Disodium 7-[[4,6-bis[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Justification for read across approach is detailed in the endpoint summary.

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)

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 all freshly prepared test media and the control were incubated under the same conditions as in the actual test (but without algae) and sampled in duplicate at the end of the test (after the 72 hours test period).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain No. 86.81 SAG
- Supplier: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universitat Gottingen, D-37073 Gottingen, F.R.G.
- Method of cultivation: the algae were cuhivated and tested in synthetic test water, prepared according to the test guidelines (detail in the 'details on test conditions' section).

TEST ORGANISM
- Common name:
- Strain:
- Source (laboratory, culture collection):
- Age of inoculum (at test initiation):
- Method of cultivation:

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.8 - 24.4 °C
During the test the temperature was measured daily in an Erlenmeyer flask, filled with water and incubated at the same conditions as the test flasks.

pH:

At the start of the test, the pH-values in the test media were between pH 8.1 and 8.2, at the end of the test pH-values were measured between pH 8.0 and 10.0.
The pH-values of the test media were measured in samples of all test concentrations and the control at the start and at the end of the test.

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10.0, 32.0 and 100 mg/l.
Average over all measurements per test concentration: 0.6, 2.0, 7.0, 27.4 and 92.4 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 aluminium foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.
- Initial biomass: the test was started (0 hours) by inoculation of a biomass of 10.000 algal cells per ml test solution. These cells were taken from an exponentially growing pre-culture, which was set up about 72 hours prior to the test at the same conditions as in the test.
- Control end cells density:
- No. of organisms per vessel: 50 ml algal suspension for each flask.
- No. of vessels per concentration: 3 flasks per test concentration.
- No. of vessels per control: 6 flasks.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: the algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. In deionized water with a conductivity lower than 0.1 µS/cm (Milli-Q-water) analytical grade salts were added to following final nominal concentrations:
Macro-nutrients:
NaHCO3 50.0 mg/l
CaCl2 x 2 H2O 18 mg/l
NH4CI 15 mg/l
MgSO4 x 7 H2O 15 mg/l
MgCl2 x 6H2O 12 mg/l
KH2PO4 1.6 mg/l

Trace elements:
Na2EDTA x 2H2O 100.0 µg/l
FeCl3 x 6 H2O 80.0 µg/l
MnCl2 x 4 H2O 415.0 µg/l
H3BO3 185.0 µg/l
Na2MoO4 x 2 H2O 7.0 µg/l
ZnCl2 3.0 µg/l
CoCI2 x 6 H2O 1.5 µg/l
CuCI2 X 2 H2O 00.1 µg/l
Calculated water hardness of the test water: 0.24 mmol/1 (= 24 mg/l) as CaCO3.

COUNTING AND EXAMINATION OF ALGAL CELLS
Small test media samples were taken out of all flasks after 24, 48 and 72 hours of exposure and not replaced. The algae cell densities in the samples were determined by counting with an electronic particle counter, three measurements per flask and time. In addition, a sample was taken from the control and from a test concentration in experimental part A with reduced algal growth (nominal 32.0 mg test substance/l) after a test period of 72 hours. The shape of the 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. The range-findingtest was not performed in compliance with the GLP-Regulations.

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Experiment A

Effect conc.:

30.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 5.7 - 165.0

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Experiment B

Effect conc.:

48.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 7.1 - 326.5

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Experiment A

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.7 - 12.6

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Experiment B

Effect conc.:

6.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 2.0 - 21.8

Details on results:

All test media down to the lowest test concentration of nominal 1.0 mg/l were slightly to strongly coloured by the test substance.
The biological results of both experimental parts A and B are nearly identical. This is demonstrated in the following by several calculated parameters, the 72-hour NOEC/LOEC, the percentage inhibition of algal growth and the EC-values.
In both experimental parts A and B, the 72-hour LOEC (lowest concentration tested with a statistically significant inhibition effect) for the growth rate amounted to 2.0 mg/l (as the mean measured terst substance concentration at the test concentration of nominal 3.2 mg/l).
The 72-hour NOEC (highest concentration tested without a significant inhibition effect) amounted to 0.6 mg/l (as the mean measured concentration at nominal 1.0 mg/l).
The percentage inhibition of the algal biomass respectively the algal growth rate after the 72 hours exposure period in experimental part B were in the same magnitude as in experimental part A. The differences between the percentages of the inhibition rates in experimental parts A and B after the 72 hours test period varied only in the range of 0.1 % to 20.5 % for the algal biomass and from 1.5 % to 11.9 % for the growth rates. Thus, under consideration of the usual
variability of the results of algal growth inhibition tests, the percentage inhibition of both the algal biomass and the algal growrth rates after the 72 hours exposure period were identical.
And also at the EC-values, calculated for both growth parameters, no significant differences are observable between the values in both experimental parts.
Thus, 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 light filter effect (experimental part B) as in experimental part A, where the algae grew in test solutions with dissolved test substance.
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 concentration of nominal 32 mg/l in experimental part A and the algal cells in the control. Thus, a modification of the shape of the algal cells, growing in the test solution with this high concentration of dissolved test substance could not be observed.
In conclusion, the modified algal test has demonstrated that the observed growth inhibition effect of the test substance was caused 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 algal cells can be excluded up to at least the highest tested concentration of nominal 100 mg/l (mean measured test substance concentration: 92.4 mg/l).

In the control the cell density has increased from nominal N = 1 x 10^4 cells/ml at the start of the test (0 hours) to N= 92.61 x 10^4 cells/ml (mean value) after 72 hours by a factor of approximately 93. Thus, the algal growth in the control was sufficiently high.

TEST CONCENTRATIONS
The analytically determined test substance concentrations in the test media samples varied in the range from 31.5 % to 97.0 % of the nominal values. The mean measured test substance concentrations (calculated as the average over all measurements per test concentration) ranged from 56.5 % to 92.4 % of nominal).
After 72 h all samples showed a concentration dependent decrease. While at c = 100 mg/l the concentration decreased from 97.0 to 87.9 % only, the recovery fell down to 31.5 % at c = 1 mg/l. Possible reasons may be adsorption or degradation due to the intense irradiation during the performance of the biological test.

EC-values

	Concentration (mg/l)	95 % conf. Limits (mg/l)
Experimental part A - growth rate
ErC 50 (0 - 72 h)	30.6	5.7 - 165.0
ErC 10 (0 - 72 h)	2.4	0.2 - 25.6
Experimental part B - growth rate
ErC 50 (0 - 72 h)	48.1	7.1 - 326.5
ErC 10 (0 - 72 h)	3.8	0.3 - 56.1
Experimental part A - biomass
EbC 50 (0 - 72 h)	3.0	0.7 - 12.6
EbC 10 (0 - 72 h)	0.3	0.0 - 5.0
EbC 90 (0 - 72 h)	31.7	3.2 - 315.1
Experimental part B - biomass
EbC 50 (0 - 72 h)	6.6	2.0 - 21.8
EbC 10 (0 - 72 h)	1.0	0.1 - 7.0
EbC 90 (0 - 72 h)	45.4	6.4 - 324.2

Influence of the test substance on algae growth: growth rates p and percentage inhibition of p in both experimental parts A and B.

Measured concentration (mg/l)	Growth rate r (l/day) and % inhibition
	24 h		48 h		72 h
	r	%	r	%	r	%
Experiment A
Control	1.68	0.0	1.65	0.0	1.51	0.0
0.6	1.32	21.4	1.46	11.7	1.49	1.5
2.0	1.04	38.1	1.26	23.5	1.38	8.5
7.0	0.61	63.6	0.98	40.5	1.18	21.6
27.4	0.61	63.7	0.48	71.2	0.67	55.5
92.4	0.73	56.3	0.49	70.3	0.51	66.1
Experiment B
0.6	1.72	-2.3	1.6	3.2	1.51	-0.1
2.0	1.58	6.3	1.51	8.6	1.4	7.0
7.0	1.05	37.7	1.22	26.1	1.33	11.8
27.4	0.71	57.6	0.68	58.8	0.85	43.6
92.4	0.65	61.5	0.44	73.6	0.58	61.3

Comparison of the inhibition effects on growth rate r in experimental parts A and B after 72 hours test period.

Measured concentration (mg/l)	% inhibition of A after 72 hrs
	Part A	Part B	A-B
0.6	1.5	-0.1	1.6
2.0	8.5	7.0	1.5
7.0	21.6	11.8	9.8
27.4	55.5	43.6	11.9
92.4	66.1	61.3	4.8

Influence of the test substance on algae growth: areas under growth curves (A) and percentage inhibition of A in both experimental parts A and B.

Measured concentration (mg/l)	Area (A) and % inhibition of A
	24 h		48 h		72 h
	A	%	A	%	A	%
Experiment A
Control	53	0.0	422	0.0	1838	0.0
0.6	34	36.4	278	34.2	1512	17.7
2.0	23	57.5	184	56.4	1063	42.1
7.0	10	80.9	94	77.7	575	68.7
27.4	10	80.9	40	90.6	137	92.6
92.4	13	75.3	46	89	110	94.0
Experiment B
0.6	55	-4.2	394	6.7	1774	3.5
2.0	46	13.0	329	22.2	1375	25.2
7.0	23	57.5	181	57.1	952	48.2
27.4	13	75.8	61	85.6	237	87.1
92.4	11	79.1	39	90.8	113	93.9

Comparison of the inhibition effects on biomass in experimental parts A and B after 72 hours test period.

Measured concentration (mg/l)	% inhibition of A after 72 hrs
	Part A	Part B	A-B
0.6	17.7	3.5	14.2
2.0	42.1	25.2	16.9
7.0	68.7	48.2	20.5
27.4	92.6	87.1	5.5
92.4	94.0	93.9	0.1

Results obtained for the concentration of test tem in test medium.

Nominal concentration	Sampling time (d)	Age of sample (hrs)	Test item
			mg/l	% of nominal	Mean
					mg/l	% of nominal
1.0	0	0	0.79	79.0	0.82	81.5
	0	0	0.84	84.0
	3	72	0.31	31.0	0.32	31.5
	3	72	0.32	32.0
total mean					0.57	56.5
3.2	0	0	2.95	92.2	2.95	92.0
	0	0	2.94	91.9
	3	72	1.14	35.6	1.14	35.6
	3	72	1.14	35.6
total mean					2.04	63.8
10.0	0	0	8.96	89.6	8.97	89.7
	0	0	8.97	89.7
	3	72	5.04	50.4	5.03	50.3
	3	72	5.02	50.2
total mean					7.00	70.0
32.0	0	0	30.9	96.6	30.81	96.3
	0	0	30.72	96.0
	3	72	23.99	75.0	24.00	75.0
	3	72	24.00	75.0
total mean					27.40	85.6
10.00 (100)	0	0	9.71	97.1	9.70	97.0
	0	0	9.69	96.9
	3	72	8.74	87.4	8.79	879
	3	72	8.83	88.3
total mean					9.24	92.4
control	0	0	n.d.	-	-	-
control	3	72	n.d.	-	-	-
Fortification samples
13.40  (134.0)	0	0	13.22	98.7	13.19	98.4
	0	0	13.16	98.2
1.34	0	0	1.58	117.9	1.58	117.9
	0	0	1.58	117.9
total mean						108.2
0 (blank)	0	0	n.d.	-	-	-

n.d. = no test article detected

Conclusions:

The observed growth inhibition effect of the test substance on Scenedesmus subspicatus was caused 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 algal cells can be excluded up to at least the highest tested concentration of nominal 100 mg test substance/l (mean measured test substance concentration: 92.4 mg/l).

Executive summary:

The influence of the test substance on the growth of the green alga Scenedesmus subspicatus Chodat was investigated in a 72-hour static test according to the OECD Guideline No. 201 and the Commission Directive 92/69/EEC, Annex Part C.3. However, the test method was modified to differentiate between a reduced growth of 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 coloured test solutions. The test was performed in compliance with Good Laboratory Practice Regulations. The nominal test concentrations were 1,0, 3.2, 10.0, 32.0 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 test media varied in the range from 31.5 % to 97.0 % of the nominal values. The mean measured test substance concentrations (calculated as the average over all measurements per test concentration) ranged from 56.5 % to 92.4 % of nominal. Therefore, all biological results are based on the mean measured test substance concentrations. The biological results of both experimental parts are identical. This could be demonstrated by several calculated parameters, the NOEC/LOEC, the percentage inhibition of algal growth and the EC-values.

In conclusion, this modified algal test has demonstrated that the observed growth inhibition effect of the test substance on Scenedesmus subspicatus was caused 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 algal cells can be excluded up to at least the highest tested concentration of nominal 100 mg test substance/l (mean measured test substance concentration: 92.4 mg/l).

Conclusion

The observed growth inhibition effect of the test substance on Scenedesmus subspicatus was caused 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 algal cells can be excluded up to at least the highest tested concentration of nominal 100 mg test substance/l (mean measured test substance concentration: 92.4 mg/l).

Description of key information

Not harmful/toxic to aquatic algae and cyanobacteria.

Additional information

No experimental, nor literature data are available about the toxicity to aquatic algae and cyanobacteria of Direct Red 253, thus the available data on the Similar Substance 01, i.e. Red DK 2740 (EC: 419-460-2), have been considered.

The influence of the Similar Substance 01 on the growth of the green alga Scenedesmus subspicatus was investigated in a 72-hour static test, according to the OECD Guideline No. 201 and the Commission Directive 92/69/EEC, Annex Part C.3. However, the test method was modified to differentiate between a reduced growth of 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 coloured test solutions. 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 test media varied in the range from 31.5 % to 97.0 % of the nominal values. The mean measured test substance concentrations (calculated as the average over all measurements per test concentration) ranged from 56.5 % to 92.4 % of nominal. Therefore, all biological results are based on the mean measured test substance concentrations.

The biological results of both experimental parts A and B are nearly identical. This is demonstrated in the following by several calculated parameters, the 72-hour NOEC/LOEC, the percentage inhibition of algal growth and the EC-values. In both experimental parts A and B, the 72-hour LOEC (lowest concentration tested with a statistically significant inhibition effect) for the growth rate amounted to 2.0 mg/l (as the mean measured terst substance concentration at the test concentration of nominal 3.2 mg/l). The 72-hour NOEC (highest concentration tested without a significant inhibition effect) amounted to 0.6 mg/l (as the mean measured concentration at nominal 1.0 mg/l). The percentage inhibition of the algal biomass respectively the algal growth rate after the 72 hours exposure period in experimental part B were in the same magnitude as in experimental part A. The differences between the percentages of the inhibition rates in experimental parts A and B after the 72 hours test period varied only in the range of 0.1 % to 20.5 % for the algal biomass and from 1.5 % to 11.9 % for the growth rates. Thus, under consideration of the usual variability of the results of algal growth inhibition tests, the percentage inhibition of both the algal biomass and the algal growrth rates after the 72 hours exposure period were identical; furthermore, at the EC-values, calculated for both growth parameters, no significant differences are observable between the values in both experimental parts. Thus, 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 light filter effect (experimental part B) as in experimental part A, where the algae grew in test solutions with dissolved test substance (Memmert and Kellner, 1995).

READ ACROSS APPROACH

Both Direct Red 253 and Similar Substance 01 are Secondary Disazo Dyes (portion represented by sodium 4-[4-methyldiazenyl]phenyl]diazenyl]benzene-1-sulfonate group), containing I-acid moiety bearing a triazinic ring on the amine. The only difference occurring between chemical structures of Direct Red 253 and Similar Substance 01 regards the functionalization of the amines in positions 2 and 4 of triazine ring: Similar Substance 01 has a diethylpropylamine functional group instead of the ethanol one, as in the case of Direct Red 253. The difference is expected to not significantly impact the toxicity to aquatic algae and cyanobacteria potential.

Both substances are very water soluble (solubility > 100 mg/l); they are expected to be non-readly biodegradable and, based on the chemical stucture, hydrolysis can be considered as a negligible mechanism of degradation. The substances are expected undergoing to the same environmental fate and pathway, including possible degradation/transformation products that are expected to be comparable.

Based on the partition coefficient octanol-water, Direct Red 253 and Similar Substance 01 are expected to possess a low potential for bioaccumulation and a low potential to cross biological membranes (high molecular weight, high polarity).