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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 2009-20 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Biomass residue (liquid) obtained after B2 production
IUPAC Name:
Biomass residue (liquid) obtained after B2 production

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for Total Organic Carbon (TOC) analysis were taken from the highest test concentration and the control without algae according to the schedule below.
Frequency: at t=0 h and t=72 h
Volume: 40 ml
Storage: Samples were stored in a refrigerator until analysis
At the end of the exposure period, the replicates without algae were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of Biomass residue (liquid) obtained after B2 production tested was a brownish-yellow turbid liquid . The test substance was not completely soluble in M2-medium at loading rates of 1.0, 10 and 100 mg/l. This is probably due to the salts in the M2-medium.

Preparation of the test solutions started with loading rates of 1.0, 10 and 100 mg/l applying 1 day of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixtures, of which the 1.0 mg/l loading rate was observed to be colourless with a few particles while the higher loading rates were turbid and increasingly yellow, were filtered through a glass fibre pre-filter (Whatman). The Water Soluble Fractions (WSFs) were used for testing.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/ml.
- 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
Between 22.0 and 23.7°C.
pH:
Between 8.0 and 8.2
Nominal and measured concentrations:
Nominal concentrations:Control and WSFs prepared at loading rates of 1.0, 10 and 100 mg/l.
Measured concentrations: Control and WSF prepared at 100 mg/l
Based on the TOC content of Biomass residue (liquid) obtained after B2 production (i.e. 13%) the concentration Biomass residue (liquid) obtained after B2 production in the aquatic samples was calculated and corrected for the control. The initial concentration in the WSF prepared at a loading rate of 100 mg/l was calculated to be 42 mg/l. This concentration decreased to 30 mg/l at the end of the test. Based on these results the average exposure concentration was calculated to be 36 mg/l.
Details on test conditions:
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1470000 cells/ml
- Replicates:
6 replicates of the control and the highest test concentration
3 replicates of each lower test concentration
One or more replicates of all test solutions without algae

GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps of the type Cool-white of 30 Watt, with a light intensity within the range of 63 to 76 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: control and WSFs prepared at loading rates of 1.0, 10 and 100 mg/l
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) converted to testmaterial
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon converted to testmaterial
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 other: mg/l (WSF prepared at 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 other: mg/l (WSF prepared at 100 mg/l)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) converted to test material
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Remarks:
Total Organic Carbon (TOC) convereted to test material
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 other: mg/l (WSF prepared at 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 other: mg/l (WSF prepared at 100 mg/l)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Effect concentrations exceeding solubility of substance in test medium: Yes
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):
The EC50 for growth rate reduction (ErC50: 0-72h) was 1.6 mg/l with a 95% confidence interval ranging from 1.1 to 2.3 mg/l.
The EC50 for yield inhibition (EyC50: 0-72h) was 0.62 mg/l with a 95% confidence interval ranging from 0.50 to 0.78 mg/l.
Reported statistics and error estimates:
Statistical analysis of the data for growth rate reduction was not performed as the effects recorded were not significant (<10%).
Slight but statistically significant inhibition of yield was found at the highest test concentration (Bonferroni t and Tukey test, a = 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study Biomass residue (liquid) obtained after B2 production did not induce growth rate reduction at a loading rate of 100 mg/l (NOEC), corresponding to an average exposure concentration of 36 mg/l.

The EC50 for growth rate reduction (ErC50: 0-72h) was above a loading rate of 100 mg/l. Based on non-specific analysis the 72h-EC50 was determined to be >36 mg/l.

Biomass residue (liquid) obtained after B2 production induced significant inhibition of yield at the highest loading rate tested, i.e. the NOEC was below a loading rate of 100 mg/l.

The EC50 for yield inhibition (EyC50: 0-72h) was above a loading rate of 100 mg/l. Based on non-specific analysis the 72h-EC50 was determined to be >36 mg/l.