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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:
Experimental Starting Date: 16 May 2012. Experimental Completion Date: 5 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19-21 July 2011. Date of signature: 31 August 2011
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control (replicates R1 – R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 96 hours for quantitative analysis. All samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 96 hours and stored at approximately 20 ºC for further analysis if necessary.

Additional samples of each test concentration were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24, 48 and 72 hours.

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Saturated solutions were prepared by adding test material to culture medium, stirring with the aid of propeller at approximately 1500 rpm for 24 hours, followed by filtration of undissolved material through a 0.2 µm Sartorius Sartopore filter to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions. 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.

The concentration and stability of the test item in the test preparations verified by chemical analysis at 0, 24, 48, 72 and 96 hours.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source: Obtained from the 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.
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24 ± 1 ºC
Details on test conditions:
TEST SYSTEM
250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of between 6.66 x 10^5 and 8.23 x 10^5 cells per mL. Inoculation of 1 litre of test medium with between 12.2 and 15 mL of this algal suspension gave an initial nominal cell density of 1 x 10^4 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 96 hours.

GROWTH MEDIUM
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Samples were taken at 0, 24, 48, 72 and 96 hours
- Determination of cell concentrations: Cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution for a period of 96 hours.

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of confidence limits
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
7.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It was not possible to calculate 95% confidence limits for the ErC50 values as the data generated did not fit the models available for the calculation of confidence limits
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Range finding test:
No effects on growth were found in the 1.0 and 10% v/v saturated solutions. However, growth was observed to be reduced (100 % inhibition) at 100% v/v saturated solution.

Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.

Chemical analysis of the 1.0, 10 and 100% v/v saturated solutions at 0 hours showed concentrations of 0.23, 1.7 and 18 mg/L, respectively. A decline in measured test concentration was observed at 96 hours in the range of 0.14 to 0.15 mg/L.

Definitive test:
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.72 to 16 mg/L. At 24 hours concentrations decline to <0.087 mg/L to 11.6 mg/L, at 48 hours <8.3 mg/L, at 72 hours to <0.087 mg/L to 2.3 mg/L and at 96 hours to <0.087 mg/L for all of the test treatments.

Time-weighted mean measured test concentrations were calculated and used to determine effect concentrations. In cases where the measured concentration was less than the LOQ of the analytical method a value of half the LOQ (i.e. 0.044 mg/L) was used. The time-weighted mean measured test concentrations were 0.11, 0.12, 0.23, 0.68 and 7.2 mg/L for the 6.25, 12.5, 25, 50 and 100 % v/v saturated solutions.

Observations on cultures:
All test and control cultures were inspected microscopically at 96 hours. After 96 hours there were no abnormalities detected in the control or test cultures at 0.11, 0.12, 0.23 and 0.68 mg/L, however cell debris was observed to be present in the test cultures at 7.2 mg/L.

Observations on test item solubility:
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 96-Hour test period all control, 0.11, 0.12 and 0.23 mg/L test cultures were observed to be pale green dispersions. The 0.68 mg/L test cultures were observed to be very pale green dispersions whilst the 7.2 mg/L test cultures were observed to be slightly hazy dispersions.

Physico-chemical measurements:
The pH value of the control cultures was observed to range from pH 7.8 at 0 hours to pH 7.6 at 96 hours. The pH deviation in the control cultures was less than 1.5 pH units after 96 hours and therefore was within the limits given in the Test Guidelines

Re-growth test:
Re-growth was observed to have occurred in the control and all test cultures after 72 hours. These results indicate that the test item was algistatic in effect.


Reported statistics and error estimates:
Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 0.11 and 0.12 mg/L test concentrations (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 0.12 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.23 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The 72 and 96 h EC50 (mg/L) for growth rate was 6.7 and 7.6 mg/L respectively. The 72 h and 96 h NOEC based on growth rate was determined to be 0.12 mg/L and the LOEC was 0.23 mg/L.

Description of key information

Key study performed according to GLP and OECD Guideline 201:

The 72 and 96 h EC50 (mg/L) for growth rate was 6.7 and 7.6 mg/L respectively. The 72 h and 96 h NOEC based on growth rate was determined to be 0.12 mg/L and the LOEC was 0.23 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
7.6 mg/L
EC10 or NOEC for freshwater algae:
0.12 mg/L

Additional information

Introduction

A study was performed to assess the effect 9-decenoic acid, methyl ester (purity 99 %) on the growth of the green algae Psuedokirchneriell subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” references as Method C.3 of Commission Regulation (EC) No 761/ 2009 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400.

 

Methods

In the range-finding test, Pseudokirchneriella subcapitata was exposed to a series of nominal test concentrations 1.0, 10 and 100 % v/v saturated solutions at a temperature of 24 ± 1 °C under constant illumination for a period of 96 h. After 96 h the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Following the preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at 0-Hour measured concentrations of 0.72, 1.4, 3.5, 7.5 and 16 mg/L (three replicate flasks per concentration) for 96 h, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 h. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, with the first approximate 1 L discarded in order to pre-condition the filter) to produce a saturated solution of the test item with a 0-Hour measured concentration of 16 mg/L. Media preparation trials conducted using a similar method of preparation indicated a water solubility value of approximately 22 mg/L when using this method of preparation. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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

 

Results

In the range-finding test, there was no effect on growth at the test concentrations of 1.0 and 10 % v/v saturated solution. However, growth was observed to be reduced at 100 % v/v saturated solution. Therefore, test concentrations of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution were selected for the definitive test.

In the definitive test, chemical analysis of the test preparations at 0 h showed measured test concentrations to range from 0.72 to 16 mg/L. A decline in measured test concentration was observed at 24 h in the range of less than the limit of quantitation (LOQ, 0.087 mg/L) of the analytical method employed, to 11.6 mg/L, at 48 h in the range of less than the LOQ to 8.3 mg/L, at 72 h in the range of less than the LOQ to 2.3 mg/L and at 96 h to less than the LOQ for all test concentrations.

Where a measured concentration of less than the limit of quantitation (LOQ) of the analytical method was determined, a value of ½ the LOQ was substituted into the equation:

Area= ((C0.C1)/(ln(C0).ln(C1))) x days

TWM= Total area/ Total number of days of the test

Where Area= area under the exponential curve for each renewal period, C0= measured concentration at the start of each renewal period (mg/L), C1= measured concentration at the end of each renewal period (mg/L), Days= number of days in the renewal period, TWM= time-weighted mean measured test concentration (mg/L) .

Exposure o f Pseudokirchneriella subcapitata to the test item gave the following results based on the time-weighted mean measured test concentrations:

Time Point (Hours)

Response Variable

EC50(mg/L)

95 % Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

72

Growth rate

6.7

*

0.12

0.23

Yield

0.60

0.48 - 0.74

0.12

0.23

Biomass

0.59

0.49 – 0.71

0.12

0.23

96

Growth rate

7.6

*

0.12

0.23

Yield

0.41

0.34 – 0.51

0.12

0.23

Biomass

0.52

0.42 – 0.64

0.12

0.23

*it was not possible to calculate 95 % confidence limits for the ErC50values as the data generated did not fit the models available for the calculation of confidence limits.

A re-growth test performed which showed the test item to be algistatic in effect.