reaction mass of: tetrasodium 7-(4-(4-fluoro-6-(4-(2-sulfonatoethylsulfonyl)phenylamino)-1,3,5-triazin-2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate;tetrasodium 7-(4-(4-hydroxy-6-(4-(2-sulfonatoethylsulfonyl)phenylamino)-1,3,5-triazin-2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

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:

17 February 1998 to 18 February 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU & OECD test guidance in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

88/302/EEC

Deviations:

no

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:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000mg/L

Vehicle:

no

Details on test solutions:

Addition of the test material into the test system Stock solution
Date of preparation : February 17, 1998
Addition of the test substance: 2.0002 g in 800 mL highly purified water
The batch was stirred over : 1 hour
At a temperature of: : 23 °C
pH after this period : 5.4
The pH was not adjusted.
The clear solution of the test substance was filled up to 1000 ml and used as stock solution.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Origin: Sewage treatment plant at Frankfurt am Main-Sindlingen
Type of inoculum: Domestic activated sludge
Preparation of the suspension: sediment from activated sewage sludge was washed three times by centrifuging and resuspending in water with 0.9 % NaCl.
Amount used: 450 ml/L sludge suspension
Content of suspended solids: 22.1 g/L (centrifuged wet matter)
Content of suspended solids: 3.56 g/L (dry matter)
Content in the final test volume: 1.6 g/L (dry matter)
Pretreatment: corresponding to the cited test guideline.
State of acclimatization: not pre-adapted to the test substance or the reference substance.
Storage: The concentrated inoculum suspension was aerated over 1 day. Nutrition solution was added during this period, as mentioned in the test guideline.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation

Hardness:

No data

Test temperature:

The incubation temperature was controlled at 18.5°C to 20.8°C

pH:

7.9-8.2 after the incubation of the test batches.

Dissolved oxygen:

No data

Salinity:

Not applicable.

Nominal and measured concentrations:

250, 500 and 1000 mg/L (nominal)

Details on test conditions:

Incubation vessels: Glasbottles with a diameter of 60 mm and a grounded neck (NS 29), height 170 mm and a total volume of approx. 480 mL. The bottles contained magnetic stirrer bars and were incubated in a water bath on magnetic stirrers.
Test volume: 200 mL each batch
Temperature control: The temperature control was performed by a water circulating thermostate.
Aeration rate/test vessel: 50 litres per hour, only during the incubation period.
Oxygen measurements: With oxygen electrodes, connected to oximeters and two-channel recorders.
Monitoring: During the measuring period the oxygen concentration curves were plotted by the recorders.
Respiration rate: The slope of the linear part of the respiration curves accorded to the corresponding respiration rates.
Measurements of the pH: pH-meters

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The following effect concentrations were calculated from the measured values of inhibition: EC-values Concentrations:

EC20 > 1000 mg/L (nominal)
EC50 > 1000 mg/l (nominal)
EC80 > 1000 mg/l (nominal)

A significant inhibition could not be observed. The documented EC-values were above the highest tested concentration.

The results of testing the toxicity of Reaktive-Yellow FD08064 in the Activated sludge: respiration inhibition test revealed that: The EC50 of the substance to activated sludge (domestic sewage treatment plant) is higher than 1000 mg/1 (nominal), which was the highest concentration in the test.
The results of this test are only valid for batch.

Results with reference substance (positive control):

Testing the respiration inhibition of activated sludge proceeded without disturbances. The EC50 of the reference substance 3,5-Dichlorophenol was between 5 mg/L and 30 mg/L and amounted to 14 mg/L. The range of confidence could not be determined. Raw data were calculated with Probit Analysis according to Finney (1971).

Reported statistics and error estimates:

Not reported.

Table 1: Results after 3 hours incubation time

Controls / tests [mg/L]	Start values [mg O2/L]	End values [ mg O2/L ]	Recording time [ minutes ]
Blank 1	6.7	3.2	5.0
Blank 2	6.5	3.1	4.67
Blank 3	6.6	3.0	5.0
DCP, 5 mg/L	7.7	5.0	5.0
DCP, 20 mg/L	8.4	7.0	5.0
DCP, 30 mg/L	8.5	7.3	5.0
1000.0(A)	6.5	2.8	5.0
1000.0 (B)	6.9	3.5	5.0
500.0 (A)	7.2	3.0	6.0
500.0 (B)	6.8	2.8	6.0
250.0 (A)	6.7	3.1	5.0
250.0 (B)	6.5	2.7	5.0
125.0 (A)	6.9	3.4	5.0
125.0(B)	7.6	4.0	5.0
62.5 (A)	7.4	3.8	5.0
62.5 (B)	7.0	3.4	5.0
31.3(A)	7.0	3.3	5.0
31.3 (B)	6.8	2.8	5.0
15.6 (A)	7.2	3.5	5.0
15.6(B)	7.2	3.8	5.0
7.8 (A)	6.7	2.6	5.0
7.8 (B)	7.2	3.4	5.0

Blanks: Batches without test substance to determine the respiration activity of the used inoculum,

DCP: reference controls with 3,5-Dichlorophenol to test the sensitivity of the inoculum,

(A)/(B): parallel batches. The corresponding amounts of test substance in the batches are given in mg/1

Table 2:	Results after 3 hours incubation time
Controls / tests [mg/L]	Activity (A) [mg O2/L*h]	Respiration inhibition (I)               [%]	Means of blanks 1-3(A) and tests A+B (I)
Blank 1	42.0	-	(Activity)
Blank 2	43.7	-	43.0
Blank 3	43.2	-
DCP. 5 mg/L	32.4	24.6	-
DCP. 20 mg/L	16.8	60.9	-
DCP. 30 mg/L	14.4	66.5	-
1000.0 (A)	44.4	-3.3	(Inhibition)
1000.0 (B)	40.8	5.0	0.8
500.0 (A)	42.0	2.2	(Inhibition)
500.0 (B)	40.0	6.9	4.6
250.0 (A)	43.2	-0.6	(Inhibition)
250.0 (B)	45.6	-6.1	-3.3
125.0 (A)	42.0	2.2	(Inhibition)
125.0 (B)	43.2	-0.6	0.8
62.5 (A)	43.2	-0.6	(Inhibition)
62.5 (B)	43.2	-0.6	-0.6
31.3 (A)	44.4	-3.3	(Inhibition)
31.3 (B)	48.0	-11.7	-7.5
15.6(A)	44.4	-3.3	(Inhibition)
15.6 (B)	40.8	5.0	0.8
7.8 (A)	49.2	-14.5	(Inhibition)
7.8 (B)	45.6	-6.1	-10.3

Blanks: Batches without test substance to determine the respiration activity of the used inoculum.

DCP: reference controls with 3,5-Dichlorophenol to test the sensitivity of the inoculum.

(A)/(B): parallel batches. The corresponding amounts of test substance in the batches are given in mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of the substance to activated sludge (domestic sewage treatment plant) is higher than 1000 mg/L (nominal), which was the highest concentration in the test.

Executive summary:

A study was condcuted according to EU test guidance 88/302/EEC part C and OECD guideline 209 in compliance with GLP.

No inhibition compared to blanks were observed at any concentration tested. The EC10 and EC50 of the substance to activated sludge (domestic sewage treatment plant) is higher than 1000 mg/L (nominal), which was the highest concentration in the test.

EC50 > 1000 mg/L. The substance is not considered to inhibit the respiration of sewage sludge micro-organisms.

Description of key information

The EC10 and EC50 of the substance to activated sludge (domestic sewage treatment plant) is higher than 1000 mg/L (nominal), which was the highest concentration in the test.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A study was condcuted according to EU test guidance 88/302/EEC part C and OECD guideline 209 in compliance with GLP.

No inhibition compared to blanks were observed at any concentration tested. The EC10 and EC50 of the substance to activated sludge (domestic sewage treatment plant) is higher than 1000 mg/L (nominal), which was the highest concentration in the test.

EC50 > 1000 mg/L. The substance is not considered to inhibit the respiration of sewage sludge micro-organisms.