4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxy-N-(2-methylphenyl)naphthalene-2-carboxamide

Sediment toxicity

Administrative data

Endpoint:

sediment toxicity: long-term

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2008-05-26 to 2008-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study compliant to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment (October 2007)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-(acetylamino)phenyl]-4-[[5-(aminocarbonyl)-2-chlorophenyl]azo]-3-hydroxynaphthalene-2-carboxamide

EC Number:

235-464-5

EC Name:

N-[4-(acetylamino)phenyl]-4-[[5-(aminocarbonyl)-2-chlorophenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Cas Number:

12236-64-5

IUPAC Name:

N-(4-acetamidophenyl)-4-[(5-carbamoyl-2-chlorophenyl)diazenyl]-3-hydroxy-2-naphthamide

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Details of spiking: The test substance concentration was weighed out, dissolved in 15 mL acetone and mixed thoroughly with 60 g quartz sand (10 g quartz sand per replicate) in a glass vessel. After complete evaporation of the solvent, the dry quartz sand was given to the sediment. Residues of the dry quartz sand sticking at the glas vessel were rinsed off with 50 mL demineralised water per treatment. Thereafter, the sediment was thoroughly mixed with a long handled blender for at least three minutes and filled into the test vessels.
- Equilibration time: 2 days prior to insertion of test organisms
- Equilibration conditions: Same as test
- Controls: Controls with test sediment and quartz sand. Vehicle controls with test sediment and quartz sand treated with acetone.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): organic solvent, acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 15 mL per 60 g quartz sand (6 replicates)
- Evaporation of vehicle before use: Yes, complete evaporation

Test organisms

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Lumbriculus variegatus (Müller), Lumbriculidae, Oligochaeta
- Source: Bundesanstalt für Gewässerkunde (BfG), Am Mainzer Tor 1, 56068 Koblenz, Germany
- Age of test organisms (mean and range, SD): Synchronized from cultured organisms 13 days before test insertion
- Breeding conditions: 20 +/- 2 deg C, diffuse light (100 - 1000 lx, 16 h photoperiod) dissolved oxygen concentration > 60 % of the air saturation value
- Handling of worms: 13 days before insertion into the test, the worms were synchronized. Therefore, a sufficient amount of worms was taken from the breeding and cut into half.. After regeneration of heads, the worms were inserted into the test.
- Age of animals at beginning of exposure: 13 days after synchronisation, completely regenerated head region. Synchronization was done to minimize uncontrolled regeneration and reproduction during the test.
- Feeding during test
- Food type: Powder of Urtica dioica (Heinrich Klein GmbH & Co KG, 97525 Schwebheim, Germany)
- Amount: 0.5 % Urtica powder
- Frequency: Urtica powder was mixed into the sediment once at test start.


ACCLIMATION
- Acclimation period: 13 days (after synchronization)
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: Same as test
- Feeding frequency: Same as test (Once)
- Health during acclimation (any mortality observed): No mortality observed

Study design

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

yes

Exposure duration

Duration:

28 d

Exposure phase:

total exposure duration

Test conditions

Hardness:

Day 0: Control: > 356 mg/L CaCO3, Solvent Control: 340 mg/L CaCO3, 1000 mg test substance/kg sediment DW: > 356 mg/L CaCO3
Day 28: Control: > 356 mg/L CaCO3, Solvent Control: > 356 mg/L CaCO3, 1000 mg test substance/kg sediment DW: > 356 mg/L CaCO3

Test temperature:

Day Parameter Treatment
Control Solvent Control 1000 mg/kg sediment DW
0 Temperature [°C] 21.1 21.0 20.9
7 Temperature [°C] 21.9 21.2 21.9
14 Temperature [°C] 22.0 20.2 20.2
21 Temperature [°C] 21.8 21.8 21.4
28 Temperature [°C] 21.8 21.2 20.8

pH:

Day Parameter Treatment
Control Solvent Control 1000 mg/kg sediment DW

0 pH 7.74 7.82 7.88
7
pH 7.86 7.86 7.88
14
pH 7.76 7.79 7.86
21
pH 7.58 7.87 8.01
28
pH 8.22 8.27 8.29

Dissolved oxygen:

Day Parameter Treatment
Control Solvent Control 1000 mg/kg sediment DW
0 O2 [mg/L] 6.16 6.78 6.37
7 O2 [mg/L] 6.06 6.05 6.40
14 O2 [mg/L] 6.90 6.22 6.71
21 O2 [mg/L] 7.37 7.11 7.05

28

O2 [mg/L] 7.30 8.04 7.96

Salinity:

Not measured, freshwater conditions

Ammonia:

Day Parameter Treatment
Control Solvent Control 1000 mg/kg sediment DW
0 NH4-N [mg/L] 0.803 0.622 0.797

2 NH4-N [mg/L] 0.114 < 0.02* < 0.02*
5 NH4-N [mg/L] 0.039 0.031 0.113
8 NH4-N [mg/L] 0.045 0.039 0.044
9 NH4-N [mg/L] 0.091 0.050 0.031
12 NH4-N [mg/L] 0.064 0.060 0.068
15 NH4-N [mg/L] 0.089 0.241 0.063
16 NH4-N [mg/L] 1.03 0.715 0.121
19 NH4-N [mg/L] > 2.5* > 2.5* > 2.5*
NH4-N [mg/L] 5.71 5.35 4.29
23 NH4-N [mg/L] 4.84 4.75 5.42
26 NH4-N [mg/L] 6.91 6.01 5.85
28 NH4-N [mg/L] 6.76 6.58 4.30

Nominal and measured concentrations:

ominal concentration limit concentration: 1000 mg test substance/kg sediment DW.
The submission substance has a low water solubility and due to its sorption to particular matter, low volatility and very low biotic and abiotic degradation. It resides predominantly in the solid phase of the sediment. The fraction of the pigment in the overlying water as well as the porewater can be neglected. As no loss of pigment was expected to occur during the exposure period an analytical determination of the pigment in the overlying water and the sediment has been omitted. Based on the fact that the test system consists of 150 mL overlying water 0.008 % pigment would be in the overlying water at saturation and thus 99.992 % remains in the sediment (solids & porewater).

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 250 mL glass beakers, 6 cm diameter
- Sediment volume: 45 g DW per vessel, ca. 2 cm from the ground
- Overlying water volume: 150 mL
- Depth of sediment and overlying water: Sediment depth ca. 2 cm, water depth ca. 10 cm
- Aeration: yes
- Aeration frequency and intensity: Gentle aeration (2 bubbles per second)
- Replacement of evaporated test water, if any: Yes, once weekly


EXPOSURE REGIME
- No. of organisms per container (treatment): 10 per container (60 per treatment)
- No. of replicates per treatment group: 6 replicates per treatment group
- No. of replicates per control / vehicle control: 6 replicates per control/vehicle control
- Feeding regime: Once at test start
- Type and preparation of food: Powder of Urtica dioica mixed into the sediment before insertion of test organisms
- Amount of food: 0.5 %



OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): Reconstituted water acc. to OECD 203


CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- Composition (if artificial substrate): 5 % peat air dried and finely ground, 20 % kaolin, 75 % quartz sand, 0.33 % calcium carbonate
Table 1: Sediment Properties at Test Start
Total Organic Carbon [%] pH-value Moisture [%]
Sediment at test start 2.15 6.74 8.29



OTHER TEST CONDITIONS
Room temperature [°C] Photoperiod [h] Light intensity [lx]
Nominal 20 +/- 2 16 100 - 1000
Actual 18 - 21 16 472 +/- 47.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality and behaviour changes (Day 0-28), reproduction and biomass at test end (28 d)

VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
1000 mg test substance/kg sediment DW

- Range finding study
- Test concentrations: 10 - 100 - 1000 mg/kg sediment DW
- Results used to determine the conditions for the definitive study:
Concentration Mortality Mean number of worms Mean dry weight
[mg test item/kg sediment DW] [%] [mg]
Control 0 13.5 14.7
Solv. Control 0 14.5 16.0
10 0 13.0 35.4
100 0 11.5 16.8
1000 0 12.0 15.9

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, behaviour, biomass reproduction

Details on results:

- Mortality of test animals at end of exposure period: No mortality
- Total mass of test animals at beginning of test: 10 worms were equal to ca. 45.8 mg wet mass
- Morphological abnormalities: None
- Behavioural abnormalities: None

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA, Dunnett´s Method) was carried out with worm numbers and biomass data for the determination of statistically significant differences compared to the pooled control. Prior to the ANOVA a normality test and an equal variance test were conducted. P-values for both normality and equal variance test were 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) for ANOVA was alpha = 0.05.

Any other information on results incl. tables

Mortality/Reproduction of worms:

After 28 days of exposure to the limit test item concentration the total number of worms did not differ significantly compared to the pooled control (Table 1). No mortality of complete worms was observed due to the exposure to the limit test item concentration of 1000 mg/kg sediment DW over a period of 28 days (Table 2). Also, the number of incomplete worms and thus the reproduction did not significantly differ to the pooled control at test end (Table 3).

Table 1:                 Total Worm Number after 28 days of Exposure

Nominal conc.	Total Number of
	Day 28
[mg/kg sediment DW]	1	2	3	4	5	6	MV	SD	% Inhibition	Significance*
Control	17	19	31	20	23	22	22.9	4.76	-	-
Solvent Control	21	32	25	26	19	20
1000	20	20	22	20	22	18	20.3	1.51	11.3	No

Table 2:                 Mortality after 28 days of Exposure

Nominal conc. [mg/kg sediment DW]	Total Number of Complete
	1	2	3	4	5	6	MV	SD	% Mortality	Significance
Control	10	10	10	10	10	10	10	0	0	-
Solvent Control	10	10	10	10	10	10
1000	10	10	10	10	10	10	10	0	0	No

Table 3:                 Reproduction after 28 days of Exposure

Nominal conc. [mg/kg sediment DW]	Number of incomplete worms
	1	2	3	4	5	6	MV	SD	% Inhibition	Significance*
Control	7	9	21	10	13	12	12.9	4.76	-
Solvent Control	11	22	15	16	9	10
1000	10	10	12	10	12	8	10.3	1.51	20.0	No

No difference in the worm behaviour was observed at the limit test item concentration of 1000 mg/kg sediment DW compared to the pooled control during the entire study (Table 4).

Table 4:                 Worm Behaviour during 28 Days of Exposure

Nominal conc.             [mg/kg sediment DW]	Effect	Days after insertion (pooled replicates)
		0	2	5	7	9	12	14	16	19	21	23	26	28
Control	s)	60	60	60	60	60	60	60	60	60	60	60	60	60
Solvent Control	s)	60	60	58	60	60	60	60	59	60	60	60	60	60
	a)	0	0	2	0	0	0	0	1	0	0	0	0	0
1000	s)	60	60	60	60	60	60	60	60	60	60	60	60	60

conc. = concentration                         DW = Dry Weight

s) = worm burrowing in sediment       a) = worm on sediment surface           t) = dead

Dry biomass of the worms

The dry biomass of the worms at the limit test item concentration of 1000 mg/kg sediment DW did not differ significantly to the pooled control (Table 5).

Table 5:                 Worm Dry Biomass after 28 Days of Exposure

Nominal conc. [mg/kg sediment DW]	Dry weight [mg]
	Replicate						per Repl.	Significance*	per worm
	1	2	3	4	5	6	MV ± SD		MV ± SD
Control	7.10	12.7	18.2	14.0	13.5	13.0	14.8 ± 3.27	-	0.65 ± 0.12
Solvent Control	15.5	20.3	14.9	15.7	16.7	16.0
1000	13.7	14.4	19.0	12.3	16.8	13.3	14.9 ± 2.51	No	0.73 ± 0.08

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this sedmient-water Lubriculus toxicity test according to OECD 225 the submission substance caused no significant effects on mortality, behaviour, biomass and reproduction at the limit test item concentration of 1000 mg/kg sediment DW. Thus, the NOEC was determined to be equal or larger than 1000 mg/kg sediment DW.

Executive summary:

The effects of the test item on the oligochaete Lumbriculus variegatus in a water-sediment system were determined. The study was carried out according to OECD Guideline 225 (2007) compliant to GLP (reliability category 1). Test duration was 28 days after insertion of the test organisms. A limit test with the nominal test concentration of 1000 mg/kg sediment dry weight (DW) was performed by spiking the test item into the sediment. Six replicates per control, vehicle control and limit test item concentration with ten worms per replicate were set up. Water quality parameters as temperature, pH-value, O₂-concentration, ammonia and total hardness were determined.

Due to the chemical properties of the test item the fraction of the pigment in the overlying water

as well as in the porewater can be neglected. Thus, no analytical verification of the test item

was performed.

After 28 days no mortality or significant behavioural changes of L. variegatus were observed at the limit test item concentration of 1000 mg/kg sediment DW. Also, the biomass was not significantly reduced compared to the pooled control after 28 days of exposure to the test item. L. variegatus reproduced without any significant differences between the pooled control and the limit test item concentration of 1000 mg/kg sediment DW (Table 1).

Table 1:           Summary of all Significant Effects

Effects	Test item concentration Significance of effects
	[mg/kg sediment DW]
Mortality	> 1000
Behaviour	> 1000
Biomass	> 1000
Reproduction	> 1000
NOEC (mortality, behaviour, biomass, reproduction)	>= 1000