Reaction mass of lithium sodium hydrogen 4-amino-6-({5-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-sulfonatophenyl}diazenyl)-5-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate and lithium sodium hydrogen 4-amino-6-({5-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-sulfonatophenyl}diazenyl)-5-hydroxy-3-{[4-(vinylsulfonyl)phenyl]diazenyl}naphthalene-2,7-disulfonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 14, 2001 to August 17, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

to show absence of growth inhibitory effect of the test substance in plants

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

0 and 72 h

Vehicle:

no

Details on test solutions:

PREPARATION OF STOCK SOLUTION
- Method: A stock solution was prepared by dissolving 250.3 mg of the test substance in 2 L of dilution water and treating for 1 h on a magnetic stirrer.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Strain: CHODAT (non-axenic strain)
- Source (laboratory, culture collection): Obtained from the 'Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Gottingen (Germany)
- Age of inoculum (at test initiation): Pre-cultures were set up three days before the start of the test.
- Method of cultivation: The algal cultures for the test were taken from an exponentially-growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 10E4 cells/mL in the test medium.

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): Same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21-25°C

pH:

Control (Part III) - 7.6 at 0 h, to 9.1 at 72 h
Part I - 7.7 at 0 h, to 8.9 at 72 h
Part II - 7.6 at 0 h, to 9.0 at 72 h

Nominal and measured concentrations:

- Nominal concentrations: 0.4, 1.0, 2.3, 4.7, 10.3, 22.7 and 50 mg/L
- Measured concentrations ranged from 102-117% of nominal values at 0 h, and from 73-113% of nominal values at 72 h.

Details on test conditions:

TEST SYSTEM
- Test vessel: 200 mL glass beakers
- Culturing apparatus: Light chamber in which a temperature in the range 21-25°C could be maintained and continuous uniform illumination could be provided in the spectral range 400 to 700 nm.
- Initial cell density: 10E4 cells/mL

- Experimental design:
Part I: 7 test concentrations with the algae (a test flask at the highest test concentration without algae was run in parallel to check whether or not significant amount of the test substance was incorporated into the algal biomass during the test period)
Part II: Glass beakers containing the algae, placed under glass vessels containing the above 7 test concentrations
Part III: Glass beakers containing the algae, placed under glass vessels containing the test medium (control group)
- Test concentrations (nominal): 0.4, 1.0, 2.3, 4.7, 10.3, 22.7 and 50 mg/L
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Duration of exposure: 72 h

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

EFFECT PARAMETERS MEASURED:
- Criteria for effects: Adverse effects were monitored at 72 h by determining the test substance induced inhibition of growth [b] and growth rate [r] of the algal population
- Determination of cell density: Microcell counter or microscopic counting chamber
- Observation points: 24, 48 and 72 h

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

other: EC10/EC50

Remarks on result:

not determinable

Remarks:

No algicidal effect observed. The growth inhibition results from algistatic light absorption by the discolouration of the test medium. Therefore, a true algicidal effect of the test substance was not observed in this study, i.e. the true ErC50 was >50 mg/L.

Details on results:

No algicidal effect observed. The growth inhibition results from algistatic light absorption by the discolouration of the test medium. Therefore, a true algicidal effect of the test substance was not observed in this study, i.e. the true ErC50 was >50 mg/L.

Reported statistics and error estimates:

- EC10/EC50 determination by Probit analysis
- NOEC/LOEC determination by Dunnett's test

Percentage inhibition of cell growth (integral of biomass) and growth rate:

Part	Nominal concentration of test substance (mg/L)	Percentage inhibition of cell growth [b]	Percentage inhibition of mean growth rate [r]
III (Control)	0.0	0.0	0.0
I (Test substance in contact with the algae)	0.4	10.2	0.7
	1.0	27.1	10.6
	2.3	42.3	18.0
	4.7	64.3	32.6
	10.3	76.7	53.5
	22.7	90.8	71.7
	50	98.5	100.0
II (Test substance not in contact with the algae. It was present in the glass vessel that was placed over the beaker containing the algae)	0.4	5.0	0.1
	1.0	24.7	5.2
	2.3	41.0	12.7
	4.7	56.1	25.5
	10.3	77.3	50.5
	22.7	91.4	70.5
	50	95.1	85.3

It can be clearly seen that there is no difference between treatment I and II. Therefore, the substance has no algicidal effect.

Validity criteria fulfilled:

yes

Conclusions:

A true algicidal effect of the test substance was not observed in this study, i.e. the true ErC50 was >50 mg/L. Hence, under the conditions of the study, EC50 for gowth inhibition could not be determined up to the highest tested concentration (50 mg/L).

Executive summary:

A study was conducted to determine the acute toxicity of the test substance to the algae Desmodesmus subspicatus according to EU Method C.3, in compliance with GLP. Triplicate inocula of the algae were exposed to nominal concentrations of the test substance (0.4, 1.0, 2.3, 4.7, 10.3, 22.7 and 50 mg/L) for 72 h under static conditions (Part I). Analytical validation of the applied concentrations was carried out using HPLC. A second series (Part II) of test vessels treated in the same way as the 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 algicidal (phytotoxic) effects. The cell densities were measured at 24, 48 and 72 h. Inhibition of the algal population was measured as reduction in biomass and growth rate, relative to control cultures grown under identical conditions. The measured concentrations of the test substance ranged from 102 -117% of nominal values at 0 h, and from 73 -113% of nominal values at 72 h. The growth inhibition observed in this study results from algistatic light absorption by the discolouration of the test medium. Therefore, a true algicidal effect of the test substance was not observed in this study, i.e. the true ErC50 was >50 mg/L. Hence, under the conditions of the study, ErC50 for gowth inhibition could not be determined up to the highest tested concentration (50 mg/L) (Bruns, 2001b). In the terrestrial plant growth inhibition test, there was no effect up to 500 mg/kg dw soil, clearly showing that the substance has no phytotoxic properties.

Description of key information

A true algicidal effect of the test substance was not observed in this study, i.e. the true ErC50 was >50 mg/L. Hence, under the conditions of the study, ErC50 for growth inhibition could not be determined up to the highest tested concentration (50 mg/L). For this reason, the study cannot be used for the classification and PNEC derivation purposes.

Additional information

A study was conducted to determine the acute toxicity of the test substance to the algae Desmodesmus subspicatus according to EU Method C.3, in compliance with GLP.Triplicate inocula of the algae were exposed to nominal concentrations of the test substance (0.4, 1.0, 2.3, 4.7, 10.3, 22.7 and 50 mg/L) for 72 h under static conditions (Part I). Analytical validation of the applied concentrations was carried out using HPLC. A second series (Part II) of test vessels treated in the same way as the 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 algicidal (phytotoxic) effects. The cell densities were measured at 24, 48 and 72 h. Inhibition of the algal population was measured as reduction in biomass and growth rate, relative to control cultures grown under identical conditions.The measured concentrations of the test substance ranged from 102 -117% of nominal values at 0 h, and from 73 -113% of nominal values at 72 h.The growth inhibition observed in this study results from algistatic light absorption by the discolouration of the test medium. Therefore, a true algicidal effect of the test substance was not observed in this study, i.e. the true ErC50 was >50 mg/L. Hence, under the conditions of the study, ErC50 for gowth inhibition could not be determined up to the highest tested concentration (50 mg/L) (Bruns, 2001b).

In the terrestrial plant growth inhibition test, there was no effect up to 500 mg/kg dw soil, clearly showing that the substance has no phytotoxic properties.