C.I. Pigment Red 282

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:

2009-01-19 to 2009-02-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP. The study is well documented and results are plausilbe.

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 (October 2007) Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment

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:

5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

EC Number:

213-561-3

EC Name:

5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

Cas Number:

980-26-7

Molecular formula:

C22H16N2O2

IUPAC Name:

2,9-dimethyl-5,12-dihydroquino[2,3-b]acridine-7,14-dione

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.
The test vessels were prepared two days prior to the insertion of the test organisms. First, the treated sediment was filled into the glass beakers. Second, the water was added to the beakers without disturbing the sediment. Third, aeration was provided through a glass PASTEUR pipette fixed ca. 3 cm above the sediment layer (ca. two bubbles / sec.). Water levels were topped up weekly during the study to prevent accumulation of the test item. Water levels did not change by more than 10 %.
- Equilibration time: 2 d
- Equilibration conditions: same as test
- Controls: 6 replicates with the test sediment mixed with quartz sand as described above.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone, organic solvent
- Concentration of vehicle in test medium (stock solution and final test solution): 2.5 mL per replicate
- Evaporation of vehicle before use: Yes

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 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: 9 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: 9 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: 9 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 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: 3.3 mmol/L CaCO3, Solvent Control: 3.1 mmol/L CaCO3, 993 mg test substance/kg sediment DW: 3.2 mmol/L CaCO3 Day 28: Control: 3.3 mmol/L, Solvent Control: 3.7 mmol/L, 993 mg test substance/kg sediment DW: 3.1 mmol/L CaCO3

Test temperature:

Day Parameter Actual test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 993 mg/kg sediment dry weight
0 Temperature [°C] 20.2 20.1 20.4
7 Temperature [°C] 20.7 20.6 20.2
14 Temperature [°C] 20.9 20.5 20.6
21 Temperature [°C] 20.2 20.2 20.0
28 Temperature [°C] 20.6 20.0 20.1

pH:

Day Parameter Actual test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 993 mg/kg sediment dry weight
pH 7.64 7.72 7.74
7
pH 8.32 8.26 8.30
14
pH 8.23 8.36 8.30
21
pH 8.46 8.49 8.45
28
pH 8.33 8.40 8.34

Dissolved oxygen:

Day Parameter Actual test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 993 mg/kg sediment dry weight
O2 [mg/L] 8.01 8.57 8.35

7
O2 [mg/L] 8.72 8.44 8.71
14
O2 [mg/L] 8.26 8.16 8.29
21
O2 [mg/L] 8.60 8.61 8.66
28
O2 [mg/L] 8.74 8.82 8.77

Salinity:

Not measured (Freshwater)

Ammonia:

Water Parameters (Ammonium-N)
Day Actual test item concentration [mg/kg sediment dry weight]
Control Solvent Control 993 mg/kg sediment dry weight
Ammonium (NH4-N) in mg/L
0 1.35 1.35 1.27
2 2.04 2.44 1.93
5 1.47 1.31 1.08
7 1.33 1.06 0.882
9 1.28 1.27 1.11
12 0.824 1.47 1.08
14 1.32 1.77 1.44
16 0.683 2.04 1.90
19 2.34 2.35 2.24
21 2.02 2.19 2.16
23 1.57 2.7 2.36
26 2.25 2.17 2.26
28 3.37 2.71 2.27

Nominal and measured concentrations:

Nominal concentration: 1000 mg test substance/kg sediment DW
Actual concentration: 993 mg/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: 0.4 %



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


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 1.79 6.57 7.60



OTHER TEST CONDITIONS

Table 2: Environmental Conditions
Room temperature [°C] Photoperiod [h] Light intensity [lx]
Nominal 20 +/- 2 16 100 - 1000
Actual 17.4 ¿ 22 16 499 ± 82.0



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: 500 - 1000 mg test substance/kg sediment DW
- Results used to determine the conditions for the definitive study:
Range Finding Test (14 d): Mortality and Biomass
Nominal test item concentration
[mg/kg sediment dry weight] Mortality Mean number of worms Mean dry weight
[%] [mg]
Control 0 35.0 26.4
Solvent Control 0 28.3 19.4
500 0 29.3 16.0
1000 0 34.7 26.0

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

993 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

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. 16 mg dry mass
- Changes in body weight of live adults (% of initial weight) at end of exposure period: See "remarks on results including tables anf figures"
- No. of offspring produced: See "remarks on results including tables anf figures"
- 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 a-value (acceptable probability of incorrectly concluding that there is a difference) for ANOVA was a=0.05.

Any other information on results incl. tables

Mortality/Reproduction of worms:

No mortality of complete worms was observed in the controls as well as at the actual limit test item concentration of 993 mg/kg sediment dry weight after an exposure for 28 days.

At test end, the total worm number did not statistically significantly differ compared to the pooled control at the actual limit test item concentration of 993 mg/kg sediment dry weight.

Also, the number of incomplete worms and thus the reproduction did not statistically significantly differ to the pooled control at the actual limit test item concentrations of 993 mg/kg sediment dry weight after 28 days of exposure

Mortality after 28 days of Exposure

Actual test item concentration [mg/kg sediment dry weight]	Total Number of Complete
	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
993	10	10	10	10	10	10	10 ± 0	0

Total Worm Number after 28 days of Exposure

Actual test item concentration	Total Number of
[mg/kg sediment dry weight]	1	2	3	4	5	6	MV ± SD	% Inhibition	Significance*
Control	39	35	32	28	31	31	32.3 ± 2.99	¿
Solvent Control	35	33	30	30	33	30
993	30	29	31	30	30	28	29.7 ± 1.03	8.01	No

Reproduction after 28 days of Exposure:

Actual test item concentration	Number of incomplete worms
[mg/kg sediment dry weight]	1	2	3	4	5	6	MV ± SD	% Inhibition	Significance*
Control	29	25	22	18	21	21	22.3 ± 2.99	¿
Solvent Control	25	23	20	20	23	20
993	20	19	21	20	20	18	19.7 ± 1.03	11.6	No

MV = mean value, SD = Standard deviation, *)Statistical significance compared to pooled control (P<0.05)

Behaviour:

No difference in the worm behaviour was observed at the actual limit test item concentration of 993 mg/kg sediment dry weight compared to the pooled control during the entire study

Worm Behaviour during 28 Days of Exposure

Actual 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	59	60	60	60	60	60	59	60	60	60	60
	a)	-	-	1	-	-	-	-	-	1	-	-	-	-
	f)	-	-	-	+	+	+	+	+	+	+	+	+	+
Solvent Control	s)	60	60	60	60	60	60	60	60	60	60	60	60	60
	f)	-	-	-	+	+	+	+	+	+	+	+	+	+
993	s)	60	60	60	60	60	60	60	60	60	60	60	60	60
	f)	-	-	-	+	+	+	+	+	+	+	+	+	+

The single worm dry biomass was not statistically significantly reduced at the limit test item concentration compared to the pooled control. However, the dry biomass of worms per replicate was statistically significantly reduced at the actual limit test item concentration of 993 mg/kg sediment dry weight compared to the pooled control

Worm Dry Biomass after 28 Days of Exposure per Worm

Actual test item concentration             [mg/kg sediment dry weight]	Dry weight [mg]
	Replicate							Significance*
	1	2	3	4	5	6	MV ± SD
Control	0.65	0.64	0.76	0.89	0.63	0.79	0.70 ± 0.12	-
Solvent Control	0.64	0.42	0.77	0.80	0.68	0.77
993	0.64	0.51	0.60	0.56	0.73	0.66	0.62 ± 0.08	No

Worm Dry Biomass after 28 Days of Exposure per Replicate

Actual test item concentration             [mg/kg sediment dry weight]	Dry weight [mg]
	Replicate							Significance*
	1	2	3	4	5	6	MV ± SD
Control	25.2	22.3	24.3	25.0	19.4	24.5	22.5 ± 3.13	-
Solvent Control	22.4	13.9	23.0	24.1	22.5	23.2
993	19.1	14.7	18.5	16.7	22.0	18.6	18.3 ± 2.45	Yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item (C.I. Pigment Red 122) caused no significant effects on mortality, behaviour and reproduction of L. variegatus at the actual limit test item concentration of 993 mg/kg sediment dry weight. The single worm biomass at the limit test item concentration did not statistically differ from the control data. However, the worm biomass per replicate was significantly reduced at the limit test item concentration. Since the significant reduction of worm biomass per replicate results from the not significant lower total worm number per replicate and not from a significant decrease of the single worm biomass it is concluded that the worm biomass is not affected by the limit test item concentration.

Thus, the NOEC id determined to be at 993 mg/kg sediment dry weight.
All validity criteria recommended by the test guidelines were fulfilled.

Executive summary:

The effects of the test item (C.I. Pigment Red 122) on the oligochaete Lumbriculus variegatus in a water-sediment system were determined at Dr.U.Noack-Laboratorien, Germany from January 19^thto February 20^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 actual test item concentration of 993 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.

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 behavioural changes of L. variegatus were observed at the actual limit test item concentration of 993 mg/kg sediment dry weight .

Also, L. variegatus reproduced without any significant differences between the pooled control and the actual limit test item concentration of 993 mg/kg sediment dry weight.

The biomass per worm was not statistically significantly reduced at the limit test item concentration, whereas the worm biomass per replicate was statistically significantly reduced at the limit test item concentration compared to the control. The significant reduction of worm biomass per replicate results from the not significant lower total worm number per replicate and not from a significant decrease of the single worm biomass. Thus, it is concluded that the worm biomass is not affected by the limit test item concentration.

  

Overall, the NOEC of the test item concerning mortality, reproduction and biomass is determined to be at 993 mg/kg sediment dry weight .

All validity criteria recommended by the test guidelines were fulfilled.

Summary of all Significant Effects

Effects	Hostaperm-Rosa E Significance of effects
	[mg/kg sediment dry weight]
Worm Mortality	> 993
Worm Behaviour	> 993
Worm Biomass	> 993
Worm Reproduction	> 993
NOEC (mortality, behaviour, biomass, reproduction)	993