Pentasodium 7-(4-(4-(5-amino-4-sulfonato-2-(4-((2-(sulfonato-ethoxy)sulfonyl)phenylazo)phenylamino)-6-chloro-1,3,5-triazin-2-yl)amino-2-ureidophenylazo)naphtalene-1,3,6-trisulfonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 1997 to 28 November 1997

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:

yes

Remarks:

The test method was modified following a method developed by RCC to quantify the algicidal effect of coloured test substances, but also the growth inhibition effect caused by reduced light intensities in the coloured test solutions

Qualifier:

according to guideline

Guideline:

other: Commission Directive 92/69/EEC, Annex Part C, C.3: "Algal Inhibition Test", Official Journal of the European Communities No. L 383 A, dated December 29, 1992.

Deviations:

yes

Remarks:

The test method was modified following a method developed by RCC to quantify the algicidal effect of coloured test substances, but also the growth inhibition effect caused by reduced light intensities in the coloured test solutions

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40'549/A
Batch No: TV1
Expiration date: 01 Jan 2001
Stability in water: for atleast 48 hours
Solubility in water: approximately 90 mg/L at 20 degree celcius.
Aggregate state under storage conditions: solid (powder)
Colour: yellow brown.
Storage conditions: at room temperature
Safety precautions: Routine hygenic procudures are sufficient to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

The pH-values of the test media were measured in all test concentrations and the control at the start and the end of the test.

Small volumes of the test media (1.0 mL) were taken out of all test flasks after 24, 48 and 72 hours of exposure and were not replaced. The algae cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), three measurements per sample.

In addition, a sample was taken from the control and from the highest test concentration of nominal 100 mg test substance/I in experimental part A with reduced algal growth after the test period of 72 hours. The shape of these treated algal cells was microscopically examined and compared with the cells in the control.

Vehicle:

no

Details on test solutions:

The test medium of the highest test substance concentration of nominal 100 mg/L was prepared by dissolving 60 mg of the test substance in 600 mL test water. Adequate volumes of this intensively mixed test medium were added to test water to prepare the test media of the following nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg test substance/L. Additionally, a control was tested in parallel (test water without addition of the test substance). The test media were prepared just before the start of the test.
The test concentrations were based on the results of a range-finding test. However, concentrations in excess of nominal 100 mg test substance/I have not been tested in compliance with the Commission Directive 92/69/EEC. The enlarged spacing factor of 3.2 between the test concentrations was chosen, since according to the results of the range-finding test a large concentration range had to be tested in the definitive test.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricomutum), Strain No. 61.81 SAG, supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", D-37073 Göttingen. The algae are cultivated in the laboratories of RCC 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:

7.9 - 8.4

Nominal and measured concentrations:

The test substance FAT 40549/A degraded into a defined reaction product during the performance of the biological test. The analytically determined mean test substance concentrations in the non-aged test media at the start of the test ranged from 83 % to 98 % of the nominal values (based on the measured concentrations of the main compound). After 72 hours the concentrations of the main compound had decreased to 16% - 42% of the nominal values. The average over all measurements in the aged and nonaged test media per test concentration ranged from 49% to 70% of the nominal concentrations, if the quantification is based on the main compound.

If the measured test substance concentrations are based on the sum of the main compound plus degradation product, the average over all measurements in the aged and non-aged test media per test concentration ranged from 80% to 89% of the nominal concentrations. Since a toxic effect can be caused by the main compound as well as by the degradation product, and since the sum of these concentrations are within the acceptable range of 80% -120% of nominal, the reported biological results are based on the nominal test substance concentrations.

Details on test conditions:

The test was started (0 hours) by inoculation of a biomass of 10.000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up about 3 days prior to the test at the same conditions as in the test.

The test was performed in Erlenmeyer flasks (50 ml), each with 15 ml algal suspension, continuously stirred by magnetic stirrers, 3 flasks per test concentration and 6 flasks in the control. Each Erlenmeyer flask was placed in a black cylinder, coated inside with aluminium foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.

The test included two experimental parts:
Experimental part A:
The algae grew in test media with dissolved test substance in the Erlenmeyer flasks (five test concentrations and a control, see "Dosage and concentrations"). All glass dishes above the cylinders contained untreated test water. Thus, the inhibition of algal growth in this experimental part was caused due to a real toxic effect of the test substance and in addition to the reduced light intensities in the coloured test media in the Erlenmeyer flasks.
Experimental part B:
In this experimental part the glass dishes above the cylinders contained the coloured test media with the same five test concentrations as in part A, however without algae (3 replicates per test concentration). In the Erlenmeyer flasks below, the algae grew in test water without test substance (as in the control), however under changed light conditions due to the filter effect of the coloured test media in the glass dishes. Thus, the growth inhibition in part B was caused due to light absorption only. The depth of the test media in the glass dishes was 4 mm, i.e. half the depth of the test media in the Erlenmeyer flasks, because the algae in the stirred test media stay in the statistical mean in this mean depth.
All flasks were incubated in a temperature controlled water bath and continuously illuminated at a mean light intensity of about 8300 Lux, range 7800 - 8800 Lux. The light intensity was measured just before the start of the test below the coating cylinders at nine places in the area, where the Erlenmeyer flasks were placed in the test. This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the algal flasks.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

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 percentage inhibition of the growth rate µA minus the percentage inhibition of the growth rate µB after the 72 hours test period. At all test concentrations these differences were lower than 10%..
As another measure of difference the quotient of the growth rates µA/µB was calculated for each test concentration. At all test concentrations this quotient was at least 0.9 or higher.
Differences in growth rates up to the magnitude of 10% are accepted to be caused by pure chance in the used algal toxicity test. Thus, according to the recommendations of the Ad-hoc working group of experts on algal growth inhibition tests for coloured substances the differences between inhibition in experimental part A and B should be lower than 10%, respectively the quotient µA/µB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates µA and µB are essentially the same.
At all test concentrations of this test the differences of the growth rates µA and µB are lower than 10% and the quotients µA/µB are at least 0.9.

Reported statistics and error estimates:

The EbC 50 and EµC50 (the concentrations of the test substance corresponding to 50% inhibition of algal biomass respectively growth rate compared to the control), and the corresponding EC10 and as far as possible their 95%-confidence limits were calculated for both experimental parts by Probit Analysis. For the determination of the LOEC and NOEC, the calculated mean growth rates u at the test concentrations were tested on significant differences to the control values by Dunnett-Tests.

Analytical results:

The test substance FAT 40'549/A degraded into a defined reaction product during the performance of the biological test. The analytically determined mean test substance concentrations in the non-aged test media at the start of the test ranged from 83 % to 98 % of the nominal values (based on the measured concentrations of the main compound. After 72 hours the concentrations of the main compound had decreased to 16 % - 42 % of the nominal values. The average over all measurements in the aged and non-aged test media per test concentration ranged from 49 % to 70 % of the nominal concentrations, if the quantification is based on the main compound.

If the measured test substance concentrations are based on the sum of the main compound plus degradation product, the average over all measurements in the aged and non-aged test media per test concentration ranged from 80 % to 89 % of the nominal concentrations. Since a toxic effect can be caused by

the main compound as well as by the degradation product, and since the sum of these concentrations are within the acceptable range of 80 % - 120 % of nominal, the reported biological results are based on the nominal test substance concentrations.

Validity criteria fulfilled:

yes

Conclusions:

This modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance FAT 40'549/A on Pseudokirchneriella subcapitata was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a real toxic effect of the test substance on the growth of Pseudokirchneriella subcapitata can be excluded up to the highest test concentration of 100 mg test substance/I.

Executive summary:

The influence of the test substance FAT 40549/A on the growth of the green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to the Commission Directive 92/69/EEC, Annex Part C.3, dated December 29, 1992, and the OECD Guideline No. 201, adopted June 7, 1984. However, the test method was modified to quantify the algicidal effect of the test substance, but also the growth inhibition effect caused by reduced light intensities in the coloured test solutions.

The nominal test concentrations were 1, 3.2, 10, 32 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were slightly to strongly coloured by the test substance.

The test substance FAT 40549/A degraded into a defined reaction product during the performance of the biological test. The analytically determined mean test substance concentrations in the non-aged test media at the start of the test ranged from 83% to 98% of the nominal values (based on the measured concentrations of the main compound). After 72 hours the concentrations of the main compound had decreased to 16 % - 42 % of the nominal values. The average over all measurements in the aged and nonaged test media per test concentration ranged from 49% to 70% of the nominal concentrations, if the quantification is based on the main compound. If the measured test substance concentrations are based on the sum of the main compound plus degradation product, the average over all measurements in the aged and non-aged test media per test concentration ranged from 80% to 89% of the nominal concentrations. Since a toxic effect can be caused by the main compound as well as by the degradation product, and since the sum of these concentrations are within the acceptable range of 80% -120% of nominal, the reported biological results are based on the nominal test substance concentrations.

In the experimental part, where the algae grew in the test media with dissolved test substance as in a usual algal growth inhibition test, FAT 40'549/A had a statistically significant inhibition effect on the growth of Pseudokirchneriella subcapitata after the exposure period of 72 hours first at the concentration of 32 mg test substance/L (= 72-hour LOEC: lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects) was 10 mg test substance/L, since at this test concentration the mean growth rates of the algae were statistically not significant lower than in the control.

However, the same growth inhibition of Pseudokirchneriella subcapitata was observed when the algae grew in test water without test substance, but under reduced light intensities by the filter effect of the coloured test media as in the second parallel experimental part, where the algae grew in the test media with dissolved test substance.

Thus, in conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance FAT 40'549/A on Pseudokirchneriella subcapitata was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a real toxic effect of the test substance on the growth of Pseudokirchneriella subcapitata can be excluded up to the highest test concentration of nominal 100 mg test substance/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April 1996 to 07 June 1996

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:

yes

Remarks:

he test method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algai cells or due to an indirect effect, a reduced algal growth by light absorption in coloured test media.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

he test method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algai cells or due to an indirect effect, a reduced algal growth by light absorption in coloured test media.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40'549/A
Batch No: TV1
Expiration date: 01 Jan 2001
Stability in water: for atleast 48 hours
Solubility in water: approximately 90 mg/L at 20 degree celcius.
Aggregate state under storage conditions: solid (powder)
Colour: yellow brown.
Storage conditions: at room temperature
Safety precautions: Routine hygenic procudures are sufficient to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

The pH-values of the test media were measured in samples from all test concentrations and the control at the start and at the end of the test.

Small test media samples (0.1 - 1.0 ml) were taken out of all flasks after 24, 48 and 72 hours of exposure and were not replaced. The algae cell densities in the samples were determined by counting with an electronic particle counter (AL CELLCOUNTER, Model 871, AL-Systeme, D-76149 Karlsruhe), three measurements per sample.

In addition, a sample was taken from the control and from the test concentration of nominal 100 mg/L in experimental part A with reduced algal growth after a test period of 72 hours. The shape of the treated algal cells was microscopically examined and compared with the cells in the control.

Vehicle:

no

Details on test solutions:

The test medium of the highest test substance concentration was prepared by dissolving the test substance in test water (100 mg/L). Adequate volumes of the intensively mixed test medium were added to test water to prepare the test media of the following nominal concentrations: 1.0, 3.2, 10, 32, and 100 mg test substance/L. Additionally, a control was tested in parallel (test water without addition of the test substance). The test media were prepared just before the start of the test.
The test concentrations were based on the results of a range-finding test. The range-finding test was not performed in compliance with the GLP-Regulations, but the raw data of the range-finding test will be archived under the RCC Project number of the present study.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, D-37073 Göttingen, F.R:G. The algae were grown in the laboratories of RCC under standardized conditions according to the OECD Guideline No. 201.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCÛ3

Test temperature:

23.4 - 23.5 °C

pH:

8.2 - 9.5

Nominal and measured concentrations:

The analytically determined test concentrations in the test media ranged from 77% to 97% of the nominal values (calculated as the average over all measurements per test concentration). The test substance FAT 40'549/A degrades into a defined reaction product during the performance of the biological test. The mean measured concentrations were determined to be 20% to 47% of the nominal values, if the quantification is based on the main compound. Since a toxic effect can be due to the main compound as well as to a reaction product the reported results are related to the mean measured concentrations of the sum of the main compound and the reaction product.

Details on test conditions:

The algae were cultivated and tested in synthetic test water, prepared according to the mentioned test guidelines. In deionized water with a conductivity lower than 0.1 µS cm/L (Milli- Q-water) analytical grade salts were added.

The test was started (0 hours) by inoculation of a biomass of 10000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up about 72 hours prior to the test at the same conditions as in the test.

The test was performed in Erlenmeyer flasks (50 ml), each with 50 ml algal suspension, continuously stirred by magnetic stirrers, 3 flasks per test concentration and 6 flasks in the control. Each Erlenmeyer flask was placed in a black cylinder, coated inside with aluminium foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.

The test included two experimental parts:
Experimental part A:
The algae grew in test media with dissolved test substance in the Erlenmeyer flasks (5 test concentrations and a control, see "Dosage and concentrations"). All glass dishes above the cylinders contained untreated test water. Thus, the inhibition of algal growth in this experimental part was caused due to a real toxic effect of the test substance and in addition to the reduced light intensities in the coloured test media in the Erlenmeyer flasks.

Experimental part B:
In this experimental part the glass dishes above the cylinders contained the coloured test media with the same five test concentrations as in part A, however without algae (3 replicates per test concentration). In the Erlenmeyer flasks below, the algae grew in test water without test substance (as in the control), however under changed light conditions due to the filter effect of the coloured test media in the glass dishes. Thus, the growth inhibition in part B was caused due to light absorption only. The depth of the test media in the glass dishes was 20 mm, i.e. half the depth of the test media in the Erlenmeyer flasks, because the algae in the stirred test media stay in the statistical mean in this mean depth.

All flasks were incubated in temperature controlled water baths and continuously illuminated at a mean light intensity of about 8500 Lux, range 7900 - 8900 Lux. The light intensity was measured just before the start of the test below the coating cylinders at nine places in the area, where the Erlenmeyer flasks were placed in the test. This illumination was achieved by fluorescent tubes (universal white L 25, 36 W) installed above the algal flasks.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

89.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: the sum of main compound and reaction product

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: the sum of main compound and reaction product

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: the sum of main compound and reaction product

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: the sum of main compound and reaction product

Basis for effect:

biomass

Details on results:

According to the recommendations of the Ad-hoc meeting of experts on algal growth inhibition tests for coloured substances (Vienna, 30-31 January 1996) 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 percentage inhibition of the growth rate µA minus the percentage inhibition of the growth rate µB after the 72 hours test period. Only at the test concentrations of nominal 1.0 and 10 mg/l these differences were lower than 10%. At all other test concentrations the differences between experimental part A and B were higher than 10%.

As another measure of difference the quotient of the growth rates uA/uB was calculated for each test concentration. These quotients were also high (at least 0.9) at the test concentrations of 1.0, 10 and 32 mg/l but lower than 0.9 at the test concentrations of 3.2 and 100 mg/l.

Differences in growth rates up to the magnitude of 10% are accepted to be caused by pure chance in the used algal toxicity test. Thus, according to the recommendations of the Adhoc meeting of experts on algal growth inhibition tests for coloured substances (Vienna, 30- 31 January 1996) the differences between inhibition in experimental part A and B should be lower than 10%, respectively the quotient uA/uB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates µA and µB are essentially the same.

At some test concentrations of this test the differences of the growth rates µA and µB are higher than 10% and the quotients µA/µB lower than 0.9. Thus, a real toxic effect of the test substance on the growth of Scenedesmus subspicatus can not be excluded at these test concentrations.

Reported statistics and error estimates:

The EbC 50 and EµC 50 (the concentrations of the test substance corresponding to 50% inhibition of algal biomass respectively growth rate compared to the control), and the corresponding EC 10 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 growth rates u at the test concentrations were tested on significant differences to the control values by DUNNET T-TEST.

Analytical results:

The analytically determined test concentrations in the test media ranged from 77 % to 91 % of the nominal values (calculated as the average over all measurements per test concentration). The test substance FAT 40'549/A degrades into a defined reaction product

during the performance of the biological test.

The mean measured concentrations were determined to be 20 % to 47 % of the nominal values, if the quantification is based on the main compound. Since a toxic effect can be due to the main compound as well as to a reaction product the reported results are related to the

mean measured test concentrations of the sum of the main compound and the reaction product.

Validity criteria fulfilled:

yes

Conclusions:

The 72 h EC50 of FAT 40549/A was determined to be 89.9 mg/L based on the growth rate. The corresponding EC10 was determined to be 2.8 mg/L.

Executive summary:

The influence of the test substance FAT 40549/A on the growth of the green alga Scenedesmus subspicatus was investigated in a 72-hour static test according to the OECD Guideline No. 201, adopted June 7, 1984, and the Commission Directive 92/69/EEC, Annex Part C.3, dated December 29, 1992. However, the test method was modified to quantify the algicidal effect of the test substance, but also the growth inhibition effect due to reduced light intensities in the coloured test media. The test was performed in compliance with Good Laboratory Practice Regulations.

The nominal test concentrations tested were 1.0, 3.2, 10, 32 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were slightly to strongly coloured by the test substance.

The analytically determined test concentrations in the test media ranged from 77 % to 97 % of the nominal values (calculated as the average over all measurements per test concentration). The test substance FAT 40'549/A degrades into a defined reaction product during the performance of the biological test. The mean measured concentrations were determined to be 20% to 47% of the nominal values, if the quantification is based on the main compound. Since a toxic effect can be due to the main compound as well as to a reaction product the reported results are related to the mean measured concentrations of the sum of the main compound and the reaction product.

The growth inhibition effect of FAT 40549/A on Scenedesmus subspicatus was caused in a high degree due to the indirect effect, the light absorption in the coloured test solutions. However, a real toxic effect of the test substance on the growth of Scenedesmus subspicatus can not be excluded. Therefore, the results of this experimental part, where the algae grew in the test media with dissolved test substance as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic effect of the test substance on the growth of Scenedesmus subspicatus.

In this experimental part FAT 40549/A had a statistically significantly inhibition effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 3.0 mg test substance/l (= 72-hour LOEC: lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects) was 0.9 mg test substance/l, since up to this test concentration the mean growth rates of the algae were statistically not significantly lower than in the control.

This modified algal test has demonstrated that the observed growth inhibition effect of the test substance FAT 40549/A on Scenedesmus subspicatus was caused in a high degree due to 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 substance as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic effect of the test substance on the growth of Scenedesmus subspicatus.

Description of key information

A real toxic effect of the test substance on the growth of Pseudokirchneriella subcapitata can be excluded up to the highest test concentration of nominal 100 mg test substance/l. However, using Scenedesmus subspicatus as test species an EC50 of 89.9 mg/l and an EC10 of 2.8 mg/l were found, which are taken forward for hazard and risk assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

89.9 mg/L

EC10 or NOEC for freshwater algae:

2.8 mg/L

Additional information

Two reports for algal growth inhibition test were available according to a modified OECD test guidance under GLP compliance.

The influence of the test substance FAT 40'549/A on the growth of the green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to OECD Guideline No. 201 and used as the key study. The test method was modified to quantify the algicidal effect of the test substance, but also the growth inhibition effect caused by reduced light intensities in the coloured test solutions. This modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance FAT 40'549/A on Pseudokirchneriella subcapitata was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a real toxic effect of the test substance on the growth of Pseudokirchneriella subcapitata can be excluded up to the highest test concentration of nominal 100 mg test substance/l.

The influence of the test substance FAT 40549/A on the growth of the green alga Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the OECD Guideline No. 201 and used as a supportive study. This modified algal test has demonstrated that the observed growth inhibition effect of the test substance FAT 40549/A on Scenedesmus subspicatus was caused in a high degree due to 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 substance as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic effect of the test substance on the growth of Scenedesmus subspicatus. The EC50 determined was 89.9 mg/l whereas the EC10 was 2.8 mg/l in this study.

In conclusion, the observed growth inhibition effect of the test substance FAT 40'549/A on Pseudokirchneriella subcapitata was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a real toxic effect of the test substance on the growth of Pseudokirchneriella subcapitata can be excluded up to the highest test concentration of nominal 100 mg test substance/l. However, for Scenedesmus subspitatus the results could not be fully attributed to the light absorption effect and the result of this study is taken forward for hazard and risk assessment.