4-[[5-[[[4-(aminocarbonyl)phenyl]amino]carbonyl]-2-methoxyphenyl]azo]-N-(5-chloro-2,4-dimethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

According to German Chemikaliengesetzt and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: Test sediment was artificial soil acc. to OECD 207, modified, consisting of 5 % peat, 20 % kaolin, 75 % quartz sand and 0.33 % calcium carbonate
- 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 per replicate). After complete evaporation of the solvent, the dry quartz sand was given to the sediment. Residues of the dry quartz sand sticking at the glass 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: Two days before insertion of the 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 acetone per 6 replicates
- Evaporation of vehicle before use: Yes completely evaporated

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 parental stock (mean and range, SD): Synchronized from cultured organisms 13 days before test insertion
- Breeding conditions:20 +/- 2 °C, diffuse light (100 - 1000 lx, 16 h photoperiod), dissolved oxygen concentration > 60 % of the air saturation value
- Handling of egg masses and larvae: 13 days before insertion into the test, the worms were synchronized. Therefore a sufficient amount of worms was taken from the breeding and cutted 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. Synchronisation 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 synchronisation)
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: same as test
- Feeding frequency: Once, same as test
- Health during acclimation (any mortality observed): No mortality observed

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

yes

Duration:

28 d

Exposure phase:

total exposure duration

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

Test temperature:

Day Parameter Treatment
Control Solvent Control 1000 mg/kg sediment DW
2 Temperature [°C] 21.1 21.0 21.0
7 Temperature [°C] 21.9 21.2 21.3
14 Temperature [°C] 22.0 20.2 20.9
21 Temperature [°C] 21.8 21.8 21.5
28 Temperature [°C] 21.8 21.2 21.2

pH:

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

2 pH 7.74 7.82 7.84
7
pH 7.86 7.86 7.83
14
pH 7.76 7.79 7.84
21
pH 7.58 7.87 7.92
28
pH 8.22 8.27 8.28

Dissolved oxygen:

Day Parameter Treatment
Control Solvent Control 1000 mg/kg sediment DW
0 O2 [mg/L] 6.16 6.78 6.93
7 O2 [mg/L] 6.06 6.05 6.33
14 O2 [mg/L] 6.90 6.22 6.91
21

28 O2 [mg/L] 7.37 7.11 7.01

O2 [mg/L] 7.30 8.04 8.47

Salinity:

Not measured (Freshwater)

Ammonia:

Day Parameter Treatment
Control Solvent Control 1000 mg/kg sediment DW
0 NH4-N [mg/L] 0.803 0.622 0.759

2 NH4-N [mg/L] 0.114 < 0.02* 0.247
5 NH4-N [mg/L] 0.039 0.031 0.026
8 NH4-N [mg/L] 0.045 0.039 0.039
9 NH4-N [mg/L] 0.091 0.050 0.035
12 NH4-N [mg/L] 0.064 0.060 0.041
15 NH4-N [mg/L] 0.089 0.241 0.185
16 NH4-N [mg/L] 1.03 0.715 0.154
19 NH4-N [mg/L] > 2.5* > 2.5* > 2.5*
NH4-N [mg/L] 5.71 5.35 3.23
23 NH4-N [mg/L] 4.84 4.75 4.71
26 NH4-N [mg/L] 6.91 6.01 5.47
28 NH4-N [mg/L] 6.76 6.58 5.28

*) below or above range of analysis of the used cuvette detection test

Nominal and measured concentrations:

Nominal concentration: 1000 mg test substance/kg sediment DW

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 250 mL glass beakers (6 cm diameter)
- Sediment volume: 45 g dry weight per vessel, ca. 2 cm from the ground
- Overlying water volume: 150 mL
- Depth of sediment and overlying water: Sediment: ca. 2 cm, overlying water ca. 10 cm
- Aeration: yes
- Aeration frequency and intensity: Gentle aeration, ca. two bubbles/sec.
- 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

Table 2: Environmental 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 behavioural changes (Day 0 - 28), Biomass and reproduction (Day 28)

VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS

- Range finding study
- Test concentrations: 10 - 100 - 1000 mg test substance/kg sediment DW
- Results used to determine the conditions for the definitive study:
Table 3: Range Finding Test (14 d): Mortality and Biomass
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 14.0 19.3
100 0 11.5 7.95
1000 0 14.0 10.4

Reference substance (positive control):

no

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:

reproduction

Remarks:

& biomass, behaviour, mortality

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
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
Table 1: Worm Dry Biomass after 28 Days of Exposure
Nominal conc. [mg/kg sediment DW] Dry weight [mg]
Replicate per Repl. 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 0.63 ± 0.11
Solvent Control 15.5 20.3 14.9 15.7 16.7 16.0
1000 13.4 12.9 22.5 21.0 12.8 13.5 16.0 ± 4.47 0.71 ± 0.08


- No. of offspring produced:
Table 2: 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 11 8 18 25 6 12 13.3 7.03 -3.23 No


- Morphological abnormalities: None
- Behavioural abnormalities: None

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA, Kruskal-Wallis) 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 ¿-value (acceptable probability of incorrectly concluding that there is a difference) for ANOVA was ¿=0.05.

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 significant 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 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 Worms
	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	19	18	28	35	16	21	22.8	7.25	0.36	No

Table 2:                 Mortality after 28 days of Exposure

Nominal conc. [mg/kg sediment DW]	Total Number of Complete Worms
	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	8	10	10	10	10	9	9.5	0.84	5	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	11	8	18	25	6	12	13.3	7.03	-3.23	No

Behaviour and pathological symptoms:

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	59	57	57	57	57	57	57	57	56
	a)	0	0	0	0	0	0	0	0	0	0	0	0	1
	t)	0	0	0	0	1	3	3	3	3	3	3	3	3

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.		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		0.63 ± 0.11
Solvent Control	15.5	20.3	14.9	15.7	16.7	16.0
1000	13.4	12.9	22.5	21.0	12.8	13.5	16.0 ± 4.47		0.71 ± 0.08

Validity criteria fulfilled:

yes

Conclusions:

In this study 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 >/= 1000 mg/kg sediment DW.

Executive summary:

Effects of the submission substance 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 significant mortality or 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 Significance of effects
	[mg/kg sediment DW]
Mortality	> 1000
Behaviour	> 1000
Biomass	> 1000
Reproduction	> 1000
NOEC (mortality, behaviour, biomass, reproduction)	>=  1000

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-06 to 2010-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-study compliant to GLP

Qualifier:

according to guideline

Guideline:

other: Draft ISO/DIS 10872 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetz and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates:
Modified artificial sediment:
- 70 % quartz sand (with > 50 % particles sized between 50 and 200 µm)
- 20 % Al2O3
- 4.5 % Fe2O3
- 4 % peat, air dried and finely ground
- 0.3 % CaCO3
- 0.5 % dolomite (clay)
The pH-value and the moisture of the artificial sediment were determined. One day prior to introduction of the nematodes the sediment was moistened with M9 medium to a moisture of 44 %.
- Details of spiking: The respective test item amount for the limit concentration was weighed out and dissolved in acetone. Subsequently, the solution was thoroughly mixed with quartz sand (20 % of the entire sediment amount) in a glass vessel. After complete evaporation of the solvent, the dry quartz sand was given to the sediment and thoroughly mixed with the sediment.
Additional M9 medium* was added to the sediment to obtain a water content of 44 %. Subsequently, 0.5 g sediment wet weight was filled into each test well.
- Equilibration time: 1 day
- Equilibration conditions: 8 +/- 2 °C, dark
- Controls: Moistened test sediment was tested under the same conditions as the test replicates.

*) M9 medium: 13.95 g Na2HPO4+ 12 H2O corresponding to 6 g Na2HPO4, 3 g KH2PO4, 5 g NaCI and 0.25 g MgSO4 x 7H2O dissolved in 1000 mL demineralised water and autoclaved.

Test organisms (species):

Caenorhabditis elegans

Details on test organisms:

TEST ORGANISM
- Common name: Nematode
- Strain/clone: Wilde type strain N2 var Bristol
- Source: Caenorhabditis Genetics Center, Minneapolis
- Breeding conditions:Breeding is performed at the test facility at 20 +/- 2 °C in the dark on agar plates containing NGM (nematode growth medium) agar inoculated with Escherichia coli OP 50.
- Age of animals at beginning of exposure: First stage juveniles were obtained by filtering nematode solutions from culture plates through nylon nets (10 µm and subsequently 5 µm mesh size). The size of 30 representative juvenile nematodes, which were not used in the test, was determined prior to nematode insertion.
- Feeding during test
LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 21.5 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.



ACCLIMATION
- Acclimation period: None, test conditions = breeding conditions

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

yes

Duration:

96 h

Exposure phase:

total exposure duration

Nominal and measured concentrations:

Nominal: 1000 mg test item/kg sediment dry weight

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Nunc Polystyrol Multiwells (12 well microtiter plates)
- Sediment volume: 0.5 g sediment wet weight per replicate
- Overlying water volume: 0.5 mL food medium (see above)
- Aeration: no
- Replacement of evaporated test water, if any: No

Controls:
Control: Moistened test sediment with the addition of quartz sand (20% of entire sediment amount) was tested under the same conditions as the test replicates.
Solvent control: Moistened test sediment with the addition of quartz sand with acetone was tested under the same conditions as the test replicates.
Liquid Control: Tap water (0.55 ml) without sediment was tested under the same test conditions as the test replicates.



EXPOSURE REGIME
- No. of organisms per container (treatment): 40
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 6
- No. of replicates for the liquid control: 4
- Type and preparation of food: LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 21.5 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.


CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- Composition (if artificial substrate):
Modified artificial sediment:
- 70 % quartz sand (with > 50 % particles sized between 50 and 200 µm)
- 20 % Al2O3
- 4.5 % Fe2O3
- 4 % peat, air dried and finely ground
- 0.3 % CaCO3
- 0.5 % dolomite (clay)
The pH-value and the moisture of the artificial sediment were determined. One day prior to introduction of the nematodes the sediment was moistened with M9 medium to a moisture of 44 %.
- Maturation of artificial substrate (if any): no

OTHER TEST CONDITIONS
- Continuous dark
The initial pH of the artificial sediment at test start was 6.7. The initial sediment moisture was
5.5 %. The room temperature ranged from 19.0 to 21.0 °C.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The recovery / mortality of the inserted nematodes was analysed by counting the adult nematodes under a microscope. The body length of each female adult nematode was determined with a microscale.
The reproduction was analysed by inserting the extracted nematodes in a counting chamber. The produced number of offspring was determined in the specified volume of the counting chamber under a microscope. The total number of offspring per replicate was calculated by using the entire volume of extract.


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: none
- Range finding study
Range Finding Test (96 h): Recovery, Mortality and Reproduction
Test Item Concentration Recovery of adult nematodes Fertility of adult nematodes Reproduction
[mg test item/kg soil dry weight] [%] [%] [Offspring per inserted test organism]
Control 83.3 100 42.0
Solv. control 90.0 100 44.8
10 90.0 100 46.0
100 96.7 100 51.7
1000 93.3 100 42.6

Reference substance (positive control):

yes

Remarks:

Benzyldimethylhexadecylammonium chloride

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortaliy, fertility, reproduction, growth

Details on results:

- Mortality of test animals at end of exposure period: In the artificial sediment a recovery of 90 % was observed in the pooled control after 4 days of exposure. At the limit test item concentration of 1000 mg test item/kg sediment dry weight the recovery was also 90 %. The resulting mortality was not considered to be of biological significance, and was within the range of experimental variation considered acceptable for the control group.

- No. of offspring produced: All female nematodes in the control as well as at the limit test item concentration were observed to be fertile. This also applies to the surviving nematodes in the reference item concentrations.
The reproduction in artificial sediment was not statistically significant reduced compared to the pooled control at the limit test item concentration of 1000 mg test item/kg sediment dry weight. The reproduction rates at the reference item concentrations of 10 and 15 mg reference item/L were statistically significant reduced compared to the pooled control by 40.7 % at 10 mg/L and by 97.0 % at 15 mg/L

- Growth: The initial mean size of the introduced juvenile nematodes was 230.4 µm.
In artificial sediment the growth of the adult nematodes did not statistically significantly differ compared to the pooled control at the limit test item concentrations of 1000 mg test item/kg sediment dry weight.
At 10 and 15 mg reference item/L the growth of the nematodes was statistically significantly reduced compared to the pooled control

- Morphological abnormalities: Not observed
- Behavioural abnormalities: Not observed

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- % recovered test organisms in the control: 100%
- Relevant effect levels:


Effects Reference Item Benzyldimethylhexadecylammonium chloride [mg/L]

LOEC (Mortality, Fertility, Reproduction, Growth) 5
NOEC (Mortality, Fertility, Reproduction, Growth) < 5
EC50 (Mortality) 15.0 (14.2 - 15.8)
EC50
(Reproduction) 10.2 (9.75 - 10.9)
EC50
(Size) 15.9 (11.5 - 23.4)

Reported statistics and error estimates:

t-tests were used for LOEC and NOEC calculations. Prior to running the t-tests a Normality and an Equal Variance Test were done. P-values for both Normality and Equal Variance Test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

Recovery / Mortality after 4 Days of Exposure to submission substance

Nominal test item concentration [mg test item/kg sediment dry weight]	[%] Recovered test organisms								Mortality [%]
	Replicate						MV	SD
	1	2	3	4	5	6
Liquid Control	100	90	90	100	-	-	95.0	5.77	5.00
Control	90	80	90	100	100	90	90.0	7.39	10.0
Solvent Control	90	80	80	90	90	100
1000	90	90	90	90	100	80	90.0	6.32	10.0

Nematode Reproduction after 4 Days of Exposure to submision substance

Nominal test item concentration [mg test item/kg sediment dry weight]	Number of offspring per inserted nematode
	Replicate						MV	SD	Inhibition [%]
	1	2	3	4	5	6
Control	38	35	35	45	35	34	36.7	3.61	-
Solvent Control	35	34	32	38	41	38
1000	35	40	34	38	35	32	35.7	2.98	2.72

Nematode Growth after 4 Days of Exposure to submission substance

Nominal test item concentration [mg test item/kg sediment dry weight]	Mean Growth [µm]
	Replicate						MV	SD	Inhibition [%]
	1	2	3	4	5	6
Control	772	802	799	799	816	794	798	11.8	-
Solvent Control	799	796	808	784	810	804
1000	810	804	826	760	802	760	793	27.5	0.62

Validity criteria fulfilled:

yes

Conclusions:

In this study the submission substance did not induce significant mortality of C. elegans after an exposure to the limit test item concentration of 1000 mg test item/kg sediment dry weight for 96 hours. Also, adult nematode fertility as well as nematode reproduction was not affected at 1000 mg test item/kg sediment dry weight.
No reduction of growth was observed at the limit test item concentration of 1000 mg test item/kg sediment dry weight after four days of exposure to the submision substance compared to the pooled control.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set at > 1000 mg test item/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentrations of Pigment Red 187.

Executive summary:

Effects of the submission substance on the bacterivorous nematode Caenorhabditis elegans in a sediment system were determined. The study was carried out according to the Draft guideline ISO/DIS 10872 (2008). Test duration was 96 hours after insertion of the test organisms. The study was performed by spiking the test item into the sediment with the limit test item concentration of 1000 mg test item/kg sediment dry weight. Six replicates per control, solvent control and limit test item concentration were set up.

After four days of exposure to the limit test item concentration of 1000 mg test item/kg sediment dry weight the submission substance did not induce biologically significant mortality of C. elegans. Also, adult nematode fertility as well as nematode reproduction was not affected at 1000 mg test item/kg sediment dry weight.

Accordingly, no statistically significant reduction of growth was observed at the limit test item concentration of 1000 mg test item/kg sediment dry weight after an exposure of 96 hours to the submission substance.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set at > 1000 mg test item/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentration of the submission substance.

Summary of all Effects and resulting LOEC, NOEC based on nominal concentrations

Effects	Submission substance
	[mg test item/kg sediment dry weight]
Mortality	> 1000
Fertility	> 1000
Reproduction	> 1000
Growth	> 1000
LOEC (Mortality, Fertility, Reproduction, Growth)	> 1000
NOEC (Mortality, Fertility, Reproduction, Growth)	1000

Description of key information

Effects of the submission substance 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.

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 >= 1000 mg/kg sediment DW.

 

Effects of the submission substance on the bacterivorous nematode Caenorhabditis elegans in a sediment system were determined. The study was carried out according to the Draft guideline ISO/DIS 10872 (2008). Test duration was 96 hours after insertion of the test organisms. The study was performed by spiking the test item into the sediment with the limit test item concentration of 1000 mg test item/kg sediment dry weight.

the submission substance did not induce significant mortality of C. elegans after an exposure to the limit test item concentration of 1000 mg test item/kg sediment dry weight for 96 hours. Also, adult nematode fertility as well as nematode reproduction was not affected at 1000 mg test item/kg sediment dry weight.
No reduction of growth was observed at the limit test item concentration of 1000 mg test item/kg sediment dry weight after four days of exposure to the submision substance compared to the pooled control.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set at > 1000 mg test item/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentrations of Pigment Red 187.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

1 000 mg/kg sediment dw