Cuprate(3-), [3-[2-[5-(hydroxy-.kappa.O)-4-[2-[1-(hydroxy-.kappa.O)-6-(phenylamino)-3-sulfo-2-naphthalenyl]diazenyl-.kappa.N1]-2-methylphenyl]diazenyl]-1,5-naphthalenedisulfonato(5-)]-, sodium (1:3)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The complete read across justification is detailed in section 13. Test substance is an isomer of the substance under registration; structural difference is not expected to significantly impact the toxicity to algae.

Justification for type of information:

Details for read across approach are included into the IUCLID section 13

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: experimental study: source of read-across study record

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The complete read across justification is detailed in section 13. Test substance is an isomer of the substance under registration; structural difference is not expected to significantly impact the toxicity to algae.

Justification for type of information:

Details for read across approach are included into the IUCLID section 13

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Principles of method if other than guideline:

The method described in the Council Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 'Algal inhibition test' assesses adverse effects (inhibition of growth and growth rate) of various concentrations of a test substance to a unicellular planktonic freshwater algal species. The instruction for the performance of a modified algal growth inhibition test published by Memmert & Knoell (RCC Umweltchemie, August 1992) is the description of a test method suitable to distinguish between algistatic (indirect effects caused by light absorption) and algicide (toxic) effects.

GLP compliance:

yes

Analytical monitoring:

yes

Remarks:

TOC determination

Details on sampling:

Control: at 0 and 72 hours
Test concentrations: at 0 and 72 hours

Vehicle:

no

Details on test solutions:

A stock solution was prepared to give the desired series of test concentrations. To achieve this 249.9 mg of the test substance were added to 2 litres of dilution water and treated for 1 h on a magnetic stirrer.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Source (laboratory, culture collection): Non-axenic strain of the test species obtained from the Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Gottingen (Gennany)
- Maintenance of stock cultures: Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions (23 +/- 2°C) at a light intensity in the range 60 - 120 µE. x m·2 x s·1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KOHN (1977) is renewed once a week. Cell density measurements are made using a microcell counter.
- Preparation of pre-cultures: Pre-cultures are set up three days before the start of a test. They are grown under identical exposure conditions as the stock cultures, except from the use of a different nutrient medium (annex 1).
- Test cultures: The algal inocula for a test are taken from an exponentially-growing pre-culture and are mixed with the nutrient medium (annex 1) to make up to a final cell density of about 104 cells per millilitre in the test concentrations and the control medium ( compare figure 1).

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 °C to 25°C, maintained at +/- 2°C

pH:

The pH is measured at the beginning of the test and at 72 hours.

Nominal and measured concentrations:

1.0, 2.1, 4.6, 10.3, 22.7 and 50 mg/I (nominal)

Details on test conditions:

- Test vessel: 200 ml glass beakers, height: 9.5 cm, diameter: 5.5 cm;
Glass vessel, height 4 cm, diameter 8 cm
Black plastic container, height 13 cm, diameter 9.5cm
- Light intensity and quality: At the average of the test solutions, a light intensity in the range 60 to 120 µE. x m-2 x s-1, or an equivalent range of 6.000 to 10.000 Ix, is recommended to use.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities are measured in a microcell counter or alternatively, are determined by means of a microscopic counting chamber.

TEST CONCENTRATIONS.
- 6 test concentrations ( for part I and II) plus I control 3 replicates per concentration, 6 replicates per control
- Initial cell density in the test cultures approximately 10E4 cells per millilitre. Cell densities are recorded at 24-hour intervals. The cell density in the control cultures should increase by a factor of at least 16 within 72 hours.
- additionally highest test concentration without algae
- Test concentrations: 1.0, 2.1, 4.6, 10.3, 22.7 and 50 mg/I (nominal)
- Results used to determine the conditions for the definitive study: The criteria of adverse effects used in this study were the substance-induced inhibition of growth [b] and growth rate [r], respectively, of the algal populations.

CULTURING APPARATUS
-Details on culturing apparatus used: Light chamber in which a temperature in the range 21 °C to 25°C can be maintained at +/- 2°C, and continuous uniform illumination is provided in the spectral range 400 to 700 nm.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

All results are expressed in terms of nominal concentrations. Measured concentrations ranged from 86 - 156% of nominal values at 0 hours, and from 63 - 104% of nominal values at 72 hours, respectively.

Analysis of the growth and growth rate of the algal population within the 72 h exposure period (by probit analysis and DUNNETT' s test, respectively) give the following results:

Results (mg/L):

	Part I	Part II
EbC50 (0-72h)	3.4	4.3
EbC50 (0-72h)	9.4	12
NOEC [b]	1.0	1.0
LOEC [b]	2.1	2.1
NOEC [r]	1.0	1.0
NOEC [r]	2.1	2.1

 

 

 

Conclusions:

EC50(biomass) = 4.3 mg/l
EC50(growth rate) = 12 mg/l
The growth rates for the indirect and direct exposure were within 10 % at all concentrations, thus proving that algal growth was affected by light intensity. On these bases, EC50 values were not used to the purpose of classification.

Executive summary:

A study was performed to assess adverse effects of the substance on the growth and the growth rate of freshwater algal species Scenedesmus subspicatus over several generations. 

The study was conducted in accordance with EEC Methods described in Annex to Directive 92/69/EEC Part C.3 which is in most parts equivalent to the OECD guideline 201.

Exponentially growing algal cells were exposed for a period of 72h to a range of concentrations, nominally 1.0,  2.1, 4.6, 10.3, 22.7 and 50.0 mg/L of the test item dissolved in water (Part I). A second series (Part II) of test vessels treated in the same way as controls (Part III), but placed below glass vessels containing the same range of concentrations of the test substance as described above, but without algae, was set up to distinguish between algistatic (indirect effect caused by light absorption) and algicide (toxic) effects.

 

The cell densities were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth (index b - biomass) and growth rate (index r), relative to control cultures grown under identical conditions. Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Dunnet (1955, 1964).

A comparison of the results for the parameter "percentage inhibition of the growth rate" gained in part I and II of the study results in no differences exceeding 10 %. Therefore the inhibition of growth observed in this study seemed to be caused by light absorption only.

 

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

analogue substance (refer to IUCLID chapter 13)

IUPAC Name:

analogue substance (refer to IUCLID chapter 13)

Test solutions

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: magnetic stirrer stock solution
(249.9 mg to 2 l water)

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Study design

Test type:

not specified

Water media type:

not specified

Limit test:

no

Total exposure duration:

72 h

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 4.3 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 12 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 mg/L

Nominal / measured:

not specified

Conc. based on:

not specified

Basis for effect:

biomass

Details on results:

% concentration loss over test: 37 ... 0

Any other information on results incl. tables

The test was conducted to the ETAD (Ecological and Toxicological Association of Dyestuffs and Organic Pigments Manufacturers) design. The growth rates for the indirect and direct exposure were within 10 % at all concentrations. This indicates that algal growth was affected by light intensity, thus EC50 value of the algae study should not be used for the purpose of classification.

Applicant's summary and conclusion

Conclusions:

EC50(biomass) = 4.3 mg/l
EC50(growth rate) = 12 mg/l

Executive summary:

Method

The test has been conducted according to the 92/69/EC C.3 (modified algal growth inhibition test for dyestuffs) and in compliance to the GLP principles. Available information derived from migrated NONS file, as per Article 25(3) request, with permission to refer granted by ECHA.

 

Results

EC50(biomass) = 4.3 mg/l

EC50(growth rate) = 12 mg/l.

The growth rates for the indirect and direct exposure were within 10 % at all concentrations, thus proving that algal growth was affected by light intensity. On these bases, EC50 values were not used to the purpose of classification.