation parameter
a) no obvious pathological symptoms
b) no reaction to touching
c) no negative phototactical reaction
d) spasmodic winding
e) spontaneous segmentation and separation
f) yellow excretion from the oral aperture
g) ulcer and skin bleedings
t) dead earthworms


VEHICLE CONTROL PERFORMED: Yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: -
- Range finding study No

Nominal and measured concentrations:

Nominal concentration: 1000 mg/kg soil DW

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide

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:

mortality

Remarks:

loss of body weight, other symptoms

Details on results:

- Mortality at end of exposure period: No mortality observed in the control, no signiificant mortality in the test item concentration
- Total mass of adults at beginning of test: 0.50 g/worm
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: Control: 0.47 g/worm, 1000 mg/kg: 0.48 g/worm
- Morphological abnormalities:One earthworm showed ulcer and skin bleedings at the limit test item concentration of 1000 mg/kg soil dry weight after 14 days of exposure to the test item. All other earthworms in the control and at the limit test item concentration showed no symptoms or behavioural changes.
- Behavioural abnormalities: No
- Other biological observations: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: LC50: 50.1 (CI: 45.8 - 55.5) mg/kg doil DW, NOEC: 30.0 mg/kg soil DW

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA, DUNNETT’S Method) as well as a t-test were carried out for the determination of statistically significant differences compared to control replicates. A Normality Test and an Equal Equal Variance Test were conducted prior to ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA (acceptable probability of incorrectly concluding that there is no difference) is a=0.05.

No mortality occurred at the limit test item concentration of 1000 mg/kg soil dry weight as well as in the controls after 14 days of exposure to the test item.

During the test the biomass loss of the earthworms was below 20 % in the controls. After 14 days of exposure the earthworm biomass was not statistically significant different at the limit test item concentration of 1000 mg/kg soil dry weight compared to the pooled control

Validity criteria fulfilled:

yes

Conclusions:

No mortality as well as no noteworthy pathological symptoms or changes in the earthworm behaviour were observed at the limit test item concentration of 1000 mg/kg soil dry weight after 14 days of exposure to PV Echtviolett RL. Compared to the control the biomass of earthworms exposed to test item at the limit test item concentration of 1000 mg/kg soil dry weight was not statistically significantly reduced at test end.
Consequently, the No Observed Effect Concentration (NOEC) of the test item with respect to mortality, loss of body weight and other symptoms was determined to be at 1000 mg/kg soil dry weight.

Executive summary:

The acute effects of the test item (Batch: DEB 2060256) on the earthworm Eisenia fetida were determined according to the OECD Guideline 207 and DIN ISO 11268-1 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from April 9^th to April 24^th, 2009. The study was conducted with a limit test item concentration of 1000 mg/kg soil dry weight throughout 14 days. Artificial soil without test item was used as control and artificial soil without test item but treated with the solvent was used as solvent control. 40 test organisms with an individual weight between 0.3 - 0.6 g, divided into four replicates, were tested at the limit test item concentration and controls in artificial soil containing 10 % peat.

No mortality as well as no noteworthy pathological symptoms or changes in the earthworm behaviour were observed at the limit test item concentration of 1000 mg/kg soil dry weight after 14 days of exposure (Table 1). Compared to the pooled control, the biomass of the earthworms at the limit test item concentration of 1000 mg/kg soil dry weight was not statistically significantly reduced at test end.

Consequently, the no observed effect concentration (NOEC) with respect to mortality, loss of biomass and other symptoms was determined to be at 1000 mg/kg soil dry weight.

All validity criteria of the test guideline were fulfilled.

Summary of all Effects

Effects	Test item [mg/kg soil dry weight]
LOEC Mortality	> 1000
LOEC Loss of body weight	> 1000
LOEC behaviour and pathological symptoms	> 1000
NOEC mortality, loss of body weight, other symptoms	1000

Description of key information

The acute effects of the test item (Pigment violet 23) the earthworm Eisenia fetida were determined according to the OECD Guideline 207. The study was conducted with a limit test item concentration of 1000 mg/kg soil dry weight throughout 14 days. The no observed effect concentration (NOEC) with respect to mortality, loss of biomass and other symptoms was determined to be at 1000 mg/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

1 000 mg/kg soil dw

Additional information

The acute effects of the test item (Pigment Violet 23) on the earthworm Eisenia fetida were determined according to the OECD Guideline 207. The study was conducted with a limit test item concentration of 1000 mg/kg soil dry weight throughout 14 days.

No mortality as well as no noteworthy pathological symptoms or changes in the earthworm behaviour were observed at the limit test item concentration of 1000 mg/kg soil dry weightafter 14 days of exposure.Compared to the pooled control, the biomass of the earthworms at the limit test item concentration of 1000 mg/kg soil dry weight was not statistically significantly reducedat test end.

Consequently, the no observed effect concentration (NOEC) with respect to mortality, loss of biomass and other symptoms was determined to be at 1000 mg/kg soil dry weight.