Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

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

Study period:

19 March 2014 - 24 April 2014

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

None

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant of 75172 Pforzheim, Germany, collected from the aeration tank, was used as the microbial inoculum for the test. This plant was predominantly treating domestic sewage.

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

the test assays were aerated during the whole test period

Hardness:

no data

Test temperature:

19.8 – 21.5 °C (measured during O2-determination at 3 h after start of respiration)

pH:

7.38 – 7.87

Dissolved oxygen:

Oxygen content in the control after 3 h (total oxygen uptake assay)
1.15 to 7.57 mg O2/L

Salinity:

no data

Nominal and measured concentrations:

no data

Details on test conditions:

Two litre glass beakers were used; the test volume was 500 mL; the test period was 3 h; 16 mL synthetic sewage feed was used.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol (DCP)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

653 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

30.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

97.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The effect of the test item on microorganisms of an aerobic microbial treatment plant was tested using the activated sludge respiration inhibition test. The test item FAT 20060/F had significant effects at 97.7, 312.5 and 1000 mg/L. Hence, the EC50 of FAT 20060/T was determined to be 653 mg/L and the NOEC is 30.5 mg/L.

Results with reference substance (positive control):

The EC50 of the total oxygen uptake for the reference item DCP was estimated between 5-25 mg/L after 3 hours. This is within the accepted range of 2 to 25 mg/L for total respiration.

Reported statistics and error estimates:

The statistical evaluation was performed for specific respiration rate using SAS® Software service pack 9.3 (2002–2010). The control and all test item concentrations were statistically tested using the Dunnetts t-test (left sided). The EC50 was calculated using probit analysis following logistic procedure. After 3 hours, statistically significant effects were observed at 97.7, 312.5 and 1000 mg/L for the total oxygen uptake. The NOEC is therefore determined to be 30.5 mg/L and the LOEC 97.7 mg/L.

Results, specific respiration rates

	Specific respiration rates [mg O2/(g × h)]
Test assay	Total
	3 h
Control 1	27.05
Control 2	27.27
Control 3	29.71
Control 4	22.45
Control 5	27.29
Control 6	22.59
Mean	26.06
Stdev	2.91
CV	11
9.54 mg/L 1	26.83
9.54 mg/L 2	25.15
9.54 mg/L 3	23.84
30.5 mg/L 1	24.71
30.5 mg/L 2	25.69
30.5 mg/L 3	27.13
97.7 mg/L 1	21.80
97.7 mg/L 2	21.39
97.7 mg/L 3	20.85
312.5 mg/L 1	18.15
312.5 mg/L 2	17.55
312.5 mg/L 3	16.22
1000 mg/L 1	10.30
1000 mg/L 2	11.09
1000 mg/L 3	10.79
DCP 0.2	20.95
DCP 1	18.15
DCP 5	17.05
DCP 25	3.57

Results, inhibition

Test assay nominal [mg/L]	Inhibition [%]
	Total
	3 h
9.54	3.0
30.5	0.8
97.7	18.1
312.5	33.6
1000	58.8
DCP 0.2 mg/L	19.6
DCP 1 mg/L	30.4
DCP 5 mg/L	34.6
DCP 25 mg/L	86.3

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of FAT 20060/T was determined to be 653 mg/L and the NOEC is 30.5 mg/L.

Executive summary:

The effect of the test item on microorganisms of an aerobic microbial treatment plant was tested using the activated sludge respiration inhibition test conducted according to OECD Guideline 209. The total oxygen uptake was measured up to a test item concentration of 1000 mg/L. After 3 hours, statistically significant effects were observed at 97.7, 312.5 and 1000 mg/L for the total oxygen uptake. The NOEC is therefore determined to be 30.5 mg/L and the LOEC 97.7 mg/L. The EC50of the total oxygen uptake is determined to be 653 mg/L. The EC50 of the total oxygen uptake for DCP was estimated within a range of 5 to 25 mg/L after 3 hours. This is within the recommended range of 2 to 25 mg/L. All validity criteria of OECD Guideline for Testing of Chemicals No. 209 were fulfilled:

•  The mean oxygen uptake rate of the blank control were determined to be 26 mg O2/(g × h) dry matter after 3 hours. Hence the rate was not less than 20 mg oxygen uptake per g of activated sludge (dry weight of suspended solids) per hour.

•   The EC50of the total oxygen uptake for DCP was estimated between 5 -25 mg/L after 3 hours. This is within the accepted range of 2 to 25 mg/L for total respiration.

•  The coefficient of variation of the control for total oxygen uptake were 11 % after 3 hours and did not exceed 30 % at the end of the test.

Description of key information

The EC50 was determined to be 653 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

653 mg/L

EC10 or NOEC for microorganisms:

30.5 mg/L

Additional information

The effect of the test item on microorganisms of an aerobic microbial treatment plant was tested using the activated sludge respiration inhibition test. The test item FAT 20060/F had significant effects at 97.7, 312.5 and 1000 mg/L. Hence, the EC50 of FAT 20060/T was determined to be 653 mg/L and the NOEC is 30.5 mg/L. All validity criteria according to OECD Guideline for Testing Chemicals 209 were fulfilled.