1,8-bis[(4-methylphenyl)amino]anthraquinone

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

LANXESS Deutschland GmbH has registered five mono-constituent anthraquinone dyes of similar chemical structure using a category approach: Solvent Violet 36 (CAS No 82-16-6), Solvent Green 3 (CAS No 128-80-3), Reinblau RLW (CAS No 41611-76-1), Reinblau BLW (CAS No 32724-62-2) and Solvent Green 28 (CAS No 4851-50-7). Additional data were taken from another registered anthraquinone dye, Solvent Blue 104 (CAS No 116-75-6), leading to a category consisting of six members (see attached justification in IUCLID chapter 13).

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

- Concentrations: Limit test with the saturated solution with 1 mg Solvent blue 104 /L
- Sampling method: No analytical determinations were carried out, due to the low solubility of < 0.02 mg/L of the test item in water.

- Sample storage conditions before analysis: See above

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (1 mg/L test item were weighed out) were prepared with dilution water two days prior to each renewal day. The stock solution was stirred with approximately 1100 rpm for 48 hours at room temperature. Undissolved particles were removed by centrifugation with 10000 rpm (corresponding to 15317 g) for 10 minutes at 20 °C.

- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: 10 replicates of dilution water without test item

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness as CaCO3 [mg/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
2012-09-26 2012-09-28 2012-10-03 2012-10-05 2012-10-10 2012-10-12
1.00
(Saturated
solution) 162 164 171 166 176 183
Control 171 151 164 166 180 166

Test temperature:

Temperature [°C]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
2012-09-26 2012-09-28 2012-10-03 2012-10-05 2012-10-10 2012-10-12
1.00
(Saturated
solution) 21.5 21.8 20.6 21.6 21.4 20.9
Control 21.2 21.8 21.3 21.1 20.1 21.0

pH:

Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
2012-09-26 2012-09-28 2012-10-03 2012-10-05 2012-10-10 2012-10-12
1.00
(Saturated
lution) 7.99 7.99 7.92 7.91 7.98 7.88
Control 8.80 7.96 8.10 8.04 8.34 7.80

Dissolved oxygen:

Dissolved Oxygen Concentration [mg O2/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
2012-09-26 2012-09-28 2012-10-03 2012-10-05 2012-10-10 2012-10-12
1.00
(Saturated
solution) 8.27 8.81 8.76 8.22 8.65 8.26
Control 8.63 8.79 8.25 7.31 8.35 7.96

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): glass beaker closed with watch glasses
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control and pH-control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
Immobilization Rates in the Preliminary Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Concentration
of the Test Item[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
(Saturated
solution)
1.00 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Details on results:

- Mortality of parent animals:
No adult mortality or immobilization of parental daphnids was observed in the control and the saturated solution with 1.00 mg Solvaperm Blue 2B/L.
- No. of offspring produced per day per female:
IIn the control and the saturated test item test solution (limit concentration), four broods were released during the test period by all daphnids. The first brood was released on days 8 and 9 by all parent daphnids in the control and the saturated solution.
The mean number of living offspring produced per parent daphnid surviving the test were 84 for the control and 83 for the saturated test item solution* with 1.00 mg Solvaperm Blue 2B/L after 21 days. The reproductive output of daphnids in the saturated test item solution* (limit concentration) was comparable to the output of daphnids in the control (t-test, p = 0.05).
- Body length and weight of parent animals:
please refer to the respective table
- Type and number of morphological / behavioural abnormalities:
please refer to the respective table
- Time to first brood release or time to hatch:
The first brood was released from all parent daphnids in all treatments until day 9. Four broods were observed during the test period for all parental daphnids.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.44 (CI 1.29 - 1.52) mg/L

Reported statistics and error estimates:

The NOEC and LOEC for reproduction as measured by the intrinsic rate of natural increase and total number of living neonates per surviving female as well as adult survival was derived directly from the test data.
Significant deviations were determined in comparison to the control using statistical standard procedures as normality test, equal variance test and t-test. Prior to running an analysis of variance test, a normality test and an equal variance test were performed. P-values for the equal variance tests were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) was =0.05. For the determination of significant deviations of the reproduction rates and the intrinsic rates of natural increase t-test (p = 0.05) was used. The coefficients of variation of living offspring produced per parent were calculated for the control and the saturated test item solution.
The EC50-value was calculated for the reference dose-response test by sigmoidal dose-response regression. The confidence interval for the EC50-value was calculated from the best-fit dose-response curve, standard error and t-distribution using the software GraphPad prism.

Number of Juveniles in the Control and Test Group after 21 d

Nominal Test Item Concentration [mg/L]												No. of Parents producing Juveniles	Mean No. of Juveniles per Parent producing Juveniles			Compared versus Control
	Number of Juveniles in Replicate No.										Total
											No.
														CV		Red.		Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD	[%]		[%]
1.00 (Saturated solution)	79	77	72	83	92	85	75	94	91	86	834	10	83 ± 8	9		0.832		no
Control	90	87	86	81	82	92	82	80	80	81	841	10	84 ± 4		5		—

First Appearance of Living Juveniles in the Individual Groups

Nominal Test Item Concentration [mg/L]	Day of First Appearance of Living Juveniles										First Appearance Mean Day	Number of Broods per Adult [N]
	in Replicate No.
	1	2	3	4	5	6	7	8	9	10
1.00 (Saturated solution)	9	9	8	8	9	8	8	9	9	9	8.6	4
Control	9	9	8	9	8	8	8	8	8	9	8.4	4

Mortality [%] of the Adult Daphnids after 7, 14 and 21 d of Exposure

(n = 10)

Nominal Test Item Concentration [mg/L]	Mortality [%]
	7 days	14 days	21 days
1.00 (Saturated solution)	0	0	0
Control	0	0	0

Total Length and Dry Weight of the Parental Daphnids

Nominal Test Item Concentration [mg/L]	Total Length of the Parental Daphnids [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
1.00 (Saturated solution)	5.00	5.00	5.50	5.50	5.50	5.75	5.50	5.25	5.00	5.25	5.33	10	10.3	1.03
Control	5.50	5.00	5.75	5.00	5.75	5.50	5.75	5.50	5.50	5.75	5.50	10	10.5	1.05

Validity criteria fulfilled:

yes

Conclusions:

At the saturated test item solution with 1.00 mg/L of Solvent Blue 104 no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) of 1.00 mg/L and the and the Lowest Observed Effect Concentration (LOEC) of higher than 1.00 mg/l are concerning Solvent Blue 104 in saturated solution. All provided effect values are based on the nominal loading rate of Solvent Blue 104.

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of Solvent Blue 104 (batch number ESB0044477) was conducted as a limit test with the saturated solution* with 1.00 mg/L Solvaperm Blue 2B according to OECD 211 (2008) from 2012-09-24 to 2012-10-19, with the definitive exposure phase from 2012-09-26 to 2012-10-17 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids individually held were used for the saturated solution* and the control. At the start of the exposure, the daphnids were 2 to 24 h old. The test method was semi-static with renewal of the test solutions three times per week. The aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects on parameters such as intrinsic rate of natural increase, occurrence of aborted eggs and stillborn juveniles, time of production of the first brood, adult mortality, dry body weight and length of the parental daphnids.

A specific analytic of the test item Solvent Blue 104 was not possible because of the low solubility of 0.02 mg/L of the test item in water. The results given are based on the nominal concentration of 1.00 mg/L of the test item Solvent Blue 104 in the saturated solution*.

·   The average number of juveniles per parental daphnid were 84 for the control and 83 for the saturated test item solution* with 1.00 mg Solvent Blue 104/L after 21 days. The reproductive output of daphnids in the saturated test item solution* (limit concentration) was comparable to the output of daphnids in the control (t-test, p = 0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 5 % for the control and 9 % for the saturated test item solution*.

·   The intrinsic rates of natural increase (IR) of the surviving parent daphnids accounting for generation time and number of offspring were calculated as a measure for population growth and maintenance. The IR of the saturated solution* was comparable to the control group (t-test, p = 0.05).

·   The first brood was released from all parent daphnids in all treatments until day 9. Four broods were observed during the test period for all parental daphnids.

·   No dead juveniles or aborted eggs were observed in the control and the saturated solution.

·   No adult mortality or immobilization of parental daphnids was observed in the control and the saturated solution*.

*)    Saturated solution: The maximum dissolved 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. 

·   The mean dry body weight and total body lengths of all parental daphnids of the saturated solution and the control were determined at the end of the study.
The mean dry body weight of the parental daphnids was 1.05 mg per daphnid for the control and 1.03 mg per daphnid for the saturated test item solution.
The mean body length of the parental daphnids was 5.50 mm per daphnid for the control and 5.33 mm for the saturated test item solution.

·   No malesand ephippia (winter eggs) were observed in the control or test groups.

A summary of all test item related effects is given in Table 1. No biologically or statistically significant effect could be determined in the saturated test item solution with 1.00 mg/L Solvent Blue 104 in comparison to the control.

Summary of all Test Item Related Effects

(Based on the nominal concentration of the test item Solvaperm Blue 2B of 1.00 mg/L)

		Solvaperm Blue 2B
Effects	Control	Nominal concentration 1.00 mg/L	Significance
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	84 ± 4	83 ± 8	no^1)
Coefficient of Variation of the Mean Numberof Juveniles per Producing Parent	5	9	no
Intrinsic Rate of Natural Increase (Mean ± SD)	0.45 ± 0.02	0.45 ± 0.02	no^1)
Appearance of First Brood (Mean Day ± SD)	8.4 ± 0.5	8.6 ± 0.5	no
Number of Broods per Adult	4	4	no
Stillborn Juveniles and Aborted Eggs	0	0	no
Adult Mortality after 21 Days [%]	0	0	no
Parental Daphnids: Mean Dry Weight [mg]	1.05	1.03	no
Parental Daphnids: Mean Body Length [mm] ± SD	5.50 ± 0.29	5.33 ± 0.26	no
NOEC	1.00 mg/L

The water quality parameters (pH-value, dissolved oxygen, water hardness and temperature) were within the acceptable limits.

In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 is carried out with potassium dichromateas reference item once per month.
The EC₅₀ of the reference item at 1.44 mg/L (95% confidence limits: 1.29 - 1.52 mg/L) after 24 hours is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC₅₀-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Description of key information

No toxic effects against daphnia were observed under exposure conditions (21 d-NOEC 1.0 mg/L/21-d LOEC >1.0 mg/L). All provided effect values are based on the nominal loading rate.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 mg/L

Additional information

At the saturated test item solution with 1.00 mg/L of Solvent blue 104 (CAS 116 -75 -6) no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) was 1.00 mg/L and the Lowest Observed Effect Concentration (LOEC) was higher than 1.00 mg/L. All provided effect values are based on the nominal loading rate of the test item.