3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

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:

December 20, 2001 - March 14, 2002

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

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

other: EU Commission Directive 87/302/EEC, Part C, Activated Sludge Respiration Inhibition Test, 1988

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

No data

Vehicle:

no

Details on test solutions:

Please refer to 'Details on test conditions' and table in 'Any other information in MM'

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzerland) treating predominantly domestic wastewater.
- Preparation of inoculum for exposure: During the entire holding period, the sludge was kept at room temperature under continuous aeration. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was adjusted from 8.4 to 6.9 with a diluted sulfuric acid solution.
- Pretreatment: The sludge was washed once with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During holding, the sludge was fed three times with 50 ml synthetic wastewater* per liter. On the fourth day of holding immediately prior to use, the sludge was fed once again with 25 ml synthetic wastewater* per liter.

*Synthetic wastewater: [16 g peptone; 11 g meat extract; 3 g urea; 0.7 g NaCl; 0.4 g CaCl2 · 2H20; 0.2 g MgS04 · 7H20; 2.8 g K2HP04] filled up to a final volume of 1 liter with deionized water

Test type:

static

Water media type:

other: tap water and synthetic wastewater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

none

Hardness:

Not specified

Test temperature:

The water temperature was measured in one control at the start and end of the incubation period, and was continuously monitored in all test media and the controls during measurement of the respiration rate.

pH:

The pH values was determined in all test media and the controls at the start and at the end of the 3-hour incubation period.

Dissolved oxygen:

During the incubation period of exactly 3 hours all test media and the controls were continuously aerated by intense stirring on magnetic stirrers.
The dissolved oxygen concentrations was determined in all test media and the controls at the start and at the end of the 3-hour incubation period.
The concentration of dissolved oxygen did not drop below 2.5 mg/l during the incubation period, and just before measurement of the respiration rates the dissolved oxygen concentrations were at least 6.5 mg/l.

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations: 0, 32, 100, 348 and 1000 mg/L

Based on the results of a pre-test, no stock solution was prepared due to the insufficient water solubility of the test item.
Therefore, test item amounts of 5.12, 16.36, 50.53, 174.05 and 518.60 mg were weighed (by means of an analytical balance) and transferred into the respective test flasks with 284 ml of tap water. The test item was mixed into the tap water by ultrasonic treatment for ten minutes and intense stirring for 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. Then, 16 ml of synthetic wastewater and 200 ml of the activated sludge inoculum were added. The final nominal test item concentrations were 10, 32, 100, 348 and
1000 mg/L. Concentrations exceeding 1000 mg test item per liter were not tested.

Additionally, two controls (tap water, synthetic wastewater and activated sludge inoculum, without test item) and the reference item 3,5-dichlorophenol (positive control) at nominal concentrations of 5, 16 and 50 mg/l were tested in parallel under otherwise identical test conditions.

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL glass flasks
- Aeration: During the incubation period of exactly 3 hours all test media and the controls were continuously aerated by intense stirring on magnetic stirrers. The concentration of dissolved oxygen did not drop below 2.5 mg/l during the incubation period, and just before measurement of the respiration rates the dissolved oxygen concentrations were at least 6.5 mg/l.
- Sludge concentration (weight of dry solids per volume): At the start of the test 200 ml activated sludge inoculum with a sludge concentration of 2.3 g dry weight/l (corresponding to about 0.9 g dry material per liter test medium) was added. The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, then to the test solutions of the reference item, then to the test solutions of the test item, and finally to the second control.

TEST CONCENTRATIONS
- Test concentrations: 10, 32, 100, 348 & 1000 mg/L
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: higher than highest tested concentration

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

Remarks on result:

other: higher than highest tested concentration

Details on results:

The test item BLUE GS 5664.80 had no significant inhibitory effect ( <15%) on the respiration rate of activated sludge after the incubation period of three hours up to and including the highest test item concentration of 1000 mg/L. Thus, the 3-hour NOEC of BLUE GS 5664.80 to activated sludge microorganisms was at least 1000 mg/L. This value might be even higher, but concentrations exceeding 1000 mg/L were not tested. The 3-hour EC20, EC50and EC80 could not be calculated, but were clearly higher than 1000 mg/L.
The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed only by 2.5% (guideline-recommended maximum variation: 15%).

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 19 mg/L (the 95% confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming suitability of the activated sludge used.

Influence of 3,5-dichlorophenol and BLUE GS 5664.80 on oxygen consumption of activated sludge:

Flask	Test chemical	Nominal concentration of test chemical (mg/L)	Oxygen consumption rate (mg O2/L min-1)	Inhibition (%)	pH values		Oxygen Concentration (mg O2/L)
					Start (0h)	End (3h)	Start (0h)	End (3h)
1	Control	0	1.395	-	7.3	8.3	8.5	8.0
10	Control	0	1.430	-	7.8	8.3	8.5	7.9
Control: Mean (deviation %)			1.413 (2.5)	-
2	3,5-dichlorophenol	5	1.330	5.9	7.6	8.3	8.8	8.5
3	3,5-dichlorophenol	16	0.771	45.4	7.6	8.3	8.9	8.4
4	3,5-dichlorophenol	50	0.243	82.8	7.6	8.4	8.9	8.6
5	BLUE GS 5664.80	10	1.480	-4.7	7.7	8.3	8.7	8.1
6	BLUE GS 5664.80	32	1.535	-8.6	7.7	8.2	8.2	8.2
7	BLUE GS 5664.80	100	1.410	0.2	7.7	8.0	8.8	7.4
8	BLUE GS 5664.80	348	1.400	0.9	7.8	8.2	8.8	6.5
9	BLUE GS 5664.80	1000	1.228	13.1	8.0	8.1	8.8	7.7

Validity criteria fulfilled:

yes

Conclusions:

Up to and including the concentration of 1000 mg/L the test item BLUE GS 5664.80 had no significant inhibitory effect ( <15%) on the respiration rate of activated sludge after the incubation period of three hours.

Executive summary:

The inhibitory effect of the test item BLUE GS 5664.80 on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 87/302/EEC, and the OECD Guideline for Testing of Chemicals, No. 209.

The following nominal concentrations of BLUE GS 5664.80 were tested: 10, 32, 100, 348 and 1000 mg/L. Concentrations exceeding 1000 mg/L were not tested.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

Up to and including the concentration of 1000 mg/L the test item BLUE GS 5664.80 had no significant inhibitory effect ( <15%) on the respiration rate of activated sludge after the incubation period of three hours. Thus, the 3-hour NOEC of BLUE GS 5664.80 to activated sludge microorganisms was at least 1000 mg/L. This value might be even higher, but concentrations exceeding 1000 mg/L were not tested. The 3-hour EC20, ECSO and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

Description of key information

Up to and including the concentration of 1000 mg/L the test item BLUE GS 5664.80 had no significant inhibitory effect ( <15%) on the respiration rate of activated sludge after the incubation period of three hours.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L