Diammonium 5-(hexylsulfonyl)-2-{[3-methyl-2,7-dioxo-1-(3-sulfonatobenzoyl)-2,7-dihydro-3H-naphtho[1,2,3-de]heterocycle-6-yl]amino}benzenesulfonate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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 relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

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)

Remarks:

Inspection date: 21 August 2007; Date of signature: 15 October 2007

Test material

Test material information

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations:
- Sampling method: Not applicable as no analysis conducted. Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the test, the test material was dissolved directly in water. An amount of test material (2000 mg) was dissolved in water with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution from which serial dilutions were made to give 200 and 20 mg/l stock solutions. An aliquot (250 ml) of the 20 mg/l stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 10 mg/l. Similarly, aliquots (80 ml and 250 ml) of the 200 mg/l and 2000 mg/l stock solutions were used to prepare the test concentrations of 32, 100, 320 and 1000 mg/l. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions. The control group was maintained under identical conditions but not exposed to the test material.

- Controls: At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 500 ml conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. Finally a second control was prepared.

Reference material preparation
For the purpose of the test a reference material, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 15402DH-367) was used. Two stock solutions of 50 and 160 mg/l were prepared by dissolving the reference material directly in water with the aid of ultrasonication for approximately 30 minutes. Aliquots (10 and 100 ml) of the 160 mg/l stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/l. Similarly, a 100 ml aliquot of the 50 mg/l stock solution was used to prepare the 10 mg/l concentration. The volumetric flasks containing the reference material were inverted several times to ensure homogeneity of the solutions.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Method of cultivation: A mixed population of activated sewage sludge micro-organisms was obtained 19 January 2009 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.6 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter WTW pH 320 pH meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/l prior to use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Hardness:

140 mg/l as CaCO3

Test temperature:

Approximately 21ºC.

pH:

7.6 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter WTW pH 320 pH meter.

Dissolved oxygen:

2.0 -2.8 mg O2/l (final)

Salinity:

Not applicableas freshwater study.

Nominal and measured concentrations:

Nominal:
10, 32, 100, 320 and 1000 mg/l

Measured:
>1000 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml conical flask
- Aeration: yes, the mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately
0.5 – 1 litre per minute.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not stated.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: 0.810 - 1.380, mean: 1.041 mg/l
- Pesticides: 0.00 - 0.001, mean: 0.00 µg/l
- Chlorine: Free chlorine: 0.030 - 0.550, mean: 0.231; Total chlorine: 0.80 - 0.610, mean: 0.297 mg/l
- Conductivity: 421.385 µS/cm at 20°C

OTHER TEST CONDITIONS
- Photoperiod: Not applicable

TEST CONCENTRATIONS
- Test concentrations: 10, 32, 100, 320 and 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:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

640 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Please see Table 1 below for further details.

Any other information on results incl. tables

Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1. 

Table1              Oxygen Consumption Rates and Percentage Inhibition Values in the Definitive Test after 3 Hours Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	4.6	5	1.8	0.56	-
	R2	5.2	5	2.6	0.52	-
Test Material	10	4.3	4	2.0	0.58	[7]
	32	3.8	3	2.3	0.50	7
	100	4.7	4	2.7	0.50	7
	320	4.9	6	2.1	0.47	13
	1000	5.5	6	2.8	0.45	17
3,5-dichlorophenol	3.2	5.3	6	2.3	0.50	7
	10	7.1	10	4.5	0.26	52
	32	8.1	10	7.1	0.10	81

[Increase in respiration rate as compared to controls]

R₁– R₂= Replicates 1 to 2

Applicant's summary and conclusion

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 640 mg/l.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Council Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods. Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 10, 32, 100, 320 and 1000 mg/l for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact tiand compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀ of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was640mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/l.

The reference material gave a 3-Hour EC₅₀value of 11 mg/l, 95% confidence limits 9.3 - 13 mg/l.

 Conclusion.

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC₅₀of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 640 mg/l.