Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Ambient at room temperature

Analytical monitoring:

no

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 (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: 22 Aug 2016
- Arrival in the test facility: 22 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18.7 - 19.8 °C

pH:

7.5 - 8.3

Dissolved oxygen:

- Oxygen concentration during aeration: > 2 mg/L
- Oxygen concentration immediately before measurement: > 7 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg/L without correction of purity and blank controls.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glas-beakers (nominal volume 1L)
- Test volume: 500 mL
- Aeration: Yes
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- No. of vessels per reference substance: 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
- Measurements of conditions: The temperature was measured for seven times in a separate vessel filled with aerated deionized water during incubation phase.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Nominal concentrations of reference substance: 100, 10 and 1 mg/L.

EFFECT PARAMETERS MEASURED: oxygen uptake, for 8 - 10 minutes

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 deionised 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.

EXPOSURE
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 the mixtures in the test vessels were placed subsequently for oxygen measuring. 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 (blank 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 last. No abiotic control was tested.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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

Key result

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

Details on results:

No respiration inhibition was observed after 3 hours exposure at any of the tested concentrations.

VALIDITY CRITERIA
- The coefficient of variation of the six replicates of blank control was 10.8 % O2 consumption.
- The EC50 of the reference substance was in the range of 2-25 mg/L (see ‘Results with reference substance (positive control'), indicating that the microorganisms are responding normally to toxicant stress.
- The mean oxygen uptake of the blank controls was 22 mg/g*h.
In conclusion, the test conformed to all validity criteria and is valid.

Results with reference substance (positive control):

The 3-h EC50 value of the reference substance was determined to be 9.7 mg/L.

ADDITIONAL EFFECT VALUES

- 3-h EC20: > 1000 mg/L

- 3-h EC80: > 1000 mg/L

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.

Description of key information

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

Additional information

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (BASF SE, 2017). In this study non-adapted activated sludge from a domestic sewage was exposed to nominal test substance concentrations of 0 (control), 62.5, 125, 250, 500 and 1000 mg/L for 3 hours. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. No inhibition of respiration rates was observed up to the highest concentrations. Therefore, the 3-h EC50 and 3-h EC10 values were both determined to be > 1000 mg/L.