2,9-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-08-20 - 2008-09-3

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:

yes

Remarks:

Only 2 tested concentrations in the manner of a limit test (recommmended: 5); recommended pH-value by 0.2-0.3 lower as recommended, T-range 17-21°C (20 +/- 2°C recommended): without influence on results.

GLP compliance:

yes (incl. QA statement)

Remarks:

2004; GLP according to German Chemikaliengesetz and Directive 88/320/EEC;

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on preparation and application of test substrate:

The test item is poorly soluble in water and insoluble in suitable organic solvents (e.g. hexane, acetone or chloroform); therefore, a mixture of finely ground quartz sand and the test item was produced for each test item concentration separately. All concentrations to be tested were based on the test item.
The test item was mixed in an amount of quartz sand sufficient to prepare a mixture for each test item concentration separately. An appropriate amount of these test item/quartz sand mixtures served to prepare the different concentrations of the test item in the artificial soil.
The test item was incorporated into the remainder of the artificial soil by thoroughly mixing in the respective test item/quartz sand mixtures for approx. 10 minutes as described in the test guideline.
Finally, the treated artificial soil was filled into the test vessels. Per test vessel an amount corresponding to 500 g dw was used

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.
Breeding and keeping of the test organisms follows the guideline.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Remarks:

Other than with an usual limit test, two concentrations had been tested: 100 and 1000 mg/kg soil dry weight

Post exposure observation period:

not applicable

Test temperature:

17-21°C

pH:

Control: 5.3 (Start) - 5.3 (End)
100 mg/kg soil dw: 5.2 (Start) - 5.2 (End)
1000 mg/kg soil dw: 5.2 (Start) - 5.2 (End)

Moisture:

Soil moisture: 40-70 % of the WHCmax (70.9%) of the artificial soil = 28.4 - 42.5% of the dry weight of the aritificial soil.
[% WHCmax]
Control: 54.9 (Start) - 59.1 (End)
100 mg/kg soil dw: 57.5 (Start) - 59.3 (End)
1000 mg/kg soil dw: 56.8 (Start) - 56.0 (End)

Details on test conditions:

The test was performed with two concentrations and an untreated deionised water control. Per concentration and control four test vessels (= replicates) each containing 10 earthworms were used.

Preparation of the artificial soil:
- The dry constituents of the artificial soil were prepared as described in the guideline. 2.86% of the quartz sand was separated for the application of the test item. Calcium carbonate was used to adjust the pH-value to 6 ± 0.5. The maximum water capacity (WHCmax) of the artificial soil was determined.
- One day before starting the test the artificial soil (per replicate 500 g dw) was pre-moistened with deionised water (20 ¿ 40% 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:
- Preparation of the test item mixtures. An appropriate amount of the test item/quartz sand mixtures was incorporated into the remainders of the artificial soil to prepare the different test item soil concentrations.
- At the same time an appropriate amount of deionised water was added to adjust the final soil moisture (40 ¿ 60% of WHCmax).
- The respective batches of test substrate were mixed thoroughly and the artificial soil was filled into the test containers (corresponding to 500 g dw).
- Determination of the pH-value and the moisture of the artificial soil once for each concentration (samples are taken from one test vessel).

Introduction of worms into the test containers:
- The acclimatised worms were weighed (individually) immediately after application of the test item. Sets of 10 worms were placed onto the soil surface of each test container randomly.
- The test vessels were kept into an incubator or a climate chamber with controlled abiotic conditions (esp. temperature) and permanent light according to the guideline (640-779 Lux).

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. The combined weight of surviving worms from each replicate was determined. Afterwards the 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 individual weight of the surviving worms from each replicate was determined.
- Determination of the pH-value and the moisture of the artificial soil once in each concentration (samples were taken from one test vessel).

Nominal and measured concentrations:

The test item was tested in two concentrations. The test item concentrations (specified in a previous Range-Finding Test not covered by the study plan) were as follows:
1000 mg test item/kg soil dw (dry weight) 100 mg test item/kg soil dw (dry weight) nominal concentration.

Reference substance (positive control):

yes

Remarks:

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

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:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Adult Biomass

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Adult Biomass

Details on results:

Mortality:
No mortality was observed at the control and at all concentrations of the test item tested.

Adult biomass:
Statistical analysis (Student-t test; 2-sided, p ¿ 0.05) showed no significant difference in the biomass development of individual adults after 14 days between the control and the two concentrations of the test item tested.
Therefore, the NOECBiomass was considered to be ¿ 1000 mg test item/kg artificial soil (dw).
Cocoons were observed in all test vessels of the control and of both concentrations of the test item tested. Other effects on the morphology and the behaviour of the worms have not been observed.

Results with reference substance (positive control):

Dates of work: July 18, ¿ August 01, 2008.
Mortality: In the control, 0% of the adult worms died at the end of the test. At the
concentrations of 10 and 20 mg/kg soil (dw) 0% and at the concentration of 40 mg/kg soil (dw) 5% mortality was observed. At the concentration of 80 mg/kg soil (dw) 100% mortality was observed.
Comments: The LC50 value for the toxic reference item, chloroacetamide, was determined as 50.2 mg/kg. The LC50 value is within the range given in the ISO guideline 11268-1 (1997) and within the order of magnitude published in scientific literature (Kula 1998).

Reported statistics and error estimates:

Determination of the NOECBiomass
To evaluate the biomass development statistically, firstly the biomass of day 0 was subtracted from the biomass of day 14 for each of the four replicates of each treatment separately. These four values for each treatment were used for the statistical analysis of the biomass development.

One-way Analysis of Variance
p(F) (0.104) is greater than the selected significance level of 0.050; therefore, treatments are not significantly different.
R/s test procedure confirmed the correspondence with normal distribution. The hypothesis of homogeneity of the biomass data was accepted by the Cochran¿s test. Therefore, the pairwise comparison of the concentrations with the control test by the Student-t test was used for the statistical analysis.

STUDENT-t test for homogeneous Variances
Pair-wise comparison of treatments with "Control" by the t test procedure. Significance was Alpha = 0.05, two-sided;
The ANOVA and the Student-t test (2-sided, p ¿ 0.05) showed no significant difference in biomass development of individual adults after 14 days between the control and both concentrations of the test item tested.
Other effects on the morphology and the behaviour of the worms have not been observed. Therefore, the NOEC was considered to be ¿ 1000 mg test item/kg artificial soil (dw).

Mean live weight per surviving worm at all concentrations and the control at the beginning of the test, after 7 and 14 days (n = 40). Data are given as absolute values (biomass) and in percent of the initial weight:

Concentration [mg test item/kg soil (dw)]	Biomass (mg fresh weight)			Percent of the initial weight
	Day 0	Day 7	Day 14	Day 7	Day 14
Control	401.9 ± 21.9	430.9 ± 11.5	405.1 ± 19.0	107.2	100.8
100	407.0 ± 17.4	429.3 ± 17.4	407.3 ± 18.9	105.5	100.1
1000	404.3 ± 25.3	428.0 ± 4.0	400.6 ± 10.5	105.9	99.1

Validity criteria fulfilled:

yes

Conclusions:

An acute toxicity test on earthworm Eisenia fetida had been performed with the submission substance (realibility category 1, compliant to GLP). Of the two tested concentrations (100 and 1000 g/kg soil dry weight), neither caused any death nor statistically significant changes in body weight compared to the control. Therfore, the
NOEC (death / body weight, 14d) = 1000 mg/kg soil dry weight,
LC50 (14 d) > 1000 mg/kg soil dry weight,
EC50 (body weight, 14d) > 1000 mg/kg soil dry weight.

Executive summary:

An acute toxicity test according to OECD 207 on earthworm Eisenia fetida had been performed with the submission substance (realibility category 1, compliant to GLP). No mortality was observed at the control and at all concentrations of the test item tested.

Statistical analysis (Student-t test; 2-sided, p ¿ 0.05) showed no significant difference in the biomass development of individual adults after 14 days between the control and the two concentrations of the test item tested.

Therefore, the NOEC was considered to be ¿ 1000 mg test item/kg artificial soil (dw).

Description of key information

One key study with the substance is available. An acute toxicity test on earthworm Eisenia fetida had been performed with the submission substance (reliability category 1, compliant to GLP). Of the two tested concentrations (100 and 1000 g/kg soil dry weight), neither caused any death nor statistically significant changes in body weight compared to the control. Therefore, the

NOEC (death / body weight, 14d) = 1000 mg/kg soil dry weight,

LC50 (14 d) > 1000 mg/kg soil dry weight,

EC50 (body weight, 14d) > 1000 mg/kg soil dry weight.

Based on the result of the short term test a long term toxicity test was waived.

Waiving according to "column 2" in Annex IX and X of REGULATION (EC) No 1907/2006 (CSA does not indicate need for further investigations)

Key value for chemical safety assessment

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

1 000 mg/kg soil dw