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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-08 to 2010-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study meets requirements of OECD Guideline 211 and GLP requirements with no deviations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
No analytical determination of the test item was carried out due to the low water solubility of the test item.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution* was prepared. A dispersion of 1 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: The saturated solution was tested as limit concentration.
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
The test medium had a nominal hardness of 0.24 mmol CaMg/L
Test temperature:
Mean: 21.5 °C, Min: 21 °C, Max: 22 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
Saturated solution (1 mg/L) 8.04 8.35
Control 8.13 8.34
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
See above
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 636336 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test
- Range finding study:
Results of the Range Finding Test (0-72 h)

Saturated solution [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
Filtration of the saturated solution
20 - 3 - 19
1 - 3 - 19

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 other: mg/L (saturated solution)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.60 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.28 (CI 0.266 - 0.299) mg/L
Reported statistics and error estimates:
The NOEC and LOEC were determined by calculation of
statistical analyses statistical significance of growth rates and yield.
t-test was carried out for the determination of statistically significant differences compared to control replicates. When running a t-test a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value was a=0.05. Detection of statistically significant differences were considered negligible when inhibition values were = 10 %. These findings were marked biological not significant.

NOEC, LOEC and EC - values and 95 % Confidence Intervals of the test item (0-72 h)

                   based on nominal test item concentration [mg/L]

                               

Rate-related Inhibition

NOEC

Saturated solution (1 mg/L)

LOEC

> Saturated solution (1 mg/L)

ErC10

> Saturated solution (1 mg/L)

ErC20

> Saturated solution (1 mg/L)

ErC50

> Saturated solution (1 mg/L)

Inhibition of Yield

NOEC

Saturated solution (1 mg/L)

LOEC

> Saturated solution (1 mg/L)

EyC10

> Saturated solution (1 mg/L)

EyC20

> Saturated solution (1 mg/L)

EyC50

> Saturated solution (1 mg/L)

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

Saturated solution              (1 mg/L)

1

5074

19924

172088

811286

 

2

5074

22729

170534

778264

 

3

5074

24729

147336

721561

 

4

5074

22023

140071

772979

 

5

5074

23323

172006

761032

 

6

5074

21941

177028

809346

 

Mean

5074

22445

163177

775745

 

Control

1

5074

22589

146618

743013

 

2

5074

22778

126409

607983

 

3

5074

21527

162481

677384

 

4

5074

19227

151563

600533

 

5

5074

25234

125864

562982

 

6

5074

23782

161637

626122

 

Mean

5074

22523

145762

636336

 

Evaluation after 72 h

              Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

                              

Nominal test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

Saturated solution              (1 mg/L)

1

1.69

-5.12

806212

-27.7

2

1.68

-4.26

773190

-22.5

3

1.65

-2.69

716487

-13.5

4

1.68

-4.11

767905

-21.7

5

1.67

-3.79

755958

-19.8

6

1.69

-5.07

804272

-27.4

Mean

(+)*

1.68

-4.17

(+)*

770671

-22.1

Control

1

1.66

737939

2

1.60

602909

3

1.63

672310

4

1.59

595459

5

1.57

557908

6

1.61

621048

Mean

1.61

631262

Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

1.49

1.87

1.62

1.66

0.192

11.5

14.4

2

1.50

1.71

1.57

1.60

0.108

  6.78

3

1.45

2.02

1.43

1.63

0.338

20.7

4

1.33

2.07

1.38

1.59

0.411

25.8

5

1.60

1.61

1.50

1.57

0.062

  3.95

6

1.55

1.92

1.35

1.61

0.286

17.8

Mean

1.61

 

± SD

0.03

 

CV [%]

2.04

 

Validity criteria fulfilled:
yes
Conclusions:
In this study the test item was found not to inhibit the freshwater green algae Desmodesmus subspicatus after 72 h at the saturated solution (prepared with 1 mg/L). All effect levels are given based on the nominal concentration of Pigment Red 112.
Executive summary:

The toxicity of Pigment Red 112 (batch number 2468) to the unicellular freshwater green algae Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from January 08 to 14, 2010 with the definitive exposure phase from January 11 to 14, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 103cells/mL. Based on a preliminary range finding test, a limit test with a saturated solution*  was carried out. The saturated solution was prepared out of the dispersion of 1 mg/L. Six replicates were tested for the limit concentration and control. Environmental conditions were determined to be within the acceptable limits.

All effect values are given based on the nominal concentration of the test item.

NOEC, LOEC and EC - values and 95 % Confidence Intervals of the tets item (0-72 h)

                   based on nominal test item concentration [mg/L]

                               

Rate-related Inhibition

NOEC

Saturated solution (1 mg/L)

LOEC

> Saturated solution (1 mg/L)

ErC10

> Saturated solution (1 mg/L)

ErC20

> Saturated solution (1 mg/L)

ErC50

> Saturated solution (1 mg/L)

Inhibition of Yield

NOEC

Saturated solution (1 mg/L)

LOEC

> Saturated solution (1 mg/L)

EyC10

> Saturated solution (1 mg/L)

EyC20

> Saturated solution (1 mg/L)

EyC50

> Saturated solution (1 mg/L)

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:
2010-01-08 to 2010-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study meets requirements of OECD Guideline 211 and GLP requirements with no deviations.
Justification for type of information:
See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 184 (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
No analytical determination of the test item was carried out due to the low water solubility of the test item.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution* was prepared. A dispersion of 1 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: The saturated solution was tested as limit concentration.
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
The test medium had a nominal hardness of 0.24 mmol CaMg/L
Test temperature:
Mean: 21.5 °C, Min: 21 °C, Max: 22 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
Saturated solution (1 mg/L) 8.04 8.35
Control 8.13 8.34
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
See above
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 636336 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test
- Range finding study:
Results of the Range Finding Test (0-72 h)

Saturated solution [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
Filtration of the saturated solution
20 - 3 - 19
1 - 3 - 19

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 other: mg/L (saturated solution)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.60 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.28 (CI 0.266 - 0.299) mg/L
Reported statistics and error estimates:
The NOEC and LOEC were determined by calculation of
statistical analyses statistical significance of growth rates and yield.
t-test was carried out for the determination of statistically significant differences compared to control replicates. When running a t-test a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value was a=0.05. Detection of statistically significant differences were considered negligible when inhibition values were = 10 %. These findings were marked biological not significant.

NOEC, LOEC and EC - values and 95 % Confidence Intervals of the test item (0-72 h)

                   based on nominal test item concentration [mg/L]

                               

Rate-related Inhibition

NOEC

Saturated solution (1 mg/L)

LOEC

> Saturated solution (1 mg/L)

ErC10

> Saturated solution (1 mg/L)

ErC20

> Saturated solution (1 mg/L)

ErC50

> Saturated solution (1 mg/L)

Inhibition of Yield

NOEC

Saturated solution (1 mg/L)

LOEC

> Saturated solution (1 mg/L)

EyC10

> Saturated solution (1 mg/L)

EyC20

> Saturated solution (1 mg/L)

EyC50

> Saturated solution (1 mg/L)

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

Saturated solution              (1 mg/L)

1

5074

19924

172088

811286

 

2

5074

22729

170534

778264

 

3

5074

24729

147336

721561

 

4

5074

22023

140071

772979

 

5

5074

23323

172006

761032

 

6

5074

21941

177028

809346

 

Mean

5074

22445

163177

775745

 

Control

1

5074

22589

146618

743013

 

2

5074

22778

126409

607983

 

3

5074

21527

162481

677384

 

4

5074

19227

151563

600533

 

5

5074

25234

125864

562982

 

6

5074

23782

161637

626122

 

Mean

5074

22523

145762

636336

 

Evaluation after 72 h

              Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

                              

Nominal test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

Saturated solution              (1 mg/L)

1

1.69

-5.12

806212

-27.7

2

1.68

-4.26

773190

-22.5

3

1.65

-2.69

716487

-13.5

4

1.68

-4.11

767905

-21.7

5

1.67

-3.79

755958

-19.8

6

1.69

-5.07

804272

-27.4

Mean

(+)*

1.68

-4.17

(+)*

770671

-22.1

Control

1

1.66

737939

2

1.60

602909

3

1.63

672310

4

1.59

595459

5

1.57

557908

6

1.61

621048

Mean

1.61

631262

Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

1.49

1.87

1.62

1.66

0.192

11.5

14.4

2

1.50

1.71

1.57

1.60

0.108

  6.78

3

1.45

2.02

1.43

1.63

0.338

20.7

4

1.33

2.07

1.38

1.59

0.411

25.8

5

1.60

1.61

1.50

1.57

0.062

  3.95

6

1.55

1.92

1.35

1.61

0.286

17.8

Mean

1.61

 

± SD

0.03

 

CV [%]

2.04

 

Validity criteria fulfilled:
yes
Conclusions:
In this study the test item was found not to inhibit the freshwater green algae Desmodesmus subspicatus after 72 h at the saturated solution (prepared with 1 mg/L). All effect levels are given based on the nominal concentration of Pigment Red 112.
Executive summary:

The toxicity of Pigment Red 112 (batch number 2468) to the unicellular freshwater green algae Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from January 08 to 14, 2010 with the definitive exposure phase from January 11 to 14, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 103cells/mL. Based on a preliminary range finding test, a limit test with a saturated solution*  was carried out. The saturated solution was prepared out of the dispersion of 1 mg/L. Six replicates were tested for the limit concentration and control. Environmental conditions were determined to be within the acceptable limits.

All effect values are given based on the nominal concentration of the test item.

NOEC, LOEC and EC - values and 95 % Confidence Intervals of the tets item (0-72 h)

                   based on nominal test item concentration [mg/L]

                               

Rate-related Inhibition

NOEC

Saturated solution (1 mg/L)

LOEC

> Saturated solution (1 mg/L)

ErC10

> Saturated solution (1 mg/L)

ErC20

> Saturated solution (1 mg/L)

ErC50

> Saturated solution (1 mg/L)

Inhibition of Yield

NOEC

Saturated solution (1 mg/L)

LOEC

> Saturated solution (1 mg/L)

EyC10

> Saturated solution (1 mg/L)

EyC20

> Saturated solution (1 mg/L)

EyC50

> Saturated solution (1 mg/L)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Remarks:
Pigment Red 022
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From December 13, 2000 to April 27, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guideline and compliant to GLP, well documented translation of original report (in Japanese)
Justification for type of information:
See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 184 (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
yes
Principles of method if other than guideline:
Deviating from the guideline, growth rate related inhibition of growth was determined for the interval of 24 to 72 hours only, instead of zero to 72 hours, as recommended by the guideline. From evaluating the raw data, this deviation leads to a slight overestimation of growth rate related inhibition and therefore does not put into question the reliability of the study.
GLP compliance:
yes (incl. QA statement)
Remarks:
The study was conducted in accordance with "Eco-toxicity test Standards" by Japanese Ministry of the Environment
Analytical monitoring:
yes
Details on sampling:
At the initiation of exposure, the samplings were made from the remaining test solutions for all groups after distributing to test vessels. At the termination of exposure, the sampling of about the same volume of test solutions was made from three test vessels of each test group for analysis. After sampling, algae were removed by centrifugation (2500 rpm, 10 minutes), the supernatants were analyzed by HPLC.
Vehicle:
yes
Details on test solutions:
After weighing of a necessary amount of test substance using an electronic balance, the same amount of hydrogenated castor oil (HCO-40) was added to the test substance as co-solvent and mixed well. Then, the solution was made up to the volume with culturing medium to prepare the stock solution (100 mg/L). After a necessary aliquot was weighed and sampled from the stock solution for the preparation of each test concentration, the shortfall of co-solvent was added to each solution to adjust the constant concentration of co-solvent (100 mg/L) in the test solution and made up with
medium. In the preparation of test solutions, the amount was considered to cover the necessary solutions for pH measurement and analytical sampling, since the sampling volume for analytical samples at the initiation of the exposure was expected to be more than 5% of test solution volume. The stock solution was used as the test solution of 100 mgIL in the test as it is.
The prepared test solutions showed strong red color due to test substance original in proportion to the test concentration but no visible precipitate was observed. The solution of solvent control was observed to be clear and colorless without visible undissolved test substance.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Scientific name: Selenastrum capricornutum
- Supplier: American Type Culture Collection
- Strain No.: ATCC22662 strain
- Date of receipt: August 1, 1996
- Maintenance after received: Aseptically sub-cultured using agar slant medium.
- Pre-cultivation period (definitive test): January 19, 2001 - January 22, 2001. During this period, exponential growth of algae was observed (the
environmental conditions were the same as in the test).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Test temperature:
23 deg C for all test vessels
pH:
7.5 to 7.9 for control and treatment groups during the 72 hours of incubation.
Nominal and measured concentrations:
The following nominal test item concentrations were tested (mg/L):
0.23, 1.0, 3.2, 10, 32, 100;
The measured concentrations of the test substance in the test solution were 94 - 100% and 69 - 96% of the nominal at the initiation of exposure and
termination, respectively. The concentration of test substance in the control and solvent control were less than quantification limit (0.06 mg/L) at the initiation and termination of exposure.
Please see table under section "Any other information on results including tables" for details on analytical results.
Details on test conditions:
- Type of exposure: Static, shaking culture (100 rpm)
- Volume of test solution: 100 mL (OECD medium) / vessel
- Replicates: 3 vessels per test group
- Initial cell concentration: 1 x 10^4 cells/mL
- Lighting: Continuous lighting of 4000 - 5000 lx (within +/- 20%, near the liquid level in flask)
- In both of the pre-cultivation and the study, OECD medium recommended by OECD Guideline was used with sterilization and filtration before use.
- Test vessel: 500 mL glass Erlenmeyer flask (air-permeable silicon stopper)
- Algal culturing apparatus: AGP-50RL (Daikin Plant Co., Ltd.; continuous shaking culture. Able to maintain constant lighting intensity during the culturing)
- Hemocytometer: Burker-Turk
- Particle counter: Coulter Multisizer II (Beckman Coulter, Inc.)
- Electrolyte solution for particle counter: ISOTON II (Beckman Coulter, Inc.)
- Illuminometer: SPI-6A (Topcon Corporation)

- Selection of test concentration:
A range-finding test was conducted at three concentration levels of 1.0 and 10 mg/L, and 100 mg/L (which was the maximum attainable homogeneous dispersion concentration using the co-solvent at 100 mg/L). At the termination (72-hr) of exposure, growth inhibitions (by comparison of areas under the growth curve) were 5.1, ca. 60 and ca. 81% at 1.0, 10 and 100 mg/L treatment level, respectively.
Based on these results, the nominal test concentration in the definitive test was fixed to be performed at six concentration levels of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (common ratio: 3.2). As reference, a control group with dilution water only as well as a solvent control group (with solvent concentration of 100 mg/L identical to the treatment groups) were allocated in the study.

- Test operation procedure:
The number of cells of pre-cultivated algae was counted with a particle counter and an aliquot of pre-cultivated solution was added to each test solution to make 1 x 10^4 cels/mL of cell concentration. After mixing well, 10 mL of the solution, each was transferred into three test vessels per group.
The test was started by placing each of the test vessels in a culturing apparatus maintained at 23 +/- 2 deg C. After 24, 48 and 72 hours of exposure, cell concentrations in all three vessels of all groups were measured. A part of test solution was sampled from each test vessel and the cell concentrations were measured with a hemocytometer.
The pH at the initiation of exposure was measured for the remaining test solutions after distributing into test vessels. At the termination of exposure, pH was measured for one vessel (No. 1) out of three vessels in each test group, since difference in growth rate of each test group were within double. The temperature, lighting intensity and rotation speed in algal culturing apparatus were measured once a day during the exposure period.

Growth curve
The growth curve was drawn by plotting the mean cell concentration of each test group against the time.
Calculation of growth inhibition concentration

Growth inhibition rates were calculated in accordance with the methods described below (area method and growth rate method).
1) Growth inhibition rates (IA) by comparison of areas under the growth curve (Area-based method)
Area under the growth curve was calculated using the following equitation:
A = [(N_1 - N_0)/2]*t_1 + [(N_1 + N_2 - 2*N_0)/2]*(t_2 - t_1) + ... + [(N_n-1 + N_n - 2*N_0)/2]*(t_n - t_n-1)
whith
N_0: Nominal cell concentration at the initiation of exposure (cells/mL)
N_i: Measured cell concentration at t_i (cells/mL)
N_n: Measured cell concentration at to (cells/mL)
t_1: Time of first measurement of cell concentration after initiation of exposure
t_n: Time of nth measurement of cell concentration after initiation of exposure
Calculation of inhibition based on area under the growth curve [%]:
I_A = [(A_c - A_t)/A_c]*100
where A_c: Area under the growth curve for solvent control; A_t: Area under the growth curve for the treatment group.
Calculation of growth rate based inhbition was performed according to formulas given in OECD 201.






Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
corresponding to 97 mg/L gemetric mean measured concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
corresponding to 2.85 mg/L gemetric mean measured concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Test item concentration analytically verified
Basis for effect:
biomass
Remarks:
determined from area under the growth curve
Remarks on result:
other: 95%CI: 14-20
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
corresponding to 0.92 mg/L gemetric mean measured concentration
Basis for effect:
biomass
Remarks:
determined from area under the growth curve
Details on results:
Based on results of the preliminary verification for the difference of means of growth in two-sample (Student's t-test) between control and solvent control test group, the solvent control vales were used for statistical analysis, since the growth in solvent control group was increased compared to control group, significantly.
During 72 hours of incubation, the cell concentrations in the control and the solvent control group increased to 305 and 331 fold on average, respectively.
It was observed that the growth in the 0.32 mg/L test group was almost the same as that in the solvent control group. However, in the other test groups, decreased growth was observed depending upon the test concentration. In the comparison of 24-48 hours growth rate, 40% of inhibition was observed at the highest attainable concentration (100 mg/L) compared to solvent control. In case of growth rate during 24 - 72 hours, statistically significat inhibition between 14 and 34% was observed in the test groups exposed to 10 mg/L and above.
The prepared test solutions showed strong red color in proportion to test item concentration, but no visible precipitate was observed. The solution of solvent control was observed to be clear and colorless.

Results with reference substance (positive control):
The 50% algal growth inhibition concentration (EbC50) at 72 hr with the reference substance (potassium dichromate, guaranteed grade, Lot No. ACQ2610, Wako Pure Chemical Industries) was 0.50 mg/L, which was comparable to the historical EbC50 of 0.42 - 0.50 mg/L (n=6) obtained at the testing facility since August, 1996.
Reported statistics and error estimates:
The EC50 was determined by the Logit method (biomass based value only).
The no-observed-effect concentration (NOEC) was determined to be the highest test concentration which had no significant difference (a = 0.05) compared with the solvent control group using statistical analysis (homogeneity of variance (Bartlett), one-way analysis of variance and multiple comparison analysis (Dunnett).

Growth rates and rate related inhibition of growth: Selenastrum capricornutum during 72 hour exposure.

Nominal

Vessel

Growth rate per day

Concentr.

No.

 

 

 

Mean

SD

CV

 

% Inhibition

mg/L

 

0 to 24 h

24 to 48 h

48 to 72 h

0 to 72 h

24 to 72 h

24 to 72 h

 

1

2.0229

1.8014

1.8260

1.8834

0.1214

6.4448

1.8137

 

2

2.1725

1.7111

1.8954

1.9263

0.2322

12.0549

1.8033

Control

3

2.2690

1.6430

1.8124

1.9081

0.3238

16.9703

1.7277

 

Average

2.1599

1.7141

1.8453

1.9064

Mean CV

11.8233

1.7797

 

SD

SD

0.0215

 

 

CV

1.1295

 

1

1.8976

2.0381

1.8671

1.9343

0.0912

4.7150

1.9526

 

2

2.0516

1.9300

1.8446

1.9421

0.1040

5.3545

1.8873

Solvent Control

3

1.9459

2.0541

1.7741

1.9247

0.1412

7.3359

1.9141

 

Average

1.9671

2.0057

1.8285

1.9338

Mean CV

5.8018

1.9180

 

SD

SD

0.0087

 

 

CV

0.4492

 

1

2.0229

2.0025

1.7193

1.9149

1.8609

 

2

2.2332

1.6748

1.8806

1.9295

1.7777

0.32

3

1.9459

2.0812

1.7851

1.9374

1.9332

 

Average

2.0748

1.9135

1.7939

1.9274

1.8572

3.2

 

SD

 

1

2.2214

1.7163

1.9068

1.9481

1.8115

 

2

2.0069

1.9858

1.5776

1.8568

1.7817

1

3

1.8810

2.1263

1.5689

1.8587

1.8476

 

Average

2.0464

1.9333

1.6924

1.8907

1.8136

5.4

 

SD

 

1

2.0373

2.1058

1.4679

1.8704

1.7869

 

2

1.9459

1.8585

1.5977

1.8007

1.7281

3.2

3

1.9769

1.8733

1.6837

1.8446

1.7785

 

Average

1.9874

1.9567

1.5752

1.8398

1.7645

8.0

 

SD

 

1

2.1199

1.5256

1.3051

1.6502

1.4153

 

2

1.7156

1.9454

1.4487

1.7032

1.6971

10

3

1.8810

1.6857

1.4543

1.6737

1.5700

 

Average

1.9194

1.7059

1.4040

1.6764

1.5608

18.6**

 

SD

 

1

1.4907

2.0119

1.2718

1.5915

1.6419

 

2

1.4907

1.8903

1.4117

1.5976

1.6510

32

3

1.4398

1.9171

1.3661

1.5743

1.6416

 

Average

1.4740

1.9415

1.3482

1.5879

1.6448

14.2**

 

SD

 

1

1.4398

1.1174

1.4206

1.3259

1.2690

 

2

1.4398

1.1328

1.3253

1.2993

1.2290

100

3

1.2698

1.3905

1.2377

1.2993

1.3141

 

Average

1.3863

1.2114

1.3275

1.3084

1.2707

33.7**

 

SD

1.8137

% Inhibition is related to solvent control, as growth was increased compared to control.

**: Significant difference from solvent control (Dunnett´s Test, alpha = 0.01)

Solvent control: HCO-40 (100 mg/L)

Nominal and measured test item concentrations:

Nominal

 

Measured Concentration (mg/L)

 

Concentration
(mg/L)

0 Hour

Percent of
Nominal

72 Hours

Percent of Nominal

Control

<0.06

--

<0.06

--

Solvent Control

<0.06

--

<0.06

--

0.32

0.32

100

0.22

69

1.0

0.99

99

0.86

86

3.2

3.0

94

2.7

84

10

9.8

98

7.2

72

32

30

94

29

91

100

98

98

96

96

Validity criteria fulfilled:
yes
Conclusions:
In a reliable study (reliability category 1) on green alga Selenastrum capricornutum growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:
EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);
NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).
These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.
Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.
Thus, observed effects are to be regarded as not relevant because they are
1) most probably not due to intrinsic toxicity of the test item and
2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).
Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.
The test item is to be regarded as non-toxic to freshwater green algae.
Executive summary:

In a reliable study (reliability category 1) on green alga Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata) growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:

EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);

NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).

These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.

Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.

Thus, observed effects are to be regarded as not relevant because they are

1) most probably not due to intrinsic toxicity of the test item and

2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).

Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.

The test item is to be regarded as non-toxic to freshwater green algae.

All validity criteria of the guideline are fulfilled: The average biomass increase in the control culture ant the solvent control was 305 and 331 -fold, respectively. The mean coefficient of variation for section-by-section specific growth rates in control cultures were below 35% (11.8 and 5.8 % for control and solvent control, respectively) and the coefficient of variation of average specific growth rate of replicate control cultures was below 7 % (1.13 and 0.45 % for control and solvent control, respectively).

The robust study summary is based on the translation of the original study report performed by the same company involved with performance of the test (Sumika TechnoService Corporation, Japan).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Remarks:
Pigment Red 022
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 13, 2000 to April 27, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guideline and compliant to GLP, well documented translation of original report (in Japanese)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
See 'principles of method' field below
Principles of method if other than guideline:
Deviating from the guideline, growth rate related inhibition of growth was determined for the interval of 24 to 72 hours only, instead of zero to 72 hours, as recommended by the guideline. From evaluating the raw data, this deviation leads to a slight overestimation of growth rate related inhibition and therefore does not put into question the reliability of the study.
GLP compliance:
yes (incl. QA statement)
Remarks:
The study was conducted in accordance with "Eco-toxicity test Standards" by Japanese Ministry of the Environment
Analytical monitoring:
yes
Details on sampling:
At the initiation of exposure, the samplings were made from the remaining test solutions for all groups after distributing to test vessels. At the termination of exposure, the sampling of about the same volume of test solutions was made from three test vessels of each test group for analysis. After sampling, algae were removed by centrifugation (2500 rpm, 10 minutes), the supernatants were analyzed by HPLC.
Vehicle:
yes
Details on test solutions:
After weighing of a necessary amount of test substance using an electronic balance, the same amount of hydrogenated castor oil (HCO-40) was added to the test substance as co-solvent and mixed well. Then, the solution was made up to the volume with culturing medium to prepare the stock solution (100 mg/L). After a necessary aliquot was weighed and sampled from the stock solution for the preparation of each test concentration, the shortfall of co-solvent was added to each solution to adjust the constant concentration of co-solvent (100 mg/L) in the test solution and made up with
medium. In the preparation of test solutions, the amount was considered to cover the necessary solutions for pH measurement and analytical sampling, since the sampling volume for analytical samples at the initiation of the exposure was expected to be more than 5% of test solution volume. The stock solution was used as the test solution of 100 mgIL in the test as it is.
The prepared test solutions showed strong red color due to test substance original in proportion to the test concentration but no visible precipitate was observed. The solution of solvent control was observed to be clear and colorless without visible undissolved test substance.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Scientific name: Selenastrum capricornutum
- Supplier: American Type Culture Collection
- Strain No.: ATCC22662 strain
- Date of receipt: August 1, 1996
- Maintenance after received: Aseptically sub-cultured using agar slant medium.
- Pre-cultivation period (definitive test): January 19, 2001 - January 22, 2001. During this period, exponential growth of algae was observed (the
environmental conditions were the same as in the test).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Test temperature:
23 deg C for all test vessels
pH:
7.5 to 7.9 for control and treatment groups during the 72 hours of incubation.
Nominal and measured concentrations:
The following nominal test item concentrations were tested (mg/L):
0.23, 1.0, 3.2, 10, 32, 100;
The measured concentrations of the test substance in the test solution were 94 - 100% and 69 - 96% of the nominal at the initiation of exposure and
termination, respectively. The concentration of test substance in the control and solvent control were less than quantification limit (0.06 mg/L) at the initiation and termination of exposure.
Please see table under section "Any other information on results including tables" for details on analytical results.
Details on test conditions:
- Type of exposure: Static, shaking culture (100 rpm)
- Volume of test solution: 100 mL (OECD medium) / vessel
- Replicates: 3 vessels per test group
- Initial cell concentration: 1 x 10^4 cells/mL
- Lighting: Continuous lighting of 4000 - 5000 lx (within +/- 20%, near the liquid level in flask)
- In both of the pre-cultivation and the study, OECD medium recommended by OECD Guideline was used with sterilization and filtration before use.
- Test vessel: 500 mL glass Erlenmeyer flask (air-permeable silicon stopper)
- Algal culturing apparatus: AGP-50RL (Daikin Plant Co., Ltd.; continuous shaking culture. Able to maintain constant lighting intensity during the culturing)
- Hemocytometer: Burker-Turk
- Particle counter: Coulter Multisizer II (Beckman Coulter, Inc.)
- Electrolyte solution for particle counter: ISOTON II (Beckman Coulter, Inc.)
- Illuminometer: SPI-6A (Topcon Corporation)

- Selection of test concentration:
A range-finding test was conducted at three concentration levels of 1.0 and 10 mg/L, and 100 mg/L (which was the maximum attainable homogeneous dispersion concentration using the co-solvent at 100 mg/L). At the termination (72-hr) of exposure, growth inhibitions (by comparison of areas under the growth curve) were 5.1, ca. 60 and ca. 81% at 1.0, 10 and 100 mg/L treatment level, respectively.
Based on these results, the nominal test concentration in the definitive test was fixed to be performed at six concentration levels of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (common ratio: 3.2). As reference, a control group with dilution water only as well as a solvent control group (with solvent concentration of 100 mg/L identical to the treatment groups) were allocated in the study.

- Test operation procedure:
The number of cells of pre-cultivated algae was counted with a particle counter and an aliquot of pre-cultivated solution was added to each test solution to make 1 x 10^4 cels/mL of cell concentration. After mixing well, 10 mL of the solution, each was transferred into three test vessels per group.
The test was started by placing each of the test vessels in a culturing apparatus maintained at 23 +/- 2 deg C. After 24, 48 and 72 hours of exposure, cell concentrations in all three vessels of all groups were measured. A part of test solution was sampled from each test vessel and the cell concentrations were measured with a hemocytometer.
The pH at the initiation of exposure was measured for the remaining test solutions after distributing into test vessels. At the termination of exposure, pH was measured for one vessel (No. 1) out of three vessels in each test group, since difference in growth rate of each test group were within double. The temperature, lighting intensity and rotation speed in algal culturing apparatus were measured once a day during the exposure period.

Growth curve
The growth curve was drawn by plotting the mean cell concentration of each test group against the time.
Calculation of growth inhibition concentration

Growth inhibition rates were calculated in accordance with the methods described below (area method and growth rate method).
1) Growth inhibition rates (IA) by comparison of areas under the growth curve (Area-based method)
Area under the growth curve was calculated using the following equitation:
A = [(N_1 - N_0)/2]*t_1 + [(N_1 + N_2 - 2*N_0)/2]*(t_2 - t_1) + ... + [(N_n-1 + N_n - 2*N_0)/2]*(t_n - t_n-1)
whith
N_0: Nominal cell concentration at the initiation of exposure (cells/mL)
N_i: Measured cell concentration at t_i (cells/mL)
N_n: Measured cell concentration at to (cells/mL)
t_1: Time of first measurement of cell concentration after initiation of exposure
t_n: Time of nth measurement of cell concentration after initiation of exposure
Calculation of inhibition based on area under the growth curve [%]:
I_A = [(A_c - A_t)/A_c]*100
where A_c: Area under the growth curve for solvent control; A_t: Area under the growth curve for the treatment group.
Calculation of growth rate based inhbition was performed according to formulas given in OECD 201.






Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
corresponding to 97 mg/L gemetric mean measured concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
corresponding to 2.85 mg/L gemetric mean measured concentration
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Test item concentration analytically verified
Basis for effect:
biomass
Remarks:
determined from area under the growth curve
Remarks on result:
other: 95%CI: 14-20
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
corresponding to 0.92 mg/L gemetric mean measured concentration
Basis for effect:
biomass
Remarks:
determined from area under the growth curve
Details on results:
Based on results of the preliminary verification for the difference of means of growth in two-sample (Student's t-test) between control and solvent control test group, the solvent control vales were used for statistical analysis, since the growth in solvent control group was increased compared to control group, significantly.
During 72 hours of incubation, the cell concentrations in the control and the solvent control group increased to 305 and 331 fold on average, respectively.
It was observed that the growth in the 0.32 mg/L test group was almost the same as that in the solvent control group. However, in the other test groups, decreased growth was observed depending upon the test concentration. In the comparison of 24-48 hours growth rate, 40% of inhibition was observed at the highest attainable concentration (100 mg/L) compared to solvent control. In case of growth rate during 24 - 72 hours, statistically significat inhibition between 14 and 34% was observed in the test groups exposed to 10 mg/L and above.
The prepared test solutions showed strong red color in proportion to test item concentration, but no visible precipitate was observed. The solution of solvent control was observed to be clear and colorless.

Results with reference substance (positive control):
The 50% algal growth inhibition concentration (EbC50) at 72 hr with the reference substance (potassium dichromate, guaranteed grade, Lot No. ACQ2610, Wako Pure Chemical Industries) was 0.50 mg/L, which was comparable to the historical EbC50 of 0.42 - 0.50 mg/L (n=6) obtained at the testing facility since August, 1996.
Reported statistics and error estimates:
The EC50 was determined by the Logit method (biomass based value only).
The no-observed-effect concentration (NOEC) was determined to be the highest test concentration which had no significant difference (a = 0.05) compared with the solvent control group using statistical analysis (homogeneity of variance (Bartlett), one-way analysis of variance and multiple comparison analysis (Dunnett).

Growth rates and rate related inhibition of growth: Selenastrum capricornutum during 72 hour exposure.

Nominal

Vessel

Growth rate per day

Concentr.

No.

 

 

 

Mean

SD

CV

 

% Inhibition

mg/L

 

0 to 24 h

24 to 48 h

48 to 72 h

0 to 72 h

24 to 72 h

24 to 72 h

 

1

2.0229

1.8014

1.8260

1.8834

0.1214

6.4448

1.8137

 

2

2.1725

1.7111

1.8954

1.9263

0.2322

12.0549

1.8033

Control

3

2.2690

1.6430

1.8124

1.9081

0.3238

16.9703

1.7277

 

Average

2.1599

1.7141

1.8453

1.9064

Mean CV

11.8233

1.7797

 

SD

SD

0.0215

 

 

CV

1.1295

 

1

1.8976

2.0381

1.8671

1.9343

0.0912

4.7150

1.9526

 

2

2.0516

1.9300

1.8446

1.9421

0.1040

5.3545

1.8873

Solvent Control

3

1.9459

2.0541

1.7741

1.9247

0.1412

7.3359

1.9141

 

Average

1.9671

2.0057

1.8285

1.9338

Mean CV

5.8018

1.9180

 

SD

SD

0.0087

 

 

CV

0.4492

 

1

2.0229

2.0025

1.7193

1.9149

1.8609

 

2

2.2332

1.6748

1.8806

1.9295

1.7777

0.32

3

1.9459

2.0812

1.7851

1.9374

1.9332

 

Average

2.0748

1.9135

1.7939

1.9274

1.8572

3.2

 

SD

 

1

2.2214

1.7163

1.9068

1.9481

1.8115

 

2

2.0069

1.9858

1.5776

1.8568

1.7817

1

3

1.8810

2.1263

1.5689

1.8587

1.8476

 

Average

2.0464

1.9333

1.6924

1.8907

1.8136

5.4

 

SD

 

1

2.0373

2.1058

1.4679

1.8704

1.7869

 

2

1.9459

1.8585

1.5977

1.8007

1.7281

3.2

3

1.9769

1.8733

1.6837

1.8446

1.7785

 

Average

1.9874

1.9567

1.5752

1.8398

1.7645

8.0

 

SD

 

1

2.1199

1.5256

1.3051

1.6502

1.4153

 

2

1.7156

1.9454

1.4487

1.7032

1.6971

10

3

1.8810

1.6857

1.4543

1.6737

1.5700

 

Average

1.9194

1.7059

1.4040

1.6764

1.5608

18.6**

 

SD

 

1

1.4907

2.0119

1.2718

1.5915

1.6419

 

2

1.4907

1.8903

1.4117

1.5976

1.6510

32

3

1.4398

1.9171

1.3661

1.5743

1.6416

 

Average

1.4740

1.9415

1.3482

1.5879

1.6448

14.2**

 

SD

 

1

1.4398

1.1174

1.4206

1.3259

1.2690

 

2

1.4398

1.1328

1.3253

1.2993

1.2290

100

3

1.2698

1.3905

1.2377

1.2993

1.3141

 

Average

1.3863

1.2114

1.3275

1.3084

1.2707

33.7**

 

SD

1.8137

% Inhibition is related to solvent control, as growth was increased compared to control.

**: Significant difference from solvent control (Dunnett´s Test, alpha = 0.01)

Solvent control: HCO-40 (100 mg/L)

Nominal and measured test item concentrations:

Nominal

 

Measured Concentration (mg/L)

 

Concentration
(mg/L)

0 Hour

Percent of
Nominal

72 Hours

Percent of Nominal

Control

<0.06

--

<0.06

--

Solvent Control

<0.06

--

<0.06

--

0.32

0.32

100

0.22

69

1.0

0.99

99

0.86

86

3.2

3.0

94

2.7

84

10

9.8

98

7.2

72

32

30

94

29

91

100

98

98

96

96

Validity criteria fulfilled:
yes
Conclusions:
In a reliable study (reliability category 1) on green alga Selenastrum capricornutum growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:
EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);
NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).
These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.
Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.
Thus, observed effects are to be regarded as not relevant because they are
1) most probably not due to intrinsic toxicity of the test item and
2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).
Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.
The test item is to be regarded as non-toxic to freshwater green algae.
Executive summary:

In a reliable study (reliability category 1) on green alga Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata) growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:

EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);

NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).

These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.

Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.

Thus, observed effects are to be regarded as not relevant because they are

1) most probably not due to intrinsic toxicity of the test item and

2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).

Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.

The test item is to be regarded as non-toxic to freshwater green algae.

All validity criteria of the guideline are fulfilled: The average biomass increase in the control culture ant the solvent control was 305 and 331 -fold, respectively. The mean coefficient of variation for section-by-section specific growth rates in control cultures were below 35% (11.8 and 5.8 % for control and solvent control, respectively) and the coefficient of variation of average specific growth rate of replicate control cultures was below 7 % (1.13 and 0.45 % for control and solvent control, respectively).

The robust study summary is based on the translation of the original study report performed by the same company involved with performance of the test (Sumika TechnoService Corporation, Japan).

Description of key information

Algae


PR022 


In a reliable study (reliability category 1) on green alga Selenastrum capricornutum growth inhibition (static, 72 hours, analytical verification of test item concentration) performed according to GLP the following growth rate related (no)effect concentrations were determined for the test item:
EC50 (72 hours) > 100 mg/L (97 mg/L geometric mean measured concentration);
NOEC (72 hours) = 3.2 mg/L (2.85 mg/L geometric mean measured concentration).
These values were determined in relation to the solvent control (100 mg/L HCO-40, equal concentration to treatment groups), as growth was significantly enhanced compared to the control without solvent.
Tested concentrations were far above water solubility of the test item (11.8 µg/L) and were achieved by use of hydrogenated castor oil (HCO-40) at equal weight as co-solvent. The strong red coloring of test solutions might have resulted in light absorption causing reduced growth of algae due to reduced effective illumination strength.
Thus, observed effects are to be regarded as not relevant because they are
1) most probably not due to intrinsic toxicity of the test item and
2) far above the true water solubility of the test item (NOEC is a factor of 270 above water solubility).
Thus, the NOEC is not appropriate to derive a PNEC, as light absorption due to the coloring of the test item is not relevant in the range of actual water solubility.
The test item is to be regarded as non-toxic to freshwater green algae.


PR122


In this study the test item was found not to inhibit the freshwater green algae Desmodesmus subspicatus after 72 h at the saturated solution (prepared with 1 mg/L). All effect levels are given based on the nominal concentration of Pigment Red 112.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
1 mg/L

Additional information