Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[(2-cyanoethyl)[2-(2-hydroxyethoxy)ethyl]amino]-1,3,5-triazine-4,2-diyl]imino]]bis[benzene-1,4-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 September the 28th to October the 08th, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Justification for read across is detailed in 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 in 2006

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling: duplicate samples taken from the test media of all test concentrations (plus control) at the start of the test (without algae) and at the end of the test (containing algae).
- Determination: test item concentration was measured only at the nominal concentration of 1000 mg/l plus the control.
- Storing: at -20 °C immediately after sampling.

Vehicle:

no

Details on test solutions:

The test medium of the highest nominal concentration of the test item of 1000 mg/l was prepared by dissolving 300.4 mg of the test item completely in 300 ml of test water using ultrasonic treatment (5 minutes) and intense stirring (10 minutes at room temperature). Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media of the lower test item concentrations. The test media were prepared just before the start of the test, i.e. addition of algae.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Strain No. 86.81 SAG
- Source: collection of Algal Cultures (Institute for Plant Physiology, University of Gottingen).
- Cultivation: algae were cultivated in the testing laboratory under standardized conditions, according to the testing guideline.
- Aget at inoculum: 3 days
- Acclimation: no.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/l

Test temperature:

Range from 22 to 23 °C

pH:

Range from 7.9 to 8.8

Nominal and measured concentrations:

22, 46, 100, 220, 460 and 1000 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml Erlenmeyer flasks; 15 ml algal suspension for each flask.
- Aeration: covered with glass dishes; continuously stirred by magnetic stirrers.
- Initial cells density: 5000 cells per ml test medium.
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 6 replicates

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: synthetic test water, according to guideline.

OTHER TEST CONDITIONS
- Incubation temperature: 22 - 23 °C
- Photoperiod: 24 h
- Light intensity and quality: range from 6500 to 7300 Lux (mean 6900 Lux); fluorescent tubes installed above the test flasks (Philips TLD 36W/840).
- Other: the appearence of the test media was recorded daily.

EVALUATION OF ALGAL BIOMASS
Small volumes of the test media and the control were taken from all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal biomass in the samples was determined by fluorescence measurement (BIOTEK Multi-Detection Microplate Reader, Model FLx800). The measurements were performed in duplicate.
In addition, after 72 hours of exposure, a sample was taken from the control and from the test medium of the highest nominal test concentration of 1000 mg/l. The shape and size of the algal cells in these samples were examined using a microscope.

Reference substance (positive control):

yes

Remarks:

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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 170 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 170 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The test item had no statistically significant inhibitory effect on the growth (growth rate and yield) of the algae during the test period of 72 hours up to and including the highest (mean measured) concentration of the test item of 170 mg/l.
- Exponential growth in the control: yes; factor of 156 over 72 hours.
- Section-by-section variation coefficient: 12 %
- Growth rate variation troughtout the test: 1.6 %
- Observation of abnormalities: microscopic examination did not show any difference in both appearance, shape and size between exposed algae and control algae.
- Other: all test media were clear solutions throughout the test period.

MEASURED CONCENTRATIONS
At the start of the test, the measured concentration of the test item at the highest nominal test concentration of 1000 mg/l was 81 % of the nominal value. During the test period of 72 hours the concentration of the test item decreased to 4% of the nominal concentration. The mean measured concentration of the test item at the nominal concentration of 1000 mg/l (calculated as the geometrical mean of the concentrations measured at the start and the end of the test) was 170 mg/l.

Results with reference substance (positive control):

The latest result of the positive control test in 2007 showed that the toxic performance was valid and within the historical range of the testing laboratory.

Growth rate and its inhibition % during test period

Nominal test item concentration [mg/l]	Measured test item concentration [mg/l]	Growth rate µ and inhibition of µ (Ir)
		0 - 24 h		0 - 48 h		0 - 72 h
		µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	-	1.77	0	1.61	0	1.68	0
22	n.d.	1.9	-7.3	1.67	-3.9	1.7	-0.9
46	n.d.	1.84	-4.3	1.67	-3.4	1.69	-0.3
100	n.d.	1.87	-6.1	1.69	-5	1.71	-1.4
220	n.d.	1.88	-6.6	1.68	-4.6	1.67	1
460	n.d.	1.82	-3.2	1.68	-4.5	1.66	1.6
1000	170	1.8	-1.9	1.72	-6.9	1.66	1

Yield and its inhibition % during test period

Nominal test item concentration [mg/l]	Measured test item concentration [mg/l]	Yield y and inhibition of y (Iy)
		0 to 24 h		0 to 48 h		0 to 72 h
		Yield y and inhibition of y (Iy)	Iy (%)	Yield y and inhibition of y (Iy)	Iy (%)	Yield y and inhibition of y (Iy)	Iy (%)
Control	-	5.4	0	26.6	0	171	0
22	n.d.	6.3	-17	30.3	-14	179	-4.5
46	n.d.	5.9	-9.2	29.9	-12	173	-1.3
100	n.d.	6.1	-14	31.5	-18	184	-7.2
220	n.d.	6.2	-15	31.1	-17	163	5.1
460	n.d.	5.8	-6.8	30.9	-16	158	7.8
1000	170	5.6	-4.3	33.5	-26	162	5.3

Temperature values

	Temperature [C°]
Day 0	23
Day 1	23
Day 2	22
Day 3	22

pH values

Nominal test item concentration [mg/l]	pH values
	Start	End
Control	8	7.9
22	8.3	7.9
46	8.5	7.9
100	8.3	7.9
220	8.5	7.9
460	8.7	8.1
1000	8.8	8.1

MEASURED CONCENTRATIONS

Results of test item samples concentrations

Sample age [day]	Nominal test item concentration [mg/l]	Measured concentration [mg/l]			Sample preparation factor	Test item average concentration
		Sample 1	Sample 2	Average		mg/l	%
0	Control	n.d	-	n.a.	10	n.a.	n.a.
	1000	82	79.9	80.9	10	809	81
3	Control	n.d	-	n.a.	1	n.a.	n.a.
	1000	35.9	35.8	35.9	1	35.9	3.6

Validity criteria fulfilled:

yes

Remarks:

Biomass increment factor of control >16; section-by-section mean coefficient <35 %; growth rate variation coefficient <7 %.

Conclusions:

ErC50 (72 h) > 170 mg/l (geom mean)
EyC50 (72 h) > 170 mg/l (geom mean)

Executive summary:

Test item influence on algal growth was investigated in a 72-hour static test according to OECD 201. The nominal concentration tested were 22, 46, 100, 220, 460 and 1000 mg/l, plus control. Analytical assessment of the test item was performed only on the highest nominal concentration. At the beginning, the measured value was determined to be 81 % (80.9 mg/l) of its nominal; while, at the end of the test, the concentration decreased to 4 % of its nominal value (35.9 mg/l). The decrease might have been caused by photolytical degradation. The mean measured concentration of the highest nominal value was 170 mg/l, calculated as the geometrical mean of the concentration measured at the start and the end of the test.

Test item did not show any statistically significant inhibitory effect on the growth rate, yield and appearance of algae, up to the highest tested concentration. Therefore, the NOEC (72 h) was determined to be 170 mg/l.

Conclusion

ErC50 (72 h) > 170 mg/l (geom mean)

EyC50 (72 h) > 170 mg/l (geom mean)

Description of key information

Not harmful/toxic to aquatic algae and cyanobacteria.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

170 mg/L

Additional information

There is no information about the toxicity potential to algae and cyanobacteria of Optical Brightener 380, therefore, the available data on the structural analogous Similar Substance 01 have been taken into consideration. The read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13).

The Similar Substance 01 influence on algal growth was investigated in a 72 -hour static test, according to the OECD guideline 201. The nominal concentration tested were 22, 46, 100, 220, 460 and 1000 mg/l, plus control.

Duplicate samples were taken from the test media of the highest test concentration at the start of the test and at the end of the test period. The average recoveries found in the treatment samples at test start were 81 % and at test end 3.6 % of the nominal concentrations; the mean measured concentration was 170 mg/l, calculated as the geometrical mean. All test media were clear solutions throughout the test period.

The test item did not show any statistically significant inhibitory effect on the growth rate, yield and appearance of algae, up to the highest tested concentration. Therefore, the NOEC (72 h) was determined to be 170 mg/l, for both growth rate and yield.