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 aquatic algae and cyanobacteria

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The range-finding test was conducted between 18 November 2002 and 21 November 2002 and the definitive test between 3 February 2003 and 6 February 2003. The final report was issued 28th 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Method C3 of Commission Directive 92/69/EEC

Deviations:

yes

Remarks:

Further refined for coloured test substances, to differentiate between a reduced growth of algae due to a true toxic effect of the chemical or due to an indirect effect, a reduction in growth by light absorption of the coloured test substance.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Refined for coloured test substances, to differentiate between a reduced growth of algae due to a true toxic effect of the chemical or due to an indirect effect, a reduction in growth by light absorption of the coloured test substance.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each test group from Experiment A alone at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at each occasion and stored frozen (approximately -20°C) for further analysis if necessary.The concentration and stability of the test material in the test preparations (Experiment A only) were verified by chemical analysis at 0 and 72 hours. Analysis of the test preparations from Experiment B was not performed as this was not a requirement of the test guidelines.

Test solutions

Vehicle:

no

Details on test solutions:

The test material was dissolved directly in culture medium.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Scenedesmus subspicatus strain CCAP 276120. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Feny House, Far Sawrey, Ambleside, Cumbria.Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 + 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24+/- 1ºC

pH:

7.3-7.8

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations ranging from 90% to 108% of nominal and so it was considered justifiable to estimate the EC50 values in terms of nominal test concentrations only.

Details on test conditions:

250 ml glass conical flasks were used.For Experiment A three flasks each containing 100 ml of algal suspension plus the relevant test material concentration were prepared for the control and each treatment group. Glass petri dishes containing 80 ml of culture medium were placed above each of the conical flasks. For Experiment B one flask containing 100 ml of algal suspension was prepared for the control and each treatment group. Glass petri dishes containing 80 ml of each test preparation were placed above each of the conical flasks.The depth of preparation (15 mm) in the petri dishes for both Experiment A and B was half the depth of the preparation in the conical flasks (30 mm). The control groups for both Experiment A and B were maintained under identical conditions but not exposed to the test material.Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.14^6 cells per ml. This suspension was diluted to a cell density of 1.96^4 cells per ml prior to use. At initiation of the study the culture contained a nominal cell density of 10^4 cells per ml.The flasks were incubated at 24 plus or minus 1°C under continuous illumination (intensity approximately 7000 lux) and constantly stirred at approximately 100 rpm for 72 hours. Samples were taken at 0 and 24 hours and the cell densities determined using a coulter Multisizer II Particle Counter. The Coulter counts performed at 24 hours showed that interference was occurring in the small particle diameter range in both control and test cultures. Microscopic examination of the cultures showed that there was a large number of extremely small algal cells present, and it was considered that it was these small cells that were being detected. It was therefore considered appropriate to monitor algal growth using a haemocytometer and light microscope for the remainder of the study period (24, 48 and 72 hours) in order to obtain an accurate count of the number of algal cells present.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

39 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the area under the growth curve data for Experiment A at 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups.

Any other information on results incl. tables

Exposure of Scenedesmus subpicatus to the test material in Experiment A gave a EbC50 (72h) value of 9.4 mg/l and a ErC50 (0 -72h) value of 39 mg/l. The No Observed Effect Concentration at 72hrs was 1.0 mg/l. Given that significant differences (greater than 10%) in the inhibition values between Experiments A and B were observed, it was considered that the effect of the test material on algal growth was not only due to a reduction in light intensity, but also due to the intrinsic toxic properties of the test material.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Given that significant differences (greater than 10%) in the inhibition values between Experiments A and B were observed, it was considered that the effect of the test material on algal growth was not only due to a reduction in light intensity, but also due to the toxicity of the test material.

Executive summary:

Introduction

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC) and further refined for coloured test substances, to differentiate between a reduced growth of algae due to a true toxic effect of the chemical or due to an indirect effect, a reduction in growth by light absorption of the coloured test substance (Memmert et al 1994).

Methods

Following a preliminary range-finding test, Scenedesmus subspicatus was exposed to an aqueous solution of the test material for 72 hours, under constant illumination and stirred continuously via magnetic stirrer at a temperature of 24 ± 1 °C. The test was conducted using two experimental methods performed in parallel.

Experiment A

In Experiment A the algae were exposed to test material concentrations of 3.2, 10, 32, 100 and 320 mg active ingredient ai/l. Glass petri dishes above the test vessels contained culture medium alone. Therefore, inhibition of algal growth in these test vessels was due to a combination of both the toxic effects of the test material and reduction in light intensity.

Experiment B

In Experiment B the glass petri dishes above the test vessels contained test material solutions at concentrations of 3.2, 10, 32, 100 and 320 mg ai/l. The test vessels contained algal cells in culture medium alone. Therefore inhibition of algal growth was due to a reduction in light intensity alone.

Results

Exposure of Scenedesmus subspicatus to the test material in Experiment A gave an EbC50 (72 h) value of 9.4 mg ai/l and an ErC50 (0-72 h) value of 39 mg ai/l. The No Observed Effect Concentration at 72 hours was 1.0 mg ai/l.

In Experiment B, reduction in light intensity resulted in reduction of algal growth. The EC50 values were calculated based on the concentration of the test material in the glass petri dishes above the test cultures. The reduction in growth of Scenedesmus subspicatus exposed to light passed through the test material solutions gave an EC50 (72 h) value of 14 mg ai/l and an ErC50 (0 -72 h) value of 46 mg ai/l. The No Observed Effect Concentration was 1.0 mg ai/l.

Conclusion

Given that significant differences (greater than 10%) in the inhibition values between Experiments A and B were observed, it was considered that the effect of the test material on algal growth was not only due to a reduction in light intensity, but also due to the intrinsic toxic properties of the test material.