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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:
key study
Study period:
09/08/2010-12/08/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP, Guideline study with slight deviation in temperature during the test and slight deviation of pH of the control that have been assessed with respect to study validity criteria and considered to have no influence on the results of the study.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
slight deviation of temperature by one degree outside of specified range during hours 24 and 48. slight deviation of pH of the control, with pH increase by 1.79 which is more than 1.5.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
At test initiation, hour 24 and experimental completion samples were removed from each test solution and the control and analyzed for concentrations of Trifluoroethanol. Samples analyzed on day 0 were taken from the excess test solutions in the volumetric flasks. Samples analyzed at 72 hours of exposure were removed from the composite of the replicate vessels for each treatment and the control. The samples were put into a screw cap vial. Additionally, a sample from the abiotic control which did not contain algae was removed.
Vehicle:
no
Details on test solutions:
A 1000 mg test item/L stock solution was prepared prior to test initiation by dissolving 1.00419 g of Trifluoroethanol in 1000 mL of algal medium. To avoid loss of test item, due to its volatility, precautions were taken that the algal medium was added immediately after the test item was weighed. The resulting stock solution was observed to be clear, colorless and with no visible undissolved test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source: test laboratory culture
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Continuously measured temperature of 22 to 26 deg C. Between hours 24 and 48, the temperature ranged from 25 to 27 deg C.
pH:
pH value after 0h: 8.0 - 8.2
pH value after 72h: 9.80 to 9.93
pH of the control changed from 8.04 up to 9.83
Salinity:
OECD medium
Nominal and measured concentrations:
Nominal test concentrations: 10, 32, 100, 320 and 1000 mg test item/L (selected based on a preliminary range-finding test).
Time-weighted mean measured concentrations: 9.99, 30.0, 74.4, 240 and 974 mg test item/L.
Details on test conditions:
Light intensity of the test area: 4547 to 8057 lux
Shaking rate: 100 rpm
Reference substance (positive control):
not required
Remarks:
not included during this study
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 480 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 974 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
At hours 24, 48 and 72, cells exposed to all treatment levels and the control were observed to be normal.
The control algae fulfilled the validity criteria given for the green algae during the test, therefore the slight deviation of the temperature is considered to have no influence on the results of the study.
The pH of the control changed from 8.04 up to 9.83, therefore the pH increased by 1.79, which is more than 1.5. Since the pH at test initiation was correct and the control algae fulfilled the validity criteria given for green algae during the test, this slight deviation of the pH is considered to have no influence on the results of the study.
Results with reference substance (positive control):
Positive controls routinely performed by the testing laboratory but not conducted during this study
Reported statistics and error estimates:
ANOVA and Bonferroni t-Test were used to determine the NOEC and LOEC values. All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilks' and Bartlett's Tests, where the 99% level of certainty was applied.
The EC values for hour 72 were calculated based on time-weighted mean measured concentrations by using bootstrap analysis.

Analysis of the quality control samples resulted in measured concentrations with recoveries ranging from 93.5 to 121% (n = 8 of 9) of nominal concentration at hour 0, 24 and 72. One QC had a recovery of 161%, which was considered to be an outlier. Based on the results of these analyses, it was established that the appropriate quality control was maintained during the analyses of the exposure solutions.

During this study, the 72 hour cell growth in the control was 134x 104cells/mL, which surpasses the above criterion of 16x104cells/mL.The mean daily CV for growth rate through 72 hours for the control was 12.2%, which is below the limit given in the guideline. The average overall growth rate of the control for the 0 to 72 hour period was 2.55%.Therefore, this study is considered acceptable and the results accurately reflect the sensitivity ofPseudokirchneriella subcapitata toTrifluoroethanol.

Validity criteria fulfilled:
yes
Conclusions:
At hours 24, 48 and 72, cells exposed to all treatment levels and the control were observed to be normal.
The results were determined to be:
EC50 (72h) growth rate >974 mg/L
NOEC growth rate = 974 mg/L
LOEC growth rate >974 mg/L
Executive summary:

The study determined the effects of Trifluoroethanol on the growth of Pseudokirchneriella subcapitata. Based on a range-finding test, the following nominal test concentrations were selected for the definitive exposure: 10, 32, 100, 320 and 1000 mg test item/L. Time-weighted mean measured concentrations of 9.99, 30.0, 74.4, 240 and 974 mg test item/L were used for the evaluation of the biological data.

At hours 24, 48 and 72, cells exposed to all treatment levels and the control were observed to be normal.

The results were determined to be:

EC50 (72h) biomass >974 mg/L

NOEC biomass = 974 mg/L

LOEC biomass >974 mg/L

EC50 (72h) growth rate >974 mg/L

NOEC growth rate = 974 mg/L

LOEC growth rate >974 mg/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test conducted following OECD guideline 201, but not under GLP. Furthermore, no substance purity was provided and no analytical analysis were done.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Light intensity was not high enough (4000 lux), and pH increased too much during the experiment (from 7.9 to 10.3-10.5)
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES

- Vapour pressure: No data
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular green alga
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): No data
- Method of cultivation: No data

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): culture medium (OECD medium)
- Any deformed or abnormal cells observed: No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No data
Hardness:
No data
Test temperature:
23+-2°C
pH:
from 7.9 to 10.5
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0, 10, 100, and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL glass, stoppered Erlenmeyer flask (sealed vessel used because volatility expected)
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 500 mL
- Aeration: No data
- Type of flow-through (e.g. peristaltic or proportional diluter): No data
- Renewal rate of test solution (frequency/flow rate): No data
- Initial cells density: 10e4 cell/mL
- Control end cells density: 56.3 x 10e4 cell/mL
- No. of organisms per vessel: 5 x 10e6 cell/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)
- Detailed composition if non-standard medium was used: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: No data
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: no
- Intervals of water quality measurement: No data

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No data
- Photoperiod: 24:0 (continuous illuminating)
- Light intensity and quality: 4000 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: particle counter (Toa Medical Electronics Compagny CDA-500)
- Chlorophyll measurement: No data
- Other: None

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 1000 mg/mL
- Results used to determine the conditions for the definitive study: preliminary test was carried out with 3 concentrations: 10, 100, and 1000 mg/L (1 replicate each).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
No data
Results with reference substance (positive control):
- Results with reference substance valid: yes
- EC50: 0.41 mg/L at 72 hours
- Other: none
Reported statistics and error estimates:
No data

Table: Cell density of S. capricornutum

Nominal concentration (mg/L) Vessel No. Cell density (cells/mL)
0 24 48 72
Control 1 10000 71200 359300 622400
2 10000 58000 304300 558400
3 10000 53800 264300 508400
average 10000 61000 309300 563100
SD 0 9100 47700 57100
1000 1 10000 70300 365300 619400
2 10000 73100 363300 666400
3 10000 67400 325300 580400
average 10000 70300 351300 622100
SD 0 2900 22500 43100
Validity criteria fulfilled:
yes
Conclusions:
Toxicity of trifluoroethanol has been tested on Selenastrum capricornutum, on a growth inhibition limit test following OECD guideline 201. A concentration of 1000 mg/L and control has been tested in 3 replicates. Either for growth rate and biomass, EC50(72h) = NOEC(72h) > 1000 mg/L of substance.
Executive summary:

Toxicity of TFEA has been tested on Selenastrum capricornutum, on a growth inhibition limit test following OECD guideline 201 without GLP statement. The report doesn't provide the purity of the test substance and no analytical measurement was performed during the study. All validity criteria were fulfilled and the report is sufficiently well documented. A concentration of 1000 mg/L and control has been tested in 3 replicates. No toxicity was observed and therefore for growth rate and biomass, EC50 (72h) and NOEC (72h) were determined above 1000 mg/L of test substance.

Description of key information

Two studies performed according to OECD 201 guideline are available for Selenastrum capricornatum.

One is a key study performed without major deviation to the guideline and according to GLP. Based on a range-finding test, the following nominal test concentrations were selected for the definitive exposure: 10, 32, 100, 320 and 1000 mg test item/L. Time-weighted mean measured concentrations of 9.99, 30.0, 74.4, 240 and 974 mg test item/L were used for the evaluation of the biological data. At hours 24, 48 and 72, cells exposed to all treatment levels and the control were observed to be normal. The results were determined to be: EC50 (72h) biomass >974 mg/L; NOEC biomass = 974 mg/L; LOEC biomass >974 mg/L; EC50 (72h) growth rate >974 mg/L and NOEC growth rate = 974 mg/L.

The second one is a supportive study without GLP statement, with no purity of the test substance reported and no analytical measurement performed during the study. All validity criteria were fulfilled and the report is sufficiently well documented. A concentration of 1000 mg/L and control has been tested in 3 replicates. No toxicity was observed and therefore for growth rate and biomass, EC50 (72h) and NOEC (72h) were determined above 1000 mg/L of test substance.

Key value for chemical safety assessment

EC50 for freshwater algae:
974 mg/L
EC10 or NOEC for freshwater algae:
974 mg/L

Additional information