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Ecotoxicological information

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:
The study was conducted between 05 November 2018 and 19 December 2018.
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
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Batch: Y180510A
Purity: 95.0%
Physical State/Appearance: Black powder
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and the 100 mg/L loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. A set of duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Experimental Design and Study Conduct
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.

Range-Finding Test
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 1.0, 10 and 100 mg/L for a period of 72 hours.
Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 liters of culture medium to give the 1.0, 10 and 100 mg/L loading rates respectively.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.0 mL) to give the required test concentrations of 1.0, 10 and 100 mg/L loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed.
A nominal amount of test item (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate.
An aliquot (1 liter) of the WAF was inoculated with algal suspension (8.6 mL) to give the required test concentration of 100 mg/L loading rate WAF.

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. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test.

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

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
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 type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ±1 °C
pH:
The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.9 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.
Nominal and measured concentrations:
Range-Finding Test
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 1.0, 10 and 100 mg/L for a period of 72 hours.
Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 liters of culture medium to give the 1.0, 10 and 100 mg/L loading rates respectively.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.0 mL) to give the required test concentrations of 1.0, 10 and 100 mg/L loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed.
A nominal amount of test item (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate.
An aliquot (1 liter) of the WAF was inoculated with algal suspension (8.6 mL) to give the required test concentration of 100 mg/L loading rate WAF.
Details on test conditions:
Experimental Design and Study Conduct
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.

Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of the marker component silicon in the WAF.

Range-Finding Test
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.

The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 1.0, 10 and 100 mg/L for a period of 72 hours.

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 prepared for each control and test concentration.

Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 liters of culture medium to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for
1-Hour. Microscopic observations made on the 1.0 mg/L loading rate WAF showed there to be no micro-dispersions of test item present, however, visual inspection of the 10 and
100 mg/L loading rate WAFs showed there to be dispersed test item present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Further filtration through one sheet of filter paper was employed to ensure all undissolved test item was removed. Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions of test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.0 mL) to give the required test concentrations of 1.0, 10 and 100 mg/L loading rate WAF.

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 to 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 loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis. Only the concentration to be used for the definitive test was analyzed.

Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed.

Experimental Preparation
A nominal amount of test item (200 mg) was added to the surface of 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixture allowed to stand for 1-Hour. Visual observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded). Further filtration through one sheet of filter paper was employed to ensure all undissolved test item was removed. Microscopic observations of the WAF were performed after filtering and showed no micro-dispersions of test item to be present.
An aliquot (1 liter) of the WAF was inoculated with algal suspension (8.6 mL) to give the required test concentration of 100 mg/L loading rate WAF.

The concentration and stability of the marker component silicon 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 test preparation were used for the control and 100 mg/L loading rate WAF 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 5.81 x 105 cells per mL. Inoculation of 1 liter of test medium with 8.6 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 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Assessments
Test Organism Observations
Samples were taken at 25, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria
The pH of the control and 100 mg/L loading rate WAF was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.

The appearance of the test media was recorded daily.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.

Chemical Analysis of Test Loading Rates
Samples were taken from the control and the 100 mg/L loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. A set of duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Remarks:
ErL50
Effect conc.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Remarks:
EyL50
Effect conc.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of yield
Details on results:
Validation of Mixing Period
Preliminary investigational work indicated that there was a significant increase in the amount of silicon present in the WAF when the preparation period was extended from 24 to 96 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period.

Range-finding Test
The results showed no effect on growth at 1.0, 10 and 100 mg/L loading rate WAF.
Based on this information a single loading rate of six replicates of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.
Whilst samples were taken for analysis from the range-finding test, the results are not reported due to the failure of the corresponding procedural recoveries that were analyzed concurrently. The procedural recoveries were outside the acceptable range of 80-120%.

Definitive Test
Chemical Analysis of Test Loading Rates
Chemical analysis of the test preparations was conducted using inductively coupled plasma with mass spectrometric detection (ICP-MS) for the presence of silicon in solution.

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured silicon concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.051 mg/L. This does not infer that no silicon was present in solution, just that the levels of silicon were below the quantifiable level.

As the measured concentration obtained from the definitive test was significantly below that obtained from the validation of mixing period trial, it was considered appropriate to analyze the duplicate test samples in order to confirm the levels of silicon present. Analysis of these additional test samples also showed measured concentrations of silicon to be less than the LOQ.

Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Growth Data
From the data given, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErL10 (0 to 72 hour) : >100 mg/L loading rate WAF
ErL20 (0 to 72 hour) : >100 mg/L loading rate WAF
ErL50 (0 to 72 hour) : >100 mg/L loading rate WAF

Where ErLx is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant decreases in growth rate (P≥0.05), between the control and 100 mg/L loading rate WAF test group and therefore the NOEL based on growth rate was 100 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 to 72 hour) : >100 mg/L loading rate WAF
EyL20 (0 to 72 hour) : >100 mg/L loading rate WAF
EyL50 (0 to 72 hour) : >100 mg/L loading rate WAF

Where EyLx is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out. There were no statistically significant decreases in yield between the control and 100 mg/L loading rate WAF test group (P≥0.05) and therefore the NOEL based on yield was 100 mg/L loading rate WAF.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 308 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 1.54 x 10^6 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

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 value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.9 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.

Temperature was maintained at 24 ±1 ºC throughout the test.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAF.

At the start of the mixing period the 100 mg/L preparation was observed to have formed a clear colorless media column with test item floating at the media surface. After 95 hours stirring the WAF was observed to have formed a hazy media column with test item floating at the media surface, dispersed throughout and settled on the bottom of the mixing vessel. After a 1-Hour standing period a slightly hazy media column with test item floating at the media surface, dispersed throughout and settled on the bottom of the mixing vessel was observed.

As visual observations made showed dispersed test item to be present the aqueous phase was removed by filtration through a glass wool plug and one sheet of filter paper to remove as much undissolved test item as possible. Microscopic inspection of the WAF after filtration showed no micro-dispersions of test item to be present.

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 and test cultures were observed to be green dispersions, with the exception of control replicate R4 which was observed to be an extremely pale green dispersion.
Results with reference substance (positive control):
A positive control (Envigo study number YJ31TQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 12 November 2018 and 03 December 2018

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 to 72 hour) : 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
EyC50 (0 to 72 hour) : 0.76 mg/L; 95% confidence limits 0.69 to 0.85 mg/L

No Observed Effect Concentration based on growth rate: 0.50 mg/L
No Observed Effect Concentration based on yield: 0.50 mg/L L
owest Observed Effect Concentration based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration based on yield: 1.0 mg/L

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

Cell Densities and Percentage Inhibition of Growth from the Range-findingTest

 

Nominal Loading Rate (mg/L)

Cell Densities*(cells per mL)

Inhibition Values (%)

 

0 Hours

 

72 Hours

 

Growth Rate

 

Yield

 

Control

R1

5.78E+03

1.09E+06

 

-

 

-

R2

6.63E+03

9.47E+05

Mean

6.20E+03

1.02E+06

 

1.0

R1

6.31E+03

1.64E+05

 

[8]

 

[60]

R2

6.37E+03

1.61E+06

Mean

6.34E+03

1.62E+06

 

10

R1

5.43E+03

1.26E+06

 

[8]

 

[34]

R2

5.60E+03

1.47E+06

Mean

5.51E+03

1.36E+06

 

100

R1

6.83E+03

1.15E+6

 

[4]

 

[14]

R2

4.46E+03

1.18E+06

Mean

5.65E+03

1.16E+06

* 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

R = Replicate

-  = Not applicable

[ ] = Increase in growth as compared to the control

Cell Densities and pH Values in the Definitive Test

 

Nominal Loading Rate (mg/L)

pH

Cell Densities*(cells per mL)

pH

0 Hour

25 Hour

46 Hours

72 Hours

72 Hour

 

 

 

 

 

Control

R1

 

 

 

 

 

7.9

3.73E+04

1.81E+05

1.66E+06

 

 

 

 

 

8.9

R2

3.87E+04

1.80E+05

1.69E+06

R3

3.63E+04

1.70E+05

1.35E+06

R4

1.14E+04

3.78E+04

1.70E+05

R5

3.73E+04

1.61E+05

1.52E+06

R6

3.57E+04

1.67E+05

1.49E+06

Mean**

3.71E+04

1.72E+05

1.54E+06

 

 

 

 

 

100

R1

 

 

 

 

 

7.9

4.63E+04

2.16E+05

2.04E+06

 

 

 

 

 

9.1

R2

4.64E+04

2.21E+05

1.97E+06

R3

4.47E+04

2.30E+05

1.83E+06

R4

4.37E+04

2.12E+05

1.82E+06

R5

4.48E+04

1.76E+05

1.76E+06

R6

5.54E+04

2.24E+05

1.63E+06

Mean

4.69E+04

2.13E+05

1.84E+06

* 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.

R = Replicate

** = Values calculated excluding control replicate R4

Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

 

Treatment

Daily Specific Growth Rate (cells/mL/hour)

Day 0 to 1

Day 1 to 2

Day 2 to 3

 

 

 

 

Control

R1

0.080

0.075

0.085

R2

0.082

0.073

0.0836

R3

0.079

0.073

0.080

R4

0.033

0.057

0.058

R5

0.080

0.070

0.086

R6

0.079

0.074

0.084

Mean*

0.080

0.073

0.084

R = Replicate

* Values calculated excluding control replicate R4

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

 

 

 

 

 

Control

R1

0.081

 

 

 

 

-

1.65E+06

 

 

 

 

-

R2

0.081

1.68E+06

R3

0.078

1.34E+06

R4

0.049

1.65E+05

R5

0.079

1.52E+06

R6

0.079

1.49E+06

Mean**

0.080

1.54E+06

SD**

0.001

1.36E+05

 

 

 

 

 

100

R1

0.083

[4]

2.03E+06

 

 

 

 

 

 

 

[19]

R2

0.083

[4]

1.97E+06

R3

0.082

[3]

1.83E+06

R4

0.082

[3]

1.82E+06

R5

0.081

[1]

1.75E+06

R6

0.080

0

1.62E+06

Mean

0.082

[2]

1.84E+06

SD

0.001

 

1.48E+05

 *          In accordance with the OECD test guideline only the mean value for yield is calculated

R =Replicate

SD = Standard deviation

** = Values calculated excluding control replicate R4

[] = Increase in growth compared to controls

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The NOEL was 100 mg/L loading rate WAF.
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) 761/2009.

 

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a WAF of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) 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.

 

Results

Chemical analysis of the test preparations was conducted using inductively coupled plasma with mass spectrometric detection (ICP-MS) for the presence of silicon in solution.

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured silicon concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.051 mg/L. This does not infer that no silicon was present in solution, just that the levels of silicon were below the quantifiable level.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) 761/2009.

 

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a WAF of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) 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.

 

Results

Chemical analysis of the test preparations was conducted using inductively coupled plasma with mass spectrometric detection (ICP-MS) for the presence of silicon in solution.

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured silicon concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.051 mg/L. This does not infer that no silicon was present in solution, just that the levels of silicon were below the quantifiable level.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information