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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:
23 February 2017 - 08 September 2017
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
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
not specified
Details on test solutions:
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions

Preliminary Media Preparation Trial
Saturated Solution Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)

Examination of the centrifuged test samples showed that tiny particles of undissolved test item remained, as such the use of centrifugation was considered inappropriate for this test item. There was no significant increase in the dissolved test item concentration obtained from the filtered test samples when the preparation period was extended beyond 24 hours.
Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a nominal test concentration of approximately 0.23 mg/L. A reduction in the initial loading rate of the saturated solution to 50 mg/L would still ensure an excess of test item was present to allow complete saturation of the test media.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °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 104 - 105 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Nominal and measured concentrations:
The geometric mean measured test concentrations were determined to be 0.018, 0.048, 0.075, 0.17 and 0.30 mg/L.
Details on test conditions:
Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.23 mg/L could be obtained using a saturated solution method of preparation.
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 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.
A nominal amount of test item (550 mg) was dispersed in 11 liters 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 item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (1.3 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
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 control group was maintained under identical conditions but not exposed to the test item.
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 (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) 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.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive Test
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.


Experimental Preparation
A nominal amount of test item (550 mg) was dispersed in 11 liters 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 item was removed by filtration through a 0.2 µm Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 7.5 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
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 were verified by chemical analysis at 0 and 72 hours

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.33 x 105 cells per mL. Inoculation of 500 mL of test medium with 7.5 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 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 72 hours.


Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.
The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced 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 10 and 100% v/v saturated solution test preparations at 0 hours (see Annex 5) showed measured test concentrations of 0.0087 and 0.34 mg/L respectively were obtained. A decline in measured test concentration occurred at 72 hours to between 0.0020 and 0.28 mg/L indicating that the test item was unstable under the conditions of the test.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations at 0 hours (see Annex 5) showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.036 mg/L, to 0.42 mg/L. A decline in measured test concentration was observed at 72 hours to between less than the LOQ and 0.21 mg/L (42% to 62% of the 0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.018, 0.048, 0.075, 0.17 and 0.30 mg/L.

Growth Data
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3.
From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 - 72 h): 0.062 mg/L
ErC20 (0 - 72 h): 0.15 mg/L
ErC50 (0 - 72 h): >0.30 mg/L

Where ErCx is the test concentration that reduced growth rate by x%.
It was not possible to calculate an ErC50 value as no more than 32% inhibition of growth rate was observed at the maximum attainable dissolved test item concentration.
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 (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 and 0.018 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 0.018 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.048 mg/L.

6.2.4 Inhibition of Yield
EyC10 (0 - 72 h): 0.028 mg/L
EyC20 (0 - 72 h): 0.040 mg/L
EyC50 (0 - 72 h): 0.077 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences between the control and 0.018 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 0.018 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.048 mg/L.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Water Quality Criteria
The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.6 at 0 hours to pH 7.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the
Test Guidelines.

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 72-Hour test period all control, 0.018 and 0.048 mg/L test cultures were observed to be green dispersions. The 0.075 mg/L test cultures were observed to be pale green dispersions whilst the 0.17 and 0.30 mg/L test cultures were observed to be very pale green dispersions.



Results with reference substance (positive control):
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 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 (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) 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 (SAS, 1999 - 2001).

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

Growth Rate
EC50->0.30 mg/L
NOEC-0.018 mg/L
LOEC-0.048mg/L

Yield
EC50->0.077 mg/L
NOEC-0.018 mg/L
LOEC-0.048mg/L
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.23 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding testPseudokirchneriella subcapitatawas exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, 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 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. 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

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.036 mg/L, to 0.42 mg/L. A decline in measured test concentration was observed at 72 hours to between less than the LOQ and 0.21 mg/L (42% to 62% of the        0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.018, 0.048, 0.075, 0.17 and 0.30 mg/L.

Exposure ofPseudokirchneriella subcapitatato the test item gave the following results based on the geometric mean measured test concentrations:

Response Variable

EC50(mg/L)

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

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

Growth Rate

>0.30*

0.018

0.048

Yield

0.077

0.018

0.048


*It was not possible to calculate an ErC50value as no more than 32% inhibition of growth rate was observed at the maximum attainable dissolved test item concentration.

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

Growth Rate

EC50->0.30 mg/L

NOEC-0.018 mg/L

LOEC-0.048mg/L

Yield

EC50->0.077 mg/L

NOEC-0.018 mg/L

LOEC-0.048mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
0.3 mg/L
EC50 for marine water algae:
0.3 mg/L
EC10 or NOEC for freshwater algae:
0.018 mg/L
EC10 or NOEC for marine water algae:
0.018 mg/L

Additional information