Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

Based on the specified water solubility the application of the test substance via an aqueous stock solution was prepared with deionized water.
The amount of 5000.1 mg test substance was weighed into a 1 L volumetric flask and dissolved by adding of deionized water. After a shortstirring, the test substance was completely dissolved and the stock solution was blue.
Subsequently, the stock solution made up to the mark. The stock solution was prepared on 19 July 2016 (at day of exposure).
For preparation of the scheduled test substance concentrations, required aliquots of the stock solution were filled in a measuring cylinder. Enough deionized water was added to reach to volume for adjustment of the water content in the prepared test substrate. These test substance solutions were added to 2.5 kg test substrate dry weight.

Preparing the test substance assays
For preparation of the test assays with test substance amounts of about 2525 g dry test substrate (depend on the water content of the dry test substrate) were added to a bowl.
Required aliquots of the test substance stock solution were added to a measuring cylinder and the volume was completed to 725 mL with deionized water. After mixing the solution with the test substance was added to the test substrate in the mixing bowl and thoroughly mixed with the test substrate. Four portions of approx. 715 g of the treated wet test substrate for each test concentration were distributed to preserving jars.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Agricultural Center Limburgerhof of BASF SE, 67056 Ludwigshafen, Germany
- Age at test initiation (mean and range, SD): approx. 8 months
- Weight at test initiation (mean and range, SD): 300 mg – 587 mg

ACCLIMATION
A transport box containing approx. 1950 g test substrate was provided to the supplier. Therefore, the amount of approx. 1515 g dry test substrate was moistened with 435 mL deionized water, in order to adjust the content of water to 30 %.
One day before start of exposure a sufficient number of worms were sorted out by the supplier from the breeding box and placed in the transport box with the prepared test substrate. For acclimatization to the prepared test substrate and to the test temperature the transport box was stored in the incubation room for one day under the temperature of 20 ± 2°C and continuous light without feeding.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

21.1 – 21.4 °C

pH:

5.9 mean value of 2 single values (5.8 and 5.9)

Moisture:

Moisture content of the current test substrate: 1.0 % (mean value of two single values, 1.0%/1.0%)
Water content of the test substrate at start of exposure: 29.4 % (mean of 2 single determinations: 29.5% and 29.2 %)
Water content of the test substrate at the end of the exposure period: 28.4 % (mean of 2 single determinations: 28.7 % and 28.0 %)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Preserving jars, volume 1 L, covered with glass lids
- Amount of soil or substrate: 715 g ± 1 g artificial soil (wet 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 (if soil)
Actual mixture:
23.1 kg quartz sand (70 % of total test substrate mass)
Supplier: Gebrüder Willersinn GmbH &Co. KG, 67071 Ludwigshafen, Germany
6.6 kg kaolin clay (19.9 % of total test substrate mass)
Supplier: Bassermann minerals GmbH, 68169 Mannheim, Germany
3.3 kg sphagnum peat (10.0 % of total test substrate mass), finely grounded
Supplier: Raiffeisen Mannheim eG, 68307 Mannheim, Germany
150 g CaCO3, p.A. (0.5 % of total test substrate mass)
Supplier: Merck
Total mass of the test substrate: 33.15 kg

OTHER TEST CONDITIONS
- Photoperiod: Continuously over the exposure period
- Light intensity:
Measurement of light intensity,once at start of exposure: 517 Lux, mean value of 5 single values (583, 446, 513, 532, 511 [Lux])
Measurement of light intensity, once at start of adaptation: 783 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality and behaviour of the worms were observed on day 7 and 14 of exposure. The body weight change was assessed at the end of exposure by determination of the single weights of the live worms at start and end of exposure. Observations on day 7 and day 14 of exposure were documented. After 7 and 14 days, the number of dead worms for each test concentration was determined.
The body weights were determined after 14 days of exposure and the weight change was calculated. Additionally the behavioral effects were recorded after 7 and 14 day of
exposure.


TEST CONCENTRATIONS
- Concentrations of the test substance: 0 (control), 62.5, 125, 250, 500, 1000 mg/kg soil dry weight (nominal value, factor 2)
- Reason for the selection of the test concentrations: The test guidelines recommends a range of five concentrations, up to 1000 mg/kg soil dry weight

Nominal and measured concentrations:

0 (control), 62.5, 125, 250, 500, 1000 mg/kg soil dry weight (nominal value, factor 2)

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide

Key result

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 on result:

other: no toxic effects were observed up to the highest tested concentration

Key result

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

Remarks on result:

other: no toxic effects were observed up to the highest tested concentration

Details on results:

No abnormal behaviour or toxic symptoms of the worms were observed up to the highest tested test concentration (1000 mg/kg dw) during the exposure. After an exposure period of seven days, a growth of fungi was observed at all test container. At the end of exposure, only a slight blue discolouration of the worms was observed at the highest tested concentration of 1000 mg/kg dry weight.

Results with reference substance (positive control):

The reference substance 2-Chloroacetamide was tested by a non-GLP project (start of exposure 19 July 2016). The raw data and the report are archived. The result of the reference substance is presented below.
LCx values [mg/kg soil (dw)], reproduction, conf. limits 95%
LC10: 21.0
LC50: 25.5
The LC50 value of the reference substance was in the recommended range of 20 mg/kg to 80 mg/kg. The value shows that the test system was sufficiently sensitive and suitable to determine the acute toxicity of the test substance based on the test guideline.

Validity criteria fulfilled:

yes

Conclusions:

No toxic effects have been observed in studies with soil living organisms. No effects have been observed at the highest test concentration.

Description of key information

No toxic effects have been observed in studies with soil living organisms. No effects have been observed at the highest test concentration.

Additional information

An acute toxicity study according to OECD 207 and GLP has been conducted using Eisenia fetida. After 14 d no abnormal behaviour or toxic symptoms of the worms were observed in the control group and at the test substance groups up to the highest tested concentration of 1000 mg/kg dw) during the exposure. Therefore, the NOEC was determined to be >= 1000 mg/kg soil dw. The LOEC was determined to be > 1000 mg/kg soil dw.