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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:
2015-04-08 - 2015-09-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
All original samples were stored at room temperature.


Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A dispersion was prepared with dilution water two days prior to the start of the exposure (0 hours). 100 mg/L of the test item was weighed on a glass slide and inserted in a brown glass bottle with an appropriate amount of dilution water. This dispersion was stirred for 48 hours with 1100 rpm at room temperature. Then undissolved particles were removed by membrane filtration (membrane filter 0.2 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL filtrate was discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered.

The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution corresponding to nominal concentration levels of 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
The concentrations were based on the results of a preliminary range finding test (non GLP).



Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: Pseudokirchneriella subcapitata HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkultutren (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Method of cultivation: fresh stocks were prepared every month on Z-Agar, light intensity amounted 35 - 70 µE/m²s for 24 h per day; Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
21 - 24 °C, controlled at +- 2 °C
pH:
8.1 +- 0.2
Dissolved oxygen:
no data
Salinity:
freshwater test according to guideline
Nominal and measured concentrations:
Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
Measured: < LOQ for all nominal concentrations
Details on test conditions:
TEST SYSTEM
- Initial cells density: approximately 6153 cells/mL
- Control end cells density: cells/ml (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM
- Dilution water: according to OECD test guideline 201

OTHER TEST CONDITIONS
- Photoperiod: 24 h/d light
- Light intensity and quality: 60 - 120 µE/m²s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell density: chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm, Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test.
- Measurement intervall: at 0, 24, 48 and 72 hours

Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L

- Range finding study
Results used to determine the conditions for the definitive study:

1. - Test concentrations: 10; 1; 0.1 mg/L
10 mg/L: -2 % growth rate inhibition, means growth stimulation
1 mg/L: -2 % growth rate inhibition, means growth stimulation
0.1 mg/L; 1 % growth inhibtion

2. - Test concentrations: 100 mg/L
100 mg/L: 4 % growth rate inhibition
Reference substance (positive control):
yes
Remarks:
potassium dichromate (experimental date 2015-04-14 to 2015-04-17)
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
>= 100 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 50 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: yield inhibition
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no (slight increase of growth rate in lower concentrations, but within coefficient of variation of average growth in replicate control cultures)
- 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: n.a.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 0.613 mg/l/72h
- EyC50: 0.281 mg/l/72h
Reported statistics and error estimates:
No statistics performed

Table 1: Cell denisities

 Nominal loading rate [mg/L]  Cell density x 104[cells/mL]          
=  % saturated solution  0 h 24 h   48 h  72 h
 control   0.6153  4.6464  47.0455  256.7209
 100   0.6153  4.9139  50.0845  214.2281
 50   0.6153  4.6634  47.4828  250.8441
25    0.6153  3.9596  36.6645  247.0453
 12.5   0.6153  3.7144  37.8064  247.6934
 6.25   0.6153  4.3601  42.6939  269.0287

Table 2: Evaluation after 72 h

 Nominal test item concentration [mg/L] = % saturated solution  Growth rate [d-1]  Inhibition of growth rate [%]  Yield [cells/mL]  Inhibition of yield [%]
 control  2.01    2561056  
 100 1.95   3  2136128 17
 50 -2.00  0  2502288 2
 25 -2.00 1  2464300 4
 12.5  -2.00 1  2470781 4
 6.25  -2.01 -1  2684134 -5
Validity criteria fulfilled:
yes
Conclusions:
In this study static algae test according to OECD-guideline 201 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester was found to slightly inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 50.0 % of the saturated solution. The EC50-values for inhibition of both growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 % of the saturated solution (100 mg/L nominal loading rate WAF).

The growth rate inhibition at 100 % saturated solution was found to be statistical significant at 3 % inhibition compared to control. Due to this low inhibitory effect (< 10 %, biological significance) it may be justified to assume this effect as unspecific.

No further effects are expected at higher concentrations of nominal 100 mg/L due to the calculated water solubility of ≤ 0.739 ng/L.



Executive summary:

The toxicity of 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A saturated solution of 100 mg/L was prepared with dilution water and stirred for 48 hours. Then undissolved particles were removed by membrane filtration (membrane filter 0.2 µm, RC, MACHEREY-NAGEL). Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 2.50 - 50.0 - 100 %. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. The concentrations of the test item 1,2,3 -Propanetricarboxylic acid, 2 -Hydroxy-, tris(C12 -C13 -branched-alkyl)ester in all test concentrations and the control were analytically verified by LC-MS/MS at the start and the end of exposure. The saturated solution with a nominal concentration of 100 mg/L was diluted to prepare different concentrations. To account for the low calculated water solubility of the substance, all effect values given are based on nominal test item concentrations.

Only slight effects of the test item 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester on the green alga Pseudokirchneriella subcapitata in terms of inhibition of growth or yield was found up to the 100 % saturated solution. The growth rate inhibition at 100 % saturated solution was found to be statistical significant at 3 % inhibition compared to control. Due to this low inhibitory effect (10 %, biological significance) it may be justified to assume this effect as unspecific. No further effects are expected at higher concentrations of nominal 100 mg/L, due to the calculated water solubility of ≤ 0.739 ng/L.

Description of key information

In an algae test according to OECD-guideline 201 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester was found to slightly inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 50.0 % of the saturated solution. The EC50-values for inhibition of both growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 % of the saturated solution (100 mg/L nominal loading rate WAF).

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

The toxicity of 1,2,3-Propanetricarboxylic acid, 2-Hydroxy-, tris(C12-C13-branched-alkyl)ester to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3. The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A saturated solution of 100 mg/L was prepared with dilution water and stirred for 48 hours. Then undissolved particles were removed by membrane filtration (membrane filter 0.2 µm, RC, MACHEREY-NAGEL). Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 2.50 - 50.0 - 100 %. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. The concentrations of the test item 1,2,3 -Propanetricarboxylic acid, 2 -Hydroxy-, tris(C12 -C13 -branched-alkyl)ester in all test concentrations and the control were analytically verified by LC-MS/MS at the start and the end of exposure. To account for the low calculated water solubility of the substance, all effect values given are based on nominal test item concentrations.