Tetrasodium 2-[[8-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]benzoyl]amino]-1-hydroxy-3,6-disulphonato-2-naphthyl]azo]naphthalene-1,5-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 December 17 to January 05, 2000

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:

1984

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sample replicates: duplicate samples from the test medium (without algae) and duplicate samples from the control (without algae). The concentrations of the test item were measured in the duplicate test media samples from all test concentrations from both sampling times (0 and 72 hours). From the control samples only one of the duplicate samples was analyzed from each of both sampling times (0 and 72 hours).
- Frequency of sampling: samples were taken just before test start and after 72 hours.
- Samples for stability: for the 72-hour stability samples additional flasks with adequate 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.
- Sample storage: all samples were deep-frozen (at about -20 °C) immediately after sampling. Based on preexperiments for investigation of the storage stability the test item is sufficiently stable in the test medium under these storage conditions.

Vehicle:

no

Details on test solutions:

The test medium of the highest test item concentration of nominal 110 mg/l was prepared by dissolving 55 mg of the test item completely in 500 ml test water by intensive stirring for 10 minutes at room temperature. Adequate volumes of this intensively mixed test medium were added to test water to prepare the test media with the lower test concentrations. The test media were prepared just before addition of algae (=start of the 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: supplied by the "sammlung von Algenkulturen, Pflänzenphysiologisches lnstitut der Universität Göttingen", D-37073 Göttingen.
- Method of cultivation: tne algae had been grown in the testing laboratories, under standardized conditions, according to the test guideline. The algae were cultivated and tested in synthetic test water.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

All flasks were incubated in a temperature controlled water bath at 22 - 23 °C.

pH:

8.0 - 8.3

Nominal and measured concentrations:

Nominal, 1.1, 3.5, 11, 35 and 110 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (50 ml).
- Type: each Erlenmeyer flask was placed in a black cylinder, coated inside with aluminum foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.
- Fill volume: 15 ml algal suspension.
- Stirring: continuously stirred by magnetic stirrers.
- Initial cells density: the test was started (0 hours) by inoculation of 10000 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.
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 6 replicates

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Hardness: 0.24 mmol/l (= 24 mg/l) as CaCO3

OTHER TEST CONDITIONS
- Photoperiod: continuously illuminated; illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the algal flasks.
- Light intensity and quality: measured light intensity of about 8400 Lux (mean value; range: 7600 to 8900 Lux) The light intensity was measured just before the start of the test below the coating cylinders.

STUDY DESIGN
The test included two experimental parts.

Experimental part A: the algae grew in test media with dissolved test item in the Erlenmeyer flasks (five test concentrations and a contro). All glass dishes above the cylinders contained purified water. Thus, the inhibition of algal growth in this experimental part was caused due to a real toxic effect of the test item and in addition to the reduced light intensities in the colored test media in the Erlenmeyer flasks.

Exoerimental part B: the glass dishes above the cylinders contained the colored test media with the same test concentrations as in part A, however without algae (3 replicates
per test concentration). ln the Erlenmeyer flasks below, the algae grew in test water without test item (as in the control), however under changed light conditions due to the filter effect of the colored 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.

MEASUREMENTS
The pH was determined in each test concentration and the control at the start and at the end of the test.
The water temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
The appearance of the test media was recorded daily.

TEST CONCENTRATIONS
The test concentrations were based on the results of a range-finding test and on a preexperiment to the selection of suitable methods for the test media. 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.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

COMPARISON BETWEEN RESULTS IN EXP. A AND B
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 (IµA) minus the percentage inhibition of the growth rate µB (IµB) after the72 hours test period. At all 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. At all test concentrations this quotient was at least 0.9 or higher.
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 Ad-hoc working group of experts on algal growth inhibition tests for colored substances the differences between inhibition in experimental part A and B should not be higher 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. At all test concentrations of the test the differences of the growth rates µA and µB are lower than 10 % and the quotients µA/µB are at least 0.9.

The modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item on Scenedesmus subspicatus was caused only by the indirect effect, the light absorption in the colored test solutions. Thus, a real toxic effect of the test item on the growth of Scenedesmus subspicatus can be excluded up to the highest test concentration of 110 mg/l.

EXPERIMENTAL PART A - usual algal toxicity test.
The algal growth inhibition in this experimental part was caused by a possible toxic effect of the test item and/or by the reduced light intensities due to the light absorption in the colored test media.
In experimental part A, the test item had a statistically significant inhibitory effect on the growth rate of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 11 mg/l (results of a Dunnett-test, one-sided, α = 0.05); thus, this test concentration was determined as the 72-hour LOEC. The 72-hour NOEC was determined at the concentration of 3.5 mg/l, since up to and including this test concentration the mean growth rate p of the algae was statistically not significantly lower than in the control.
At the microscopical examination of the shape of the algal cells alter 72 hours incubation period no difference was observed between the algae growing in the test item concentration of 35 mg/l in experimental part A and the algal cells in the control. Thus, the shape of the algal cells, growing at this concentration of dissolved test item was obviously not atfected.
ErC50 (72h): 137 (82 - 326) mg/l // EbC50 (72h): 24 (9.8 - 86) mg/l
ErC10 (72h): 11 (5.7 - 18) mg/l // EbC10 (72h): 2.5 (0.1 - 6.7) mg/l
ErC90 (72h): >> 110 mg/l // EbC90 (72h): > 110 mg/l

EXPERIMENTAL PART B
ln experimental part B the algal growth inhibition caused by the pure light effect (the reduced light intensities in the colored test media) was quantified. In this experimental part a very similar inhibition effect on the algal growth was observed compared to experimental part A.
The calculated mean growth rates were statistically significantly reduced compared to the control after 72 hours test period first at the test concentration of 11 mg/l. The EC values in experimental part B were in the same magnitude as the corresponding EC values in experimental part A.
ErC50 (72h): 151 mg/l // EbC50 (72h): 38 (29 - 52) mg/l
ErC10 (72h): 7.4 (4.3 - 11) mg/l // EbC10 (72h): 19 (0 - .43) mg/l
ErC90 (72h): >> 110 mg/l // EbC90 (72h): > 110 mg/l

MEASURED CONCENTRATIONS
The analytically determined mean test item concentrations in the analyzed test media varied in the range from 94 to 102 % of the nominal values. The test item was stable in the test media under the test conditions during the test period ol 72 hours. Therefore, all biological results are related to the nominal concentrations of the test item.

Conclusions:

ErC50 (72h) > 110 mg/l (nominal)
EbC50 (72h) > 110 mg/l (nominal)

Executive summary:

The influence of the test item 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, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify the algicidal etfect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions.

The nominal test concentrations were 1.1, 3.5, 11, 35 and 110 mg/l and a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item.

The analytically determined mean test item concentrations in the anatyzed test media varied in the range from 94 to 102 % of the nominal values. The test item was 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 item.

The same growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test item, but under reduced light intensities by the filter effect of the colored test media as in the second parallel experimental part, where the algae grew in the test media with dissolved test item.

Thus, in conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition etfect of the test item on Scenedesmus subspicafus was caused only due to an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of 110 mg/l.

Conclusion

ErC50 (72h) > 110 mg/l (nominal)

EbC50 (72h) > 110 mg/l (nominal)

Description of key information

Not harmful/toxic to aquatic algae (ErC50 (72h) > 110 mg/l (nominal); EbC50 (72h) > 110 mg/l (nominal)).

Additional information

There is no information about the potential toxicity of Reactive Red 147 to acquatic algae, thus the available information on the structural analogous Similar Substance 01 has 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 growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test, according to the EU method C.3 and the OECD Guideline No. 201. The test method was modified to quantify the algicidal etfect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions. The analytically determined mean test item concentrations in the anatyzed test media varied in the range from 94 to 102 % of the nominal values.; thus, the test item was stable in the test media under the test conditions during the test period of 72 hours.

The differences between the results of experimental parts conducted in accordance to standard protocol and the experimental part conducted to assess the pure light effects, were described for each test concentration as percentage inhibition of the growth rate µA (IµA) minus the percentage inhibition of the growth rate µB (IµB) after the72 hours test period; at all 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 and it resulted to be at least 0.9 or higher, 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. The modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item on Scenedesmus subspicatus was caused only by the indirect effect, the light absorption in the colored test solutions. Thus, a real toxic effect of the test item on the growth of Scenedesmus subspicatus was excluded up to the highest test concentration of 110 mg/l.