Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(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:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 18 August 2006;
Experiment completion date - 02 September 2006;
Study completion date - 29 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010

Analytical monitoring:

yes

Details on sampling:

For the analysis of the actual test item concentrations the following samples were taken:
Just before the start of the test:
- duplicate samples from each test medium (without algae)
- duplicate samples from the control (without algae)
After 72 hours:
- duplicate samples from each test medium (without algae) (stability samples)
- duplicate samples from the control (without algae)
For the 72-hour stability samples additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae.
The concentrations of the test item FAT 40825/A were measured in the duplicate test media samples of all test concentrations from both sampling times (0 and 72 hours). From the control samples only one of the duplicate samples was analysed from each of the sampling times (0 and 72 hours). The analytical procedure and results are described in the attached analytical phase report.

Vehicle:

no

Details on test solutions:

The test medium of the highest nominal test item concentration of 100 mg/L was prepared by dissolving 60.5 mg of the test item completely in 600 mL of test water using stirring for 15 minutes at room temperature. In a series of dilution steps the test medium was diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before addition of algae (= start of the test).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany). The algae had been grown in RCC's laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L as CaC03)

Test temperature:

23 °C

pH:

At the start of the test, the pH values in the test media and the control were in the range of 8.0 to 8.2. At the end of the test, pH values between 8.1 and 8.9 were measured.

Nominal and measured concentrations:

nominal: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
measured: from 86 to 108 % of the nominal values

Details on test conditions:

Experimental conditions:
The test was started (0 hours) by inoculation of 10,000 algal cells per mL of test medium. These cells were taken from an exponentially growing pre-culture, which was set up three days prior to the test under the same conditions as in the test. The test was performed in Erlenmeyer flasks (50 mL), each filled with 15 mL algal suspension. The flasks were continuously stirred by magnetic stirrers. Per test concentration, 3 flasks were prepared. For the control, 6 flasks were prepared. Each flask was placed in a black cylinder, coated inside with aluminium foil. On the top of each cylinder, glass dishes covered with watch glass dishes were placed to reduce the loss of water and to avoid the entry of dust into the solutions. All flasks were incubated in a temperature-controlled water bath at 23 °C and continuously illuminated at a measured light intensity of about 8400 Lux (mean value), range: 7100 to 9420 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area). The light intensity was measured just before the start of the test below the coating cylinders. The illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the algal flasks. The test vessels were labelled with the RCC study number and all necessary additional information to ensure unmistakable identification.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 26-86 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 5.8-13 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Experimental part A
Experimental part A corresponds to the usual algal toxicity test. This means that the algal growth inhibition in this experimental part was caused by a possible toxic effect of the test item and/or by the reduced light intensities due to the light absorption in the coloured test media. In experimental part A, the test item had a statistically significant inhibitory effect on the growth rate r of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 3.2 mg/L (results of a Dunnett-test, one-sided, a = 0.05). Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined at the concentration of 1.0 mg/L, since up to and including this test concentration the mean growth rate r of the algae was statistically not significantly lower than in the control. For the biomass b, the same LOEC and NOEC values were determined. The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in test medium with reduced algal growth (nominal 32 mg/L) in experimental part A and the algal cells in the control. There were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at up to and including this nominal concentration.

Reported statistics and error estimates:

The EbC50 and ErC50 values (the respective concentrations of the test item corresponding to 50 % inhibition of algal biomass (b) and growth rate (r) compared to the control), and the corresponding EC 10 values and EC90 values and their 95 %-confidence limits were calculated for both experimental parts by Probit Analysis. For the determination of the LOEC and NOEC, the calculated mean biomass and the mean growth rate at the test concentrations were tested for significant differences when compared to the control values by a Dunnett-test.

Comparison between the results in experimental parts A and B:

According to the recommendations of the Ad-hoc working group of experts on algal growth inhibition for the interpretation of test results of coloured substances the comparison between the results in experimental parts A and B was based on the growth rates. The differences between the results of experimental parts A and B were described for each test concentration as the percentage inhibition of the growth rate rA (IrA) minus the percentage inhibition of the growth rate rB (lrB) after the 72 hours test period. At the nominal test concentrations of 0.32-3.2 and 32 mg/L these differences were lower than 10 %. However, at the test concentrations of 10 and 100 mg/L the differences were higher than 10 %. As another measure of difference, the quotient of the growth rates rA/rB was calculated for each test concentration. This quotient was also high (at least 0.9) at the nominal test concentrations of 0.32-3.2 and 32 mg/L, but lower than 0.9 at the test concentrations of 10 and 100 mg/L. Differences in growth rates up to the magnitude of 10 % are within the range of natural variability of algal growth. Thus, according to the recommendations of the Ad-hoc working group of experts on algal growth inhibition tests for coloured substances the difference between inhibition in experimental part A and B should be not higher than 10 %, and the quotient rA/rB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates rA and rB are essentially the same. At the nominal test concentrations of 10 and 100 mg/L, the difference between the inhibition of growth rates rA and rB is higher than 10 %, and the quotient rA/rB is lower than 0.9. Thus, a real toxic effect of the test item on the growth of Scenedesmus subspicatus cannot be excluded at these test concentrations.

 

Conclusion:

This modified algal test has demonstrated that the observed growth inhibition effect of the test item FAT 40825/A on Scenedesmus subspicatus was caused in part by the indirect effect, the light absorption in the coloured test solutions. 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. Therefore, the results of experimental part A, where the algae grew in the test media with dissolved test item as in a usual algal growth inhibition test should be taken into consideration for the determination of the inhibitory effect of the test item on the growth of Scenedesmus subspicatus.

Validity criteria fulfilled:

yes

Conclusions:

Algae 72 h EC50: 43 mg/L based on growth rate

Executive summary:

The influence of the test item FAT 40825/A on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify not only the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions.

The nominal test concentrations were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item.

The analytically determined test item concentrations in the analyzed test media varied in the range from 86 to 108 % of the nominal values. Consequently, the test item was stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.

The test design consisted of two experimental parts. In experimental part A, the algae were grown in the presence of different test item concentrations as in the usual algal toxicity test. In experimental part B, the algal growth inhibition caused by the pure light effect (the reduced light intensities in the colored test media) was quantified: 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 glass dishes above the algal suspensions. These dishes contained test medium with same test item concentrations as in part A, however without algae. The results in experimental parts A and B based on growth rates were compared.

The growth inhibition effect of FAT 40825/A on Scenedesmus subspicatus was caused in part by the indirect effect, the light absorption in the colored test solutions. However, at the higher test concentrations a real toxic effect of the test item on the growth of Scenedesmus subspicatus can not be excluded. Therefore, the results of the experimental part, where the algae grew in the test media with dissolved test item as in a usual algal growth inhibition test are taken into consideration for the determination of the toxic effect of the test item on the growth Scenedesmus subspicatus.

FAT 40825/A had a statistically significant inhibition effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 3.2 mg/L (= 72 hour LOEC: lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects) was 1.0 mg/L, since at this test concentration the mean biomass and the mean growth rate r of the algae was statistically not significant lower than in the control. The 72 h EC50 value was 43 mg/L based on the growth rate with the 95 % confidence limit of 26-86 mg/L.

Description of key information

The 72-hour NOEC (highest concentration tested without toxic effects) was 1.0 mg/L, since at this test concentration the mean biomass and the mean growth rate r of the algae was statistically not significantly lower than in the control. The 72 h EC50 value was 43 mg/L based on the growth rate with the 95 % confidence limit of 26-86 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

43 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

The influence of the test item FAT 40825/A on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify not only the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions. The nominal test concentrations were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item. The analytically determined test item concentrations in the analyzed test media varied in the range from 86 to 108 % of the nominal values. Consequently, the test item was stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item. The test design consisted of two experimental parts. In experimental part A, the algae were grown in the presence of different test item concentrations as in the usual algal toxicity test. In experimental part B, the algal growth inhibition caused by the pure light effect (the reduced light intensities in the colored test media) was quantified: 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 glass dishes above the algal suspensions. These dishes contained test medium with same test item concentrations as in part A, however without algae. The results in experimental parts A and B based on growth rates were compared. The growth inhibition effect of FAT 40825/A on Scenedesmus subspicatus was caused in part by the indirect effect, the light absorption in the colored test solutions. However, at the higher test concentrations a real toxic effect of the test item on the growth of Scenedesmus subspicatus cannot be excluded. Therefore, the results of the experimental part, where the algae grew in the test media with dissolved test item as in a usual algal growth inhibition test are taken into consideration for the determination of the toxic effect of the test item on the growth Scenedesmus subspicatus. FAT 40825/A had a statistically significant inhibition effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 3.2 mg/L (= 72-hour LOEC: lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects) was 1.0 mg/L, since at this test concentration the mean biomass and the mean growth rate r of the algae was statistically not significantly lower than in the control. The 72 h EC50 value was 43 mg/L based on the growth rate with the 95 % confidence limit of 26-86 mg/L.