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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:
2017-09-12 to 2017-09-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
There were no deviations and no amendments to this study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Batch/Lot no: 160306
- Purity: >99%



Analytical monitoring:
yes
Details on sampling:
- Sampling method: Sampling was performed on initial test solutions and on final solutions collected at test completion. Samples of the initial test solutions were collected from the vessels used to prepare the bulk test solutions. Samples of the final test solutions were collected from the extra flasks prepared for confirmatory analysis. Approximately 40 mL of each test solution and the negative control were collected in glass containers of appropriate size. Approximately 1L of negative control water was also collected at this time.
- Sample storage conditions before analysis: stored refrigerated (2°C to 8°C) until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was prepared at 100 mg test item/L and test solutions were prepared as dilutions of this stock. To prepare a stock solution (100 mg/L), 0.1003 g of test item was weighed and added to approximately 500 mL of nutrient media (dilution water) in a 1000 mL glass volumetric flask. The solution was then brought up to the 1000 mL mark using nutrient media (dilution water) and mixed well by inversion. The pH of the stock solution was 4.3 (outside of ±1.5 pH units of the nutrient media) and required a pH adjustment. The stock solution was adjusted to 7.4 (within ±0.2 pH of nutrient media which had a pH of 7.6) using 1N NaOH prior to use. The test solutions were prepared in a laminar flow hood.
- Differential loading: six additional test item centrations were prepared from the pH-adjusted stock solution.
- Controls: negative control (dilution water only; tested in parallel), positive control (reference substance: zinc sulphate heptahydrate; tested in a separate study)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Comment on nutrient media: As the test item contains metals, the nutrient media used for testing was prepared with a reduced amount of the chelating agent Na2EDTA•2H20, compared to the amount in the nutrient media used for culturing. Reducing the concentration of Na2EDTA•2H20 from 300 µg/L to 75 µg/L allows for increased bioavailability of the test item. The pH of the nutrient medium was 7.6 and no additional pH adjustment was needed. The media was filter sterilised through a 0.2 µm filter prior to use.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CPCC 37
- Source: Canadian Phycological Culture Centre (formerly known as University of Toronto Culture Collection)
- Age of inoculum (at test initiation): exponential growth phase
- Method of cultivation: Erlenmeyer flasks, nutrient medium
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
not specified
Test temperature:
21 -22°C
pH:
7.0 – 7.8
Dissolved oxygen:
not specified
Salinity:
not applicable
Conductivity:
test solutions (at start of the test): 94 - 110 µS/cm
Nominal and measured concentrations:
nominal: 0 (negative control), 0.41, 1.02, 2.56, 6.4, 16, 40, 100 mg/L
Tungsten (as proxy), measured arithmetric mean concentrations (standard deviation, SD): N/A (negative control), 0.3 (SD: 0.02), 0.6 (SD: 0.03), 1.7 (SD: 0.03), 4.0 (SD: 0.14), 9.5 (SD: 0.86), 19.6 (SD: 1.49), 47.2 (SD: 1.30) mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed, covered with a foam stopper
- Material, size, headspace, fill volume: 250 mL nominal volume, test volume 50 mL
- Aeration: mixed on a horizontal shaker at approximately 75 rpm
- Initial cells density: 0.99 x 104 cells/mL
- No. of vessels per concentration (replicates): 3 replicates for each testconcentration
- No. of vessels per control (replicates): six replicates for negative control
- No. of vessels per vehicle control (replicates): none

GROWTH MEDIUM
- Standard medium used: no; similar to AAP Medium (US EPA)
- Detailed composition if non-standard medium was used: sterile nutrient growth media

Nutrient Final Concentration (µg/L)
NaNO3 25,500
MgCl2*6H2O 10,000
CaCl2*2H2O 4,420
H3BO3 186
MnCl2*4H2O 416
ZnCl2 3.28
CoCl2*6H2O 1.43
CuCl2*2H2O 0.012
Na2MoO4*2H2O 7.26
FeCl3*6H2O 160
Na2EDTA*2H20 300a / 75b
MgSO4*7H20 14,700
K2HPO4 1,044
NaHCO3 15,000

a Concentration used for culturing; b Concentration used for reduced-EDTA nutrient media (testing only)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water (Millipore)
- Particulate matter: none
- Culture medium different from test medium: yes, as the test item contains metals, the nutrient media used for testing was prepared with a reduced amount of the chelating agent Na2EDTA•2H20, compared to the amount in the nutrient media used for culturing. Reducing the concentration of Na2EDTA•2H20 from 300 µg/L to 75 µg/L allows for increased bioavailability of the test item.
- Intervals of water quality measurement: pH of the test solutions at test initiation and at test completion; conductivity measured at test initiation only; temperature of the test chamber: monitored daily

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no, pH of the nutrient medium was 7.6 and no additional pH adjustment was needed
- Photoperiod: continuous (24-h light)
- Light intensity and quality: 5797 - 6325 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cellometer X4HM Auto counter
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Range finding study: yes (non-GLP)
- Test concentrations: 0 (negative control), 0.1, 1.0, 10, 100 mg/L in nominal
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
zinc sulphate heptahydrate (CAS no. 7446-20-0)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Confidence Limits: 1.9, 13.9 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Confidence Limits: 1.1, 1.7 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Confidence Limits: 0.7, 1.2 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Confidence Limits: 0.7, 1.7 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Confidence Limits: 0.5, 0.8 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Confidence Limits: 0.4, 0.6 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: yes; at test completion (~72 hours), the negative control and 0.41 mg/L test solutions appeared green, the 1.02 mg/L test solutions appeared light green, and the 2.56 mg/L test solutions appeared very light green. The 6.4 mg/L to 100 mg/L test solutions all appeared clear and colourless.

Other:
- Any stimulation of growth found in any treatment: Yes, yield was stimulated at the lowest nominal test item concentration of 0.41 mg/L
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
72-hour EC50 = 0.079 mg/L Zn2+ (95% confidence interval: 0.063 - 0.098 mg/L)
Reported statistics and error estimates:
The mean alga cell concentration for the control and each test item concentration was graphically plotted against time using Microsoft Excel to examine the effect of the test item on the growth curves. The NOEC and LOEC values for cell yield and SGR were calculated using Dunnett Multiple Comparison Test. All statistical analyses and comparisons were performed against the negative control. The 72-h EC50, EC20, EC10 along with their 95% confidence limits (based on nominal concentrations) and the NOEC and LOEC values for growth inhibition were determined using a computerised statistical package, CETIS™ (Version 1.9.2.4), a Comprehensive Environmental Toxicity Information System, recommended by Environment Canada. All reported values were rounded.

Table 1: Summary of mean cell counts in each 24 -hour period

Nominal Concentration of Test Item
(mg/L)

Mean Cell Counts (x104cells/mL)

0-h

24-h

48-h

72-h

Negative Control (0)

0.99

2.7

16.2

72.9

0.41

0.99

2.6

17.8

80.7

1.02

0.99

3.1

14.3

43.9

2.56

0.99

3.7

6.7

15.6

6.4

0.99

2.5

4.3

9.9

16

0.99

3.8

3.3

6.0

40

0.99

2.8

3.6

5.6

100

0.99

2.4

5.4

5.6

Table 2: Summary of the mean specific growth rate over time

Nominal Concentration of Test Item (mg/L)

 Mean Specific Growth Rate Over Time (day^-1)

Negative Control (0)

1.43

0.41

1.46

1.02

1.26

2.56

0.92

6.4

0.76

16

0.60

40

0.54

100

0.58

Table 3: Summary of the mean inhibition (%) in cell yield and specific growth rate

Nominal Concentration of Test Item (mg/L)

Mean Inhibition In Yield (%)

Mean Inhibition In Specific Growth Rate (%)

Negative Control (0)

N/A

N/A

0.41

-10.9

-2.35

1.02

40.3

11.78

2.56

79.7

35.79

6.4

87.5

47.18

16

93.1

58.16

40

93.6

62.07

100

93.6

59.65

Note: a negative value represents stimulation when compared to the control

Table 4: Summary of Test Validity Criteria Results

Validity Criteria

Results at 72-h

The biomass in the control cultures must increase by a factor of at least 16 over the course of the 72 hour test period

The biomass in the control increased by a factor of 74

The coefficient of variation of average specific growth rates over the test period in replicate control cultures must not exceed 7%

The %CV of mean SGR in the control was 3.06%

The mean coefficient of variation (%CV) for the specific growth rates of each time period (days 0-1, 1-2, and 2-3) in the control cultures must not exceed 35%

The highest mean %CV in the control was 12.90%

Table 5: Measured concentrations of Tungsten (proxy for the test item) in test solutions

Nominal concentration of test item [mg/L] Tungsten [mg/L]
Initial Final Mean SD
Negative control (0) BQL BQL N/A N/A
0.41 0.3 0.2 0.3 0.02
1.02 0.7 0.6 0.6 0.03
2.56 1.7 1.6 1.7 0.03
6.4 3.9 4.1 4.0 0.14
16 8.8 10.1 9.5 0.86
40 18.6 20.7 19.6 1.49
100 46.3 48.2 47.2 1.30

BQL: Below Quantitation Limit (<0.002 mg/L)

N/A: Not Applicable

Validity criteria fulfilled:
yes

Description of key information

The 72-hour EC10 and EC50 (growth rate) of the test item was determined to be 0.9 mg/L and 7.8 mg/L (based on nominal concentrations), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
7.8 mg/L
EC10 or NOEC for freshwater algae:
0.9 mg/L

Additional information

The test item was assessed for its potential to cause effects on the growth of the green algae, Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata as cited in the study and Selenastrum capricornutum), using the procedures outlined in OECD Guideline 201 (2011). The effects were followed by observing the growth of test organisms over a 72-hour exposure period, under controlled conditions, using the following nominal concentrations: 0.41, 1.02, 2.56, 6.4, 16, 40, and 100 mg test item/L, along with a negative control (0 mg/L) and a positive control with the reference substance zinc sulphate heptahydrate (CAS no. 7446-20-0; tested in a separate study). Tungsten was analysed as a proxy for the test item. Concentration of Tungsten in test solutions was consistent between test initiation and test completion, with values within ± 20% of the mean Tungsten concentration for each nominal test item concentration. Therefore, the biological test endpoints were calculated using the nominal concentrations of the test item. The 72-hour EC50, EC20, and EC10 with 95% confidence limits and NOEC/LOEC for cell yield and specific growth rate are based on the nominal concentrations of the test item. In result, the 72-hour EC10 and EC50 (with confidence limits) for the cell yield was determined to be 0.5 (0.4, 0.6) mg/L and 1.2 (0.7, 1.7) mg/L, respectively. For the specific growth rate, the 72-hour EC10 and EC50 of the test item was determined to be 0.9 (0.7, 1.2) mg/L and 7.8 (1.9, 13.9) mg/L, respectively. The 72-hour NOEC and LOEC for the cell yield was established to be 0.41 and 1.02 mg/L, respectively. For the specific growth rate, the NOEC and LOEC was determined to be 1.02 and 2.56 mg/L, respectively. All validity criteria of the test guideline were fulfilled.