C.I. Pigment Red 282

Long-term toxicity to fish

Administrative data

Endpoint:

fish, juvenile growth test

Type of information:

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

Adequacy of study:

key study

Study period:

2008-08-13 to 2008-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 215), well documented, results plausible

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Version / remarks:

revised draft document dated January 1999

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

Details on sampling:

Due to the low solubility of the test item, no specific control analysis was carried out.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L (total volume 20 L) was prepared for each renewal and mixed with an ultraturrax for 10 minutes. This dispersion was filtrated with a RC filter (0.45 µm) and used for the test.
- Eluate: Dilution water
- Differential loading: One test concentration
- Controls: One replicate with 16 fish was tested under the same conditions as the test item replicates but without test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No undissolved material due to filtration

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Aquarium am Aegi, 30175 Hannover, Germany
- Weight at study initiation (mean and range, SD): 0.05 - 0.1 g
- Method of breeding: Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1-10 µmol photons ¿ m-2 ¿ s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within the last 7 days before the study started was used in the test. No disease treatment was administered throughout holding and testing.



FEEDING DURING TEST
- Food type: Brine shrimp nauplii Artemia salina eggs were purchased from Silverstar Artemia Brine Shrimp Ex, Inter Ryba GmbH Germany. Fresh cultures were prepared with salt water (NaCl 40 g/L, 2 g eggs to 1 L salt water, gentle aeration). 24 h old brine shrimp nauplii were harvested and washed with a stainless steel mesh and resuspended in dechlorinated tap water.

- Amount: The fish were fed daily ad libitum. The food was given in two feeds per day separated by at least 5 hours (workdays). One feed was applied on weekends.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

28 d

Post exposure observation period:

No

Test conditions

Hardness:

Total Hardness in the Control Water

Day 0 Day 28
mg CaCO3 / L 60 64

Test temperature:

Water Parameter of the Control

Study day Temperature [°C]
new old
start 23.2 -
2 22.3 22.5
5 22.9 22.7
7 22.6 22.6
9 22.5 22.5
12 22.6 22.3
14 23.1 23.3
16 22.7 22.9
19 23.0 23.2
21 23.1 23.0
23 23.4 23.4
26 22.5 23.1
28 - 21.9

Water Parameter of the Test Item Solution 10 mg/L
(nominal test item concentration)

Study day Temperature [°C]
new old
start 23.4 -
2 22.7 22.4
5 22.5 22.9
7 22.3 22.6
9 22.2 22.5
12 22.4 22.4
14 23.1 23.2
16 22.7 22.6
19 22.9 23.2
21 22.7 23.1
23 22.8 23.3
26 22.6 23.0
28 - 21.9

pH:

Water Parameter of the Control

Study day pH-value
new old
start 7.36 -
2 7.28 7.15
5 7.24 7.21
7 7.13 7.13
9 7.27 7.23
12 7.41 7.51
14 7.23 7.23
16 7.51 7.24
19 7.13 7.14
21 7.09 7.15
23 7.23 7.23
26 7.69 7.85
28 - 7.60

Water Parameter of the Test Item Solution 10 mg/L
(nominal test item concentration)

Study day pH-value
new old
start 7.31 -
2 7.16 7.12
5 7.24 7.22
7 7.14 7.13
9 7.33 7.32
12 7.41 7.44
14 7.35 7.26
16 7.49 7.39
19 7.23 7.17
21 7.19 7.15
23 7.31 7.32
26 7.54 7.51
28 - 7.63

Dissolved oxygen:

Water Parameter of the Control

Study day Oxygen-saturation [%]
new old
start 100 -
2 100 90
5 100 97
7 100 96
9 100 95
12 100 95
14 100 97
16 100 99
19 100 98
21 99 95
23 100 100
26 95 95
28 - 99

Water Parameter of the Test Item Solution 10 mg/L
(nominal test item concentration)

Study day Oxygen-saturation [%]
new old
start 100 -
2 98 87
5 100 95
7 98 92
9 100 95
12 100 94
14 100 97
16 100 91
19 99 95
21 98 89
23 100 100
26 98 93
28 - 97

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Nominal limit test item concentration: 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 12.5 L Glass aquaria, loosely covered by glass tops
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass aquaria 10 L full volume
- Aeration: The test aquaria were gently aerated
- Renewal rate of test solution (frequency/flow rate): 3 times a week
- No. of organisms per vessel: 16 fish
- No. of vessels per concentration (replicates): one replicate
- No. of vessels per control (replicates): one replicate
- Biomass loading rate: 0.2 - 1.0 g/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
The water is analysed biannual acc. to German tap water regulation.

- Culture medium different from test medium: No



OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A daily 12 h photoperiod
- Light intensity: 0.1 - 10 µmol photonsxm-2xs-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Lethal effects: a fish was presumed to be dead if no respiratory movement and no reaction to a slight mechanical stimulus has been detected. Dead fish were removed and the feeding rate was adjusted. Non lethal effects: these include all effects on appearance, size and behaviour of the fish that made them clearly distinguishable from the control animals, e.g. different swimming behaviour, different reaction to external stimuli, changes in appearance of the fish, reduction or cessation of food intake, changes in length or body weight.
The fish were weighed to the nearest mg and measured to the nearest mm. The weighing and length measuring procedure was carried out on days 0 and 28. The fish were anaesthetised on day 0 to determine the initial weight and length. All fish were weighed as wet weights (blotted dry). They were not fed 24 h prior to test start. Only fish with weights within the range of 0.05 and 0.10 g were randomly introduced to the test vessels.




VEHICLE CONTROL PERFORMED: no

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

lethal and non lethal effects

Effect concentrations 2

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

lethal and non lethal effects

Details on results:

- Fish weights (individual and mean values) on day 0, 14 (if measured) and 28: see Table under Results
- Tank-average or pseudo-specific growth rates for periods 0-28 or 0-14 and 0-28 (if possible): see Table under Results
- Type of and number with morphological abnormalities: 0
- Type of and number with behavioural abnormalities: 0
- Other biological observations: No biological observation
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: No

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Mortality rate The mortality rate was indicated for all test concentrations.

Regression analysis No regression curve of the growth rate was calculated since no dose-related effects were found.

Statistical significance One way analysis of variance (ANOVA) was carried out with the pseudo-specific growth rates, body weights and length for the determination of statistically significant differences compared to control replicates. P-values for normality and equal variance test were 0.05. The a-value for ANOVA (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.

Any other information on results incl. tables

Lethal Effects

Throughout the study no mortality was observed

Cumulative Mortality [%]

                            (nominal test item concentration [mg/L])

Day	Control	10
0	0	0
1	0	0
2	0	0
3	0	0
4	0	0
5	0	0
6	0	0
7	0	0
8	0	0
9	0	0
10	0	0
11	0	0
12	0	0
13	0	0
14	0	0
15	0	0
16	0	0
17	0	0
18	0	0
19	0	0
20	0	0
21	0	0
22	0	0
23	0	0
24	0	0
25	0	0
26	0	0
27	0	0
28	0	0

Non Lethal Effects

No lethal or non lethal effects were found throughout the study.

Body Weight and Growth Rates

The mean body weight of the control fish increased from 59.7 mg to 128.1 mg (0-28 days). No statistically significant differences among the pseudo-specific growth rates were found. Percent inhibition values were ¿ 15 %.

Body Weight [mg] of the Fish, Day 0            

              (nominal test item concentration [mg/L])

Fish No.	Control	10
1	57.6	52.7
2	67.3	52.2
3	71.1	68.1
4	57.1	62.2
5	73.1	54.7
6	56.6	52.8
7	57.1	56.2
8	66.2	52.9
9	58.1	54.7
10	57.2	71.1
11	56.6	62.2
12	53.1	53.8
13	54.6	56.7
14	57.2	64.3
15	58.7	52.7
16	53.3	54.8
MV	59.7	57.6
CV [%]	10.0	10.1

Body Weight [mg] of the Fish, Day 28          

                 (nominal test item concentration [mg/L])

                                                                                                                                       

Fish No.	Control	10
1	113.8	111.8
2	126.5	117.6
3	139.9	124.2
4	169.4	110.9
5	88.9	144.0
6	109.1	100.3
7	116.2	120.8
8	120.6	89.9
9	150.3	148.0
10	143.2	145.0
11	123.2	120.5
12	137.6	140.7
13	113.5	120.7
14	146.0	109.0
15	141.7	111.1
16	110.2	120.0
MV	128.1	120.9
CV [%]	15.1	13.2

Pseudo-specific Growth Rate (0 ¿ 28 d) of the Test Fish          

                      (nominal test item concentration [mg/L])

Control		10
2.32		2.39
2.70		2.56
3.06		2.76
3.74		2.36
1.44		3.29
2.17		2.00
2.40		2.66
2.53		1.61
3.32		3.39
3.14		3.31
2.61		2.65
3.00		3.21
2.31		2.66
3.21		2.29
3.11		2.36
2.21		2.64
-----	No statistically significant difference to control

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Pigment Red 122 did not cause any effects to Zebrafish (growth rates, lethal and non lethal effects), at the saturated solution level (prepared from 10 mg/L). Therefore the NOEC after 28 days of exposure was the saturated solution level (prepared from 10 mg/L).

Executive summary:

The effects of the test item (Pigment Red 122) on the growth rate of juvenile Zebrafish were determined according to OECD-Guideline 215 from August 13 to September 10, 2008 at Dr.U.Noack-Laboratorien, Sarstedt, Germany. A semi-static test with 3 renewals of the test medium (a saturated solution* with a nominal test item concentration of 10 mg/L) per week was carried out. Duration of the test was 28 days. 16 fish were exposed to the saturated solution and the control. No vehicles were used for dissolving the test item. Daily observation of the fish was carried out with respect to lethal and non lethal effects. Body length and weight of the fish were determined on days 0 and 28. Water quality parameters temperature, pH value, oxygen saturation were determined to be within the acceptable limits (see part 9). Control analysis was not carried out due to the low solubility of the test item. Nominal test item concentrations were used for the evaluations of the effect levels.

*) Saturated solution: The maximum disolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6

Table 1:       Effect Level Concentrations of Test Item

                      (determined on growth rates, non lethal and lethal effects,

                      based on the saturated solution prepared with 10 mg/L)

No observed effect concentration (NOEC)	10 mg/L
The highest tested concentration of the test item at which  no statistically significant lethal or other effect is observed
Lowest observed effect concentration (LOEC)	> 10 mg/L
The lowest  tested concentration of the test item at which a statistically significant lethal or other effect is observed

Table 2:       EC- Value for the Tank Average Growth Rates

                                   (based on the saturated solution prepared with 10 mg/L)

Test duration [d]	EC50  [mg/L]	p = 95 %   [mg/L]
0 - 28	> 10	Not applicable