N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-02-02 to 2012-02-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

and ISO 11268-1 (1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

271-178-7

EC Name:

N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

68516-75-6

Molecular formula:

C44H26Cl4N6O4

IUPAC Name:

N,N'-naphthalene-1,5-diylbis{4-[(2,3-dichlorophenyl)diazenyl]-3-hydroxy-2-naphthamide}

Test material form:

solid: nanoform, no surface treatment

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used):
The limit test item concentration was weighed out and dissolved in acetone (2.5 mL per replicate). The dissolved test item was mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent, the treated quartz sand was given to the artificial soil. Additional demineralised water was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 40 - 60% of the maximum water holding capacity.
Subsequently, the test medium was thoroughly mixed to ensure a homogeneous distribution and about 555 g soil dry weight were filled into each test vessel.
- Controls: Artificial soil moistened with demineralised water without test item was used as control medium.
- Solvent Control: Artificial soil with addition of quartz sand treated with acetone and moistened with demineralised water without test item was used as solvent control medium.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm Eisenia fetida Savigny 1826
- Source: Own breeding
- Age at test initiation: Adult
- Weight at test initiation (mean and range, SD): individual live weight of 0.30 to 0.60 g
Control: 0.48 +/- 0.03, Solvent Control: 0.48 +/- 0.03; 1000 mg/kg: 0.48 +/- 0.04


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

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

No

Test conditions

Test temperature:

nominal: 20 +/- 2 °C, actual: 18 - 21 °C

pH:

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

day 0 day 14
Control 5.13 5.03
Solvent Control 5.10 4.99
1000 5.08 5.01

Moisture:

Test item concentration
[mg/kg soil dry weight]] Moisture [%] of dry weight
day 0 day 14
Control 33.9 33.3
Solvent Control 33.0 32.8
1000 32.9 33.6

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass dishes, covered with perforated plastic film
- Amount of soil or substrate: 555 g (dry weight)
- No. of organisms per container (treatment): 10 per replicate
- No. of replicates per treatment group: 4
- No. of replicates per control, solvent control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat (air dried and finely ground), 20 % kaolin (kaolinite content 84 - 87 %), 69 % quartzsand (sand with > 50 % particles size of 0.05 to 0.2 mm), 0.39% calcium carbonate (CaCO3) to adjust the pH to 6.0 ± 0.5
- Organic carbon (%): 10 % peat
- Maximum water holding capacity (in % dry weigth): 69
- Storage (condition, duration): under test conditions for 9 days
- Pretreatment of soil: Two days before earthworm insertion into the test vessels the artificial soil (10 kg) was pre-moistened with 1100 g deionised water to achieve a soil moisture of 26 % of the maximum water holding capacity of the artificial soil. Until start of the test evaporation of the artificial soil was avoided.

OTHER TEST CONDITIONS
- Photoperiod: 24 h
- Light intensity: 400 - 800 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none, limit test
- Range finding study
Range Finding Test (14 d, non - GLP): Mortality and Change of Live Weight [%]
Concentration
Test item
Mortality Change of Live weight
[mg/kg Soil dry weight] [%] [%]
Control 0 -10
Solvent Control 0 -8
10 0 -12
100 0 -11
1000 0 -7

Nominal and measured concentrations:

Nominal: 1000 mg/kg soil DW

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide

Results and discussion

Effect concentrations

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, behaviour and pathological symptoms

Details on results:

- Mortality at end of exposure period: No evident mortality (<10 %) occurred in the controls as well as at the limit test item concentration after 14 days of exposure to the test item
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
During the test the biomass loss of the earthworms was below 20 % in the control. There was no statistically or biologically significant difference between earthworm biomass development in the pooled control and in the limit test item group
- Morphological abnormalities: No pathological symptoms or changes in the earthworm behaviour were observed in the control as well as in the limit test item concentration after 7 and 14 days of exposure
- Other biological observations: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The LC50-value ranged between 20 and 80 mg/kg soil dry weight as recommended by the guideline
please refer to "Any other information on materials and methods"

Reported statistics and error estimates:

Because no biological differences were observed, both controls were pooled for statistical evaluation.
A t-test was carried out for the determination of statistically significant differences compared to the pooled control

Any other information on results incl. tables

Earthworm Mortality in [%] after 7 and 14 Days of Exposure

Mortality [%]
Test item concentration [mg/kg soil dry weight]	Day 7					Day 14
	1	2	3	4	MV	1	2	3	4	MV
Control	0	0	0	0	0	0	0	0	0	0
Solvent Control	0	0	0	10	2.5	0	0	0	10	2.5
1000	0	0	0	0	0	0	0	0	0	0

Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure

Test item concentration [mg/kg soil dry weight]	Effect	Day 7 Replicate				Day 14 Replicate
		1	2	3	4	1	2	3	4
Control	A)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
Solvent Control	A) H)	10/10	10/10	10/10	9/10 1/10	10/10	10/10	10/10	9/10 1/10
1000	A)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10

Mean Biomass and Mean Loss of Body Weight

	Mean live weight
Test item  concentration  [mg/kg soil dry weight]	Test start MV ± SD [g/worm]	Test end MV ± SD [g/worm]	Test end CV [%]	Change of  live weight [%]	Statistically significant vs. Pooled Control
Control	0.48 ± 0.03	0.48 ± 0.04	8	0	–
Solvent Control	0.48 ± 0.03	0.46 ± 0.05	11	- 4
1000	0.48 ± 0.04	0.48 ± 0.04	8	0	No

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No evident mortality (<10%) as well as no pathological symptoms or changes in the earthworm behaviour were observed in the control and solvent control as well as at the limit test item concentration of 1000 mg test substance /kg soil dry weight. Compared to the pooled control, no statistically or biologically significant difference of biomass was observed in the limit test item concentration at test end.
Consequently, the No Observed Effect Concentration (NOEC) with respect to mortality, loss of body weight and other symptoms was determined to be at 1000 mg test substance /kg soil dry weight.