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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:
1-16 May, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study has been performed according to OECD and/or EU guidelines and according to GLP principles.
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
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
During the final test samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h, t=24 h and t=48 h
Volume: 2 ml
Storage: Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Vehicle:
no
Details on test solutions:
Preparation of test solutions started with a solution of 100 (combined limit/range-finding test) or 320 mg/l (final) applying 15 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations (0.1, 1.0 and 10 mg/l in combined limit/range-finding test; 3.2, 10, 32 and 100 mg/l in final test) were prepared by subsequent dilutions of the 100 or 320 mg/l solution in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10000 cells/ml.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): not indicated
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not):
Stock culture medium: M1; according to the NPR 6505, formulated using Milli-Ro water (tap-water purified by reverse osmosis) with the following composition: NaNO3 500 mg/l
K2HPO4 40 mg/l
MgSO4.7H2O 76 mg/l
Na2CO3.10H2O 54 mg/l
C6H8O7.H2O 6 mg/l
NH4NO3 330 mg/l
CaCl2.H2O 36 mg/l
C6H5FeO7.xH2O 6 mg/l
H3BO3 2.9 mg/l
MnCl2.4H2O 1.81 mg/l
ZnCl2 0.11 mg/l
CuSO4.5H2O 0.08 mg/l
(NH4)6Mo7O24.4H2O 0.018 mg/l
Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water) preventing precipitation and with the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 µg/l
Na2EDTA.2H2O 100 µg/l
H3BO3 185 µg/l
MnCl2.4H2O 415 µg/l
ZnCl2 3 µg/l
CoCl2.6H2O 1.5 µg/l
CuCl2.2H2O 0.01 µg/l
Na2MoO4.2H2O 7 µg/l
NaHCO3 50 mg/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
pH 8.1 ± 0.2
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
see overall remarks below
Post exposure observation period:
None.
Hardness:
24 mg CaCO3 / litre
Test temperature:
22.6 - 22.9°C
pH:
8.0 - 8.4

Salinity:
Not applicable.
Nominal and measured concentrations:
Combined limit/range-finding test: Based on these results samples taken from nominal 10 and 100 mg/l were analysed and showed that the initial concentrations were 8.4 and 95 mg/l, respectively. These concentrations remained stable during the test period (88-95% of initial)

Final test: At the start of the test, the actual test concentrations were in agreement with nominal (86-95%) and these concentrations remained stable during the test period (86-105% of initial). Based on these results, the nominal concentrations were used to calculate the effect parameters.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Initial cells density: 10000 cells/ml
- Control end cells density: 504000 cells/ml
- No. of vessels per concentration (replicates): 3 replicates of each test concentration, 1 replicate of the highest concentration without algae, 1 extra replicate of each test solution for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control

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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED: EC50 for yield and growth
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 in final test (3.2, 10, 32, 100 and 320 mg/l)
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/l
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Growth rates were in the range of the controls at concentrations up to and including 10 mg/l during the 48-hour test period, whereas the growth rate of algae exposed to 32 mg/l and higher were increasingly reduced. Reduction of growth rate increased as exposure progressed.
Statistically significant reduction of growth rate was found at test concentrations of 32 mg/l and higher (Bonferroni t and Tukey test, α = 0.05).

Inhibition of yield increased with increasing concentration of Ditetrahydrofurylpropane from 32 mg/l upwards resulting in 62% inhibition at 320 mg/l. Statistically significant inhibition of yield was found at test concentrations of 32 mg/l and higher (Bonferroni t and Tukey test, α = 0.05).

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
Under the conditions of the reference study with Pseudokirchneriella subcapitata, potassium dichromate reduced growth rate of this fresh water algae species at nominally 1.0 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 2.0 mg/l with a 95 % confidence interval ranging from 1.7 to 2.4 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 1.1 mg/l with a 95 % confidence interval ranging from 0.88 to 1.3 mg/l. Historical ranges are not yet available.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, Ditetrahydrofurylpropane reduced growth rate and inhibited the yield of this fresh water algae species significantly at 32 mg/L and higher. The EC50 for growth rate reduction (ERC50: 0-48h) was beyond 320 mg/L.
The EC50 for yield inhibition (EYC50: 0-48h) was 120 mg/L with a 95% confidence interval ranging from 42 to 360 mg/L.
The NOEC for growth rate reduction and yield inhibition was 10 mg/L.

Description of key information

A study was conducted according to OECD 201. Growth rates were in the range of the controls at concentrations up to and including 10 mg/L during the 48-hour test period, whereas the growth rate of algae exposed to 32 mg/L and higher were increasingly reduced. Reduction of growth rate increased as exposure progressed.

Statistically significant reduction of growth rate was found at test concentrations of 32 mg/L and higher. EC50 was > 320 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
10 mg/L

Additional information

Under the conditions of the study with Pseudokirchneriella subcapitata, ditetrahydrofurylpropane reduced growth rate and inhibited the yield of this fresh water algae species significantly at 32 mg/L and higher. The EC50 for growth rate reduction (ERC50: 0-48h) was beyond 320 mg/L. The NOEC for growth rate reduction was 10 mg/L.