disodium 4-hydroxy-3-[2-[4-({4-[2-(1-hydroxy-4-sulfonatonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-07-04 to 2013-07-05

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

not specified

IUPAC Name:

not specified

Details on test material:

- Name of test material (as cited in study report): Acid Red RN 2949
- Molecular formula:
C41H30N4Na2O10S2 (as sodium salt)
C41H32N4O10S2 (as free acid)
- Molecular weight:
848.82 g/mol (as sodium salt)
804.86 g/mol (as free acid)
- Physical state: Dark red powder
- Analytical purity: 100 % (composition see AZ. No. 12-2302/4928 v1., dated 2012-08-28)
- Purity test date: 2013-02-21
- Lot/batch No.: Reg (=Batch 48)
- Expiration date of the lot/batch: 2015-03-05
- Stability under test conditions: see results
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Respective amount directly weighed into each test vessel
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: Extended limit test to determine the NOEC: 250 - 500 - 1000 mg/L
- Controls: Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: The sludge was washed twice with tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 3.27 g/L corresponding to 1.64 g/L in the test vessel

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

Not determined

Test temperature:

21.0 °C

pH:

pH-value of the activated sludge 7.46
pH-value of the synthetic waste water 7.45

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 3
- Composition of test medium:
Test item concentration: respective amount directly weighed into each test vessel
Test item concentration [mg/L]
250 500 1000
Inoculum [mL] 250
Synthetic waste water [mL] 16
Stock solution [g/L] 2.5
Stock solution [mL] 50 100 200
Demineralised water [mL] 184 134 34



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Range finding study
Inhibitions were as follows:
Nominal Test Item Concentration
[mg/L] pH(test item in demin. water) pH(test item in medium) Inhibition[%]
1st preliminary test
10 6.63 7.36 16
100 6.70 7.35 16
1000 6.39 7.32 5
2nd preliminary test
1000 -* 7.51 10
*) not measured

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 95.1 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Any other information on results incl. tables

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control, Test Item and Reference Item Concentrations

		Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control		1	62.0	37.8	–	–
		2	66.0	40.2
		3	66.4	40.5
		4	56.4	34.4
		5	50.8	31.0
		6	46.0	28.0
Test item	250	1	65.2		-13	-5
		2	63.6		-10
		3	60.4		-4
		4	56.0		3
		5	57.6		1
	500	1	57.2		1	7
		2	55.6		4
		3	48.8		16
		4	60.0		-4
		5	48.8		16
	1000	1	60.8		-5	19
		2	46.8		19
		3	40.8		30
		4	40.8		30
		5	44.4		23

Repl. = Replicate

Mean specific respiration rate of the control: 35.3 mg O₂/g×h

Coefficient of variation of oxygen uptake rates of the control: 14.4 %

Suspended solids in the test vessel: 1.64 g/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item Acid Red RN 2949 is 500 mg/L, the EC50 of the test item is > 1000 mg/L.
The test item is not toxic up to the concentration of 500 mg/L to activated sludge of a municipal sewage treatment plant.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Acid Red RN 2949(batch number: Reg(=Batch 48)) from 2013-07-04 to 2013-07-05, with the definitive experimental phase on 2013-07-05 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Sarstedt, Germany . The test was carried out under static conditions with the substance concentrations of 250 - 500 - 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates were - 5%, 7 % and 19 %.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 95.1 mg/L.

Table 1:           NOEC and EC-Value of Acid Red RN 2949

		EC-value [mg/L]
NOEC*		500
EC50		> 1000

                         *) No statistically significant effect (p 0.05), ANOVA, Dunnett’s Method

The NOEC of the test item Acid Red RN 2949 is 500 mg/L. The test item is not toxic up to the concentration of 500 mg/L to activated sludge of a municipal sewage treatment plant.