disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]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:

August 2, 2002 to November 12, 2002

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 standard algal growth inhibition tests with colored test substances (e.g. according to the OECD Guideline No. 201) it can not be differentiated between a reduced growth of the green algae due to real toxic effects of the test item on the algal cells or due to an indirect effect, a reduced algal growth by light absorption in the colored test solutions. Thus, the results of those tests, the EC50 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 item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions.
Exponentially growing cultures of the unicellular green algae Scenedesmus subspicatus were exposed to various concentrations of the test item in a modified study design. The inhibition of growth in relation to control cultures was determined 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:

23-24°C

pH:

7.8-8.0

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 item).

The analytically determined test item concentrations in the analyzed test media varied in the range from 98 to 112% of the nominal values at the start and the end of the test. Consequently, the test item was stable during the test period of 72 hours under the test conditions, and the reported biological results are based on the nominal concentration of the test item.

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 cells/mL
- Control end cells density:
Density of algal cells (cell number x 10,000/mL) after 72h in experiment A: 39.08 +/- 8.99 (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 dissolved in sterile purified water to obtain 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 7800 Lux (mean value), range: 7100 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 (Philips TLD 36W/840), installed above the algal flasks. The test vessels were labeled with the RCC study number and all necessary additional information to assure unmistakable identification.

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 at least two measurements per sample.
In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration of nominal 10 mg/l (experimental part A). The shape of the algal cells was microscopically examined in these samples.

TEST CONCENTRATIONS
The test concentrations were based on the results of a range-finding test and on pre-experiments to the selection of suitable methods for (without GLP). 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):

yes

Remarks:

potassium dichromate is tested as a positive control at least once per year to demonstrate satisfactory test conditions

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 item and/or by the reduced light intensities due to the light absorption in the colored test media. ln experimental part A, the test item had a statistically significant inhibitory effect on the growth rate r of Scenedes/nus subspicatus after the exposure period of 72 hours first at the concentration of 32 mg/L (results of a Dunnett-test, one-sided, o = 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/L, since up to and including this test concentration the mean growth rate r of the algae was statistically not significantly lower than in the control. For the biomass b, the same LOEC- and NOEC-values were determined. 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 item concentration of 10 mg/L in experimental partAand the algal cells in the control. Thus, the shape and size of the algal cells, growing at this concentration of dissolved test item were obviously not affected.
The EC values were calculated for the algal biomass b and the growth rate r after 72 hours test duration:
EbC50: 64 mg/L
ErC50: >100 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 med¡a) was quantified. ln this experimental part a very similar inhibition effect on the algal growth was observed compared to experimental part A. The algal growth was statistically significantly reduced compared to the control after 72 hours test period first at the test concentration of 32 mg/L (Table 5). The EC values in experimental part B were in the same magnitude as the corresponding EC values in experimental part A.
The EC values in experimental part B are listed below:
EbC50: 92 mg/L
ErC50: >100mg/L

Comparison between the results in experimental parts A and B
According to the recommendations of the Ad-hoc working group of experts on algal growth inhibition for the interpretation of test results of colored 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 rA (lrA) minus the percentage inhibition of the growth rate rB (IrB) after the 72 hours test period. At all test concentrations, these differences were lower than 10%.
As another measure of difference the quotient of the growth rates rA/rB 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 be not higher than 10%, respectively the quotient rA/rB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates rA and rB are essentially the same.
At all test concentration of this test, the differences of the inhibition of growth rates rA and rB are lower than 10% and the quotients rA/rB are at least 0.9.

Conclusion
This modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item Red Rwa 4565 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 100 mg/L.

General results:
All test media down to the lowest test concentration of nominal 1.0 mg/L were slightly to strongly colored by the test item.
In the control the cell density increased from nominal N = 1 x 10E4 cells/ml at the start of the test (0 hours) to N = 39 x 10E4 cells/ml (mean value) after 72 hours. Thus, the algal growth in the control was sufficiently high under the test conditions and the validity criterion of increase by at least a factor of 16 was fulfilled.
At the start of the test, the pH values in the test media and the control were in the range of 7.9 to 8.0. At the end of the test, pH values between 7.8 and 8.0 were measured .

Validity criteria fulfilled:

yes

Remarks:

In the control the cell density increased from 10E4 cells/ml at 0h to 39x10E4 cells/ml after 72h. The algal growth in the control was sufficiently high under the test conditions. The validity criterion of increase by at least a factor of 16 was met.

Conclusions:

The influence of the test item Red Rwa 4565 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 NOEC = 10.0 mg/L

Executive summary:

The influence of the test item Red Rwa 4565 on the growth of the green algal species Scenedesmus suþspicafus 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. However, the test method was modified to quantify the algicidal effect 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 1.0,3.2,10,32 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item.

The analytically determined test item concentrations in the analyzed test media varied in the range from 98 to 112% of the nominal values at the start and the end of the test. Consequently, the test item was stable during the test period of 72 hours under the test conditions, and the reported biological results are based on the nominal concentration of the test item.

The same growth inhibition of Scenedesrnus 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 effect of the test item Red Rwa 4565 on Scenedesmus subspicafus was ðaused only by 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 100 mg/L.

The EC values were calculated for the algal biomass b and the growth rate r after 72 hours test duration:

72h EbC50 = 64 mg/L (95%-conf. limits: 26 -801 mg/L)

72h ErC50 = >100 mg/L (95%-conf. limits: -)

72h NOEC = 10.0 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

10 mg/L