A reaction mixture containing the following components;A. Tetralithium [2(or 3), 9 (or 10), 16 (or 17), 23 (or 24)-tetrakis (3-sulfonato-propylsulfonyl) phthalocyaninato]cupurate (II)B. Trilithium [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl]tris (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)C.Dilithium bis [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl] bis (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)D. Lithium tris [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl] (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)E. Tetrakis [3-(2-hydroxpropylsulfamoyl) proppylsulfonyl]phthalocyaninatocupurate (II)In the ratio A:B:C:D:E 6.25 : 25 : 37.5 : 25 : 6.25

Toxicity to microorganisms

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The range-finding test was conducted on 21 November 2002 and the definitive test on 13 January 2003. The final report was issued 4th March 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No 209

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Remarks:

water

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge sample from a predominantly domestic sewage treatment plant with a suspended solids concentration of 4.1 g/L. 200ml of the innoculum was added to each conical flask in the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

21 degrees C

pH:

7.7 - 8.1 (at the end of the exposure period)

Dissolved oxygen:

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg 02/l and 2.5 mg 02/l respectively). This was considered to have had no adverse effect on the results of the test given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Details on test conditions:

Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/l (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.Preparation of the inoculumThe activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21°C and was used on the day of collection. The pH of the sample was measured as 7.9 using a 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 piece of pre-weighed GF/A filter paper using a Buchner funnel. The paper was then dried in an oven at approximately 1 10°C for 1 hour and allowed to cool before reweighing. The suspended solids was equal to 4.1 g/l prior to use. This method followed the Japanese Industrial Standard JISK 01 02-1 98 1.An amount of test material (2000 mg) was dissolved in water with the aid of ultrasonic disruption for approximately 5 minutes and the volume adjusted to 1000 ml to give a 2000 mg/l stock solution. An aliquot (250 ml) of this 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 1000 mg/l. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the stock solution. This method of preparation was performed for three separate replicate test vessels.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 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 also prepared. As each vessel reached 30 minutes contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace for an approximate 10 minute period (between approximately 8.1 mg 02/l and 0.8 mg 02/l). After measurement the contents of the BOD bottle were returned to the test vessel. This procedure was repeated after 3 hours contact time. The test was conducted under normal laboratory lighting in a temperature controlled room at 21°C.Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations were measured using a pH and dissolved oxygen meter at the end of the exposure period prior to measurement of the oxygen consumption rate.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 2

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

Results with reference substance (positive control):

The results for the 3,5-dichlorophenol positive control were within expected ranges and therefore the test system was considered to be operating as expected.

Any other information on results incl. tables

In some instances, the intial and final dissolved oxygen concentrations were below those recommended in the test guideline (6.5 mg O2/l and 2.5 mg O2/l respectively). This was considered to have no adverse effect on the results of the test given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

There was a relativley large increase in respiration rate observed in the test vessels after 30 minutes contact time. This was considered to be due to the posssible hormetric response of the activated sewage sludge micro-organisms to the test material, i.e. the stimulation of biological activity due to the presence of test material at concentrations below toxici concnetration.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

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 1000 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", EEC Commission Directive 87/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Method.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at 21°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 minutes and 3 hours contact time and 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 was 1000 mg/l.