1,3,6-Naphthalenetrisulfonic acid, 7-[2-[2-[(aminocarbonyl)amino]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]phenyl]diazenyl]-, sodium salt

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:

24 August 2007 to 27 September 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

yes

Details on sampling:

Test item concentration/s: 0.88, 1.9, 4.3, 9.4, 21, 45 and 100 mg/l
Method of administration: stock solution
A stock solution was prepared to give the desired series of test concentrations. To achieve this 125.2 mg of the test item were added to 1 litre of dilution water. The pH was adjusted to pH 7.8.

Vehicle:

no

Details on test solutions:

A stock solution was prepared to give the desired series of test concentrations. To achieve this 125.2 mg of the test item were added to 1 litre of dilution water. The pH was adjusted to pH 7.8.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Name : Desmodesmus subspicatus
Source : Non-axenic strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany)
Maintenance of stock cultures : Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions (23 +/- 2°C) at a light intensity in the range 60 – 120 μE. x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KÜHN (1977) is renewed once a week. Cell density measurements are made using a microcell counter.
Preparation of pre-cultures : Pre-cultures are set up three days before the start of a test. They are grown under identical exposure conditions as the stock cultures, except from the use of a different nutrient medium.
Test cultures: The algal inocula for a test are taken from an exponentially-growing pre-culture and are mixed with the nutrient medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period

Hardness:

1.3°dH, corresponding to 22.5 mg/L CaCO3.

Test temperature:

21-25C

pH:

7.3-8.2

Dissolved oxygen:

No data

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominally 0.88, 1.9, 4.3, 9.4, 21, 45 and 100 mg/L
All results are expressed in terms of nominal concentrations. Recovery rates ranged from 97.8-101.0% of nominal values at 0 hours, and from 98.0-100.9% of nominal values at 72 hours.

Details on test conditions:

Test vessels: 300 ml Erlenmeyer flasks with cotton stoppers
Culturing apparatus: Light chamber in which a temperature in the range 21°C to 25°C can be maintained at +/- 2°C, and continuous uniform illumination is provided in the spectral range 400 to 700 nm.
Light intensity: At the average of the test solutions, a light intensity in the range 60 to 120 μE. x m-2 x s-1, or an equivalent range of 4000 to 8000 lx, is recommended to use.
Cell density measurements: Cell densities are measured in a microcell counter or, alternatively, are determined by means of a microscopic counting chamber.
Experimental design: 7 test concentrations plus 1 control, 3 replicates per concentration, 6 replicates per control initial cell density in the test cultures approximately 5000 cells per millilitre
Test item concentration/s: 0.88, 1.9, 4.3, 9.4, 21, 45 and 100 mg/l
Method of administration: stock solution
Duration of exposure: 72 hours
Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of growth [b] and growth rate [r], respectively, of the algal population.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

190 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.1 mg/L

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.4 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.4 mg/L

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

All results are expressed in terms of nominal concentrations. Recovery rates ranged from 97.8-101.0% of nominal values at 0 hours, and from 98.0-100.9% of nominal values at 72 hours, respectively.

Due to somewhat faster growth on the last day (72 h)of the test, the coefficient of variation of sectional growth rates (41.9 %) was slightly higher than required by the 2006 version of the guideline (35 %). There is no indication of any growth-limiting factor. Over 48 h, an option given in the guideline when the growth rate is adequate, this coefficient of variation was only 6.4 %. At both time points, no effect of the test item was evident, therefore the test is considered valid.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

No data

SUMMARY OF RESULTS

 

Mean cell density during the tests

Test item concentration [mg/l]	Cell density [cells/ml] (initial cell density = 5000 cell/ml)
	24h	48h	72h
Control	40000	357222	923889
0.88	48889	414444	1032222
1.9	41111	381111	1012222
4.3	42222	342222	1001111
9.4	44444	288889	910000
21	33333	214444	903333
45	22222	93333	606667
100	24444	94444	512222

 

Mean growth (b) [integral of biomass]

Test item concentration [mg/l]	Area under growth curve	Effect [%] (Increase (-))
Control	846667	0.0
0.88	966944	-14.2
1.9	915833	-8.2
4.3	872500	-3.1
9.4	775833	8.4
21	686944	18.9
45	406389	52.0
100	362500	57.2

 

Mean growth rate (r)

Test item concentration [mg/l]	Growth rate [1/d]	Effect [%] (Increase (-))
Control	1.74	0.0
0.88	1.78	-2.1
1.9	1.77	-1.7
4.3	1.77	-1.5
9.4	1.73	0.3
21	1.73	0.4
45	1.60	8.1
100	1.54	11.3

 

Validity criteria fulfilled:

yes

Conclusions:

The substance is not deemed to be hazardous to algae.

Executive summary:

A study was performed to assess adverse effects of Gelb Sulfato on the growth (= increase in cell density) and the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations.

 

The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test'. Study performed in accordance with the Principle of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.88, 1.9, 4.3, 9.4, 21, 45 and 100 mg/l of Gelb Sulfato dissolved in water.

 

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r), relative to control cultures grown under identical conditions. Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Williams Multiple Sequential t-test Procedure. The following values were determined:

 

Results [mg/l]:

95% confidence limits [mg/l]

ErC 50: 190       99 - 1686

ErC 10: 13         1.5 - 24

EbC 50: 32        22 - 50

EbC 10: 6.1       1.6 - 11

NOEC [r] (tα 0.05) : 9.4

NOEC [b] (tα 0.05) : 9.4

 

All results are expressed in terms of nominal concentrations. Recovery rates ranged from 97.8-101.0% of nominal values at 0 hours, and from 98.0-100.9% of nominal values at 72 hours, respectively.

 

To reduce shading effects of the coloured test substance, the test was carried out with strong illumination (80 - 120 μE) and with a reduced test volume of 25 ml in 300 ml Erlenmeyer flasks.

The test substance is not considered to be harmful to algae.

Description of key information

An ErC50 of 190 mg/l was calculated based on the data of the study, with a corresponding ErC10 of 13 mg/l.

The test substance is not considered to be harmful to algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

190 mg/L

EC10 or NOEC for freshwater algae:

13 mg/L

Additional information

A study was performed to assess adverse effects of Gelb Sulfato (= Reactive Yellow 176 Ester) on the growth (= increase in cell density) and the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations. The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test'. Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.88, 1.9, 4.3, 9.4, 21, 45 and 100 mg/l of Gelb Sulfato dissolved in water.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r), relative to control cultures grown under identical conditions. Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Williams Multiple Sequential t-test Procedure.

An ErC50 of 190 mg/l was calculated based on the data of the study, with a corresponding ErC10 of 13 mg/l.

All results are expressed in terms of nominal concentrations. Recovery rates ranged from 97.8-101.0% of nominal values at 0 hours, and from 98.0-100.9% of nominal values at 72 hours, respectively.

 To reduce shading effects of the coloured test substance, the test was carried out with strong illumination (80 - 120 μE) and with a reduced test volume of 25 ml in 300 ml Erlenmeyer flasks.

The test substance is not considered to be harmful to algae.