Benzenesulfonic acid, 2-[2-[5-(aminocarbonyl)-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridinyl]diazenyl]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:2)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

13 November 2001 to 16 November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to EU & OECD test guidance in compliance with GLP. This study is read across to the structural analogue Reaktiv-Gelb F-97494, which is considered to be structurally equivalent to the substance to be registered. Details of structure are detailed below under "illustrations"

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

See below

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: > 300 deg C
- Boiling point: Not measured
- Vapour pressure: < 10E-05 hPa
- Water solubility (under test conditions): 108 g/l
- Solubility in organic solvents: Not measured.
- log Pow: < -4.9
- Adsorption characteristics:
- Toxicity to microorganisms: EC50 > 10000 mg/l

OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: 8% after 28-day manometric test.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Test concentration; s (nominal): 3.1,6.3, 13,25, 50 and 100mg/l
Method of administration: stock solution
In order to avoid an impairment of the test system, an additional replicate is used for analysis and pH measurement at the beginning of the test (control: replicate VII; test concentrations: replicate IV). Analysis and pH measurement at the end of the test are performed using replicate I

In order to assess whether significant amounts of the test substance are incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations. The results of the measured concentrations in these test media without algae are reported separately.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution was prepared to give the desired series of test concentrations. To achieve this 125.4 mg of the test substance were added to 1 litre of dilution water and treated for 24 h on a magnetic stirrer.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Name: Desmodasmus subspicatus CHODAT
Source: Non-axenic strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Gottingcn (Germany)
Maintenance of slock cultures: Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions (23 +/- 2°C) al a light intensity in the range 60 - 120 με. 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 & KUHN (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 .
Test cultures: The algal inocula for a test are taken from an exponenlially-growing pre-culture and are mixed with the nutrient medium to make up to a final cell density of about 104 cells per millilitre in the lest medium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure period

Test conditions

Hardness:

No data

Test temperature:

21-25C

pH:

7.7 - 10.4

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Nominal and measured concentrations:

Test concentration; s (nominal): 3.1,6.3, 13,25, 50 and 100mg/l

Details on test conditions:

Test vessels: 300 ml Erlenmeyer flasks with stoppers
Culturing apparatus: 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.
Light intensity: At the average of the test solutions, a light intensity in the range 60 to 120 uE. x m-2 x s-1, or an equivalent range of 4000 to 8000 lx, is recommended to use.
Cell density measurements: Cell densities are measured in a microcell counter or, alternatively, are determined by means of a microscopic counting chamber.
Experimental design: 6 test concentrations plus 1 control. 3 replicates per concentration, 6 replicates per control. Initial cell density in the test cultures approximately 104 cells per millilitre. Additionally highest test concentration without algae
Test concentration; s (nominal): 3.1,6.3, 13,25, 50 and 100mg/l
Method of administration: stock solution
Duration of exposure: 72 hours
Criteria of effects: 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 population.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 70 mg/L

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 70 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

18.2 mg/L

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

18.2 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

37.8 mg/L

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

37.8 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

All results are expressed in terms of measured concentrations. Measured concentrations ranged from 100 - 130% of nominal values at 0 hours, and from 93 - 106% of nominal values at 72 hours, respectively.

COMMENTS
As these organic component correspond to 66.1% of the molecular weight of the test substance, 100 mg/l of the test substance equals to 23.4 mg/l of organic C. Thus, the analytical values indicate a good recovery rate.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

No data

Any other information on results incl. tables

Table: Results [mg/l]

Parameter	Organic component of the test substance	Test substance including inorganic components
EbC50 (0-72h)	>70	>106
ErC50 (0-72h)	>70	>106
NOEC [b]	18.2	27.5
LOEC [b]	37.8	57.2
NOEC [r]	18.2	27.5
LOEC [r]	37.8	57.2

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Results are as follows:
EC50>70mg/l (organic component of test substance) ; EC50 > 106 mg/l (test substance including inorganic components).
NOEC 18.2mg/l
LOEC 37.8mg/l

Executive summary:

This study is read across to the structural analogue Reaktiv-Gelb F97-494., which is considered to be structurally equivalent to the substance to be registered.

A study was performed to assess adverse effects of Reaktivgelb F 97-494 on the growth (-increase in cell density) and the growth rate rale of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus Subspicatus CHODAT over several generations.

The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (OX No. L383A, 29.12.92) Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test'. Study conducted in accordance with the Principles of Good Laboratory Practice (GLP).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 3.1, 6.2, 13, 25, 50 and 100 mg/1 of Reaktivgelb F 97-494 dissolved in water. Auxiliaries used to prepare the test media was a magnetic stirrer. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r), relative to control cultures grown under identical conditions. Growth and growth rates were used lo calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to DUNNETT (1955, 1964). The following values were determined:

 

Table: Results [mg/l]

 

Parameter	Organic component of the test substance	Test substance including inorganic components
EbC50 (0-72h)	>70	> 106
ErC50 (0-72fi)	>70	> 106
NOEC [bl	18.2	27.5
LOEC [bl	37.8	57.2
NOEC |r|	18.2	27.5
LOEC ir|	37.8	57.2

 

All results are expressed in terms of measured concentrations. Measured concentrations ranged from 100- 130% of nominal values at 0 hours, and from 93 - 106% of nominal values at 72 hours, respectively.

 

The substance is not considered to be harmful to Algae.