Disodium {sulfonylbis[4,1-phenylenediazene-2,1-diyl(1-ethyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-5,3-diyl)]}dimethanesulfonate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 04-Oct-2010 Experimental Completion Date: 06-Oct-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies,which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

1984

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the REACH, Part C.11, Activated Sludge Respiration Inhibition Test.

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Yellow RN 2903

IUPAC Name:

Acid Yellow RN 2903

Details on test material:

The test item and the following information were provided by the Sponsor:

Identity: Acid Yellow RN 2903
Batch No.: Vers. Kilo 6, 26.05.2009
Purity: Approx. 65%
Expiration Date: 01-Aug-2015
Storage Conditions: At room temperature at about 20 °C, away from direct sunlight.

Specific details on test material used for the study:

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

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Aerobic activated sludge was incubated for three hours in the presence of the test item at a single test concentration under defined conditions (limit test).
A limit test was performed based on the EU testing guidelines [1, 2] to demonstrate that the test item has no toxic effect on activated sludge up to the nominal test concentration of 1000 mg/L.

Test solutions

Vehicle:

no

Details on test solutions:

The final nominal concentration tested was 1000 mg/L.

Based on the results of a pre-experiment (without GLP), the test item was soluble at a concentration of at least 5 g/L in tap water.

Therefore, 500.3 mg of the test item was dissolved in 284 mL tap water by ultrasonic treatment for five minutes and intense stirring for five minutes at room temperature. In this way a clear yellow solution was obtained. Then, 16 mL synthetic sewage feed and 200 mL activated sludge inoculum were added.

Additionally, two controls containing only tap water, synthetic sewage feed and inoculum were tested in parallel to the single test concentration of the test item under identical test conditions.

The reference item 3,5 dichlorophenol was tested in parallel under identical test conditions, and functioned as a positive control. The nominal concentrations tested were 5, 16 and 50 mg/L. A stock solution of 3,5 dichlorophenol was prepared according to the test guidelines under Harlan Laboratories Study C77077 as follows: 0.5 g 3,5 dichlorophenol was dissolved in 10 mL 1 M NaOH and diluted to about 30 mL with purified water. Excess NaOH was neutralized with 0.5 M H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one liter with purified water. The pH was 7.8. The final concentration of 3,5 dichlorophenol amounted to 500 mg/L. Aliquots of this 3,5 dichlorophenol stock solution were mixed with tap water, synthetic sewage feed and inoculum in the respective test vessels to obtain the desired concentrations.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The test method and the test system are recommended by the testing guidelines.

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. The sludge was washed twice 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 the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed* per liter and was kept at room temperature under continuous aeration until use. 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 7.3.

* Synthetic sewage feed:

16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl filled up to a final volume of 1 liter
0.4 g CaCl2 × 2H2O with deionized water
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

20 °C

pH:

7.4 - 8.2

Dissolved oxygen:

7.7 - 8.1

Nominal and measured concentrations:

1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: glass, 2000 mL, open, 500 mL
- Aeration: continuously aerated by intense stirring on magnetic stirrers

- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: The inoculum had a sludge concentration of 2.2 g/L dry weight (corresponding to about 0.9 g dry material per liter test
medium).


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: local tap water


OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Respiration rate (= oxygen consumption rate); measured continually.
For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. The oxygen consumption rate (in mg O2 L-1 minute-1) was determined from the linear part of the respiration curve in the range 6.5–2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer.


TEST CONCENTRATIONS
- Limit test at 1000 mg/L

- Justification for using less concentrations than requested by guideline: A limit test was performed based on the EU testing guidelines to demonstrate that the test item has no toxic effect on activated sludge up to the nominal test concentration of 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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:

respiration rate

Effect concentrations 2

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

Remarks:

respiration rate

Effect concentrations 3

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 4

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:

respiration rate

Effect concentrations 5

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The test item Acid Yellow RN 2903 had no significant inhibitory effect (≤10%) on the respiration rate of activated sludge after the incubation period of 3 hours at the loading rate of 1000 mg/L.

Thus, the 3 hour NOEC (EC10) of Acid Yellow RN 2903 to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested. The 3 hour EC20, EC50, and 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 1% (guideline-recommendation: the two control respiration rates should be within 15% of each other).

Results are presented in table 1 and figure 1 - see section any other information on results for table 1 and see in attached section for figure 1.

Evaluations

Measurement of the Respiration Rates
For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany), and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2 L-1 minute-1) was determined from the linear part of the respiration curve in the range 6.5–2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.

Determination of the Inhibitory Effect
The inhibitory effect of the test item or of the reference item on the respiration rate (oxygen consumption per minute) is expressed as a percentage of the mean respiration rate of the two controls measured at the start and at the end of the test series (vessels A and B).

1 – 2Rs
[ _____________ ] X 100% = percent inhibition
Rc1 + Rc2


where
Rs = oxygen consumption rate (in mg O2 L-1 minute-1) at tested concentration
Rc1 = oxygen consumption rate in the first control vessel
Rc2 = oxygen consumption rate in the second control vessel

The 3-hour EC20, EC50 and EC80 values of the test item Acid Yellow RN 2903 and their 95%-confidence limits could not be calculated because of the absence of a toxic effect.

The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Probit Analysis [3, 4].

All test results were based on nominal concentrations of the test and reference item.

Water Quality Criteria
The pH values and dissolved oxygen concentrations were determined after the addition of synthetic sewage feed and activated sludge in all test media and the controls at the start and at the end of the 3-hour incubation period. The water temperature was measured in one control at the start and at the end of the incubation period. Before the addition of activated sludge and synthetic sewage feed, the appearance of the test media was recorded.

Results with reference substance (positive control):

The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 11 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3 hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.

Results are presented in table 1 and figure 1 - see section any other information on results for table 1 and see in attached section for figure 1.

Reported statistics and error estimates:

The 3 hour EC20, EC50 and EC80 values of the test item Acid Yellow RN 2903 and their 95% confidence limits could not be calculated because of the absence of a toxic effect.

The 3 hour EC50 of the reference item 3,5 dichlorophenol was calculated by Probit Analysis [3, 4].

All test results were based on nominal concentrations of the test and reference item.

Any other information on results incl. tables

Table 1       Influence of Acid Yellow RN 2903 (Test Item) and 3,5-Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge

Vessel no.	Nominal concentration of test chemical	Oxygen consumption rate	Inhibition	pH values		Oxygen concentration
						(mg O2/L)
	(mg/L)	(mg O2L^-1min^-1)	(%)	start*	end*	start*	end*
	Control
A	0	0.991		7.5	8.2	7.9	7.7
B	0	1.000		7.4	8.2	8.0	8.0
Mean		0.995
Deviation (%)		0.9
	Test item
4	1000	1.013	-1.8	7.4	8.1	8.0	7.8
	Reference item
1	5	0.821	17.5	7.4	8.2	8.0	7.7
2	16	0.305	69.4	7.5	8.2	8.0	7.9
3	50	0.094	90.6	7.4	8.1	8.1	7.9

 

*                        Start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumption rate relative to control

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item Acid Yellow RN 2903 had no significant inhibitory effect (≤10%) on the respiration rate of activated sludge after the incubation period of 3 hours at the loading rate of 1000 mg/L.

Thus, the 3 hour NOEC (EC10) of Acid Yellow RN 2903 to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested. The 3 hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L.

Executive summary:

The inhibitory effect of the test item Acid Yellow RN 2903 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 88/302/EEC, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209 (1984).

 

Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed.

 

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.

 

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at the test item concentration of 1000 mg/L.

 

Thus, the 3‑hour NOEC (EC₁₀) of the test item to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested. The 3‑hour EC₂₀, EC₅₀, and EC₈₀could not be calculated but were clearly higher than 1000 mg/L.