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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:
23 February - 18 March 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was carried out according to OECD guideline 201 and GLP. However, the concentrations were not confirmed by a specific analytical method, but by DOC determinations. Analytical measurements from the chronic Daphnia study (Migchielsen, 2013) indicated a significant decrease of the test substance concentration after two days. Nevertheless, the results can be used to conclude that the daphnia is the most sensitive species and the chronic Daphnia study is critical for the dossier. Therefore, the present study can be used with restrictions and a reliability 2 was assigned.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(1984)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentration of the test item was analysed by determination of DOC content in additional test vessels without algae. The TOC content of the test item was determined with 48.85 % (measurement by IFU).
ln the first study all seven nominal concentrations of 1003; 455; 235; 97; 44; 20 and 9.1 mg/L were analysed at 0, 24, 48 and 72 hours. ln the second study only the highest concentration of 20 mg/L was analysed at 0, 24, 48 and 72 hours.
Vehicle:
no
Details on test solutions:
ln the first study for each of the test concentrations 1000; 455; 235 and 97 mg/L, the test item was added directly to the test solution. For the test concentrations 44; 20 and 9.1 the stock solution 97 mg/L was diluted with deionized water. ln the second study a stock solution of 25 mg/L was prepared and diluted with deionized water.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Tests were carried out with the green alga Desmosdesmus subspicatus CHODAT (strain No 86.81 SAG) from the University Institute of Plant Physiology, D-37073 Göttingen, Nikolausberger Weg 18, Germany.
The strain used for this study has been cultured in the Iabaratory of Hydrotox GmbH since December 2002. Once every two months the stock culture is transferred to fresh sloped-agar tubes under sterile conditions.
3 days before starting the test algae samples were transferred from the agar tubes to Erlenmeyer flasks under sterile conditions. They were incubated in approximately 50 mL of algae medium at 21.3- 24.8 °C and 8600 ± 6% lx. After 3 days the cell concentration was determined using a counter chamber.
ln the first study, the cell concentration was 13 x 10^5 algal cells/mL. 11.54 mL pre-culture was added to 138.4 mL medium to obtain an inoculum concentration of 10 x 10^4 algal cells/ml. ln the second study the cell concentration was 6.5 x 10^5 algal cells/mL. 31 mL pre-culture was added to 169 mL medium to obtain an inoculum concentration of 10 x 10^4 algal cells/mL.
As 10x concentrated medium and algal inoculum were diluted 10 times with deionised water to obtain the test solutions, the algal starting concentration in the test solutionswas 1 x 10^4 cells/mL
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
The temperature in the testwas within a range of 24.2 to 25.6°C in the first study and 21.2 to 22.5°C in the second study. ln deviation of the norm in the first study the temperature was over the Iimit of 25°C for a few hours which is without influence on the test results.
pH:
The pH of the medium was 8.5 in the first and the second study. At the end of the test the pH was measured in one flask from each concentration. Although it is stated that the differences of the pH of the test solutions and the controls from the start to the end of the experiment of 0.7 units (first study) and 0.2 units (second study) are within the Iimit of ≤1 pH unit mentioned in the guideline, the results of the pH measurements are not specified.
Dissolved oxygen:
Not reported
Nominal and measured concentrations:
First study:
Nominal concentrations: 9.1, 20, 44, 97, 235, 455 and 1003 mg/L
Mean effective concentrations (corrected for mean recovery rate from DOC measurements): 6.8, 12, 31, 77, 133, 338 and 733 mg/L

Second test:
Nominal concentrations: 0.9, 1.9, 4.2, 9.2 and 20 mg/L
Mean effective concentrations (corrected for mean recovery rate from DOC measurements) of highest concentration: 21.5 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks with a test volume of 50 mL
- Initial cells density: 1 x 10^4 algal cells/mL
- Control end cells density: First test 32 x 10^4/algal cells/mL, Second test 43 x 10^4 algal cells/mL (factor 43) which is within the range for validity for the test (required ≥16).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- 8,600 ± 6% lx.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counter chamber
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 733 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Mean concentration based on DOC measurements.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
338 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Mean concentration based on DOC measurements.
Details on results:
ln the first study with the highest tested nominal concentration of 1003 mg/L, cell growth was inhibited by 39.8 %, growth rate by 8.4%. The dose-response curve was in an u-shape. With the two lowest tested concentrations of 20 and 9.1 mg/L again an inhibition of cell growth occured. For this reason the study was repeated with five concentrations between 20 and 0.9 mg/L. The inhibiting effect of low concentrations was not reproducible. Also in a pre-test series without GLP this effect did not occur. The EC50 values are higher than the highest tested concentration. NOECs were calculated with the results of the first study. The two lowest concentrations of the first study (20 and 9.1 mg/L) were not considered.
The recovery rate at the beginning of the first test was in a range of 80%. This result coincides with first investigations on the solubility of the test item in water by DOC analysis. With a stock solution of 100 mg/L a recovery rate of 95% could be found, with a stock solution of 1000 mg/L only 81%. This result indicates the restricted water solubility of the test item in the test medium. Therefore, in the first study the evaluation of effects on algae growth are calculated with the nominal concentrations and additional with the mean effective concentrations.
The recovery rate in the second test that was only carried out with one nominal concentration of 20 mg/L is in a range of 100%. Restricted water solubility did not limit the test.
Based on nominal values, the 72h EC50 was > 1000 mg/L and the NOECr was 455 mg/L.
Results with reference substance (positive control):
The ErC50 was 1.0 mg/L (required: 0.6- 1.4 mg/L).

First study analysis. Mean recovery rates and mean effective concentrations during the test period of 72 hours.

 
 Nominal concentration (mg/L)  Mean effective concentration (mg/L)  mean recovery rate (%)
 1003.2  732.6  73
 454.7  337.6  74.2
 234.9  133.2  56.7
 96.8  77.3  79.9
 44  30.5  69.3
 20  12.4  61.8
 9.1  6.8  75
Validity criteria fulfilled:
yes
Conclusions:
The 72h-ErC50 to Desmodesmus subspicatus based on mean effective concentrations (DOC) is > 733 mg/L.
The 72h-NOErC to Desmodesmus subspicatus based on mean effective concentrations (DOC) is 338 mg/L.
Executive summary:

The toxicity of the test substance to Desmodesmus subspicatus was investigated according to OECD guideline 201 (1984) and GLP.

The 72h-ErC50 to Desmodesmus subspicatus based on mean effective concentrations (DOC) is > 733 mg/L.

The 72h-NOErC to Desmodesmus subspicatus based on mean effective concentrations (DOC) is 338 mg/L.

The validity criteria were fulfilled.

The concentrations were not confirmed by a specific analytical method, but by DOC determinations. Analytical measurements from the chronic Daphnia study (Migchielsen, 2013) indicated a significant decrease of the test substance concentration after two days. Nevertheless, the results can be used to conclude that the daphnia is the most sensitive species and the chronic Daphnia study is critical for the dossier. Therefore, the present study can be used with restrictions and a reliability 2 was assigned.

Description of key information

The 72h-ErC50 to Desmodesmus subspicatus based on mean measured concentrations (DOC) is > 733 mg/L.
The 72h-NOErC to Desmodesmus subspicatus based on mean measured concentrations (DOC) is 338 mg/L.

Key value for chemical safety assessment

Additional information

The toxicity of the test substance to Desmodesmus subspicatus was investigated according to OECD guideline 201 (1984) and GLP.

The 72h-ErC50 to Desmodesmus subspicatus based on time weighted mean measured concentrations (DOC) is > 733 mg/L.

The 72h-NOErC to Desmodesmus subspicatus based on time weighted mean measured concentrations (DOC) is 338 mg/L.

The validity criteria were fulfilled.

The concentrations were not confirmed by a specific analytical method, but by DOC determinations. Analytical measurements from the chronic Daphnia study (Migchielsen, 2013) indicated a significant decrease of the test substance concentration after two days. Nevertheless, the results can be used to conclude that the daphnia is the most sensitive species and the chronic Daphnia study is critical for the dossier. Therefore, the present study can be used with restrictions and a reliability 2 was assigned.