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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 11, 2014 - July 1, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted on March 23, 2006; Annex 5 corrected: July 28, 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION OF THE TEST MATERIAL
The test medium (reconstituted water and test item) was freshly prepared. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Desmodesmus subspicatus, strain 86.81 SAG
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany. This strain was further cultivated in the laboratories of Non-Clinical Safety.
- Method of cultivation: The permanent algae cultures, the pre-culture and the algae cultures of the study were cultivated in an air-conditioned room with a water temperature of 21.0 – 24.0°C, controlled at ± 2°C. For cultivation, 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers (Heinz Herenz Medizinalbedarf GmbH, Hamburg, Germany). The cultures were shaken continuously at about 120 rpm (Universalschüttler SM25, Edmund Bühler GmbH, Hechingen, Germany) and lit between 4440 and 8880 Lux. Fluorescent tubes (Lumilux T5 nws FLH1 HO 80W/840, Osram GmbH, München, Germany) installed above the flasks served for lighting.

ACCLIMATION
- Acclimation period: 72h
- Culturing media and conditions: same as test conditions
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
24 mg/L as CaCO3 (calculated)
Test temperature:
20.9 - 22.6°C
pH:
7.89 - 8.89
Nominal and measured concentrations:
Nominal concentration: 100.0 mg/L
Due to very low water solubility and the fact that the study was a limit test, no further analysis was initiated.
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, fill volume: glas, 300 mL, 100 mL
- Aeration: no
- Initial cells density: 10 000 cells per mL
- Control end cells density: 402 577 cells per mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to OECD TG 201
Nutrient (mg/L)
NH4Cl 15.0
MgCl2 * 6H2O 12.0
CaCl2 * 2H2O 18.0
MgSO4 * 7H2O 15.0
KH2PO4 1.60
FeCl3 * 6H2O 0.0640
Na2EDTA * 2H2O 0.100
H3BO3 0.185
MnCl2 * 4H2O 0.415
ZnCl2 0.00300
CoCl2 * 6H2O 0.00150
CuCl2 * 2H2O 0.00001
Na2MoO4 * 2H2O 0.00700
NaHCO3 50.0
- pH: 7.94
- Total hardness: 24 mg/L as CaCO3 (calculated)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The light intensity measured immediately before start of the study was 7475 Lux ± 4.5% and 7611 Lux ± 4.4% at the end of the study. Thus, the light intensity was in the anticipated range of 4440 – 8880 Lux within ± 15% from the average.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter (Coulter-Counter Z2, Beckman Coulter GmbH, Krefeld, Germany) after exposure times of 24, 48, and 72 hours
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
The EC values exceeded the water solubility of 0.085 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Any observations that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate was tested as positive control. Under the given experimental conditions, potassium dichromate showed 72h EC values which are within the recommended range (Biomass integral EC50 0.20 - 0.75 mg/L and Growth rate EC50 0.60 - 1.03 mg/L). Growth Rate: EC50 = 0.89 mg/L; Biomass Integral: EC50 = 0.42 mg/L

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, an aqueous solution of 100 mg/L of the test item in an open static system revealed no aquatic toxicity. The EC50 for growth rate and yield were >0.085 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.


Executive summary:

The objective of this study was to evaluate the influence of the test item on the growth and growth rate of the green algae species Desmodesmus subspicatus according to OECD 201 and EU Method C.3. The study design included one control group and one test item group with six replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase. The study was performed as a limit test in an open static test system. The algae were exposed to a filtrate of nominal 100 mg/L. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item group and control group. The test item concentration in the reconstituted water was not quantified at the start and the end of this study. Because of the low water solubility (<0.085 mg/L), the compound cannot be detected with standard analytical methods. The development of an analytical method with a sufficiently low detection and quantification limit is complex. Due to the absence of any adverse effects at the saturation concentration, the study was performed without analytical concentration verification. The aqueous preparation of a nominal concentration of 100 mg/L of the test item revealed no toxic effect in Desmodesmus subspicatus. The following EC values (for growth rate and yield) were determined: EC10, EC20 and EC50 >0.085 mg/L (nominal >100 mg/L), NOEC: 0.085 mg/L (nominal 100 mg/L).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2007-11-14 to 2007-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
31st July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
March 23, 2006
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test medium (reconstituted water and test material) was freshly prepared. The calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasound bath for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the preparation was filtered through a filter funnel with a pore size of 10-16 µm. The filtrate was used for the study.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: No. SAG 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen
The algae were taken from an exponentially growing pre-culture (growth rate 120.7) which was set up 3 days prior to the experimental part under the same conditions as in the final study.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23-24°C
pH:
7.71 - 9.12
Dissolved oxygen:
at least 90 %
Nominal and measured concentrations:
Nominal concentration: 100 mg/L (limit test)
due to very low water solubility and the fact that the study was a limit test, no further analysis was initiated.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: open
- Material, size, fill volume: glass, 300 mL, 100 mL
- Aeration: no
- Initial cells density: 10,000 cells/mL
- Control end cells density: 1,817,559 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Details on composition of the reconstituted water:
Macro-nutrients:
50.00 mg/L NaHCO3
18.00 mg/L CaCl2 x 2 H2O
15.00 mg/L NH4Cl
15.00 mg/L MgSO4 x 7 H2O
12.00 mg/L MgCl2 x 6 H2O
1.60 mg/L KH2PO4
Trace elements:
100.00 µg/L Na2EDTA x 2 H2O
80.00 µg/L FeCl3 x 6 H2O
415.00 µg/L MnCl2 x 4 H2O
185.00 µg/L H3BO3
7.00 µg/L Na2MoO4 x 2 H2O
3.00 µg/L ZnCl2
1.50 µg/L CoCl2 x 6 H2O
0.01 µg/L CuCl2 x 2 H2O
The pH of the reconstituted water after aeration is approximately 8.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: approximately 7500 to 9500 Lux, fluorescent tubes (Philips Master tubes TL5 80W/840 HO) installed above a rotating panel

EFFECT PARAMETERS MEASURED
- The algae cell densities were determined with an electronic particle counter (Coulter, Z2) after 24, 48 and 72h

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, no inhibition observed at a concentration of 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
Results with reference substance (positive control):
Regularly, a positive control test to check the test system is carried out with potassium dichromate (no results provided in the study report).
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 validated PC-program 511. The NOEC was determined as the highest dose without significant inhibitory effect. According to HEGER et al. (1998), an inhibition of growth rate < 10 % compared to the control is not considered to be significant.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, an aqueous solution of 100 mg/L of the test item in an open static system revealed no aquatic toxicity. The EC50 for growth rate and biomass were >0.085 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.
Executive summary:

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested according to OECD 201 and EU Method C.3. The study design included one control and one test material group with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase. The study was performed as a limit test in an open static test system. The algae were exposed to a filtrate of nominal 100 mg/L. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test medium. The test material concentration in the aqueous medium was not quantified at the start and the end of this study as a very low water solubility was calculated for this test item (< 0.085 mg/L). Therefore, the development of an analytical method with a sufficiently low detection and quantification limit would be very time and cost intensive. Due to the absence of any adverse effects at the saturation concentration, the study was performed without analytical concentration verification. For the test material the following EC50 values and no effect concentration for Desmodesmus subspicatus were determined: EC50 (72h, based on growth rate and biomass): >100 mg/L nominal; NOEC (72h, based on growth rate and biomass): 100 mg/L.

Description of key information

An aqueous solution of 100 mg/L of the test item in an open static system revealed no aquatic toxicity towards algae. The EC50 for growth rate was >0.085 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

The NOEC (based on growth rate) was determined to be 100 mg/L (reference 6.1.5 -1).

An aqueous solution of 100 mg/L of the test item in an open static system revealed no aquatic toxicity towards algae (Desmodesmus subspicatus). The EC50 and EC10 for growth rate were >0.085 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study. (reference 6.1.5 -2).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Two studies of equivalent reliability are available. These studies were evaluated in a weight of evidence (WoE) approach.

In a first study, the influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested according to OECD 201 and EU Method C.3. The study design included one control and one test material group with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase. The study was performed as a limit test in an open static test system. The algae were exposed to a filtrate of nominal 100 mg/L. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test medium. The test material concentration in the aqueous medium was not quantified at the start and the end of this study as a very low water solubility was calculated for this test item (< 0.085 mg/L). Therefore, the development of an analytical method with a sufficiently low detection and quantification limit would be very time and cost intensive. Due to the absence of any adverse effects at the saturation concentration, the study was performed without analytical concentration verification.For the test material the following EC50values and no effect concentration for Desmodesmus subspicatus were determined: EC50 (72h, based on growth rate and biomass): >100 mg/L nominal; NOEC (72h, based on growth rate and biomass): 100 mg/L (reference 6.1.5 -1).

The objective of the second study was to evaluate the influence of the test item on the growth and growth rate of the green algae species Desmodesmus subspicatus according to OECD 201 and EU Method C.3. The study design included one control group and one test item group with six replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase. The study was performed as a limit test in an open static test system. The algae were exposed to a filtrate of nominal 100 mg/L. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item group and control group. The test item concentration in the reconstituted water was not quantified at the start and the end of this study. Because of the low water solubility (<0.085 mg/L), the compound cannot be detected with standard analytical methods. The development of an analytical method with a sufficiently low detection and quantification limit is complex. Due to the absence of any adverse effects at the saturation concentration, the study was performed without analytical concentration verification. The aqueous preparation of a nominal concentration of 100 mg/L of the test item revealed no toxic effect in Desmodesmus subspicatus. The following EC values (for growth rate and yield) were determined: EC10, EC20 and EC50 >0.085 mg/L (nominal >100 mg/L), NOEC: 0.085 mg/L (nominal 100 mg/L) (reference 6.1.5 -2).