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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:
24 September - 13 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as an unpublished report, no restrictions, fully adequate for assessment.
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:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Duplicate samples were taken from the test media of all test concentrations and control at the start and end of the test.
- Sampling method: Samples taken from test media (without algae) and at the end (containing algae).
- Sample storage conditions before analysis: Deep frozen immediately and stored at -20˚C.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of the nominal concentration of 100 mg/L was prepared by dissolving 30.09 mg of the test item completely in 300 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The stock solution was serially diluted with test water to prepare the test media of the different test concentrations.
- Controls: Culture medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures, SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): 3 day old culture in exponential growth phase
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
24 mg/L as CaCO3
Test temperature:
22˚C
pH:
Test start = 8.1
Test end = 8.2 - 9.4
Dissolved oxygen:
Not reported
Nominal and measured concentrations:
Nominal concentrations: 0, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L. Mean measured concentrations: 0.020, 0.071, 0.19, 0.82, 2.7 and 7.8 mg/L.

The measured concentrations of the substance in the test media of the nominal test concentrations of 0.032 to 10 mg/L were between 89 and 108% of the nominal values at the start of the study. During the study period of 96 hours, a decrease of test item concentration in the test media occurred. At the end of the study, 11 to 65% of the nominal values were found. The mean measured test concentrations (calculated as the arithmetic means of the concentrations measured at the start and end of the test) were between 59 and 84% of the nominal values.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flask
- Type (delete if not applicable): Closed, covered with a glass dish
- Material, size, headspace, fill volume: Fill volume = 15 ml
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (OECD medium)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: 4510 to 4880 Lux

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
yes
Remarks:
potassium dichromate, tested twice per year.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.12 - 0.24
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 1.5 - 8.2 extrapolated value
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 0.11 - 0.18
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.14 - 0.24
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
8.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 5.5 - 17 extrapolated values for CL and mean
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 0.15 - 0.26
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.071 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.071 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.071 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.071 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.071 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.071 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities were observed
- Unusual cell shape: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: (March 2010) 72 h growth rate = 0.94 mg/L
- Other: Results from Harlan Laboratories study C86922
Reported statistics and error estimates:
72 and 96 h LOEC and NOEC values were determined using Williams' t-test and Welch t-test. 72 and 96 h EC50 values and their confidence intervals were calculated by probit analysis.

The test item had a significant inhibitory effect on the growth of the algae (AUC, average growth rate and yield) after the test periods of 72 and 96 hours at the concentration of 0.19 mg/L and at all higher test concentrations (results of Williams t-test and Welch t-test, one-sided, α = 0.05). Thus, this concentration was determined to be the 72- and 96-hour LOEC.

 

The 72- and 96-hour NOEC were determined to be 0.071 mg/L, since up to and including this test concentration the AUC, growth rate and yield of the algae after 72 and 96 hours were not significantly lower than in the control.

 

In the control the biomass increased by a factor of 106 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 and 96 hours was 3.7 and 24.8%, respectively. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 and 96 hours was 1.4 and 0.9%, respectively. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Validity criteria fulfilled:
yes
Conclusions:
In a guideline, 96-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the test material exhibited EC50 values of 0.18, 8.7 and 0.19 mg/L based on biomass, growth rate and yield, respectively. The 96 hour NOEC was 0.071 mg/L for all endpoints. The 96 hour LOEC was 0.19 mg/L for all endpoints. All results are based on mean measured concentrations.
Executive summary:

In a GLP-compliant, guideline 96-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the test material exhibited EC50 values of 0.18, 8.7 and 0.19 mg/L based on biomass, growth rate and yield, respectively. The 96 hour NOEC was 0.071 mg/L for all endpoints. The 96 hour LOEC was 0.19 mg/L for all endpoints. All results are based on mean measured concentrations.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Apr - 7 May 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, guideline study. No analytical monitoring.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
No analytical monitoring
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
yes
Remarks:
temp between 20 - 22°C - Guideline requires 24 ± 2°C
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
No analytical monitoring
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of nominal concentration 100 mg/L, was prepared by dissolving 30.05 mg of test item in 300 mL test water with 15 minutes ultrasonic treatment and 15 minutes intense stirring. The test series was prepared by diluting samples of this solution with appropriate volumes of test water
- Controls: Test water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Insititute for Plant Physiology, University of Göttingen / Germany)
- Age of inoculum (at test initiation): 3 days. Algae were in the exponential growth phase
- Method of cultivation: Standardized conditions according to guidelines.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
24 mg/L as CaCO3
Test temperature:
20-22 °C
pH:
At test start: 7.9 - 8.0
At test end: 8.3 - 9.1
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
0 (control),0.032, 0.1, 0.32, 1.0, 3.2 and 10 mg test item/L. All values nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: Flasks
- Type (delete if not applicable): Erlenmeyer flasks covered with a glass dish
- Material, size, headspace, fill volume: 50-mL Erlenmeyer flasks containing 15 mL of test solution
- Aeration: Stirred by magnetic stirrers
- Initial cells density: 5000 cells/mL
- Control end cells density: Not calculated
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconsituted test water according to OECD guideline 201
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: Mean: 4600, Range: 4140 - 5030 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter
- Other: Algae in the control and 0.032 mg/L test solution were compared microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate, tested twice during year
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
area under the growth curve
Remarks on result:
other: 95% CL: 0.23 - 0.38 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
area under the growth curve
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.2 - 3.6 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: Not determined
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
area under the growth curve
Remarks on result:
other: 95% CL: 0.21 - 0.28 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
area under the growth curve
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.7 - 3.7 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.21 - 0.27 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yeild
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Control and 0.032 mg/L algae compared. No differences found.
- Unusual cell shape: No
- Colour differences: At highest test concentration a slight brown turbidity through the test item observed at 96 hours.
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Any stimulation of growth found in any treatment: No significant effects
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
Results with reference substance (positive control):
- Results with reference substance valid?: Yes, tested March 2010
- EC50: 0.94 mg Potassium dichromate/L (72 hours, growth rate)
- Other: Sensitivity of test system demonstrated, internal historical range 72-h EC50 0.71 - 1.7 mg/L
Reported statistics and error estimates:
The EC50 values and their 95% confidence limits were calculated by probit analysis.
The test concentration results were compared to the control values by Welch-t test and William's tests.
Validity criteria fulfilled:
yes
Remarks:
Control biomass increased by a factor of 73 over 72 hours; mean coefficient of variation in control daily growth rates was 4.7 and 7.4% during 72 and 96 hours, respectively, and coefficient of variation of average specific growth rates in control replicat
Conclusions:
In a 96-hour algal growth inhibition test test with Pseudokirchneriella subcapitata exhibited EC50 values of 0.25, 2.4 and 0.24 mg/L based on biomass, growth rate and yield respectively. The 96 hour NOEC was 0.1 mg/L for all assessment parameters.
Executive summary:

In a GLP-compliant, guideline 96-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the substance exhibited EC50 values of 0.25, 2.4 and 0.24 mg/L based on biomass, growth rate and yield, respectively. The 96 hour NOEC was 0.1 mg/L for all assessment parameters. All results are based on nominal concentrations.

Description of key information

ErC50=2.4 mg/L, NOEC=0.1 mg/L, 96h, Pseudokirchneriella subcapitata, OECD 201, Höger 2010
ErC50=8.7 mg/L, NOEC=0.071 mg/L, 96h, Pseudokirchneriella subcapitata, OECD 201, Liedtke 2010

Key value for chemical safety assessment

EC50 for freshwater algae:
8.7 mg/L
EC10 or NOEC for freshwater algae:
0.071 mg/L

Additional information

The influence of the test substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test under GLP and according to OECD Guideline 201 (2006), by Höger (2010). The nominal concentrations of the test item of 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L were tested in parallel with a control. No analytical measurements were performed in the test. The 96h ErC50 was found to be 2.4 mg/L (1.7-3.7 mg/L, CI=95%), and a NOEC=0.1 mg/L determined, based on nominal concentrations.

 

The influence of the test substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test under GLP and according to OECD Guideline 201 (2006), by Liedtke (2010). The nominal concentrations of the test item of 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L were tested in parallel with a control. The measured concentrations of the substance in the test media with nominal test concentrations from 0.032 to 10 mg/L, were between 89 and 108% of the nominal values at the start of the study. During the study period of 96 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 11 to 65% of the nominal values were found. The mean measured test concentrations (calculated as the arithmetic means of the concentrations measured at the start and end of the study) were between 59 and 84% of the nominal values. The 72h and 96h ErC50 were found to be 2.9 mg/L (1.5-8.2 mg/L, CI=95%) and 8.7 mg/L (5.5-17 mg/L, CI=95%), respectively, and the NOEC(growth rate)=0.071 mg/L.

 

In literature studies investigating hydrolysis, the substance has been shown to be hydrolytically unstable, but under strongly acid and alkaline conditions at elevated temperatures (~100°C). No experimental information is available at relevant temperatures - see section 5.1.2. However, the study by Liedtke demonstrated a decrease in salicylamide concentration during the study period, which may be attributable to hydrolysis (the pH observed at the end of the study was 8.2-9.4). As a result, the study by Höger is not considered to be reliable, as toxicity may be underestimated due to the lack of analytical measurements of test item concentrations during the study. Despite the methodological differences both studies reported quite similar effect concentrations. However, in this case it is of particular concern because of the proximity of the observed effect concentrations to the regulatory cut-offs for classification.

 

The second study by Liedtke was conducted with analytical measurements of the test concentrations, and reported the study results as mean measured effect concentrations. This study is thus considered to be reliable, and adequate, for risk assessment and classification purposes.