Tetrasodium 2,2''-diamino-8,8''-dihydroxy-4',4'''-{4-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,6-diyldiimino}bis(naphthalene-1-azobenzene-2',6-disulfonate)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 14th to September 11th, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Principles of method if other than guideline:

the test method was modified to quantify the algicidal effect of colored test substances, but also the growth inhibition effect caused by reduced light intensities in the colored test media

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Red LF 6339

IUPAC Name:

Red LF 6339

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

samples were taken just before test start (duplicate samples from the single test medium and duplicate samples from the control without algae)
after 72 hours - stability samples (duplicate samples from the single test medium and duplicate samples from the control without algae)

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus Subspicatus CHODAT
- Strain: 86.81 SAG
- Source (laboratory, culture collection): supplied by the Collection of Algal Cultures (SAG), Institute for Plant Physiology, University of Gottingen, Germany
- Method of cultivation: grown in the laboratory under standardised conditions according to the test guidelines. Cultivated in synthetic test water prepared according to the test guidelines.

ACCLIMATION
exponentially growing pre-culture was used, which was set up 3 days prior to the test under conditions as in the test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/l as CaCO3

Test temperature:

23-24 °C

pH:

7.9-8.1 (start)
8.0-8.3 (end)

Nominal and measured concentrations:

1, 3.2, 10, 32 and 100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (50ml), each filled with 15 ml algal suspension continously stirred by magnetic stirrers. Each flask was placed in a black cylinder, coated inside witth aluminium foil
- Type (delete if not applicable): on the top of each cylinder glass dishes covered with watch glass dishes were placed to reduce the loss of water
- Initial cells density: 10000 algal cells/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: synthetic water as recommended in the test guideline
- Intervals of water quality measurement: pH was measured in each test concentration and the control at the start and at the end of the test. The water temperature was measured daily in an Erlenmeyer flask filled withwater and incubated under the same conditions as the test flask. The appearance of the media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: continously illuminated
- Light intensity and quality: 8217 lux (mean value). The light intensity was measured just before the start of the test below the coating cylinders.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: small volumes of the test media and the control were taken out of all test flasks after 24, 48 and 72 hours of exposure. The algal cell densities were determined by counting with an electronic partcile counter, with at least 2 measurements per sample. in addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth in experimental part A (nominal 32 mg/l). The shape of the algal cells were microscopically examined in these samples. For the quantification of the algicidal effect versus the growth inhibition due to reduced light intensitied, the percentage inhibition of algal biomass and growth rate (compared to the control in part A) was calculated for each concentration. The growth rate was compared between parts A and B.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Experimental Part A
The algae grew in the test media with dissolved test item in the Erlenmeyer flasks (five test concentrations and a control). All glass dishes above the cylinders contained purified water. Thus the inhibition of algal growth in this part was caused by the toxic effect of the test item an in addition by the reduced light intensities in the coloured test media in the flasks

Experimental Part B
The glass dishes above the cylinders contained the colored test media at the same test concnetrations as in part A, but without algae (3 replicates per test concentration). In the flasks below, the algae grew in test water without test item (as in the control), however, under changed light conditions, due to the filter effect of the colored test media in the glass dishes. Thus, the growth in part B, was caused by light absorption only.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

114.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: this EC-value and its 95 % confidence limits should be taken with caution, since the inhibition of r was up to the highest test concentration just 50 %.

Remarks:

experimental part A

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: experimental part A

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: experimental part A

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: this EC-value should be taken with caution, since the inhibition of r was up to the highest test concentration only 21.4 %

Remarks:

experimental part B

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: experimental part B

Details on results:

The test item was stable during the 72-hrs test period under the test conditions.
Experimental Part A: test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) after the exposure period of 72 hrs at the concentration of 3.2 mg/l. This test concentration was determined as the 72-hr LOEC. The 72-hr NOEC was determined as 1.0 mg/l. At the microscopical examination, the shape of algae cells after the 72-hr incubation, showed no difference between the algae growing in the test concentration of 32 mg/l and the algal cells in the control.
The observed growth inhibition of the test item on algae was caused in part due to the indirect effect, the light absrption in the colored test solution. However, the differences between experimental parts A and B were too high to state that the algal growth is inhibited solely as a result of a reduction in light intensity. Thereforem the results of experimental part A should be taken into consideration for the detemination of the toxic effect of the test item on the growth of the algae.
All test media were slightly colored by the test item
Exponential growth in the control (for algal test): yes

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

EC50 (72h, growth rate) = 114.6 mg/l (nominal)
EC10 (72h, growth rate) = 3.4 mg/l (nominal)
NOEC (72h, growth rate) = 1.0 mg/l (nominal)