Disodium 7-[[2-(acetylamino)-4-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]phenyl]azo]naphthalene-1,3-disulphonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

From November 08th to December 17th, 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The read across approach is detailed into the document attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted June 7, 1984

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

December 29, 1992

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analytical measurements of the test substance concentrations duplicate samples were taken at the start of the test from the freshly prepared test media (without algae) of all test concentrations and from the control.
For the determination of the stability of the test substance under the test conditions, sufficient volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test (but without algae), and were sampled in duplicate at the end of the test (after the 72 hours test period).
The concentrations of the test substance were measured in the duplicate test media samples from the nominal test concentrations of 10 - 100 mg test substance/l from both sampling dates (0 and 72 hours). The samples from the nominal test concentrations of 1.0 and 3.2 mg test substance/l were not analysed, since these concentrations were below the determined 72-hour NOEC.
From the control samples only one of the duplicate samples was analysed from each of both sampling dates (0 and72 hours).
All samples are kept stored at about -20 °C to enable additional analyses. After delivery of the final test report all samples will be discarded.

Details on test solutions:

The test medium of the highest test substance concentration was prepared by dissolving the test substance in test water (100 mgl).
The test media were preparedjust before the start ofthe test.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG.
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universitat Gottingen", D-37073 Göttingen.
- Method of cultivation: the algae had been grown in the testing laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.7 - 24.0 °C

pH:

Control: 8.1 - 7.8
Test vessels: 8.0 - 7.9

Nominal and measured concentrations:

1.0, 3.2, 10, 32 and 100 mg test substance/l, nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (50 ml).
- Type: black cylinder, coated inside with aluminium foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.
- Stirring: continuously stirred by magnetic stirrers.
- Initial cells density: the test was started (0 hours) by inoculation of a biomass of 10.000 algal cells per ml test medium.
- No. of organisms per vessel: 15 ml algal suspension.
- No. of vessels per concentration: 3 flasks per test concentration.
- No. of vessels per control: 6 flasks per control.

EXPERIMENT A - Test item
Test item was dissolved in the growth medium. All glass dishes above the cylinders contained untreated test water. Thus, the inhibition of algal growth in the experimental part was caused due to a real toxic effect of the test substance and in addition to the reduced light intensities in the coloured test media in the flasks.

EXPERIMENT B - Light adsorption
The glass dishes above the cylinders contained the coloured test media with the same five test concentrations as in part A, however without algae. In the Erlenmeyer flasks below, the algae grew in test water without test substance (as in the control), however under changed light conditions due to the filter effect of the coloured test media in the glass dishes. Thus, the growth inhibition in part B was caused due to light absorption only. The depth of the test media in the glass dishes was 4 mm, i.e. half the depth of the test media in the Erlenmeyer flasks, because the algae in the stirred test media stay in the statistical mean in this mean depth.

GROWTH MEDIUM
- Standard medium used: yes. The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines, in deionized water analytical grade salts.

INCUBATION CONDITIONS
- Photoperiod: continuously illuminated.
- Light intensity and quality: at a mean light intensrty of about 8300 Lux, range 7600 - 8600 Lux. The illumination was achieved by fluorescent tubes installed
above the algal flasks.

EFFECT PARAMETERS MEASURED
Small volumes of the test media (1.0 ml) were taken out of all test flasks afr.er 24, 48 and 72 hours of exposure and were not replaced. The algae cell densities in the samples were determined by counting with an electronic particle counter, three measurements per sample.
In addition, a sample was taken from the control and from the test concentration of nominal 100 mg test substance/l 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.

For the quantification of the algicidal effect versus the growth inhibition due to reduced light intensities, the percentage inhibition of algal biomass and gowth rate (compared to the control in experimental part A) was calculated for each test concentration and compared between both experimental parts A and B.

MEASUREMENTS
- pH: the pH-values of the test media were measured in all test concentrations and the control at the start and the end ofthe test.
- Temperature: during the test duration the test media temperatures were measured daily in an Erlenmeyer flask filled with water and incubated at the same conditions as the test flasks.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 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.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: test item vessels

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: shadowing effect

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: test item vessels

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

56.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: shadowing effect

Details on results:

All test media down to the lowest test concentration of nominal 1.0 mg test substance/l were slightly to strongly coloured by the test substance.

The comparison between the results in experimental parts A and B was based on the growth rates. Only at the test concentration of 3.2 mg test substance/l the difference of the growth rates was determined to be 13.4 % and thus higher than 10 %. However, the concentration of 3.2 mg/l 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 A/B 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, the differences between inhibition in experimental part A and B should be lower than 10 %, respectively the quotient A/B should be at least 0.9 or higher to accept that the inhibition curves of the growth rates A and B are essentially the same.
With the only exception of the test concentration of 3.2 mgl (below the NOEC) the differences of the gowth rates A and B are lower than 10 % at all other test concentrations of this test. The quotients A/B are at least 0.9 at all test concentrations.
Thus, the modified algal test demonstrated that the observed growth inhibition effect of the test substance on algae 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.

EXPERIMENT A - Test item
Experimental part A corresponds to the usual algal toxicity test. 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/l. 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/l, 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/l 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.
ErC10 (72h): 9.9 mg/l (CL 95 %: 2.4 - 21.1 mg/l), nominal
EbC10 (72h): 2.1 mg/l (CL 95 %: 0.2 - 5.5 mg/l), nominal

EXPERIMENT B - Light adsorption
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 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/l. And also the EC-values in experimental part B were nearly in the same magnitude as the corresponding EC-values in experimental part A:
ErC10 (72h): 28.8 mg/l (CL 95 %: 20.8 - 37.3 mg/l), nominal
EbC10 (72h): 10.5 mg/l (CL 95 %: 5.9 - 15.2 mg/l), nominal

MEASURED CONCENTRATIONS
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 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.

Conclusions:

ErC50 (72h) > 100 mg/l (nominal), both test item and shadowing effect
EbC50 (72h): 30.3 mg/l (CL 95 % 14.4 - 107) (nominal), test vessels
EbC50 (72h): 56.1 mg/l (CL 95 % 41.1 -84.2) (nominal), shadowing effect

Executive summary:

The influence of the test substance on the gowth 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 l00 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 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, the modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance 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.

Conclusion

ErC50 (72h) > 100 mg/l (nominal), both testi item and shadowing effect

EbC50 (72h): 30.3 mg/l (CL 95 % 14.4 - 107) (nominal), test vessels

EbC50 (72h): 56.1 mg/l (CL 95 % 41.1 -84.2) (nominal), shadowing effect

Description of key information

Not harmful/toxic to aquatic algae (ErC50 (72h) > 100 mg/l (nominal))

Additional information

There is no information about the toxicity to aquatic algae potential of Reactive Yellow 215, thus the available information on the structural analogous Similar Substance 01 have been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

The influence of the Similar Substance 01 on the gowth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test. according to the OECD Guideline No. 201. 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 l00 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. 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, the modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance 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.