disodium 7-(4,6-difluoropyrimidin-2-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate; reaction mass of: disodium 7-(2,4-difluoropyrimidin-6-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate

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:

November 08, 1996 to March 05, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Principles of method if other than guideline:

In algae growth inhibition tests with coloured test substances ( e.g. according to the OECD-Guideline 201) it can not be differentiated between a reduced growth of the green algae due to real toxic effects of the test substance on the algal cells or due to an indirect effect, a reduced algal growth by light absorption in the coloured test solutions. Thus, the results of those tests, the EC 50 and the NOEC, depend on both effects, the toxicity and the reduced light intensity. Thus, the purpose of this modified algal growth inhibition test was to quantify the algicidal effect of the test substance, but also the growth inhibition effect due to reduced light intensities in the coloured test solutions. Exponentially growing cultures of the unicellular green algae Scenedesmus subspicatus were exposed to various concentrations of the test substance in a modified test system. The inhibition of growth in relation to control cultures was detennined over a test period of 72 hours.

GLP compliance:

yes

Analytical monitoring:

yes

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: No. 86.81 SAG
- Source (laboratory, culture collection): supplied by the Sammlung von Algenkulturen Gottingen (SAG, Experimentelle Phykologie und Sammlung von Algenkulturen, Albrecht-von-Haller-lnstitut fur
Pflanzenwissenschaften, Universitat Gottingen, D-37073 Gottingen, Germany).
- Age of inoculum (at test initiation): The test was started (O hours) by inoculation of 10,000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test under the same conditions as in the test.
- Method of cultivation: grown in the RCC laboratories under standardized conditions according to the test guidelines. The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines.

Test type:

static

Water media type:

other: synthetic test water, prepared according to the test guidelines

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

23.7-24°C

pH:

7.8-8.1

Dissolved oxygen:

Not specified

Salinity:

NaHCO3: 50.0 mg/L
CaCl2x2 H20: 18.0 mg/L
NH4CI: 15.0 mg/L
MgS04X 7 H20: 15.0 mg/L
MgCl2X 6 H20: 12.0 mg/L
KH2P04: 1.6 mg/L

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel (test water without test substance).

The analytically determined test substance concentrations in the analyzed test media varied in the range from 98 to 101% of the nominal values. The test substance SCARLET RN 1165 was sufficiently stable in the test media
under the test conditions during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test substance.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (50 ml), each filled with 15 ml algal suspension.
- Type: On the top of each cylinder, glass dishes covered with watch glass dishes were placed to reduce the loss of water and to avoid the entry of dust into the solutions
- Initial cells density: 10,000/mL
- Control end cells density:
Density of algal cells (cell number x 10,000/mL) after 72h in experiment A: 45.46 +/- 14.73 (6 replicates)
- No. of organisms per vessel: The test was started (0 hours) by inoculation of 10,000 algal cells per ml test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Analytical grade salts were added to the following final nominal concentrations:
Macro-nutrients:
NaHCO3: 50.0 mg/L
CaCl2x2 H20: 18.0 mg/L
NH4CI: 15.0 mg/L
MgS04X 7 H20: 15.0 mg/L
MgCl2X 6 H20: 12.0 mg/L
KH2P04: 1.6 mg/L

Trace elements:
Na2EDTA x 2 H20: 100.0μg/L
FeCl3x 6 H20: 80.0μg/L
MnCl2x4 H20: 415.0μg/L
H3BO3: 185.0 µg/L
Na2M004 x 2 H20: 7.0μg/L
ZnCl2: 3.0μg/L
CoCl2x 6 H20: 1.5μg/L
CuCl2x2 H20: 0.01 μg/L

OTHER TEST CONDITIONS
- Light intensity and quality: continuously illuminated at a measured light intensity of about 8300 Lux (mean value), range: 7600 to 8600 Lux (minimum and maximum value of measurements at 9 places
distributed over the experimental area). The light intensity was measured just before the start of the test below the coating cylinders. The illumination was achieved by fluorescent tubes (universal white L 25, 36 W), installed above the algal flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Small volumes of the test media and the control (1.0 ml) were taken out of all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with three measurements per sample.
In addition, a sample was taken from the control and from the test concentration of nominal 100 mg test substance/I after the test period of 72 hours. The shape of these treated algal cells was microscopically examined and compared with the cells in the control.

TEST CONCENTRATIONS
The test concentrations were based on the results of a range-finding test. However, concentrations in excess of nominal 100 mg/L have not been tested in compliance with the EU Commission Directive 92/69/EEC. The enlarged spacing factor of 3.2 between the test concentrations was chosen, since according to the results of the range-finding test a large concentration range had to be tested in the definitive test.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Experimental part A
Experimental part A corresponds to the usual algal toxicity test. This means that the algal growth inhibition in this experimental part was caused by a possible toxic effect of the test substance and/or by the reduced light intensities due to the light absorption in the coloured test media.
In experimental part A, the test substance had a statistically significant inhibitory effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 32 mg test substance/I (results of a Dunnett-test, one-sided, α = 0.05). Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined at the concentration of 10 mg test substance/I, since up to and including this test concentration the mean growth rates of the algae were statistically not significantly lower than in the control. At the microscopical examination of the shape of the algal cells after 72 hours incubation period no difference was observed between the algae growing in the test substance concentration of 100 mg test substance/I in experimental part A and the algal cells in the control. Thus, the shape of the algal cells, growing in the test solution with this concentration of dissolved test substance was obviously not affected.
The EC values were calculated for the algal biomass b and the growth rate µ after 72 hours test duration:
EbC50: 30.3 mg/L
EµC50: >100 mg/L

Experimental part B
In experimental part B the algal growth inhibition by the pure light effect (the reduced light intensities in the coloured test media) was quantified. In this experimental part a nearly identical inhibition effect on the algal growth was observed compared to experimental part A.
In experimental part B the algal growth was significantly reduced compared to the control after 72 hours test period first at the test concentration of 32 mg test substance/I. And also the EC-values in experimental part B were nearly in the same magnitude as the corresponding EC-values in experimental part A:
EbC50: 56.1 mg/L
EµC50: >100mg/L

Comparison between the results in experimental parts A and B
According to the recommendations of the Ad-hoc meeting of experts on algal growth inhibition tests for coloured substances the comparison between the results in experimental parts A and B was based on the growth rates. The differences between the results of experimental parts A and B were described for each test concentration as percentage inhibition of the growth rate μA minus the percentage inhibition of the growth rate μB after the 72 hours test period. Only at the test concentration of 3.2 mg test substance/I the difference of the growth rates was determined to be 13 .4 % and thus higher than 10 %. However, the concentration of 3.2 mg/I was below the 72-hour NOEC. At all other test concentrations these differences were lower than 10 %. As another measure of difference the quotient of the growth rates μA/μB was calculated for each test concentration. These quotients were also high (at least 0.9) at all test concentrations. Differences in growth rates up to the magnitude of 10 % are accepted to be caused by pure chance in the used algal toxicity test. Thus, according to the recommendations of the Adhoc meeting of experts on algal growth inhibition tests for coloured substances (Vienna, 30- 31 January 1996) the differences between inhibition in experimental part A and B should be lower than 10 %, respectively the quotient μA/μB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates μA and μB are essentially the same. With the only exception of the test concentration of 3.2 mg/I (below the NOEC) the differences of the growth rates μA and μB are lower than 10 % at all other test concentrations of this test. The quotients μA/μB are at least 0.9 at all test concentrations.

Conclusion
This modified algal test has demonstrated that the observed growth inhibition effect of the test substance SCARLET RN 1165 on Scenedesmus subspicatus was caused with high possibility only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a real toxic effect of the test substance on the growth of Scenedesmus subspicatus can be excluded up to the highest test concentration of 100 mg test substance/I.

General results:
In the control the cell density has increased from nominal N = 1 * 10E4 cells/ml at the start of the test ( 0 hours) to N = 45. 46 * 10E4 cells/ml (mean value) after 72 hours by a factor of approximately 46. Thus, the algal growth in the control was sufficiently high. At the start of the test, the pH-values in the test media were in the range of pH 8.0 to pH 8.1, at the end of the test pH-values were measured between pH 7.8 and pH 8.0.

Validity criteria fulfilled:

yes

Remarks:

In the control the cell density increased from 10E4 cells/ml at 0h to 45.46x10E4 cells/ml after 72h. Thus the algal growth in the control was sufficiently high.

Conclusions:

The influence of the test substance SCARLET RN 1165 on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984 resulting in:
72h EµC50 : >100 mg/L
72h NOEC = 10.0 mg/L

According to CLP criteria, as the test item is not rapidely biodegradable and adequate chronic toxicity data are available, the test item is not classified for Aquatic Hazards.

Executive summary:

The influence of the test substance SCARLET RN 1165 on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the Commission Directive 92/69/EEC, Annex Part C.3, dated December 29, 1992, and the OECD Guideline No. 201, adopted June 7, 1984. However, the test method was modified to quantify the algicidal effect of the test substance, but also the growth inhibition effect due to reduced light intensities in the coloured test media. The nominal test concentrations were 1.0, 3.2, 10, 32 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were slightly to strongly coloured by the test substance. The analytically determined test substance concentrations in the analysed test media varied in the range from 98 % to 101 % of the nominal values. The test substance SCARLET RN 1165 was sufficiently stable in the test media under the test conditions during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test substance. Nearly the same growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test substance, but under reduced light intensities by the filter effect of the coloured test media as in the second parallel experimental part, where the algae grew in the test media with dissolved test substance. Thus, in conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance SCARLET RN 1165 on Scenedesmus subspicatus was caused with high possibility only due to an indirect effect, the light filter effect in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to the highest test concentration of nominal 100 mg test substance/L.

Description of key information

The influence of the test substance SCARLET RN 1165 on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984 resulting in:

72h ErC50 >100 mg/L

72h NOErC = 10.0 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

10 mg/L