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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:
17 May 2010 and 30 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Date of GLP inspection: 15 September 2009 Date of signature on GLP certificate: 26 November 2009)
Analytical monitoring:
yes
Details on sampling:
Verification of test concentrations
Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately 20ºC for further analysis if necessary.
The method of analysis, stability, recovery and test preparation analyses are described in Appendix 3 see in attached section.
Vehicle:
no
Details on test solutions:
Range-finding test
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 Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 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 used for each control and test concentration. The test item was dissolved directly in culture medium.
An amount of test item (50 mg) 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 to give further stock solutions of 10, 1.0 and 0.10 mg/l. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (2.5 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
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 ± 1ºC 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.

Definitive test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.

Experimental Preparation
For the purpose of the definitive test, the test item was dissolved directly in culture medium.
Amounts of test item (100 and 32 mg) were each separately dissolved in culture medium and the volumes adjusted to 1 litre to give 100 and 32 mg/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/l. An aliquot (500 ml) of each of the stock solutions was separately inoculated with algal suspension (10.5 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Physico-chemical measurements
The pH of each control and test flask 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.

Evaluation of data
For comparison of growth rates and comparison of Yield see in attached section.

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
-The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
-The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
-The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test Species
The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. 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 ± 1ºC.
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 ± 1ºC until the algal cell density was approximately 104 - 105 cells/ml.
A positive control (Harlan Laboratories Ltd Project Number: 0039/1127) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 1. The positive control was conducted between 12 January 2010 and 15 January 2010.

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.
The culture medium is defined in Appendix 2 see below.

Appendix 2 Culture Medium
NaNO3 25.5 mg/l
MgCl2.6H2O 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
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised 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:
no
Total exposure duration:
72 h
Post exposure observation period:

Not applicable
Hardness:

Not recorded.
Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH values of the control cultures (see Table 2 in any other information on materials and method section) were observed to increase from pH 7.4 – 7.5 at 0 hours to pH 7.7 – 7.8 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
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320pH meter.
Dissolved oxygen:

Not recorded.
Salinity:

freshwater used
Nominal and measured concentrations:
The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.
Details on test conditions:
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 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 characterised by a cell density of 1.91 x 105 cells per ml. Inoculation of 500 ml of test medium with 10.5 ml of this algal suspension gave an initial nominal cell density of 4 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.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL not stated
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL not stated
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL not stated
Details on results:
Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding test are given in Table 1 see in any other information on materials and method section.
The results showed no effect on growth rate at the test concentrations of 0.10 and 1.0 mg/l. However, growth was observed to be reduced at 10 and 100 mg/l.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l were selected for the definitive test.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 see in any other information in materials and method section. Daily specific growth rates for the control cultures are given in Table 3 see in any other information on results section. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 see in any other information in result section
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 see in attached section.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 39 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.

Mean cell density of control at 0 hours : 4.41 x 103 cells per ml
Mean cell density of control at 72 hours : 1.74 x 105 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 20% 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 – 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data
From the data given in Tables 2 see in any other information on materials and method section and 4 see in any other information in result section, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) 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) : 40 mg/l
ErC20 (0 - 72 h) : 57 mg/l
ErC50 (0 - 72 h) : >100 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 concentration tested resulted in greater than 50% inhibition of growth rate.
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, 1.0, 3.2, 10 and 32 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 32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100 mg/l.

Inhibition of yield
EyC10 (0 - 72 h) : 22 mg/l
EyC20 (0 - 72 h) : 30 mg/l
EyC50 (0 - 72 h) : 51 mg/l; 95% confidence limits 43 - 61 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 5.2.2.1. There were no statistically significant differences between the control, 1.0, 3.2, 10 and 32 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 yield was 32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100 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 1.0, 3.2, 10 and 32 mg/l, however no intact cells were observed to be present in the test cultures at 100 mg/l.

Observations on test item 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, 1.0, 3.2, 10 and 32 mg/l test cultures were observed to be green dispersions whilst the 100 mg/l test cultures were observed to be clear colourless solutions.

Physico-chemical measurements
The pH values of each test and control flask are given in Table 2 see in any other information on materials and method section. Temperature was maintained at 24 ± 1ºC throughout the test.

Verification of test concentrations
Analysis of the test preparations at 0 hours (see Appendix 3 in attached section) showed measured test concentrations to be near nominal with the exception of the 1.0 mg/l test sample which showed a measured test concentration of less than the limit of quantitation (LOQ) of the analytical method employed was obtained which was determined to be 1.2 mg/l. Analysis of the test preparations at 72 hours showed near nominal concentrations were obtained from the 10, 32 and 100 mg/l test samples. Measured concentrations of less than the LOQ and 75% of nominal were obtained from the 1.0 and 3.2 mg/l test samples respectively. The low measured concentrations obtained for the 1.0 and 3.2 mg/l test samples were considered to have had no adverse effect on the outcome of the test given that the No Observed Effect Concentration (NOEC) based on both growth rate and yield was determined to be 32 mg/l. As such the results of the study have been calculated based on nominal test concentrations only.
en in Table 2 see in any other information on materials and method section. Temperature was maintained at 24 ± 1ºC throughout the test.
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:
ErC50 (0 – 72 h) : 0.49 mg/l*
EyC50 (0 – 72 h) : 0.18 mg/l, 95% confidence limits 0.16 – 0.21 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 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:
Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Statistical analysis
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)

Table 1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.38E+03

3.20E+05

 

 

 

R2

5.22E+03

3.39E+05

-

-

 

Mean

5.30E+03

3.29E+05

 

 

0.10

R1

5.09E+03

2.82E+05

 

 

 

R2

5.77E+03

2.82E+05

4

15

 

Mean

5.43E+03

2.82E+05

 

 

1.0

R1

5.48E+03

2.96E+05

 

 

 

R2

5.48E+03

2.84E+05

4

12

 

Mean

5.48E+03

2.90E+05

 

 

10

R1

5.94E+03

1.86E+05

 

 

 

R2

5.43E+03

1.68E+05

16

47

 

Mean

5.69E+03

1.77E+05

 

 

100

R1

5.43E+03

3.66E+04

 

 

 

R2

6.02E+03

3.66E+04

54

90

 

Mean

5.73E+03

3.66E+04

 

 



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

R1and R2= Replicates 1 and 2

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.065

0.035

0.056

 

R2

0.059

0.041

0.054

 

R3

0.064

0.039

0.053

 

R4

0.051

0.047

0.065

 

R5

0.062

0.050

0.052

 

R6

0.057

0.039

0.052

 

Mean

0.060

0.042

0.055

 


R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.052

 

1.64E+05

 

 

R2

0.051

 

1.58E+05

 

 

R3

0.052

 

1.63E+05

 

 

R4

0.054

-

1.96E+05

-

 

R5

0.055

 

2.02E+05

 

 

R6

0.050

 

1.37E+05

 

 

Mean

0.052

 

1.70E+05

 

 

SD

0.002

 

2.44E+04

 

1.0

R1

0.052

0

1.64E+05

 

 

R2

0.052

0

1.64E+05

 

 

R3

0.052

0

1.63E+05

 

 

Mean

0.052

0

1.64E+05

4

 

SD

0.000

 

3.89E+02

 

3.2

R1

0.051

2

1.49E+05

 

 

R2

0.052

0

1.67E+05

 

 

R3

0.053

[2]

1.79E+05

 

 

Mean

0.052

0

1.65E+05

3

 

SD

0.001

 

1.48E+04

 

10

R1

0.052

0

1.67E+05

 

 

R2

0.054

[4]

1.93E+05

 

 

R3

0.046

12

1.04E+05

 

 

Mean

0.051

3

1.54E+05

9

 

SD

0.004

 

4.55E+04

 

32

R1

0.046

12

1.03E+05

 

 

R2

0.053

[2]

1.71E+05

 

 

R3

0.048

8

1.25E+05

 

 

Mean

0.049

6

1.33E+05

22

 

SD

0.004

 

3.47E+04

 

100

R1

0.027

48

2.28E+04

 

 

R2

0.026

50

2.09E+04

 

 

R3

0.030

42

2.80E+04

 

 

Mean

0.028

47

2.39E+04

86

 

SD

0.002

 

3.68E+03

 


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

R1– R6= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results:
EC50 (mg/l) : >100
No Observed Effect Concentration (NOEC) (mg/l): 32
Lowest Observed Effect Concentration (LOEC) (mg/l) 100 mg/L
This study has been selected as the key study because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation EC (No.) 1272/2008 (EU CLP).
Executive summary:

Introduction.

A study was perford to assess the effect of the test item on the growth of the green alga Desmodesmus subspicatus. Thethod 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 440/2008.

Methods.

Following a preliminary range-finding test,Desmodesmus subspicatuswas exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 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.

Results.

Exposure ofDesmodesmus subspicatusto the test item gave the following results:

Response Variable

EC50(mg/l)

95% Confidence Limits (mg/l)

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

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

Growth Rate

>100

 

*

 

32

100

Yield

51

43

-

61

32

100

Analysis of the test preparations at 0 hours showed measured test concentrations to be near nominal with the exception of the 1.0 mg/l test sample which showed a measured test concentration of less than the limit of quantitation (LOQ) of the analytical method employed was obtained which was determined to be 1.2 mg/l. Analysis of the test preparations at 72 hours showed near nominal concentrations were obtained from the 10, 32 and 100 mg/l test samples. Measured concentrations of less than the LOQ and 75% of nominal were obtained from the 1.0 and 3.2 mg/l test samples respectively. The low measured concentrations obtained for the 1.0 and 3.2 mg/l test samples were considered to have had no adverse effect on the outcome of the test given that the No Observed Effect Concentration (NOEC) based on both growth rate and yield was determined to be 32 mg/l. As such the results of the study have been calculated based on nominal test concentrations only.



*It was not possible to calculate 95% confidence limits for the ErC50value as the data generated did not fit the models available for the calculation of confidence limits.

Conclusion.

The effect of the test item on the growth ofDesmodesmus subspicatushas been investigated over a 72-Hour period and gave the following results:

Response Variable

EC50(mg/l)

95% Confidence Limits (mg/l)

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

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

Growth Rate

>100

 

*

 

32

100

Yield

51

43

-

61

32

100


*It was not possible to calculate 95% confidence limits for the ErC50value as the data generated did not fit the models available for the calculation of confidence limits.

Description of key information

A key study to assess the toxicity of sodium metaphosphate to algae has been performed according to OECD guideline 201 and under the conditions of GLP.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

The effect of the test item on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results:

EC50 (mg/l) : >100    

No Observed Effect Concentration (NOEC) (mg/l): 32       

Lowest Observed Effect Concentration (LOEC) (mg/l) 100 mg/L