2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(4-ethoxyphenyl)-3-oxobutyramide]

Sediment toxicity

Administrative data

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-14 to 2009-10-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 225) performed compliant to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

According to German Chemikaliengesetz and Directive 88/320/EEC

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

EC Number:

228-787-8

EC Name:

2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

Cas Number:

6358-85-6

IUPAC Name:

2,2'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis(3-oxo-N-phenylbutanamide)

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Details of spiking: The respective test item concentration was weighed out and dissolved in 2.5 mL acetone per replicate. Subsequently, the solution was thoroughly mixed with 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. Subsequently, the sediment was mixed thoroughly 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): 2.5 mL per replicate
- 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 12 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 worms: 12 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 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: 12 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: 12 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 Parameter Nominal test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 1000
0


Total Hardness [mmol/L CaCO3] 2.87 2.92 2.94
28


Total Hardness [mmol/L CaCO3] 2.57 3.08 3.07

Test temperature:

Day Parameter Nominal test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 1000
0Temperature [°C] 19.3 19.1 20.2



7Temperature [°C] 19.8 19.7 19.7


14Temperature [°C] 21.0 21.1 21.2


21Temperature [°C] 19.3 19.5 19.4


28Temperature [°C] 19.8 19.8 20.4

pH:

Day Parameter Nominal test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 1000
0
pH 7.73 7.86 7.90


7
pH 8.31 8.31 8.25

14
pH 8.48 8.46 8.52

21
pH 8.39 8.41 8.42

28
pH 8.07 8.11 8.10

Dissolved oxygen:

Day Parameter Nominal test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 1000
0

O2 [mg/L] 8.33 8.61 7.88

7

O2 [mg/L] 8.52 8.59 8.46
14

O2 [mg/L] 8.42 8.38 8.37
21

O2 [mg/L] 8.65 8.61 8.60
28

O2 [mg/L] 8.77 8.69 8.44

Salinity:

Not measured, freshwater conditions

Ammonia:

Water Parameters (Ammonium-N)
Day Nominal test item concentration [mg/kg sediment dry weight]
Control Solvent Control 1000
Ammonium (NH4-N) in mg/L
0 0.292 0.227 0.278
2 1.66 1.55 1.46
5 1.72 1.54 0.951
7 1.54 0.854 0.374
9 0.768 0.552 0.386
12 1.52 0.904 1.19
14 1.77 1.33 1.39
16 2.52 1.83 1.95
19 3.30 2.44 2.32
21 6.55 6.41 3.00
23 3.82 3.49 2.96
26 3.71 3.98 3.32
28 3.60 3.63 4.06

Nominal and measured concentrations:

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 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
Parameters of the Test Medium at Test Start
Oxygen content [mg/L] pH-value Hardness [mmol/L]
Test Medium at test start 9.09 7.49 2.66


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

Sediment Properties at Test Start
Total Organic Carbon [%] pH-value Moisture [%]
Sediment at test start 1.5 6.94 0.81


OTHER TEST CONDITIONS

Water temperature [°C] Photoperiod [h] Light intensity [lx]
Nominal 20 +/- 2 16 100 - 500
Actual 17.6 - 21.6* 333 ± 60.8



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
- Not carried out

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 9.6 mg dry mass
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
The dry biomass of worms per replicate was not statistically significantly reduced at the limit test item concentration of 1000 mg/kg sediment dry weight compared to the pooled control. For details please refer to the respective table.
- No. of offspring produced:
At test end, the total worm number, including adult and descendant worms, did not statistically significantly differ compared to the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight. Also, the number of descendant worms and thus the reproduction did not statistically significantly differ to the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight after 28 days of exposure. For details please refer to the respective table.
- Morphological abnormalities: None
- Behavioural abnormalities: None

Reported statistics and error estimates:

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

Any other information on results incl. tables

No mortality of complete adult worms was observed in the controls as well as at the limit test item concentration of 1000 mg/kg sediment dry weight after an exposure for 28 days.

At test end, the total worm number, including adult and descendant worms, did not statistically significantly differ compared to the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight

Also, the number of descendant worms and thus the reproduction did not statistically significantly differ to the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight after 28 days of exposure.

Mortality after 28 days of Exposure

Nominal test item concentration [mg/kg sediment dry weight]	Total Number of Complete Worms
	1	2	3	4	5	6	MV ± SD	% Mortality
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

Total Worm Number after 28 days of Exposure

Nominal test item concentration	Total Number of Worms
[mg/kg sediment dry weight]	1	2	3	4	5	6	MV ± SD	% Inhibition	Significance*
Control	42	41	32	35	36	35	38.0 ± 3.19	--
Solvent Control	42	38	40	40	36	39
1000	37	41	37	31	38	40	37.3 ± 3.50	1.75	No

Reproduction after 28 days of Exposure

Nominal test item concentration	Number of descendant worms
[mg/kg sediment dry weight]	1	2	3	4	5	6	MV ± SD	% Inhibition	Significance*
Control	32	31	22	25	26	25	28.0 ± 3.19	--
Solvent Control	32	28	30	30	26	29
1000	27	31	27	21	28	30	27.3 ± 3.50	2.38	No

MV= mean value; SD= standard deviation; *) Statistical significiance compared to pooled control (P< 0.05);

No difference in the worm behaviour was observed at the limit test item concentration of 1000 mg/kg sediment dry weight compared to the pooled control during the entire study

Worm Behaviour during 28 Days of Exposure

Nominal test item concentration             [mg/kg sediment dry weight]	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
	f)	-	+	+	+	+	+	+	+	+	+	+	+	+
Solvent Control	s)	60	60	60	60	60	60	60	60	60	60	60	60	60
	f)	-	+	+	+	+	+	+	+	+	+	+	+	+
1000	s)	60	59	60	60	60	60	60	60	60	60	60	60	60
	f)	-	+	+	+	+	+	+	+	+	+	+	+	+
	a)	-	1	-	-	-	-	-	-	-	-	-	-	-

s) = worm burowing in sediment; f) = faeces on sediment surface; a) = worm on sediment surface;

-) = negative observation; +) = positive observation;

The dry biomass of worms per replicate was not statistically significantly reduced at the limit test item concentration of 1000 mg/kg sediment dry weight compared to the pooled control.

Worm Dry Biomass after 28 Days of Exposure per Replicate

Nominal test item concentration             [mg/kg sediment dry weight]	Worm dry weight [mg]
	Replicate							Significance*
	1	2	3	4	5	6	MV ± SD
Control	24.6	28.9	24.1	35.9	36.4	36.8	34.8 ± 9.11	--
Solvent Control	27.7	31.5	29.5	46.8	54.4	40.6
1000	32.6	26.7	28.4	30.2	33.9	48.4	33.4 ± 7.82	No

MV= mean value; SD= standard deviation; *) Statistical significiance compared to pooled control (P< 0.05);

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item caused no significant effects on mortality, behaviour and reproduction of L. variegatus at the limit test item concentration of 1000 mg/kg sediment dry weight. Also, the worm biomass was not significantly reduced at the limit test item concentration compared to the control. Thus, the NOEC was determined to be at 1000 mg/kg sediment dry weight.

All validity criteria recommended by the test guidelines were fulfilled.

Executive summary:

The effects of the test item on the oligochaete Lumbriculus variegatus in a water-sediment system were determined at Dr.U.Noack-Laboratorien,31157 Sarstedt , Germany , from September 14^thto October 15^th, 2009. The study was carried out according to OECD Guideline 225 (2007). Test duration was 28 days after insertion of the test organisms. A limit test with the test item concentration of 1000 mg/kg sediment dry weight was performed by spiking the test item into the sediment. Six replicates per control, solvent control and limit test item concentration with ten worms per replicate were set up. Water quality parameters as temperature, pH-value, O₂-content, ammonium and total hardness were determined.

After 28 days no mortality or behavioural changes of L. variegatus were observed at the limit test item concentration of 1000 mg/kg sediment dry weight and the controls (Table 1).

Also, L. variegatus reproduced without any significant differences between the pooled control and the limit test item concentration (Table 1).

Accordingly, the worm biomass per replicate was not statistically significantly reduced at the limit test item concentration compared to the pooled control (Table 1).

  

Overall, the NOEC of the test item concerning mortality, reproduction and biomass is determined to be at 1000 mg/kg sediment dry weight (Table 1).

All validity criteria recommended by the test guidelines were fulfilled.

Summary of all Significant Effects

Effects	Test item Significance of effects
	[mg/kg sediment dry weight]
Worm Mortality	> 1000
Worm Behaviour	> 1000
Worm Biomass	> 1000
Worm Reproduction	> 1000
NOEC (Mortality, Behaviour, Biomass, Reproduction)	1000