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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:
9 August 2012 to 14 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
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:
Samples were taken from the control and the 100 % v/v saturated solution test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis. All 0-Hour samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 ºC for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Due to the poor solubility of the test material, a modification of the standard method for the preparation of aqueous media was performed, exposing organisms to a saturated solution.
A saturated solution was prepared by adding 550 mg test material to 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a 100 % v/v saturated solution. An aliquot (1 litre.) of each of the saturated solution was inoculated with 6.2 mL of algal suspension to give the required test concentrations of 100 % v/v saturated solution.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

50 mg/L was shown to be a high enough concentration to create an excess of the test material in the saturated solution.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Master cultures: 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 10^3 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
150 mg CaCO3/L
Test temperature:
24 ± 1 °C
pH:
7.6 - 7.8
Nominal and measured concentrations:
Nominal concentration: 100 % v/v saturated solution.
Measured concentration: Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of 0.0053 and 0.0052 mg Er/L respectively, equivalent to a test concentration of 0.0060 mg test material/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks.
- Type (delete if not applicable): closed (with polyurethane foam stoppers).
- Fill volume: 100 mL
- Aeration: vessels were constantly shaken at approximately 150 rpm throughout the exposure period.
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 8.04 x 10^5 cells per mL. Inoculation of 1 litre of test medium with 6.2 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. For the purposes of the second range-finding test and the definitive test, the culture medium was prepared with an additional 198.4 mg CaCl2.2H2O/L in order to increase the hardness to approximately 150 mg CaCO3/L. Such a modification of the culture medium allows avoiding the complexing effects of the test item and prevents a complexation-induced nutrient limitation which could impair the algal growth (as observed in the first range-finding test).
- Detailed composition if non-standard medium was used: the culture medium was composed of the following:
NaNO3 (25.5 mg/L), MgCl2.6H2O (12.16 mg/L), CaCl2.2H2O (4.41 mg/L), MgSO4.7H2O (14.6 mg/L), K2HPO4 (1.044 mg/L), NaHCO3 (15.0 mg/L), H3BO3 (0.186 mg/L), MnCl2.4H2O (0.415 mg/L), ZnCl2 (0.00327 mg/L), FeCl3.6H2O (0.160 mg/L), CoCl2.6H2O (0.00143 mg/L), Na2MoO4.2H2O (0.00726 mg/L), CuCl2.2H2O (0.000012 mg/L) and Na2EDTA.2H2O (0.30 mg/L)
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Dilution water: Elga Optima 15+ or Elga Purelab Option R-15 BP
- Intervals of water quality measurement: The pH of the control and 100% v/v saturated solution 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
- Photoperiod: continuous illumination.
- 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: Yes
- Test concentrations: two range-finding studies were conducted at test concentrations of 0, 1.0, 10 and 100 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: The results of the second range-finding test showed no significant effect on growth rate at the test concentrations of 1.0, 10 and 100% v/v saturated solution and so the definitive study was conducted using a 100 % v/v saturated solution.

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 at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v saturated
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Growth and Yield Data
The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test material over the 72-hour exposure period.
Mean cell densities vs. time can be seen in Figure 1.

- 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 cultures, however some misshapen cells were observed to be present in the 100 % v/v saturated solution test cultures.

- Observations on Test Material Solubility
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-hour test period all control and test cultures were observed to be green dispersions.

- Analytical Results
The results obtained for the concentrations of erbium found in the test samples are presented in Table 4.
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item, potassium dichromate, gave the following results:

ErC50 (0 – 72 h) : 1.1 mg/L; 95 % confidence limits 1.0 – 1.3 mg/L
EyC50 (0 – 72 h) : 0.70 mg/L*

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

* It was not possible to calculate 95% confidence limits for the EyC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and 100 % v/v saturated solution test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance.

There were no statistically significant differences (P ≥ 0.05), between the control and 100 % v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on both growth rate, and yield, was 100 % v/v saturated solution.

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

5.83^3

1.38^6

-

-

1.0

5.45^3

1.25^6

0

9

10

4.77^3

1.17^6

0

15

100

4.70^3

5.22^5

14

62

 * Cell densities represent the mean number of cells per mL calculated based on three counts performed of each of the duplicate test flasks.

 

Table 2: Cell Densities and Percentage Inhibition of Growth from the Second Range-Finding Test

Nominal conc. (% v/v saturated solution)

Cell densities* (cells/mL)

Inhibition values (%)

0 hours

72 hours

Growth rate

Yield

Control

5.66^3

2.99^5

-

-

1.0

4.04^3

5.23^5

[24]

[77]

10

4.26^3

5.23^5

[22]

[77]

100

4.21^3

2.17^5

0

27

* Cell densities represent the mean number of cells per mL calculated based on three counts performed of each of the duplicate test flasks.

[Increase in growth compared to controls]

Table 3: Cell Densities and Percentage Inhibition of Growth from the Definitive Test

Nominal conc. (% v/v saturated solution)

Cell densities* (cells/mL)

Inhibition values (%)

0 hours

24 hours

48 hours

72 hours

Growth rate

Yield

Control

4.06^3

1.82^4

6.71^4

3.87^5

-

-

100

3.75^3

1.64^4

6.81^4

4.09^5

0

[6]

*Cell densities represent the mean number of cells per mLcalculated based on counts from six replicate test flasks.

 [Increase in growth compared to controls] Table 4: Analytical Test Results

Time Point (hours)

Nominal Conc. of Test Item in Test Sample (% v/v saturated solution)

Measured Concentration of Er in Sample Vial (mg/L)

Sample Preparation Factor

Determined Concentration of Er in Test Sample (mg/L)

Determined Concentration of Test Item in Test Sample (mg/L)

0

Control

<LOQ

1.02

<LOQ

<LOQ

100

0.00517

1.02

0.00527

0.00602

72

Control

<LOQ

1.02

<LOQ

<LOQ

100

0.00514

1.02

0.00524

0.00599

LOQ = limit of quantitation

 

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata was investigated and gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution. This study showed that there were no toxic effects at saturation.
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.

Information indicating the test material was insoluble in water instigated pre-study solubility work. The solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation. Given the low water solubility and high purity of the test material, the use of a saturated solution method of preparation was considered most appropriate.

Following preliminary range-finding tests the algae, Pseudokirchneriella subcapitata was exposed to a solution of the test material at a concentration of 100 % v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material gave EC50 values of greater than 100 % v/v saturated solution and No Observed Effect Concentration of 100% v/v saturated solution, based on growth rate and yield.

Analysis of the test preparations at 0 and 72 hours showed measured erbium concentrations of 0.0053 and 0.0052 mg/L respectively were obtained, equivalent to a test material concentration of 0.0060 mg/L. This study showed that there were no toxic effects at saturation.

Description of key information

The 72 hour ErC50 was greater than 100 % v/v saturated solution. The No Observed Effect Concentration was superior or equal to 100 % v/v saturated solution.

Key value for chemical safety assessment

Additional information

In the key study, 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 line with the criteria set forth by Klimisch (1997).

Information indicating the test material was insoluble in water instigated pre-study solubility work. The solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation. Given the low water solubility and high purity of the test material, the use of a saturated solution method of preparation was considered most appropriate.

Following preliminary range-finding tests the algae, Pseudokirchneriella subcapitata was exposed to a solution of the test material at a concentration of 100 % v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solution was prepared by stirring an excess (50 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a 100 % v/v saturated solution of the test material. 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 erbium concentrations of 0.0053 and 0.0052 mg/L respectively were obtained, equivalent to a test material concentration of 0.0060 mg/L.

Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material gave an ErC50 value of greater than 100 % v/v saturated solution and a NOEC of 100% v/v saturated solution. Similar results were seen for both growth rate and yield. In conclusion no toxic effects were seen in freshwater algae as a result of exposure to the test material.

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