tetrasodium 3-amino-4-{[4-({4-fluoro-6-[phenyl(2-{[2-(sulfonatooxy)ethyl]sulfonyl}ethyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-5-hydroxynaphthalene-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 January 10, 2005 to January 13, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Principles of method if other than guideline:

A study was conducted in accordance with EU Method C.3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline 201, and an instruction for the performance of a modified algal growth inhibition test published by Memmert & Knoell (RCC Umweltchemie, August 1992).

The modified test was conducted in three parts:

Part I: This part of the study was conducted according to EU method C.3. In this, exponentially growing algal cells were exposed for a period of 72 h to a range of concentrations of the test substance and the effect on growth and growth rate was determined.

Part II: This independent experiment was set up to distinguish between algistatic (indirect effect caused by light absorption) and algicidal (toxic) effects. In this, a second series of test vessels were treated in the same way as the controls, but placed below glass vessels containing the same range of concentrations of the test substance (as tested under Part I), but without the algae. Following exposure, the effect on growth and growth rate was determined.

PART III: This part of the study composed of the controls (consisting of six replicates) and was also conducted according to Guideline.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling schedule
Control: at 0 and 72 h
Test concentrations: at 0 and 72 h

Vehicle:

yes

Details on test solutions:

PREPARATION OF STOCK SOLUTION
- Method: A stock solution was prepared by dissolving 250 mg of the test substance in 2 L of dilution water.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: 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 - 8.3 at 0h, to 10.2 at 72 h
Part I - 7.8 at 0 h, to 10.5 at 72 h
Part II - 8.1 at 0 h, to 10.3 at 72 h

Nominal and measured concentrations:

Nominal concentrations: 0.9, 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/L
Measured concentrations: Only the control and the test concentrations used in PART I of the study were checked by chemical analysis. Recovery rates ranged from 97-100.6% of nominal values at 0 h, and from 90.9-98.5% of nominal values at 72 h, respectively.

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 was provided in the spectral range 400 to 700 nm.
- Initial cells 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): 0.9, 1.9, 4.3, 9.4, 20.7, 45.5 and 100 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 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:

NOEC

Effect conc.:

4.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

227 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits = 152-441 mg/L

Reported statistics and error estimates:

- 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	-	-
I (Test substance in contact with the algae)	0.9	-6.8	-0.6
	1.9	-3.5	-0.1
	4.3	4.6	2.5
	9.4	21.4	5.9
	20.7	45.3	13.9
	45.5	65.7	27.2
	100	77.2	33.0
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)	0.9	-1.7	0.2
	1.9	11.6	6.6
	4.3	6.9	3.6
	9.4	13.4	3.5
	20.7	40.6	13.9
	45.5	66.7	28.1
	100	78.9	35.5

The results are expressed in terms of nominal concentrations. Only the control and the test concentrations used in PART I of the study were checked by chemical analysis.

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.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 72 h EbC50 (biomass) and ErC50 (growth rate) for Desmodesmus subspicatus were determined to be 28 and 227 mg/L (nominal concentrations), respectively. Further, the NOECs for biomass and growth rate were determined to be 4.3 mg/L (nominal concentration).

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, and an instruction for the performance of a modified algal growth inhibition test published by Memmert & Knoell (RCC Umweltchemie, August 1992), in compliance with GLP.

Triplicate inocula of the algae were exposed to nominal concentrations of the test substance (0, 0.9, 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/L) for 72 h under static conditions. Quantification 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 (toxic) effects. The cell densities were measured at 24, 48 and 72 h. Inhibition of the algal population was measured as reduction in growth and growth rate relative to control cultures grown under identical conditions.

 

The measured concentrations of the test substance ranged from 97-100.6% of nominal values at 0 h, and from 90.9-98.5% of nominal values at 72 h, respectively. Under the conditions of the study, the 72 h EbC50 (biomass) and ErC50 (growth rate) for Desmodesmus subspicatus were determined to be 28 and 227 mg/L (nominal concentrations), respectively. Further, the NOECs for biomass and growth rate were determined to be 4.3 mg/L (nominal concentration).

 

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.

Description of key information

The 72 h EbC50 (biomass) and ErC50 (growth rate) of the test substance for Desmodesmus subspicatus were determined to be 28 and 227 mg/L (nominal), respectively. Further, the NOECs for biomass and growth rate were determined to be 4.3 mg/L (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

227 mg/L

EC10 or NOEC for freshwater algae:

4.3 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance to the algaeDesmodesmus subspicatusaccording to EU Method C.3, and an instruction for the performance of a modified algal growth inhibition test published by Memmert & Knoell (RCC Umweltchemie, August 1992), in compliance with GLP. Triplicate inocula of the algae were exposed to nominal concentrations of the test substance (0, 0.9, 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/L) for 72 h under static conditions. Quantification 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 (toxic) effects. The cell densities were measured at 24, 48 and 72 h. Inhibition of the algal population was measured as reduction in growth and growth rate relative to control cultures grown under identical conditions. The measured concentrations of the test substance ranged from 97-100.6% of nominal values at 0 h, and from 90.9-98.5% of nominal values at 72 h, respectively. Under the conditions of the study, the 72 h EbC50 (biomass) and ErC50 (growth rate) forDesmodesmus subspicatuswere determined to be 28 and 227 mg/L (nominal), respectively. Further, the NOECs for biomass and growth rate were determined to be 4.3 mg/L (nominal concentration). 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 (Bruns, 2005d).