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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:
28 September 2012 to 9 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled).
- Sampling method: Samples were taken at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: All samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 °C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
50 mg of test material was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made. An aliquot of each of the stock solutions was separately inoculated with algal suspension to give the required test concentrations.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10³ cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.

ACCLIMATION
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
7.4 - 8.2
Nominal and measured concentrations:
0.10, 0.32, 1.0, 3.2, 10 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed (fasks were plugged with polyurethane foam bungs)
- Fill volume: 100 mL
- Aeration: flasks were constantly shaken at aproximately 150 rpm
- Initial cells density: 5 x 10³ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes. NaNO3 25.5 mg/L, MgCl2.2H2O 12.164 mg/L, CaCl2.2H2O 4.41 mg/L, MgSO4.7H2O 14.7 mg/L, K2HPO4 1.044 mg/L, NaHCO3 15.0 mg/L, H3BO3 0.1855 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCL2 0.00327 mg/L, FeCl3.6H2O 0.159 mg/L, CoCl2.6H2O 0.00143 mg/L, Na2MoO4.2H2O 0.00726 mg/L, CuCl2.2H2O 0.000012 mg/L and Na2EDTA 0.30 mg/L prepared in reverse osmosis purified water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: approximately 7000 lux (provided by warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Range finding study
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test material was dissolved directly in culture medium. The control group was maintained under identical conditions but not exposed to the test material. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated at 24 ± 1 ºC under continuous illumination and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
- Test concentrations: 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10 mg/L. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L.
Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

EVALUATION OF DATA
- Comparison of Growth Rates
The average specific growth rate for a specified period was calculated as the logarithmic increase in biomass from the equation:

µ = (ln Nn - ln N1) / (tn - t1)

where
μ = average specific growth rate from time t1 to tn (cells/mL/hour)
N1 = cell concentration at t1 (cells/mL)
Nn = cell concentration at tn (cells/mL)
t1 = time of first measurement (hours)
tn = time of nth measurement (hours)

The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

Ir = [(µc - µt) / µc] x 100

where
Ir = percentage inhibition of average specific growth rate
μc = mean average specific growth rate for the control cultures (cells/mL/hour)
μt = average specific growth rate for the test culture (cells/mL/hour)

- Comparison of Yield
Yield was calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn - N0

where
Y = yield (cells/mL)
N0 = cell concentration at the start of the test (cells/mL at t0)
Nn = cell concentration at the end of the test (cells/mL at tn)

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = [(Yc - Yt) / Yc] x 100

where
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group (cells/mL)
Yt = mean value for yield for the treatment group (cells/mL)
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.85 - 1.5 mg/L (95 % CL)
Key result
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:
LOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were affected by the presence of the test material over the 72-hour exposure period.

- Growth data
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant differences between the control and 0.10 mg/L test concentration (P ≥ 0.05), however, all other test concentrations were significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.32 mg/L.

- Yield data
There were no statistically significant differences between the control and 0.10 mg/L test concentration (P ≥ 0.05), however, all other test concentrations were significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.32 mg/L.

- Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.10 and 0.32 mg/L, however cell debris was observed to be present in the test cultures at 1.0, 3.2 and 10 mg/L.

- Observations on Test Material Solubility
At the start of the test all control cultures were observed to be clear colourless solutions. The test cultures were observed to range from very pale yellow solutions at 0.10 mg/L through to pale yellow solutions at 10 mg/L. After the 72-hour test period all control, 0.10 and 0.32 mg/L test cultures were observed to be pale green dispersions whilst the 1.0, 3.2 and 10 mg/L test cultures were observed to be very pale yellow solutions.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72 hour EC50 for the growth rate = 1.1 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package.

Table 1: Cell Densities and Percentage Inhibition of Growth from the Initial Range-Finding Test

Nominal conc. (% v/v saturated solution)

Cell densities* (cells/mL)

Inhibition values (%)

0 hours

72 hours

Growth rate

Yield

Control

4.57E+03

5.95E+05

-

-

0.10

4.02E+03

5.39E+05

0

9

1.0

4.25E+03

7.98E+04

40

87

10

4.12E+03

8.55E+03

85

99

100

3.62E+03

3.36E+03

101

100

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

Table 2: Cell Densities and Percentage Inhibition of Growth from the Definitive Test

Nominal conc. (% v/v saturated solution)

Cell densities* (cells/mL)

Growth rate

0 hours

24 hours

48 hours

72 hours

0- 72 hours

% inhibition

Control

5.17E+03

2.62E+04

1.55 E+05

8.20 E+05

0.071

-

0.10

5.11 E+03

3.00 E+04

1.79 E+05

7.69 E+05

0.070

1

0.32

5.04 E+03

2.79 E+04

1.35 E+05

5.62 E+05

0.066

8

1.0

5.11 E+03

1.46 E+04

3.13 E+04

6.88 E+04

0.036

49

3.2

5.03 E+03

6.42 E+03

1.50 E+04

1.65 E+04

0.016

77

10

5.39 E+03

3.40 E+03

4.66 E+03

2.67 E+03

-0.009

113

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 72 hour EC50, based on growth rate, was determined to be 1.1 mg/L. The No Observed Effect Concentration, based on growth rate, was determined to be 0.10 mg/L.
Executive summary:

The toxicity of the test material to aquatic algae was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 201 and EU Method C.3.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89 to 96 % of nominal and so the results are based on nominal test concentrations only.

Under the conditions of the study, the 72 hour EC50, based on growth rate, was determined to be 1.1 mg/L. The No Observed Effect Concentration, based on growth rate, was determined to be 0.10 mg/L.

Description of key information

The 72-hour EC50 for the test material to Pseudokirchneriella subcapitata, based on growth rate, was 1.1 mg/L (95 % C.I. 0.85 - 1.5 mg/L)

Key value for chemical safety assessment

EC50 for freshwater algae:
1.1 mg/L
EC10 or NOEC for freshwater algae:
0.1 mg/L

Additional information

The toxicity of the test material to aquatic algae was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 201 and EU Method C.3. The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as set forth by Klimisch et al (1997).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89 to 96 % of nominal and so the results are based on nominal test concentrations only.

Under the conditions of the study, the 72 hour EC50, based on growth rate, was determined to be 1.1 mg/L. The No Observed Effect Concentration, based on growth rate, was determined to be 0.10 mg/L.