4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-06 to 2012-07-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)

Version / remarks:

October 2007

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Details of spiking: The respective test item amount was weighed out and dispersed in 2.5 mL acetone per replicate (270 mg/15 mL). Subsequently, the dispersion was thoroughly mixed with 60 g quartz sand. After complete evaporation of the solvent, the dry quartz sand was given to the sediment. Subsequently, the sediment was mixed thoroughly and 45 g/replicate were filled into each test vessel.
- Equilibration time: 2 Days prior to insertion of test organisms
- Equilibration conditions: Same as test conditions
- Controls: Six replicates with untreated artificial sediment
- Solvent control: Six replicates with test sediment mixed with quartz sand and acetone.

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 14 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: 14 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: 14 days after synchronization, 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:0.4 % Urtica powder
- Frequency: Urtica powder was mixed into the sediment once at test start.


ACCLIMATION
- Acclimation period: 14 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 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

Post exposure observation period:

No

Hardness:

Day Parameter Control Solvent control 1000 mg test item/
kg sediment dry weight
0 Total hardness [mg/L CaCO3] 272 278 262
28 Total hardness [mg/L CaCO3] 223 249 271

Test temperature:

Day Parameter Control Solvent control 1000 mg test item/
kg sediment dry weight
0
(before
insertion of worms)Temperature [°C] 21.0 20.9 20.7
7 Temperature [°C] 20.5 20.5 20.3
14 Temperature [°C] 20.4 19.8 20.2
21 Temperature [°C] 20.3 20.5 20.6
28 Temperature [°C] 20.2 20.1 20.0

pH:

Day Parameter Control Solvent control 1000 mg test item/
kg sediment dry weight
0 pH 6.94 6.97 7.04
7 pH 7.32 7.51 7.64
14 pH 7.24 7.48 7.58
21 pH 7.92 7.96 7.92
28 pH 7.48 7.70 7.79

Dissolved oxygen:

Day Parameter Control Solvent control 1000 mg test item/
kg sediment dry weight
0 O2 [mg/L]* 8.50 8.73 8.87
7 O2 [mg/L]* 8.74 9.01 8.81
14 O2 [mg/L]* 8.91 9.15 8.83
21 O2 [mg/L]* 9.29 9.13 8.68
28 O2 [mg/L]* 8.98 8.74 8.86

*) 70 % of the air saturation value corresponds to 6.2 mg O2/L

Salinity:

not measured, freshwater conditions

Ammonia:

Ammonium-N [mg/L] in the Overlying Test Water
Day after worm insertion Control Solvent control 1000 mg test item/
kg sediment dry weight
0 0.31 0.18 0.53
2 2.9 2.2 2.6
5 4.1 3.4 3.4
7 4.6 4.4 5.2
9 6.5 5.3 5.5
12 9.2 9.2 7.7
14 10.0 7.7 9.4
16 11.0 9.4 8.4
19 11.6 12.4 9.7
21 12.8 11.4 10.7
23 13.3 12.2 10.0
26 13.4 12.7 10.7
28 11.9 12.9 11.8

Nominal and measured concentrations:

Nominal concentrations: Nominal limit test item concentration of 1000 mg test item/kg sediment dry weight.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 250 mL glass beakers, 6 cm in diameter
- Sediment volume: 45 g DW per vessel, ca. 3 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: 0.4 %


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
Overlying Test Water Properties at Test Start
pH-value Hardness[mgl/L CaCO3] Oxygen content[mg/L]
Overlying Test Water 7.57 233.18 9.12


CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- Composition (if artificial substrate): 5 % peat air dried and finely ground, 20 % kaolin, 75 % quartz sand, 0.30 % calcium carbonate
Sediment Properties at Test Start
Sediment Properties prior Insertion of the Worms
Total Organic Carbon [%] pH-value Moisture [%]
Sediment prior test start 2.4 6.70 2.07


OTHER TEST CONDITIONS
Environmental Conditions
Environmental Conditions
Room Temperature [°C] Photoperiod [h] Light intensity [lx]
Nominal 20 +/- 2 16 100 - 500
Actual 20.1 ± 0.35 16 278 ± 52





EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Visual observations of behavioural differences to the control were recorded three times per week. The endpoints of the study were the number of surviving worms and the reproduction per replicate and the total dry biomass of survivors per replicate.
Worms were considered to be dead by showing no response to a mechanical stimulus, signs of composition or by absence. Living worms were assigned as adult (large complete worms) or offspring (small complete worms with regenerated body regions or incomplete worms).



VEHICLE CONTROL PERFORMED: Yes

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:

and behaviour, biomass

Details on results:

- Changes in body weight of live adults (% of initial weight) at end of exposure period:
At test start the worms had a body weight of 0.0478 g dry weight/10 worms.
The biomass dry weight of the worms compared to the solvent control did not statistically significantly differ at the limit test item concentration of 1000 mg/kg sediment dry weight
- No. of offspring produced:
The total number of worms (complete and incomplete) did not statistically significant differ from the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight after 28 days of exposure.
Accordingly, the number of small complete or incomplete worms and thus the reproduction did not statistically significantly differ from the pooled control at the limit test item concentration after 28 days of exposure
- Morphological abnormalities: None
- Behavioural abnormalities: No significant changes in the worm behaviour or pathological symptoms were observed in the controls as well as at the limit test item concentration throughout 28 days of exposure

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
Summary of all Effects and resulting LOEC, NOEC of the Reference Item Study

Effects Potassium Chloride[g/kg sediment dry weight]
LOEC
Total worm number 4.0
LOEC
Reproduction
(Number of small or incomplete worms) 4.0
LOEC
Biomass 2.0
NOEC
Total worm number 2.0
NOEC
Reproduction
(Number of small or incomplete worms) 2.0
NOEC
Biomass 1.0
EC50
Total worm number 2.72 (2.36 - 3.12)
EC50
Reproduction
(Number of small or incomplete worms) 2.84 (< 1.00 - 3.89)
EC50
Biomass 2.10 (1.96 - 2.26)

Reported statistics and error estimates:

Total worm number, incomplete worm number, total biomass dry weight:
t-tests were carried out for the determination of statistically significant differences compared to the pooled control. A Normality Test and an Equal Variance Test were conducted prior to running the t-tests. P-values for both Normality and Equal Variance Test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05.

Total Worm Number after 28 Days of Exposure

Nominal concentration [mg/kg sediment dry weight]	Total Number of Worms
	1	2	3	4	5	6	MV	SD	% Inhibition	Significance*
Pooled Control	17	27	22	30	30	28
	25	26	26	27	16	20	24.5	4.72	–	–
1000	30	25	26	25	30	18	25.7	4.41	0	No

*) Statistically different compared to pooled control

Reproduction after 28 days of Exposure

Nominal concentration [mg/kg sediment dry weight]	Number of Incomplete Worms
	1	2	3	4	5	6	MV	SD	% Inhibition	Significance*
Pooled control	8	1	15	20	20	21
	17	17	17	22	12	10	16.3	4.40	–	–
1000	22	20	21	19	25	12	19.8	4.36	-17.82	No

*) Statistically different compared to pooled control

Worm Behaviour during 28 Days of Exposure

Nominal 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	a	1	-	-	1	-	-	-	-	-	-	-	-	1
	ka	+	+	+	+	+	+	+	+	+	+	+	+	+
	f	-	-	+	+	+	+	+	+	+	+	+	+	+
Solvent control	a	-	1	-	1	-	-	1	-	-	-	-	-	1
	ka	+	+	+	+	+	+	+	+	+	+	+	+	+
	f	-	-	+	+	+	+	+	+	+	+	+	+	+
1000	a	-	1	-	1	-	1	1	-	-	-	-	-	-
	ka	+	+	+	+	+	+	+	+	+	+	+	+	+
	f	-	-	+	+	+	+	+	+	+	+	+	+	+

a) = worm on sediment surface; ka) = no striking feature; f) = faeces on the sediment surface; -) negative observation ; +) positive observation

Dry Biomass of the Worms per Replicate after 28 Days of Exposure

Nominal concentration [mg/kg sediment dry weight]	Dry weight [mg] per replicate
	Replicate						MV ± SD	Significance
	1	2	3	4	5	6
Control	12.7	16.1	15.0	16.0	17.8	14.9	15.4 ± 1.69	–
Solvent control	14.9	13.8	14.5	12.5	13.1	12.3	13.5 ± 1.06	Yes*
1000	17.4	12.2	13.3	12.4	13.5	10.4	13.2 ± 2.33	No^+

MV = Mean value; SD = Standard deviation; *) Statistically different compared to control; +) Compared to solvent control

Validity criteria fulfilled:

yes

Conclusions:

After 28 days of exposure the test item induced no significant changes in behaviour at the limit test item concentration of 1000 mg/kg sediment dry weight. The total number of worms as well as the reproduction was not statistically significant reduced at the limit test item concentration compared to the pooled control. Furthermore, the biomass was not statistically significantly reduced compared to the solvent control after 28 days of exposure to the limit test item concentration.
The No Observed Effect Concentration (NOEC) was determined to be at 1000 mg/kg sediment dry weight.

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. 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. An untreated control and a solvent control were set up. Six replicates per control, solvent control and the limit test item concentration with ten worms per replicate were set up. Water quality parameters as temperature, pH-value, O₂-concentration, ammonium and total hardness were determined throughout the study.

After 28 days of exposure the test item induced no changes in behaviour at the limit test item concentration of 1000 mg/kg sediment dry weight. The total number of worms as well as the reproduction was not statistically significantly reduced at the limit test item concentration compared to the pooled control. Furthermore, the biomass of the limit test item concentration was not statistically significantly reduced compared to the solvent control after 28 days of exposure.

The No Observed Effect Concentration (NOEC) was determined to be at 1000 mg/kg sediment dry weight.

Summary of all Effects and Resulting NOEC

Effects	Test Item [mg test item/kg sediment dry weight]
NOEC Behaviour	1000
NOEC (Total number of worms)	1000
NOEC Reproduction (Number of small or incomplete worms)	1000
NOEC Biomass	1000
NOEC (overall)	1000

Description of key information

One long-term chronic toxicity studies of reliability 1 for Pigment Orange 34 on Lumbriculus variegatus is available.
Under the conditions of this test the NOEC of the submission substance after 28 days was 1000 mg/kg dw (based an nominal concentration).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

1 000 mg/kg sediment dw