2,9-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

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:

16 May 2006 to 7 July 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)

Remarks:

GLP according to German "Chemikaliengesetz", EC and OECD Guideline principles

Analytical monitoring:

yes

Details on sampling:

The saturated solution and control were analytically verified via analysis of organic carbon (DOC, according to DIN EN 1484) at test start and test end.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances): A dispersion of 10 mg/L, prepared in dilution water, was shaken with 20 rpm for 24 h at 23 ± 2 °C and centrifugated with 3000 rpm for 20 min.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Desmodesmus subspicatus CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): three day-old preculture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 ¿E·m-2·s-1 for 24 h per day.

ACCLIMATION
- Acclimation period: three days, see above
- Culturing media and conditions (same as test or not): preculture incubated at study conditions
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None. Observations were carried out during exposure at the beginning of the experiment and after 24, 48 and 72 hours.

Hardness:

0.24 mmol Ca+Mg/l for the dilution water

Test temperature:

22-24 (mean: 23) °C

pH:

Start: 7.91 (test solution), 7.81 (control)
End, 72 h: 8.33 (test solution), 8.31 (control)

Dissolved oxygen:

DOC (mg/l) (mean of two parallel determinations):
Start (without algae): 1.6 (test solution), 1.5 (control)
End, 72 h (without algae): 5.2 (test solution), 4.5 (control)

Nominal and measured concentrations:

A limit test with a saturated solution (dispersion of 10 mg/L, see above) was carried out.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile erlenmeyer flasks, volume: 250 mL
- Initial cells density: nominally 1000 - 10000 cells/mL
- Control end cells density: 580011 (mean from 6 replicates) cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water according to guidelines
- Intervals of water quality measurement: at the start and at the end (72 h)

OTHER TEST CONDITIONS
- Sterile test conditions: yes (sterile erlenmeyer flasks were used)
- Photoperiod: 24 h/d
- Light intensity and quality: 60.1 (start) and 67.7 (end, 72 h) (mean 64.4 ¿E·m-2·s-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Chlorophyll-impulsfluorometer
- Chlorophyll measurement: Chlorophyll-a-fluorescence, excitation at 435 nm, emission at 685 nm
- Other: measurements at the beginning of the test (after application) and every 24 h, dilution water was used as background signal

TEST CONCENTRATIONS
- Spacing factor for test concentrations: only one concentration (saturated solution), limit test
- Justification for using less concentrations than requested by guideline: limit test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to poor solubility of the test item, a limit test with a nominal concentration of 10 mg/L had been performed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to poor solubility of the test item, a limit test with a nominal concentration of 10 mg/L had been performed.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to poor solubility of the test item, a limit test with a nominal concentration of 10 mg/L had been performed.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to poor solubility of the test item, a limit test with a nominal concentration of 10 mg/L had been performed.

Details on results:

Biomass growth and rate-related inhibition:
- EC50: >saturated solution
- NOEC: saturated solution
- LOEC: >saturated solution

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Environmental conditions, pH-value, measured at 0 and 72 h, and room temperature, measured continuously, met the guideline requirements.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 (biomass growth): 0.31 mg/l (0-72 h), 95 % confidence interval: 0.30-0.32 mg/l
- EC50 (rate-related inhibition): 0.65 mg/l (0-72 h), 95 % confidence interval: 0.57-0.73 mg/l

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of statistical significance of biomass integrals and growth rates. One Way Analysis of Variance (ANOVA) and DUNNETT¿s test was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The ¿-value for ANOVA was ¿=0.05.

Validity criteria fulfilled:

yes

Conclusions:

No inhibiting effects on biomass growth and specific growth rate were found in the saturated solution. The EC50-values for inhibition of biomass growth (EbC50) and specific growth rate (ErC50) after 72 h were > saturated solution.

Executive summary:

The toxicity of the test item (Pigment Red 122) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD-Guideline 201 (1984) and Directive 92/69/EC Method C.3 at DR.U.NOACK-LABORATORIEN in 31157 Sarstedt, Germany from May 22 to 25, 2006. The aim of the study was to assess the effects on growth rate and biomass production over a period of 72 h. The study was conducted under static conditions with an initial cell density of nominally 103 - 104 cells/mL. A limit test with a saturated solution was carried out. A dispersion of 10 mg/L was prepared in dilution water, shaken for 24 h at 23 ± 2°C and centrifugated with 3000 rpm for 20 min. Six replicates were tested for the saturated solution and control. Environmental conditions, pH-value and room temperature, were determined to be within the acceptable limits. DOC-analysis was carried out at test start and test end from the saturated solution and control. All effect values are based on the saturated solution of the test item.

In this study the test item did not cause any inhibiting effects to Desmodesmus subspicatus after 72 h when tested with a saturated solution.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

16 May 2006 to 7 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Red 202 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

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)

Remarks:

GLP according to German "Chemikaliengesetz", EC and OECD Guideline principles

Analytical monitoring:

yes

Details on sampling:

The saturated solution and control were analytically verified via analysis of organic carbon (DOC, according to DIN EN 1484) at test start and test end.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances): A dispersion of 10 mg/L, prepared in dilution water, was shaken with 20 rpm for 24 h at 23 ± 2 °C and centrifugated with 3000 rpm for 20 min.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Desmodesmus subspicatus CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): three day-old preculture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 ¿E·m-2·s-1 for 24 h per day.

ACCLIMATION
- Acclimation period: three days, see above
- Culturing media and conditions (same as test or not): preculture incubated at study conditions
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None. Observations were carried out during exposure at the beginning of the experiment and after 24, 48 and 72 hours.

Hardness:

0.24 mmol Ca+Mg/l for the dilution water

Test temperature:

22-24 (mean: 23) °C

pH:

Start: 7.91 (test solution), 7.81 (control)
End, 72 h: 8.33 (test solution), 8.31 (control)

Dissolved oxygen:

DOC (mg/l) (mean of two parallel determinations):
Start (without algae): 1.6 (test solution), 1.5 (control)
End, 72 h (without algae): 5.2 (test solution), 4.5 (control)

Nominal and measured concentrations:

A limit test with a saturated solution (dispersion of 10 mg/L, see above) was carried out.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile erlenmeyer flasks, volume: 250 mL
- Initial cells density: nominally 1000 - 10000 cells/mL
- Control end cells density: 580011 (mean from 6 replicates) cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water according to guidelines
- Intervals of water quality measurement: at the start and at the end (72 h)

OTHER TEST CONDITIONS
- Sterile test conditions: yes (sterile erlenmeyer flasks were used)
- Photoperiod: 24 h/d
- Light intensity and quality: 60.1 (start) and 67.7 (end, 72 h) (mean 64.4 ¿E·m-2·s-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Chlorophyll-impulsfluorometer
- Chlorophyll measurement: Chlorophyll-a-fluorescence, excitation at 435 nm, emission at 685 nm
- Other: measurements at the beginning of the test (after application) and every 24 h, dilution water was used as background signal

TEST CONCENTRATIONS
- Spacing factor for test concentrations: only one concentration (saturated solution), limit test
- Justification for using less concentrations than requested by guideline: limit test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to poor solubility of the test item, a limit test with a nominal concentration of 10 mg/L had been performed.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to poor solubility of the test item, a limit test with a nominal concentration of 10 mg/L had been performed.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to poor solubility of the test item, a limit test with a nominal concentration of 10 mg/L had been performed.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to poor solubility of the test item, a limit test with a nominal concentration of 10 mg/L had been performed.

Details on results:

Biomass growth and rate-related inhibition:
- EC50: >saturated solution
- NOEC: saturated solution
- LOEC: >saturated solution

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Environmental conditions, pH-value, measured at 0 and 72 h, and room temperature, measured continuously, met the guideline requirements.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 (biomass growth): 0.31 mg/l (0-72 h), 95 % confidence interval: 0.30-0.32 mg/l
- EC50 (rate-related inhibition): 0.65 mg/l (0-72 h), 95 % confidence interval: 0.57-0.73 mg/l

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of statistical significance of biomass integrals and growth rates. One Way Analysis of Variance (ANOVA) and DUNNETT¿s test was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The ¿-value for ANOVA was ¿=0.05.

Validity criteria fulfilled:

yes

Conclusions:

No inhibiting effects on biomass growth and specific growth rate were found in the saturated solution. The EC50-values for inhibition of biomass growth (EbC50) and specific growth rate (ErC50) after 72 h were > saturated solution.

Executive summary:

The toxicity of the test item (Pigment Red 122) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD-Guideline 201 (1984) and Directive 92/69/EC Method C.3 at DR.U.NOACK-LABORATORIEN in 31157 Sarstedt, Germany from May 22 to 25, 2006. The aim of the study was to assess the effects on growth rate and biomass production over a period of 72 h. The study was conducted under static conditions with an initial cell density of nominally 103 - 104 cells/mL. A limit test with a saturated solution was carried out. A dispersion of 10 mg/L was prepared in dilution water, shaken for 24 h at 23 ± 2°C and centrifugated with 3000 rpm for 20 min. Six replicates were tested for the saturated solution and control. Environmental conditions, pH-value and room temperature, were determined to be within the acceptable limits. DOC-analysis was carried out at test start and test end from the saturated solution and control. All effect values are based on the saturated solution of the test item.

In this study the test item did not cause any inhibiting effects to Desmodesmus subspicatus after 72 h when tested with a saturated solution.

Description of key information

Two key studies with a read across substance are available. In one test no inhibiting effects on biomass growth and specific growth rate were found in the saturated solution. The EC50-values for inhibition of biomass growth (EbC50) and specific growth rate (ErC50) after 72 h were > saturated solution.

In the study with the read across substance also no toxic effects were found.

In a fresh water algal growth inhibition test with Desmodesmus subspicatus the toxicity of the submission substance to aquatic plants had been tested (reliability category 2, compliant to GLP). A static limit test had been performed at a nominal concentration of 100 mg/L due to the poor solubility of the test item. The submission substance proved to be non-toxic to Desmodesmus subspicatus under the applied test conditions:

NOEC (growth rate / yield, 72h) equal to or larger as 100 mg/L;

EC50 (growth rate / yield, 72h) > 100 mg/L;

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L