Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-20 to 2017-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

EC Number:

915-704-1

Molecular formula:

C36H28ClCrN7O10S2.Na / C34H26Cl2CrN8O8S2.Na / C38H30CrN6O12S2.Na

IUPAC Name:

Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

Test material form:

solid

Details on test material:

Appearance
- physical state. solid
- color: brown

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

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: 1000.0 mg of the reference substance was added to about 800 mL of deionized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.2 with 1 M sodium hydroxide solution. Following this, the stock solution was made up to 1 L with deionized water. The stock solution appeared yellowish and no undissolved reference substance was visible.
- Controls: blank, positive control (3,5 dichlorophenol)
- Evidence of undissolved material: no

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: aeration tank of the wastewater treatment plant of Mannheim, Germany.
- Preparation of inoculum for exposure: After arrival of the activated sludge suspension in the test facility the suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L dry weight.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

20.1 - 20.6 °C

Nominal and measured concentrations:

Test concentrations:
1000, 500, 250, 125, 62.5 mg/L as nominal concentration based on test substance without correction of purity and blank controls.
100, 10, 1 mg/L as nominal concentration based on reference substance.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glas-beakers (nominal volume 1L)
- Type (delete if not applicable): closed
- Test volume: 500 mL
- Aeration: at the beginning of the experiment
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per reference substance concentration (replicates): 2
- Sludge concentration (weight of dry solids per volume): 1.5 g/L dry weight
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Particulate matter: 16 mL/test vessel of 100-fold concentrated OECD medium

OTHER TEST CONDITIONS
- Defoamer used: yes, 25 µL/test assays Triisobutyl phosphate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of < 3.2 should be used, preferably encompassing the range from 10 - 80 % inhibition.
- Test concentrations: 1000, 500, 250, 125, 62.5 mg/L

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 2

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? yes, According to the test guideline OECD 209 the EC50 values of the reference substance 3,5-dichlorophenol should be in the range of 2 - 25 mg/L after 3 hours of incubation. The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2 - 25 mg/L in 3 hours.
- Relevant effect levels: the EC50 of 3,5-dichlorophenol was 6.0 mg/L.

Reported statistics and error estimates:

The consumption rates were used for the determination of the ECx by the probit method based on Finney [1] with the software TOXRAT Professional 2.10 [2]. The effect concentrations were given with an accuracy of 2 significant digits.

[1] Finney, D.J., Probit Analysis; Cambr. Univ. Press, 3rd ed., 1971
[2] Ratte, M.: ToxRat Professional 2.10 (2001-2010). ToxRat Solutions GmbH, Naheweg 15, 52477 Alsdorf, Germany

Any other information on results incl. tables

Validity criteria:

The coefficient of variation of the six replicates of blank control was 12.2 % O2 consumption.

The EC50 of 3,5-dichlorophenol was 6.0 mg/L

The mean oxygen uptake of the blank controls was 23 mg/g*h

In conclusion, the test conformed to all validity criteria and is valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Depending on local conditions and existing concentrations, disturbances in the biodegradation process of activated sludge are possible.