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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:
Oct 25, 2018 - Jan 28, 2019
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:
Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Two replicates of each sample were deep-frozen (≤ -20°C) directly after preparation and after 24h, 48h and 72h and stored in a freezer until analysis. Before freezing the samples after 24h, 48h and 72h exposure, the algae were separated from the medium by centrifugation at a low g-force (4000g for 20 minutes), sufficient to settle the algae. Additionally, analysis of all test item concentrations not inoculated with algae were performed. The samples were appropriately diluted with diluent (100% methanol) into the calibration range. The concentrations of the test item were determined using the calibration curve. The stability of the test item during freezing has been demonstrated in a separate non-GLP study.
Vehicle:
yes
Details on test solutions:
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

Preparation of the Test Item:
A preparation with a test item concentration of 100 mg/L was freshly prepared.
For that purpose, the test item was weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 1 hour. The pH was not adjusted.



Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Pseudokirchneriella subcapitata, strain 61.81 SAG was obtained from „Pflanzenphysiologisches Institut der Universität Göttingen“, Germany.
- 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 or not): same as test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
-
Hardness:
24 mg/L as CaCO3 (calculated)
Test temperature:
22.9 to 23.9°C
pH:
7.87 to 8.80
Nominal and measured concentrations:
Nominal concentrations: 100.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer glas flasks filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10 000 cells per ml
- Control end cells density: 1 582 200 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
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The mean light intensities were 7667 Lux ± 2.8% and 7378 Lux ± 3.1% prior to and at the end of the exposure period, respectively.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Nominal test concentration: 100 mg/L
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
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:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: >0.011 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: >0.011 mg/L
Details on results:
The recoveries of the test material concentrations with algae were between 98.8% and 99.6% of the nominal concentration. The recovery directly after preparation was 100.5%.
The recoveries of the test material concentrations without algae were between 100.8% and 102.1% of the nominal concentration. The recovery directly after preparation was 100.5%. There was no difference between the analytical concentrations of the samples inoculated with and without algae.
No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L.
As the measured concentrations of the test item in the aqueous solutions were ± 20% of the nominal concentration during the study, the EC50 values are based on the nominal concentration.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Any observations (e.g. precipitation) 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 Commission Regulation (EU) 2016/266. Therefore, potassium dichromate (Art. 104864) was tested as positive control.

Study design:
The study comprised of one control group with six replicates and five test item groups with three replicates each. The test item concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg/L were dissolved in 100 mL reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group.

Results:
The test item Art. 104864 (Potassium dichromate) showed 72h EC50 of 0.91 mg/L for growth rate and 0.48 mg/L for yield. The EC50 for growth rate and yield obtained in this study are in the range of ± two standard deviation of the current historical means.

Therefore, this study confirms the sensitivity and reliability of the test system used in the test facility.


Objective

The objective of the present study was to determine the effect of on growth rate and yield of the unicellular green alga Pseudokirchneriella subcapitata.

Study Design

The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared reconstituted water. The initial algae cell density in each replicate was 10 000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. Samples of the test and control medium were taken directly after preparation and then daily and stored at ≤ -20°C until further analysis of the test item concentration via HPLC with UV detection.

Results

The recoveries of the test material concentrations with algae were between 98.8% and 99.6% of the nominal concentration. The recovery directly after preparation was 100.5%. The recoveries of the test material concentrations without algae were between 100.8% and 102.1% of the nominal concentration. The recovery directly after preparation was 100.5%. There was no difference between the analytical concentrations of the samples inoculated with and without algae.

No significant effect on growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L.

As the measured concentrations of the test item in the aqueous solutions were ± 20% of the nominal concentration during the study, the EC50 values are based on the nominal concentration.

The following EC values (Growth rate and Yield) were determined:

 Parameter (0 -72h) Growth Rate   Yield

EC10 [mg/L]

95% confidence interval

>100 mg/L (nominal)

n.d.

 >100 mg/L (nominal)

n.d.

EC20 [mg/L]

95% confidence interval

>100 mg/L (nominal)

n.d.

>100 mg/L (nominal)

n.d.

EC50 [mg/L]

95% confidence interval

>100 mg/L (nominal)

n.d.

>100 mg/L (nominal)

n.d.

Conclusion

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.The 72h EC50 for growth rate and yield were >100 mg/L (nominal) and, thus, could not be determined in this study.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test itemrevealed no toxicity to algae.
The 72h EC50 for growth rate and yield were >100 mg/L (nominal) and, thus, could not be determined in this study.



Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201. Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.The 72h EC50 for growth rate and yield were >100 mg/L (nominal) and, thus, could not be determined in this study.

Description of key information

OECD 202: EC50 (48 h) > 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

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