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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:
Experimental start date 20 October 2016 Experimental completion date 13 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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)
Specific details on test material used for the study:
Identification: Cyclohexanone, peroxide (CAS 12262-58-7)
Physical state/Appearance: Clear colorless liquid
Batch: 1405426797
Purity: 33.9%
Expiry Date: 29 September 2017
Storage Conditions: Approximately 4 °C in darkness
Analytical monitoring:
yes
Details on sampling:
Range-finding test
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. The 0-Hour samples were stored frozen prior to analysis. The 72-Hour samples were prepared for analysis on the day of sampling.

Definitive test
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours
Vehicle:
no
Details on test solutions:
A nominal amount of test item (50 mg) was dissolved in 1 liter of culture medium, with the aid of prolonged, slow stirring for 24 hours via magnetic stirrer, to give a 50 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 3.2, 1.0, 0.32 and 0.10 mg/L. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (7.0 mL) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 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 10E+03 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 10E+04 – 10E+05 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
All test and control cultures were inspected microscopically at 72 hours
Hardness:
Not stated in report
Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test.
pH:
7.8 - 8.3 in the definitive test
Dissolved oxygen:
Not stated in report
Salinity:
Not applicable as freshwater study
Nominal and measured concentrations:
Range finding test
0.1, 1.0, 10 and 100 mg/L (nominal concentrations)

Defintive test
0.10, 0.32, 1.0, 3.2 and 10 mg/L
Details on test 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 characterized by a cell density of 6.39 x 10E+05 cells/mL. Inoculation of 900 mL of test medium with 7.0 mL of this algal suspension gave an initial nominal cell density of 5.0 x 10E+03 cells/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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.53 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.81 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.495 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.52 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
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 Figures 2 and 3.
Accordingly the following results were determined from the data:
 
Inhibition of growth rate
ErC10 (0 - 72 h) : 0.53 mg/L
ErC20 (0 - 72 h) : 0.81 mg/L
ErC50 (0 - 72 h) : 1.7 mg/L; 95% confidence limits 1.3 – 2.2 mg/L

Where: ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using a Williams Multiple Sequential t-test procedure incorporating Levene’s test for homogeneity of variance. There were no statistically significant differences (P0.05), between the control, 0.173 and 0.495 mg/L test groups and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.495 mg/L. Correspondingly, the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 1.52 mg/L.
Results with reference substance (positive control):
A positive control 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) : 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L
EyC50 (0 – 72 h) : 0.79 mg/L; 95% confidence limits 0.70 – 0.89 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) 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.
Reported statistics and error estimates:
A Williams Multiple Sequential t-test procedure incorporating Levene’s test for homogeneity of variance procedure for comparing several treatments with a control 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 ToxRat computer software package (ToxRat).

Range-finding Test

The results showed no effect on growth at the test concentration of 0.10 mg/L. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L. 

Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

Chemical analysis of the test preparations at 0 and 72 hours (see Annex 4) showed measured concentrations ranging from 88% to 101% of the nominal concentrations indicating that the test item was stable under test conditions.

Definitive Test

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.173 to 14.1 mg/L (141 to 173% of the nominal concentrations). There was no significant change in the measured concentrations at 72 hours. The frozen duplicate samples were analysed to verify the high measured concentrations, and did support the original measured concentrations (129 to 147% of the nominal concentrations). 

The measured concentrations indicated that the test item exposure was higher than intended. However, given the consistency between the 0 and 72-Hour measured concentrations, it was considered justifiable to base the results of the test on the 0-Hour measured test concentrations only.

Inhibition of Yield

EyC10(0 - 72 h)          :         0.46 mg/L

EyC20(0 - 72 h)          :          0.57 mg/L

EyC50(0 - 72 h)          :          0.82 mg/L; 95% confidence limits were not determined*

 

Where:            EyCxis the test concentration that reduced yield by x%.

There were no statistically significant differences (P³0.05), between the control and 0.173 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.173 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.495 mg/L.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 134 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         :          5.00 x 10E+03 cells/mL

Mean cell density of control at 72 hours       :          6.72 x 10E+05 cells/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 – 72 h) was 3% 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 the control or test cultures at 0.173 and 0.495 mg/L. However, some cell debris was observed to be present in the test cultures at 1.52 mg/L, and no intact cells were observed in the 4.79 and 14.1 mg/L 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.

 Observations on Test Item Solubility

At the start of the test, the control and all test cultures were observed to be clear colorless solutions.

At 72 hours, the control, 0.173 and 0.495 mg/L test cultures were observed to be pale green dispersions. The 1.52 and 4.79 mg/L test cultures were observed to be extremely pale green dispersions. The 14.1 mg/L test culture was observed to be a clear colorless solution.


*  It was not possible to calculate 95% confidence limits for the EyC50value as the data generated did not fit the models available for the calculation of confidence limits.

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

Biomass Integral/ Yield

Control

R1

7.15E+03

5.64E+05

-

-

R2

6.93E+03

5.53E+05

Mean

7.04E+03

5.59E+05

0.10

R1

7.38E+03

6.22E+05

0

1

R2

6.66E+03

4.81E+05

Mean

7.02E+03

5.52E+05

1.0

R1

7.17E+03

3.79E+05

11

43

R2

6.14E+03

2.63E+05

Mean

6.66E+03

3.21E+05

10

R1

6.27E+03

1.54E+04

85

99

R2

7.65E+03

1.14E+04

Mean

6.96E+03

1.34E+04

100

R1

6.93E+03

9.57E+03

92

99

R2

6.43E+03

1.00E+04

Mean

6.68E+03

9.81E+05

Table2            Cell Densities and pH Values in the Definitive Test

0-Hour Measured Concentration

(mg/L)

pH

Cell Densities* (cells per mL)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

7.6

5.00E+03

2.55E+04

1.34E+05

6.35E+05

8.3

R2

5.00E+03

2.47E+04

1.22E+05

5.45E+05

R3

5.00E+03

2.54E+04

1.43E+05

7.39E+05

R4

5.00E+03

2.25E+04

1.42E+05

7.01E+05

R5

5.00E+03

2.34E+04

1.40E+05

7.49E+05

R6

5.00E+03

2.32E+04

1.46E+05

6.60E+05

Mean

5.00E+03

2.41E+04

1.38E+05

6.72E+05

0.173

R1

7.6

5.00E+03

1.98E+04

1.25E+05

6.08E+05

8.3

R2

5.00E+03

2.22E+04

1.21E+05

6.66E+05

R3

5.00E+03

2.39E+04

1.46E+05

7.60E+05

Mean

5.00E+03

2.20E+04

1.31E+05

6.78E+05

 

0.495

R1

7.6

5.00E+03

2.27E+04

1.24E+05

6.54E+05

8.3

R2

5.00E+03

1.94E+04

1.11E+05

5.96E+05

R3

5.00E+03

1.80E+04

1.10E+05

4.98E+05

Mean

5.00E+03

2.00E+04

1.15E+05

5.83E+05

 

1.52

R1

7.5

5.00E+03

1.20E+04

1.95E+04

6.79E+04

8.0

R2

5.00E+03

1.27E+04

1.86E+04

5.38E+04

R3

5.00E+03

1.34E+04

1.90E+04

6.73E+04

Mean

5.00E+03

1.27E+04

1.90E+04

6.30E+04

 

4.79

R1

7.5

5.00E+03

7.41E+03

8.66E+03

9.98E+03

7.8

R2

5.00E+03

6.88E+03

8.38E+03

1.02E+04

R3

5.00E+03

6.83E+03

6.51E+03

1.07E+04

Mean

5.00E+03

7.04E+03

7.85E+03

1.03E+04

14.1

R1

7.5

5.00E+03

5.95E+03

6.21E+03

6.96E+03

7.8

R2

5.00E+03

5.97E+03

6.38E+03

6.85E+03

R3

5.00E+03

6.53E+03

6.86E+03

8.59E+03

Mean

5.00E+03

6.15E+03

6.49E+03

7.47E+03

 

Table3            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

0.068

0.069

0.065

R2

0.067

0.067

0.062

R3

0.068

0.072

0.068

R4

0.063

0.077

0.066

R5

0.064

0.075

0.070

R6

0.064

0.077

0.063

Mean

0.066

0.073

0.066

 

Table4            Inhibition of Growth Rate and Yield in the Definitive Test

0-Hour Measured Concentration
(mg/L)

Growth Rate

(cells/mL/hr)

Yield

(cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.067

-

6.30E+05

 

R2

0.065

5.40E+05

 

R3

0.069

7.34E+05

 

R4

0.069

6.96E+05

 

R5

0.070

7.44E+05

 

R6

0.068

6.55E+05

 

Mean

0.068

6.67E+05

 

SD

0.002

7.60E+04

 

0.173

R1

0.067

1

6.03E+05

 

R2

0.068

0

6.61E+05

 

R3

0.070

[3]

7.55E+05

 

Mean

0.068

[1]

6.73E+05

[1]

SD

0.002

 

7.65E+04

 

0.495

R1

0.068

0

6.49E+05

 

R2

0.066

3

5.91E+05

 

R3

0.064

6

4.93E+05

 

Mean

0.066

3

5.78E+05

13

SD

0.002

 

7.86E+04

 

1.52

R1

0.036

47

6.29E+04

 

R2

0.033

51

4.88E+04

 

R3

0.036

47

6.23E+04

 

Mean

0.035

48

5.80E+04

91

SD

0.002

 

7.97E+03

 

4.79

R1

0.010

85

4.98E+03

 

R2

0.010

85

5.16E+03

 

R3

0.011

84

5.69E+03

 

Mean

0.010

85

5.28E+03

99

SD

0.001

 

3.66E+02

 

14.1

R1

0.005

93

1.96E+03

 

R2

0.004

94

1.85E+03

 

R3

0.008

88

3.59E+03

 

Mean

0.006

92

2.47E+03

100

SD

0.002

 

9.76E+02

 

*    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.

R1and R2= Replicates 1 and 2

*    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.

R1- R6= Replicates 1 to 6

R1- R6= Replicates 1 to 6

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

R1-R6= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to the controls]

Validity criteria fulfilled:
yes
Conclusions:
Analysis of the test preparations at 0 hours showed measured test concentrations to be higher than nominal values, ranging from 141% to 173% of nominal, and did not decline significantly at 72 hours. It was therefore considered justifiable to base the results on the 0-Hour measured test concentrations only.
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. 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" referenced as Method C.3 of Commission Regulation (EC) No. 761/2009.

Methods.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to an aqueous solution of the test item at concentrations of 0.10, 0.32, 1.0, 3.2 and 10 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.

Analysis of the test preparations at 0 hours showed measured test concentrations to be higher than nominal values, ranging from 141% to 173% of nominal, and did not decline significantly at 72 hours. It was therefore considered justifiable to base the results on the    0-Hour measured test concentrations only.

Exposure ofPseudokirchneriella subcapitatato the test item gave the following results based on the 0-Hour measured test concentrations:

Response Variable

EC10(mg/L)

EC20(mg/L)

EC50(mg/L)

95% Confidence Limits

(mg/L)

No Observed Effect Concentration (NOEC)

(mg/L)

Lowest Observed Effect Concentration (LOEC)

(mg/L)

Growth Rate

0.53

0.81

1.7

1.3

-

2.2

0.495

1.52

Yield

0.46

0.57

0.82

Not determined

0.173

0.495

 

Description of key information

An OECD 201 study is available with a 72h-ErC50 for freshwater algae of 1.7 mg/L (measured concentration) and a 72h-ErC10 of 0.53 mg/l (measured concentration).

Key value for chemical safety assessment

EC50 for freshwater algae:
1.7 mg/L
EC10 or NOEC for freshwater algae:
0.53 mg/L

Additional information

One study performed under GLP according to OECD guideline 201 is available peformed with the freshwater green algae Pseudokirchneriella subcapitata. This resulted in a 72h-ErC50 of 1.7 mg/L (measured concentration) and a 72 h-ErC10 of 0.53 mg/L (measured concentration).

The study is valid without restriction and can therefore be used for C&L and Hazard- and Risk Assessment.