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Ecotoxicological information

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 JAN 2006 - 29 JUN 2006
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)
Version / remarks:
92/69/EWG, C.3
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L and a control
- Sampling method: In the test material groups one further flask per concentration without algae was exposed under the same conditions. These flasks were taken for analysis. Directly after preparation in the laboratory samples of the freshly prepared test media were given to analysis (0h). Further samples were taken for analysis after 24, 48 and 72 hours after start of the experimental part. All samples were stored under the same conditions as the vessels with Algae and afterwards sent to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock preparation with a test material concentration of 100 mg/L was prepared. Therefore, the calibrated flask with test material and vehicle (reconstituted water) was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. After that the preparation was passed through a separatory funnel for 6 hours. The separated stock preparation was diluted with reconstituted water to the different test media concentrations.
- Controls: Control group with reconstituted water only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations were made concerning the appearance of the test media in the control flasks without algae. All test media were clear solutions and remained clear throughout the entire test period.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Common name: Desmodesmus subspicatus
- Strain: strain No. SAG 86.81
- Source (laboratory, culture collection): "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", Germany
- Method of cultivation: The algae have been cultivated in the laboratories of Merck KGaA under standardized conditions.
- Age of inoculum (at test initiation): The algae were taken from an exponentially growing preculture (growth rate 23.1) which was set up 3 days prior to the experimental part under the same conditions as in the final study. At the start of the experimental part, an algae stock suspension was prepared from this preculture containing about 10,000 algae cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
250 mg/L CaCO3
Test temperature:
22 - 23 °C
pH:
7.76 - 10.62
Nominal and measured concentrations:
nominal: 0, 1.0, 1.8, 3.2, 5.6, and 10.0 mg/L
analytical: 0.12, 0.24, 0.44, 0.79 and 1.42 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type (delete if not applicable): closed (sealed with ground stoppers)
- Material, size, headspace, fill volume: Volumes of 120 mL reconstituted water or test media were used per replicate. In order to reduce the head space volume the flask size was 100 mL only.
- Aeration: no
- Initial cells density: 10 000 cells/mL
- Control end cells density: 601 624 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
Macro-nutrients:
NaHCO3: 50.0 mg/L
CaCl2 x 2 H2O: 18.0 mg/L
NH4Cl: 15.0 mg/L
MgSO4 x 7 H2O: 15.0 mg/L
MgCl2 x 6 H2O: 12.0 mg/L
KH2PO4: 1.6 mg/L

Trace elements:
Na2EDTA x 2 H2O: 100.0 µg/L
FeCl3 x 6 H2O: 80.0 µg/L
MnCl2 x 4 H2O: 415.0 µg/L
H3BO3: 185.0 µg/L
Na2MoO4 x 2 H2O: 7.0 µg/L
ZnCl2: 3.00 µg/L
CoCl2 x 6 H2O: 1.50 µg/L
CuCl2 x 2 H2O: 0.01 µg/L

The pH of this medium after equilibration with air is approximately 8.

OTHER TEST CONDITIONS
- Photoperiod: continuos illumination
- Light intensity and quality: light intensity was approx. 7500 to 9500 Lux
- Other: The flasks were incubated under standardized conditions, in an air-conditioned room under continuous rotating. The illumination was achieved by fluorescent tubes (Philips Master Rohren (TL5 80W/840 HO) installed above a rotating panel with the flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Small samples with algae were taken from all concentrations after 24, 48, or 72 hours of exposure and were not replaced.
- Determination of cell concentrations: The algae cell densities were determined with an electronic particle counter (Coulter, Z2).

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: In a pretest under closed static conditions in 100 mL flasks, an inhibition of the growth rate (Iµt) of the algae, Desmodesmus subspicatus, was observed at the concentrations between 1 and 10 mg/L nominally.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.44 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % confidence intervals: 0.43 - 0.46 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.75 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence intervals: 0.73 - 0.77 mg/L)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Reported statistics and error estimates:
With the percent values of the inhibition and growth of the cells the EC50 was calculated as a logit analysis according the procedure of UNKELBACH and WOLF (1985) using the PC-program 511.
The NOEC was determined as the highest dose without significant inhibitory effect. According to HEGER et al., an inhibition of growth rate < 10 % compared to the control is not considered to be significant.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 201 and EU Method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards algae.
The freshwater green algae Desmodesmus subspicatus with an initial cell density of 10000 cells/mL was exposed to five test material concentrations of nominal 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L beside a control with reconstituted water only under defined conditions and the growth was calculated after 24, 48 and 72 hours of exposure in the test media. In this closed static test system, algae exposed to concentrations of nominal 3.2 to 10.0 mg/L showed a significant inhibition of growth, whereas the concentrations of nominal 1.0 and 1.8 mg/L revealed no biological effect.
The limit of quantification of the analytical method was 0.07 mg/L. The analytically determined concentrations were between 12.7 - 15.0 % and 12.5 - 13.8 % at the start and at the end, respectively. During the experimental phase of the study, the analytically determined test material concentrations were below 80 % of the nominal concentrations. Therefore, the EC50 values had to be calculated with the mean analytical concentration.
Based on mean measured concentrations of the test item, the EC50 values with the 95 % confidence intervals and the no observed effect concentration (NOEC) were determined to be: 72h EbC50 = 0.44 (0.43 - 0.46) mg/L, 72h ErC50 = 0.75 (0.73 - 0.77) mg/L, NOEC = 0.24 mg/L.
Executive summary:

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested. The GLP study was performed according to OECD TG  201.

As  the test material is not stable in an open system, 100 mL Erlenmeyer flasks were filled up with test medium and sealed with ground stoppers to form a closed system. The algae were exposed to different concentrations of test material and the growth was calculated after 24, 48, or 72 hours of exposure in the test media. The study with was performed with nominal concentrations of 1.0, 1.8, 3.2, 5.6, and 10.0 mg/L test material in reconstituted water. The study design included three replicates per test material concentration, each containing about 120 mL test medium and 10,000 cells/mL at the start of the experimental phase and a control without algae. Analyses of the test material concentrations were performed with all doses at the start and than daily up to 72 hours.

The limit of quantification of the analytical method was 0.07 mg/L. The analytically determined concentrations were between 12.7 -15.0 % and 12.5 - 13.8 % at the start and at the end, respectively. During the experimental phase of the study, the analytically determined test material concentrations were below 80 % of the nominal concentrations. Therefore, the EC50 values had to be calculated with the mean analytical concentrations.

The median effective concentration (EC50), which would result in a 50 % growth inhibition and the 95% confidence interval compared to the control, was calculated from the mean analytically determined concentrations of test material as given below:

72h EbC50= 0.44 mg/L (95 % confidence intervals: 0.43 - 0.46 mg/L)

72h ErC50= 0.75 mg/L (95 % confidence intervals: 0.73 - 0.77 mg/L)
NOEC = 0.24 mg/L

Under the given conditions of this study the 72h ErC50 value was < 1 mg/L.

Description of key information

Toxicity to aquatic algae and cyanobacteria: ErC50 (72h) = 0.75 mg/L and NOEC (72h) = 0.24 mg/L for the freshwater green algae Desmodesmus subspicatus based on growth inhibition (static, closed, OECD 201, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.75 mg/L
EC10 or NOEC for freshwater algae:
0.24 mg/L

Additional information

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested. As  the test material is not stable in an open system, 100 mL Erlenmeyer flasks were filled up with test medium and sealed with ground stoppers to form a closed system. The algae were exposed to different concentrations of test material and the growth was calculated after 24, 48, or 72 hours of exposure in the test media. The study with was performed with nominal concentrations of 1.0, 1.8, 3.2, 5.6, and 10.0 mg/L test material in reconstituted water. The study design included three replicates per test material concentration, each containing about 120 ml test medium and 10,000 cells/mL at the start of the experimental phase and a control without algae. Analyses of the test material concentrations were performed with all doses at the start and than daily up to 72 hours.The GLP study was performed according to OECD TG  201.

The limit of quantification of the analytical method was 0.07 mg/L. The analytically determined concentrations were between 12.7 -15.0 % and 12.5 - 13.8 % at the start and at the end, respectively. During the experimental phase of the study, the analytically determined test material concentrations were below 80 % of the nominal concentrations. Therefore, the EC50 values had to be calculated with the mean analytical concentrations.

The median effective concentration (EC50), which would result in a 50 % growth inhibition and the 95% confidence interval compared to the control, was calculated from the mean analytically determined concentrations of test material as given below:

72h EbC50 = 0.44 mg/L (95 % confidence intervals:0.43 - 0.46 mg/L)

72h ErC50 = 0.75 mg/L (95 % confidence intervals: 0.73 - 0.77 mg/L)
NOEC = 0.24 mg/L

Under the given conditions of this study the 72h ErC50 value was < 1 mg/L.