Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

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))

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

Two deviations from the study plan and the test guidelines was realized.
1. The start value of oxygen content for measurement of oxygen consumption was lower than 7 mg/L.
2. The start value of oxygen content for measurement of oxygen consumption was lower than 7 mg/L.
3. The required incubation time did exeed and fall below 3 hours.
An influence to the results of this study can be excluded.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

EC Number:

943-133-8

Molecular formula:

C32H22CrN10O8.C10-14H21-29NH2 / C34H24CrN8O6.C10-14H21-29NH2 / C33H23CrN9O7.C10-14H21-29NH2

IUPAC Name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Test material form:

solid

Details on test material:

- Physical state/Appearance: Solid / red
- Total Organic Carbon: 592 mg/g

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient at room temperature

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Application of the test substance: Direct addition by initial weight into the test flasks. The test substance was weighed in the required amounts for the test concentrations directly to the test vessels.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Test system: municipal Activated sludge
Age: 1 day
Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
Collection of the test system: 01 Aug 2016
Arrival in the test facility: 01 Aug 2016
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 Dw.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

21.0 – 22.4 °C

pH:

7.4 - 8.2

Dissolved oxygen:

Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 mg/L with the exceptions of 4 vessels were the oxygen concentration was about 6.6 - 6.9 mg/L

Nominal and measured 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)
- Aeration: Yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per reference substance (replicates): 2
- Sludge concentration (weight of dry solids per volume): 1.5 g/L Dw

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

OTHER TEST CONDITIONS
- Adjustment of pH: No

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 1000, 500, 250, 125, 62.5 mg/L as nominal concentration
- Results used to determine the conditions for the definitive study:

PREPARATION OF THE TEST
The test substance was added in the required amounts according to the test concentrations directly to the test vessels with 234 mL deionized water. Aliquots of the reference substance stock solution were dosed to the test vessels and made up with deionized water to a volume of 234 mL. 16 mL synthetic medium were dosed to each test vessel with test substance and reference substance afterwards. To prepare the blank control assays 234 mL of deionized water and 16 mL synthetic medium were mixed. The pH-values in all test vessels were checked. An adjustment was not necessary.
After addition of 250 mL of inoculum suspension (3 g/L DW) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance.
After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The temperature was measured for seven times in a separate vessel filled with aerated deionized water during incubation phase. The content of oxygen at the start of the measurements was > 7 mg/L with the exception described in remark. The oxygen uptake was measured for a period of about 8 to 10 minutes.
The total oxygen consumption was measured in the sequence BC (blank control) 1-3 and RS1, RS (reference substance) 6, TS (test substance) 1 and RS4-5, RS2-3 and TS2-TS3, TS4-TS7, TS8-11 and TS12-15. The oxygen consumption of the blank control BC4-6 were measured at last. The change of the required measurement sequence happens because the test vessels RS2, RS3 and RS6, PS1 were interchanged at the point of measurement. The required incubation time did not exeed or fall below 3 hours with the exceptions for RS6 and TS1 with 2h45 min and for RS2/RS3 with 3h15 min.
No abiotic control was tested.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC10

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.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 3

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 4

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Validity criteria:
The coefficient of variation of the six replicates of blank control was 13.2 % O2 consumption.
The EC50 of 3,5-dichlorophenol was 3.5 mg/L
The mean oxygen uptake of the blank controls was 18 mg/g*h

The results in this study were consistent with the validity criteria with one exception. The mean oxygen uptake in the blank controls was lower than 20 mg/g*h (18 mg/g*h). The reference substance shows an EC50 in the specified range (usual range of EC50 of the last 20 values in the laboratory was 3.5 – 15.5 mg/L, with the values in this study). The used activated sludge which was collected from the same waste water treatment plant showed a maximum oxygen consumption of 36.5 mg/g*h and minimum oxygen consumption of 8.2 mg/g*h of the last 20 measurements (with the values in this study). For this the study is classified as valid.

Results with reference substance (positive control):

In order to verify that the microorganisms are responding normally to toxic stress, tests with a reference substance are conducted according to OECD 209 guideline. The results from the reference substance test are compared to EC50 values published in OECD 209 guideline, which represent the typical response range.
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.
These results indicate that the microorganisms are responding normally to toxicant stress.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of the degradation activity of activated sludge is not anticipated when introduced to biological treatment plants in appropriate low concentrations.