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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:
2015-03-04 - 2015-11-09
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. Prepared samples were stored at room temperature in an autosampler until analysis.


Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A dispersion was prepared with dilution water one day prior to the start of the exposure (0 h). 10.0 mg/L of the test item were weighed on a glass slide and inserted in a brown glass bottle with an appropriate amount of dilution water. This dispersion was stirred for 24 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.
To validate that the highest achievable concentration of the test item in dilution medium is ≤ 10.0 mg/L, non-GLP tests with different nominal concentrations and stirring times were conducted. The tested saturated solutions were prepared as follows: 10.0 – 100 mg/L, 24 h stirring. 10.0 – 100 mg/L, 48 h stirring. Undissolved particles were removed by membrane filtration as described earlier. The results of this non-GLP test shows that no higher concentrations than concentrations measured in the preliminary test and the definitive study can be achieved. The preparation procedure with a nominal loading rate of 10.0 mg/L, 24 h stirring and membrane filtration of undissolved particles will yield a saturated solution at water solubility level. Therefore, the use of a higher loading rate, longer stirring or higher temperature cannot increase amount of dissolved test item recovered in concentration control analytics. The maximum water solubility was almost achieved after 24 h stirring of a nominal test item concentration of 10 mg/L

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 0.625 - 1.25 - 2.50 - 5.00 - 10.0 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:
yes
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: 10; 5; 2.5; 1.25; 0.625 mg/l of saturated solution (WAF) means 100; 50; 25; 12.5; 6.25 % of the saturated solution
Measured: 0.205; 0.122; 0.0607; 0.0465; 0.0249 mg/L (geometric mean)
Details on test conditions:
TEST SYSTEM
- Initial cells density: approximately 7085 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 at the water accommodated fractions of 0.1 to 10 mg/L.
- 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: 10; 5; 2.5; 1.25; 0.625 mg/l of saturated solution (WAF) means 100; 50; 25; 12.5; 6.25 % of the saturated solution

- Range finding study
- Test concentrations: 10; 1; 0.1 mg/L
- Results used to determine the conditions for the definitive study:
10 mg/L: 26 % growth rate inhibition
1 mg/L: -2 % growth rate inhibition, means growth stimulation
0.1 mg/L; - 1 % growth inhibtion, means growth stimulation

Reference substance (positive control):
yes
Remarks:
potassium dichromate (experimental date 2015-04-14 to 2015-04-17)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.205 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.:
> 0.205 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.205 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 10 mg/L
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.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
other: yield inhibition
Key result
Duration:
72 h
Dose descriptor:
other: no effects in the range of water solubility
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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]          
   0 h 24 h   48 h  72 h
 control   0.6153  2.6618  25.8466  166.6858
 10   0.6153  2.3837  21.9928  142.1158
 5   0.6153  3.1029  32.7863  184.2971
2.5    0.6153  3.2184  33.4954  199.6854
 1.25   0.6153  3.0206  31.7364  192.3964
 0.625   0.6153  2.5750  28.4195  179.1520

Table 2: Evaluation after 72 h

 Nominal test item concentration [mg/L]  Growth rate [d-1]  Inhibition of growth rate [%]  Yield [cells/mL]  Inhibition of yield [%]
 control  1.86    1660705  
 10 1.81   3  1415005  15
 5 1.90  -2  1836818  -11
 2.5 1.93  -3  1990701  -20
 1.25  1.91  -3  1917811  -15
 0.625  1.88  -1  1785367  -8
Validity criteria fulfilled:
yes
Conclusions:
In this study, 2-decyltetradecanoic acid was found to not inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours.

No effects were found up to the maximum water solubility level (estimated ≤ 0.9 µg/L), which is below the nominal loading level of the saturated solution with 10 mg/L. Therefore, no effects were found in the range of water solubility of the test item.
Executive summary:

The toxicity of 2 -decyltetradecanoic acid to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 440/2008/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 10 mg/L was prepared with dilution water and stirred for 24 hours. The maximum water solubility level (estimated ≤ 0.9 µg/L) is below the nominal loading level of the saturated solution with 10 mg/L. Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 2.50 - 50.0 - 100 % corresponding to geometric mean measured concentrations of 0.0249 - 0.0465 - 0.0607 - 0.122 - 0.205 mg/L. 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 2-decyltetradecanoic acid in all test concentrations and the control were analytically verified by LC-MS/MS at the start and the end of exposure. The measured concentration of 2 -decyltetradecanoic acid in the saturated solution was 0.453 mg/L at the start of the exposure (0 h) and 0.0926 mg/L at the end of the exposure (72 h). To account for the low water solubility of the substance, all effect values given are based on the nominal test item concentrations and the geometric mean measured concentrations of 2 -decyltetradecanoic acid. In this study 2 -decyltetradecanoic acid was found to not inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. No effects were found up to the maximum water solubility level (estimated ≤ 0.9 µg/L), which is below the nominal loading level of 10 mg/L. Therefore, no effects were found in the range of water solubility of the test item. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 0.205 mg/L, corresponding to a nominal test item concentration of 10.0 mg/L. The EC50-values for inhibition of both growth rate (ErC50) and yield (EyC50) after 72 hours were > 0.205 mg/L, corresponding to a nominal test item concentration of > 10.0 mg/L. It should be considered that the measured concentrations are higher than the calculated water solubility.

Description of key information

In an OECD 201 study 2-decyltetradecanoic aicd was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours.

Key value for chemical safety assessment

Additional information

The toxicity of 2 -decyltetradecanoic acid to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 440/2008/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 10 mg/L was prepared with dilution water and stirred for 24 hours. The maximum water solubility level (estimated ≤ 0.9 µg/L) is below the nominal loading level of the saturated solution with 10 mg/L. Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 2.50 - 50.0 - 100 % corresponding to geometric mean measured concentrations of 0.0249 - 0.0465 - 0.0607 - 0.122 - 0.205 mg/L. 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 2-decyltetradecanoic acid in all test concentrations and the control were analytically verified by LC-MS/MS at the start and the end of exposure. The measured concentration of 2 -decyltetradecanoic acid in the saturated solution was 0.453 mg/L at the start of the exposure (0 h) and 0.0926 mg/L at the end of the exposure (72 h). To account for the low water solubility of the substance, all effect values given are based on the nominal test item concentrations and the geometric mean measured concentrations of 2 -decyltetradecanoic acid. In this study 2-decyltetradecanoic acid was found to not inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. No effects were found up to the maximum water solubility level (estimated ≤ 0.9 µg/L), which is below the nominal loading level of 10 mg/L. Therefore, no effects were found in the range of water solubility of the test item. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 0.205 mg/L, corresponding to a nominal test item concentration of 10.0 mg/L. The EC50-values for inhibition of both growth rate (ErC50) and yield (EyC50) after 72 hours were > 0.205 mg/L, corresponding to a nominal test item concentration of > 10.0 mg/L. It should be considered that the measured concentrations are higher than the calculated water solubility.