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-dihydro-4-[(2-hydroxy-4-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

Study period:

21 Sep 2016 to 29 Sep 2016

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 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-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

EC Number:

943-145-3

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-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

Test material form:

solid

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:

- Age: 1 day
- Origin: aeration tank of the wastewater treatment plant of Mannheim, Germany
- Collection of the test system: 21 Sep 2016
- Arrival in the test facility: 21 Sep 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:

19.0 - 19.7 °C

pH:

7.4 - 8.2

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

Details on test conditions:

TEST PREPARATION
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.

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

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 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. The total oxygen consumption was measured in the sequence BC1-3 and RS1, RS2-5, RS6 and TS1-3, 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.

EFFECT PARAMETERS MEASURED: oxygen consumption
The consumption rate (mg O2/L*x minutes) of each test concentration and the blank controls were determined after 3 hours of exposure. The consumption rates (mg O2/L*h) were used to calculate the inhibition effects at particular test concentrations compared to the mean consumption rate of all controls. The duration of the measurement was about 8 - 10 minutes.

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.:

> 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. See ‘Any other information on results incl. tables’.

Results with reference substance (positive control):

The 3-h EC50 value of the reference substance was determined to be 13.0 mg/L.

Reported statistics and error estimates:

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

Any other information on results incl. tables

Table: Measured data of oxygen content of the test assays at the start and end of the evaluation time

Assay identification	Blank control						Reference substance						Test substance
Concentration	-						1 mg/L		10 mg/L		100 mg/L		62.5 mg/L			125 mg/L			250 mg/L			500 mg/L			1000 mg/L
Sample Identification	BC1	BC2	BC3	BC4	BC5	BC6	RS1	RS2	RS3	RS4	RS5	RS6	TS1	TS2	TS3	TS4	TS5	TS6	TS7	TS8	TS9	TS10	TS11	TS12	TS13	TS14	TS15
O2 conc. Start [mg/L]	7.9	7.5	8.0	8.0	8.5	7.5	8.0	8.0	9.0	8.5	9.0	9.0	8.0	8.0	8.5	8.0	8.0	7.7	7.5	8.0	7.5	7.1	8.4	7.8	8.2	8.1	7.8
O2 conc. End [mg/L]	4.9	4.5	5.0	5.2	5.7	4.0	5.2	5.4	7.5	6.8	8.5	8.5	5.0	5.0	5.7	5.0	5.0	4.5	4.4	4.9	4.2	4.4	5.5	5.0	5.9	5.1	4.9
O2 onsumption rate [mg/L]	3.0	3.0	3.0	2.8	2.8	3.5	2.8	2.6	1.5	1.7	0.5	0.5	6.1	6.1	6.1	6.0	6.1	6.1	5.9	5.9	6.0	6.0	6.0	5.9	6.0	6.1	6.0
O2 consumption in minutes	6.0	6.0	6.3	6.0	6.0	6.3	6.0	6.0	6.0	6.0	6.5	6.5	3.0	3.0	2.8	3.0	3.0	3.2	3.1	3.1	3.3	2.7	2.9	2.8	2.6	3.0	2.9
O2 consumption rate [mg/L*h], Rt	30	30	29	28	28	33	28	26	15	17	5	5	30	30	28	30	30	31	32	32	33	27	29	28	26	30	29
Specific O2 consumption rate [mg/g*h]	20	20	19	19	19	22	19	17	10	11	3	3	20	20	19	20	20	21	21	21	22	18	19	19	17	20	19
Calculation of inhibition respiration [%], IT	-						6	12	49	43	83	83	-1	-1	6	-1	-1	-4	-8	-8	-11	9	2	6	12	-1	2

TS = test substance, RS = reference substance, BC = blank control

The respiration rate was calculated in mg O2/L*h. The oxygen consumption was measured over a period of 8-10 minutes. 6 - 6.5 (for BC and RS) and 5.9 - 6.1 (for TS) minutes from the plotted track within a linear range of the graph was used for the determination of the respiration rate. Equation: IT=(1-(RT/RTB)*100

For calculating the inhibition the results of the fractions in the equation were rounded to two decimal places.

IT= inhibition of the total respiration [%]

RT = oxygen consumption in the test assay [mg O2/L*h]

RTB= mean value oxygen consumption of all blank controls [mg O2/L*h]

ADDITIONAL EFFECT VALUES

- 3-h EC20: > 1000 mg/L

- 3-h EC80: > 1000 mg/L

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.