Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

EC Number:

915-758-6

Molecular formula:

C32H18CrN6O8.Na / C32H22CrN10O8.Na / C32H20CrN8O8.Na

IUPAC Name:

Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Test material form:

solid

Details on test material:

Appearance:
- physical state: solid
- color: dark brown

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 (especially for difficult test substances)
Direct addition by initial weight into the test flasks. The test substance was weighed in the required amounts for the test concentrations directly into the test vessels.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Age: 1 day
Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
Collection of the test system: 2016-08-06
Arrival in the test facility: 2016-08-06
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:

21.0 – 21.3 °C

pH:

7.4 - 8.1

Dissolved oxygen:

Oxygen concentration during aeration: > 2 mg/L
Oxygen concentration immediately before measurement: > 7 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: glass-beakers (nominal volume 1L)
- Test volume: 500 mL
- 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 dry weight

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

PREPARATION OF THE TEST
The test substance was added in the required amounts according to the test concentrations directly into 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 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. The oxygen uptake was measured for a period of about 8 to 10 minutes.
The total oxygen consumption was measured in the sequence BC (blanc control) 1-3 and RS (reference substance) 1, RS2-5, RS6, and TS (test substance) 1-3, TS4-7, TS8-11 and TS12-15. The oxygen consumption of the blank control BC4-6 were measured at the end. No abiotic control was tested.

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

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

310 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:

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 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 15.9 % O2 consumption.
The EC50 of 3,5-dichlorophenol was 4.3 mg/L
The mean oxygen uptake of the blank controls was 17 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 (17 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 3.4 – 15.5 mg/L). 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. For this the study is classified as valid.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2 - 25 mg/L within 3 h.

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.