1,7-Naphthalenedisulfonic acid, 4-amino-3-[(1E)-2-(2,4-disulfophenyl)diazenyl]-5-hydroxy-6-[(1E)-2-(4-nitro-2-sulfophenyl)diazenyl]-, sodium salt (1:5)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31st July 2015 - 31st July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study contducted to OECD guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

Since no sodium bicarbonate solution was available, pH on the day of the testing was adjusted using Sodium hydroxide instead of Sodium bicarbonate. The study integrity was not adversely affected by the deviation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

yes

Remarks:

Since no sodium bicarbonate solution was available, pH on the day of the testing was adjusted using Sodium hydroxide instead of Sodium bicarbonate. The study integrity was not adversely affected by the deviation.

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

yes

Remarks:

Since no sodium bicarbonate solution was available, pH on the day of the testing was adjusted using Sodium hydroxide instead of Sodium bicarbonate. The study integrity was not adversely affected by the deviation.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

pentasodium 4-amino-3-[(1E)-2-(2,4-disulfonatophenyl)diazen-1-yl]-5-hydroxy-6-[(1E)-2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

EC Number:

811-858-8

Cas Number:

2149571-68-4

Molecular formula:

C22 H11 N6 O18 S5 .5Na

IUPAC Name:

pentasodium 4-amino-3-[(1E)-2-(2,4-disulfonatophenyl)diazen-1-yl]-5-hydroxy-6-[(1E)-2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

Constituent 2

Reference substance name:

pentasodium 4-amino-3-[(2,4- disulfonatophenyl)diazenyl]-5-hydroxy-6-[(4-nitro-2- sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate

IUPAC Name:

pentasodium 4-amino-3-[(2,4- disulfonatophenyl)diazenyl]-5-hydroxy-6-[(4-nitro-2- sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification K1600 black dye
Appearance Black powder
Batch G-152
Purity/Composition 99.03%
Test substance storage At room temperature
Stable under storage conditions until 31 December 2016 (retest date) (taken from label)
Purity/composition correction factor No correction factor required
Test substance handling No specific handling conditions required
Chemical name (IUPAC), synonym or
trade name
sodium 4-amino-3-((E)-(2,4-disulfonatophenyl)diazenyl)-6-
((E)-(4-nitro-2-sulfonatophenyl)diazenyl)-5-
oxidonaphthalene-1,7-disulfonate
CAS Number 1422649-48-6
Molecular formula C22 H11 N6 O18 S5 . 5 Na
Molecular weight 922.6
Solubility in water 20-23%
Stability in water Stable

Sampling and analysis

Details on sampling:

The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/l of sludge, as used for the test).

The pH was 5.6 and was adjusted to 7.5 using 1M NaOH (Merck, Darmstadt, Germany) on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test solutions

Details on test solutions:

The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol.

A 3,5-dichlorophenol solution with a final concentration of 1 g/l in Milli-RO water was prepared. The pH as used for the test was 8. The 3,5-dichlorophenol stock solution was stored in a freezer until use. The reference substance solution was defrosted at room temperature and diluted to reach the test concentrations. Three concentrations were tested: 2.0, 5.0 and 12 mg/l.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Study design

Water media type:

freshwater

Total exposure duration:

3 h

Post exposure observation period:

After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

Test conditions

Test temperature:

The medium temperature was recorded continuously in a temperature control vessel(s). The temperature control vessel(s) was/were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate
room. The temperature continuously measured in the temperature control vessels ranged between 19 and 21°C during the test, and complied with the requirements as laid down in the protocol (20 ± 2°C).

pH:

The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations and controls. The pH in all test vessels, before addition of sludge was between 7.5 and 7.6. After the 3 hour exposure period the pH was between 7.1 and 7.8.

Dissolved oxygen:

The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.

Details on test conditions:

Contact time: 3 hours, during which aeration and stirring took place.
Vessels: All glass open bottles/vessels.
Milli-RO / Milli-RO water: Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Synthetic medium (=sewage feed): 16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered.
The pH was within 7.5 ± 0.5.
Inhibitor of nitrification: A 2.32 g/l solution of N-allylthiourea (ATU, Merck Schuchardt OHG, Hohenbrunn, Germany) was prepared. 2.5 ml of this solution was added to 500 ml final test medium (final ATU concentration: 11.6 mg/l).
Air supply: Clean, oil-free air.
Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.
Oxygen recording: Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GOSystemelektronik GmbH, Germany), a multichannel measuring and controlling system.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/L

Nominal / measured:

meas. (not specified)

Effect concentrations 2

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

meas. (not specified)

Details on results:

In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg K1600 black dye per litre (average inhibition 5%). Thus, the EC50 was above the highest concentration tested (1000 mg/l).

There was no oxygen uptake from abiotic processes and no inhibition of nitrification was observed at 1000 mg/l.

Reported statistics and error estimates:

The respiration rate (R) from each vessel, in mg O2/l.h was calculated or interpolated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/l.

R was calculated by the BlueBox software as (V1 – V2)/Δt * 60

Where:
V1=Value 1: the oxygen concentration at the start of the selected section of the linear phase (mg O2/l),
V2=Value 2: the oxygen concentration at the end of the selected section of the linear phase (mg O2/l),
Δt is the time interval between these two measurements.
Negative R values were expressed as 0 mg O2/l.h (V1
Furthermore the respiration rate was expressed as the amount of oxygen consumed per g dry weight of sludge per hour (Rs in mg O2/g.h).

Rs = R / SS
Where SS is the concentration of suspended solids in the test mixture (g/l).

The different indices of R which may be combined are:
S specific rate
T total respiration rate
N rate due to nitrification respiration (combined limit/ range-finding test)
H rate due to heterotrophic respiration (combined limit/ range-finding test)
A rate due to abiotic processes (combined limit/ range-finding test)
B rate based on blank assays (mean)

Calculation of oxygen uptake due to nitrification:
The relationship between total respiration (RT), nitrification respiration (RN) and heterotrophic respiration (RH) is given below:

RN = RT - RH

Where:
RN is the rate of oxygen uptake due to nitrification (mg O2/l.h).
RT is the measured rate of oxygen uptake (no ATU) (mg O2/l.h).
RH is the measured rate of oxygen uptake with added ATU (mg O2/l.h).

Calculation of the inhibition of the respiration rate:
The percentage inhibition, IT, of total oxygen consumption is given below:
IT = [1- (RT/RTB)] x 100%
Similarly, the percentage heterotrophic oxygen uptake, IH, is given below:
IH = [1- (RH/RHB)] x 100%
Finally, the inhibition of oxygen uptake due to nitrification (if applicable), IN, is given below:
IN = [1- (RT-RH) / (RTB-RHB)] x 100%

Any other information on results incl. tables

Evaluation was based on the inhibition of the total respiration.

ECx:

For the reference substance calculation of EC50 value was based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the substance.

For K1600 black dye no EC50-value could be calculated because the test substance proved to be nontoxic (EC50 > 1000 mg/l).

NOEC determination:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two sample t-test Procedure, α=0.05, one-sided, smaller).

The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Acceptability of the test:

1. The mean blank control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (21 mg oxygen per one gram of activated sludge). The coefficient of variation of oxygen uptake in blank control replicates did not exceed 30% at the end of the definitive test (13%).

2. The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (3.6 mg/l)

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test K1600 black dye was not toxic to waste water bacteria (activated sludge) at 1000 mg/l.

The EC50 was above 1000 mg/l

Executive summary:

The influence of K1600 black dye on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209, 2010. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

The batch of K1600 black dye tested was a black powder with a purity of 99.03%. No correction was made for the purity/composition of the test substance.

A stock solution of 10 g/l was prepared. Vigorous mixing (vortex) and stirring was applied to completely dissolve the test substance and ensure homogeneity. Volumes of the black stock solution (pH 9.2) corresponding to the test concentration were then added to the test media. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/l were tested. The concentration rate was tested in triplicate, lower concentrations consisted of one replicate. Furthermore, at 1000 mg/l an abiotic control (1 replicate) and three replicates with a nitrification inhibitor were tested. Responses were compared to the blank and nitrification controls.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a concentration rate of 1000 mg K1600 black dye per litre (average inhibition 5%). Thus, the EC50 was above the highest concentration tested (1000 mg/l).

The batch of activated sludge was tested for sensitivity with the reference substance 3,5-dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Under the conditions of this present test K1600 black dye was not toxic to waste water (activated sludge) bacteria at 1000 mg/l (NOEC).

The EC50 was above 1000 mg/l.