Trisodium hydrogen [2-[[α-[[3-[[4-chloro-6-[ethyl[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulphophenyl]azo]benzyl]azo]-4-sulphobenzoato(6-)]cuprate(4-)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

From December 06th to 08th, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines and performed according to GLP. Justification for Read Across is detailed in the endpoint summary.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

A modified Algal Growth Inhibition test for Dyestuffs - ETAD method

Deviations:

yes

Remarks:

the test was supplemented by an modified algae test which takes into account the light absorption of the dyes (modifies algae test according to Memmert & Knoell, 1992).

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Concentrations: control plus 12.5, 25, 50 and 100 mg/l where analysed at 0 and 72h.

Vehicle:

no

Details on test solutions:

Control plus 12.5, 25, 50 & 100 mg/l where administered at 125 mg/l stock solutions.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Species: Scenedesmus subspicatus CHODAT.
- Origin: institute of Plant Physiology, University of Gottingen.
- Cultivation: in a light chamber at 23 ± 2 °C and with a quantum flux which equals 120 µE/m2/S.
- Water for dilution: deionised water.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not determined

Test temperature:

23 ± 2 °C

pH:

At 0 hours: 8.6
At 72 hours: ca. 8.1

Dissolved oxygen:

Not determined

Nominal and measured concentrations:

12.5, 25, 50, 100 mg/l (nominal)

Details on test conditions:

The test for growth inhibition was supplemented by an modified algae test which takes into account the light absorption of the dyes (modifies algae test according to Memmert & Knoell (1992)). For both tests, test item concentrations of nominally 12.5, 25, 50, and 100 mg/l was used. The further experiment lets to differentiate algicidic from algistatic effects.

TEST SYSTEM
- Test vessel: beaker (diameter. 5.5 cm, height 9.5 cm)
- Initial cells density: algal suspension taken from an exponentially growing preculture which serves to adjust an initial cell density of 10 000 cells per millilitre.
- Control: inoculum in nutrient medium and water for dilution.

GROWTH MEDIUM
- Chamber: light chamber and shaker (PVP).

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: deionized water.

EFFECT PARAMETERS MEASURED
Determination of the effects on the growth of Scenedesmus subspicatus within a 72 h exposure period.
Counting: Microcellcounter Sysmex F-300 Digitana (SOP 1.401).

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.2 - < 17.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Part A, algae-culture and dye have been incubated together

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.7 - 32.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Part B, algae culture and dye have been separately incubated

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

< 9.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Part A, algae-culture and dye have been incubated together

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.2 - 17.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Part B, algae culture and dye have been separately incubated

Details on results:

The concentration response curves obtained in experimental parts A (according to guideline) and B (modified algae test) the growth rate are considered very similar. The growth inhibition, which was observed in part A, is caused by the light absorption of the dye stuff. The substance is not toxic for algae.
Part A, algae-culture and dye have been incubated together ECr10 (72h) < 9.2 mg/l (geom. mean)
Part A, algae-culture and dye have been incubated together ECb10 (72h) < 9.2 mg/l (geom. mean)

Part B, algae culture and dye have been separately incubated ECr10 (72h) < 9.2 mg/l (geom. mean)
Part B, algae culture and dye have been separately incubated ECb10 (72h) < 9.2 mg/l (geom. mean)

The analytical content of the test item resulted to be lower than 80 % of the nominal value.

Details on test results

	EC50 (at 72h) (mg/l)
	Nominal conc.	Measured (geom mean) conc. (mg/l)
Experimental parts A (according to guideline)
Growth rate	12.5 - 25.0	9.2 - 17.7
Biomass	< 12.5	< 9.2
Experimental parts B (modified algae test)
Growth rate	25 - 50.0	17.7— 32.8
Biomass	12.5 — 25.0	9.2 — 17.7

Percentage inhibition of growth rate in the modified algae test

Nominal conc. (mg/l)	Measures conc. (mg/l)	% inhibition of algae
		Part A	Part B	Part A - B
12.5	3.2	36.4	27.3	9.1
25.0	11.4	54.5	36.4	18.1
50.0	26.6	63.6	63.6	0.0
100.0	62.6	72.7	81.8	-9.1

Nominal concentration (mg/l)	N. cells/ml			pH
	24 h	48 h	72 h	0 h	72 h
First replicate 12.5	22200	47800	93300	8.6	8.1
First replicate 25	22200	25500	51100	8.6	8.0
First replicate 50	17800	22200	36700	8.6	8.0
First replicate 100	15600	16700	26700	8.6	8.0
Second replicate 12.5	20000	45600	120000	8.6	8.2
Second replicate 25	22200	34400	75600	8.6	8.2
Second replicate 50	20000	31100	37800	8.6	8.1
Second replicate 100	12200	16700	20000	8.6	8.1
Control	21700	67200	247000	8.6	8.7

EXPERIMENTAL RESULTS ANALYSIS RELATED

TOC measurement (single determinations)

Nominal concentration (mg/l)	TOC (mg/l)		Test item (mg/l)
	0 h	72 h	0 h	72 h
First replicate 12.5	3.0	4.0	7.6	10.1
First replicate 25	7.0	7.0	17.7	17.7
First replicate 50	12.0	14.0	30.4	35.4
First replicate 100	26.0	29.0	65.8	73.4
Second replicate 12.5	3.0	5.0	7.6	126
Second replicate 100	26.0	28.0	65.8	70.8
Control	2.0	3.0	5.1	7.6

Conclusions:

The substance is not toxic for algae.

Executive summary:

The test for growth inhibition on algae according to the EU-guideline, was supplemented by an modified algae test which takes into account the light absorption of the dyes (modifies algae test according to Memmert & Knoell (1992)). For both tests, test item concentrations of nominally 12.5, 25, 50, and 100 mg/l was used. Performing this further investigation, it is possible to differentiate algicidic from algistatic effects. Investigation of the effects of light absorption of the dyestuff indicate that effects noted in the algal study are due to light absorption rather than associated toxicity.

Part A, algae-culture and dye have been incubated together ECr10 (72h) < 9.2 mg/l (geom. mean); ECr50 (72h) 9.2 - 17.7 mg/l (geom. mean)

Part A, algae-culture and dye have been incubated together ECb10 (72h) < 9.2 mg/l (geom. mean); ECr50 (72h) < 9.2 (geom. mean)

Part B, algae culture and dye have been separately incubated ECr10 (72h) < 9.2 mg/l (geom. mean; ECr50 (72h) 17.7 - 32.8 mg/l (geom. mean)

Part B, algae culture and dye have been separately incubated ECb10 (72h) < 9.2 mg/l (geom. mean); ECb50 (72h) 9.2 - 17.7 mg/l (geom. mean)

The concentration response curves obtained in experimental parts A (according to guideline) and B (modified algae test) the growth rate are considered very similar. The growth inhibition, which was observed in part A, is caused by the light absorption of the dye stuff. The substance is not toxic for algae.

Conclusion

The substance is not toxic for algae.

Description of key information

Expected to be not harmful to Algae