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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-09-03 to 2012-10-03
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:
since the test item is poorly water soluble, the standard method for the preparation of aqueous media was modified. The test was performed with a saturated solution of the test item
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
since the test item is poorly water soluble, the standard method for the preparation of aqueous media was modified. The test was performed with a saturated solution of the test item
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Test Concentrations:0, 0.625, 1.25, 2.5, 5.0 and 10.0% v/v saturated soluation (nominal)
- Sampling method: at 0 and 72 hours for quantitative analysis and pooled.
- Sample storage conditions before analysis: approximately -20°C prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method of preparation: Saturation of the experimental solution:
550 mg of test item was dispersed in 11 liters of culture medium
Stirring with a propeller at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped
Filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to remove undissolved test item
Obtention of a 100% v/v saturated solution.
Series of dilution to give stock solutions of the appropriate concentration of saturated solution.
500 mL of each stock solutions was separately inoculated with 2.9 mL of algal suspension to give the final test concentrations of saturated solution after adequate mixing to ensure homogeneity.
Concentrations of the 2 ions of the test item were verified by the chemical analysis at 0 and 72 hrs.

- Application: conic flasks containing 100 mL of the saturated solution test inoculated with tha algal suspension as preparated above.

- Controls: control group maintained under identical conditions but not exposed to the test item
- Vehicle: none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name:Peudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): culture collection of algae and protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll Scotland.
- Age of inoculum (at test initiation): no data
- Method of cultivation: master cultures maintained in the laboratory by periodic replenishment of culture medium, under constant aeration and constant illumination at 21 +/- 1°C. Prior to start, master culture was added to 100 mL volumes of culture media to obtain a initial cell density of ~10^3 cells/mL. Flask are plugged and kept under constant agitation by orbital shaket (100-150 rpm) and illumination at 24 +/-1°C to give an algal density of 10^4 - 10^5 cells/ml.

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): culture media same as range finding study and definitive test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 +/- 1 °C throughout the test
pH:
Control: pH-0hr = 7.5, pH-72 hr = 7.2
0.625 % v/v: pH-0hr = 7.4, pH-72 hr = 7.3
1.25 % v/v: pH-0hr = 7.4, pH-72 hr = 7.3
2.5 % v/v: pH-0hr = 7.3, pH-72 hr = 7.6
5 % v/v: pH-0hr = 7.3, pH-72 hr = 7.3
10 % v/v: pH-0hr = 7.3, pH-72 hr = 7.3
Nominal and measured concentrations:
Nominal test concentration, in % v/v saturated solution :
a - control
b- 0.625 % v/v
c- 1.25 % v/v
d- 2.5 % v/v
e- 5.0 % v/v
f- 10 % v/v

Corresponding Measured test concentrations (Geometric mean between 0-hour and 72-hour measured test concentration), based on dosage of BV3
a - control
b- 0.0014 mg/L
c- 0.0034 mg/L
d- 0.0052 mg/L
e- 0.0078 mg/L
f- 0.012 mg/L

Corresponding Measured test concentrations (Geometric mean between 0-hour and 72-hour measured test concentration), based on dosage of AY36
a - control
b- 0.00425 mg/L
c- 0.0076 mg/L
d- 0.02613 mg/L
e- 0.0563 mg/L
f- 0.1215 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type: closed by polyurethane foam bungs
- Material, size, headspace, fill volume: glass conical flask of 250 mL filled with 100 mL of the test solution inoculated with algal suspension
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 5 x 10^3 cells/ml
- Control end cells density: yes
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates


GROWTH MEDIUM
- Standard medium used: yes (U.S. EPA medium)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, for the culture medium
- Photoperiod: continuous illumination
- Light intensity and quality: ~7000 lux, by warm white lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter multisizer)
- Chlorophyll measurement: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: spacing factor of 2
- Range finding study: yes
- Test concentrations: 0.625, 1.25, 2.5, 5.0 and 10.0 % v/v.
- Results used to determine the conditions for the definitive study: used of the results of the range finding study
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.005 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% CI 0.0048-0.0057 mg/L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.001 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.001 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.625, 1.25 and 2.5% solution, however no intact cells were was observed to be present in the test cultures at 5.0 and 10% v/v saturated solution.
- Unusual cell shape: no unusual cell shape mentionned
- Flocculation: no flocculation phenomena mentionned
- Adherence to test vessels: no adherence to test vessels mentionned
- Aggregation of algal cells: no aggregation of algal cells mentionned
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: non mentionned
- Observations on test item solution:
At the start: all control, 0.625, 1.25, 2.5 and 5 %v/v saturated solution test cultures were observed to be clear colorless solutions but the 10 % v/v was colored in pale purple.
After the 72-Hour test period all control and 0.625% v/v saturated solution test cultures were observed to be green dispersions. The 1.25% v/v saturated solution test cultures were pale green dispersions, the 2.5% v/v saturated solution test cultures were extremely pale green dispersions, the 5.0% v/v saturated solutions were clear colorless solutions and the 10% v/v saturated solutions test cultures were extremely pale purple solutions.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50 = 1.1 mg/l (CI 95% 1.0-1.3 mg/l) (based on growth rate)
- EyC50 = 0.70 mg/l (based on yield)
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 168 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

- Mean cell density of control at 0 hours : 5.25 x 103 cells per mL

- Mean cell density of control at 72 hours : 8.83 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 10% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Table on Daily Specific Groth Rates for the Control Cultures in the Definitve test

 

Daily Specific Growth Rate (cells/mL/hour)

Day 0 – 1

Day 1-2

Day 2-3

 

 

 

Control

R1

0.070

0.084

0.063

R2

0.071

0.075

0.066

R3

0.073

0.083

0.067

R4

0.067

0.076

0.069

R5

0.075

0.078

0.062

R6

0.071

0.078

0.066

Mean

0.071

0.079

0.066

Table on Inhibition of Growth Rate and Yields in the Definitive test

Nominal Concentration of Solvent Black 46 in Test Sample
[% v/v]
Saturated solution

Geometric Mean Measured Test Concentration, based on AY36
[mg/L]

Geometric Mean Measured Test Concentration, based on BV3
[mg/L]

Replicates

Growth Rate

Yield

Ratio of Geometric Mean Measured Test Concentration, between AY36 and BV3
[mg/L]

(cells/mL/hour)

(cells/mL)

0-72 h

% Inhibition

0-72 h

% Inhibition*

Control

Control

Control

R1

0,072

-

8,92E+05

-

Control

R2

0,071

8,08E+05

R3

0,074

1,04E+06

R4

0,071

8,01E+05

R5

0,072

8,67E+05

R6

0,072

8,61E+05

Mean

0,072

 

8,78E+05

 

SD

0,001

 

8,57E+04

 

0,625

0,00425

0,0014

R1

0,072

0

9,06E+05

 

3,04

R2

0,073

[1]

9,32E+05

 

R3

0,073

[1]

9,25E+05

 

Mean

0,073

[1]

9,21E+05

[5]

SD

0,001

 

1,35E+04

 

1,25

0,0076

0,0034

R1

0,060

17

3,74E+05

 

2,24

R2

0,060

17

3,85E+05

 

R3

0,065

10

5,53E+05

 

Mean

0,062

14

4,37E+05

50

SD

0,003

 

1,00E+05

 

2,5

0,02613

0,0052

R1

0,040

44

8,32E+04

 

5,03

R2

0,031

57

4,33E+04

 

R3

0,047

35

1,38E+05

 

Mean

0,039

45

8.82E+04

91

SD

0,008

 

4.75E+04

 

5

0,0563

0,0078

R1

0,009

88

4,75E+03

 

7,22

R2

-0,003

104

-3,31E+02

 

R3

0,012

83

6,63E+03

 

Mean

0,006

92

3,68E+03

100

SD

0,008

 

3,60E+03

 

10

0,1215

0,012

R1

-0,015

121

-3,11E+03

 

10,13

R2

-0,020

128

-2,54E+03

 

R3

-0,007

110

-1,03E+03

 

Mean

-0,014

120

-2,23E+03

100

SD

0,007

 

1,07E+03

 

[Increase in growth as compared to controls]

Table on Analytical results for Test Samples based on AY36 and BV3 analysis

Time Point

 

[hours]

Nominal Concentration of Solvent Black 46 in Test Sample

[% v/v]

Saturated solution

Analysis based on AY 36

Analysis based on BV3

Measured Concentration of Solvent Black 46 in Test Sample,

[mg/L]

Calculated concentration of AY 36 in Test Sample

[mg/L]

Measured Concentration of Solvent Black 46 in Test Sample,

[mg/L]

Calculated concentration of BV3 in Test Sample

[mg/L]

0

Control

< LOQ (0.0085)

< LOQ (0.0041)

< LOQ (0.0028)

< LOQ (0.0015)

 

0.625

< LOQ (0.0085)

< LOQ (0.0041)

< LOQ (0.0028)

< LOQ (0.0015)

 

1.25

0.0138

0.00661

0.00817

0.00426

 

2.5

0.0281

0.0135

0.0194

0.0101

 

5

0.0597

0.0286

0.0432

0.0225

 

10

0.123

0.0589

0.103

0.0537

 

100

1.43

0.685

1.18

0.613

72

Control

< LOQ (0.0085)

< LOQ (0.0041)

< LOQ (0.0028)

< LOQ (0.0015)

 

0.625

< LOQ (0.0085)

< LOQ (0.0041)

< LOQ (0.0028)

< LOQ (0.0015)

 

1.25

< LOQ (0.0085)

< LOQ (0.0041)

< LOQ (0.0028)

< LOQ (0.0015)

 

2.5

0.0243

0.0116

< LOQ (0.0028)

< LOQ (0.0015)

 

5

0.0531

0.0254

< LOQ (0.0028)

< LOQ (0.0015)

 

10

0.120

0.0575

< LOQ (0.0028)

< LOQ (0.0015)

Validity criteria fulfilled:
yes
Conclusions:
the Solvent Black 46 has a EC50-72 hr based on growth rate of 0.0053 mg/L and based on yield of 0.0034 mg/l (geometric mean measured test concentration)
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Information provided by the Sponsor indicated the water solubility of the test item to be negligible.

Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

The test item is a precipitated reaction product of Acid Yellow 36 (AY36) (47.9% w/w) and Basic Violet 3 (BV3) (52.1% w/w). Assuming that the test item was a 1:1 stoichemetric mixture of the two components, based on the percentage content of each component, equivalent test item concentrations could be determined.

A pre-study media preparation trial indicated that the use of a saturated solution method of preparation followed by the removal of any undissolved test item by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) was appropriate for this test item.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 0.625, 1.25, 2.5, 5.0 and 10% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured AY36 concentrations to range from less than the limit of quantitation (LOQ) of the analytical method employed, which was determined to be 0.0041 mg/L, to 0.059 mg/L (equivalent to test item concentrations of less than 0.0085 to 0.12 mg/L). A decline in measured AY36 concentrations was observed at 72 hours in the range of less than the LOQ to 0.058 mg/L (equivalent to test item concentrations of less than 0.0085 to 0.12 mg/L).

Analysis of the test preparations at 0 hours showed measured BV3 concentrations to range from less than the limit of quantitation (LOQ) of the analytical method employed, which was determined to be 0.0015 mg/L, to 0.054 mg/L (equivalent to test item concentrations of less than 0.0028 to 0.10 mg/L). A decline in measured BV3concentrations was observed at 72 hours to less than the LOQ at all test concentrations employed.

Given that a decline in measured concentration was observed for both components, it was considered appropriate to base the results on the equivalent test item concentrations from analysis of BV3 only in order to give a worst case analysis of the data.

- Growth rate - EC 50 = 0.0053 mg/l (IC95%: 0.0048 - 0.0057 mg/l)

- Growth rate - NOEC = 0.0014 mg/l

- Growth rate - LOEC = 0.0034 mg/l

- Yield - EC 50 = 0.0034 mg/l (IC95%: 0.0031 - 0.0037 mg/l)

- Yield - NOEC = 0.0014 mg/l

- Yield - LOEC = 0.0034 mg/l

Description of key information

LC50(72h) for freshwater algae (Pseudokirchneriella subcapitata): 0.0053 mg/L (OECD TG 201; static)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.005 mg/L

Additional information

The EC50 (72 hours) was 0.0053 mg/L (geometric mean of the measured concentrations) with 95% confidence limits of 0.0048 - 0.0057 mg/L.

The NOEC (72 hours) was 0.0014 mg/L

To perform the test, a stock solution 550 mg of test item was dispersed in 11 liters of culture medium and stirred for 24 hours. The substances contains impurities (CAS 122-39-4, CAS 548-62-9 and CAS 587-98-4) which are much more solubles than the substance (>> 20 mg/L) and which are classified acute and chronic tox 1 (CAS 122-39-4, CAS 548-62-9) or chronic tox 2 with ecotoxicological data on algea (in there respective published REACH dossiers).

To remove undissolved test item, a filtration through a 0.2 µm filter was performed.

During the dissolution step, impurities were most probably solubilized more easily and to greater proportions than the substance. Therefore, during the filtration, only the very small proportion of the solubilized fraction of the substance and and all the solubilized impurities passed ghrough the filter. Doing so, the impurities were “concentrated” in the test solutions. This may have had an impact on the outcome of the test, although the substance was under the limit of quantification.