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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:
11 December 2017 to 10 January 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:
NA
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each 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. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a WAF of the test item.

Range-finding test:
The loading rates used in the definitive test were determined by a preliminary range-finding test, in which Pseudokirchneriella subcapitata cells were exposed to nominal loading rates of 1.0, 10 and 100 mg/L for a period of 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.


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


Experimental preparation:
Nominal amounts of test item (20, 32, 20, 64 and 200 mg) were each separately added to the surface of 20, 10, 2, 2 and 2 litres of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. Due to the viscous nature of the test item, the 1.0 mg/L loading rate was prepared by dispensing the test item onto a glass slide which was then suspended within the media column. 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 23 hours and the mixtures allowed to stand for 1-Hour. Visual observations made on the 3.2, 10, 32 and 100 mg/L loading rate WAFs indicated that a significant amount of dispersed test item was 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 a second glass wool plug and two sheets of filter paper was conducted in order to remove as much undissolved test item as possible. Microscopic observations of the 1.0 and 3.2 mg/L loading rate WAFs were performed after filtering and showed there to be no micro-dispersions of test item present. Microscopic observations of the 10 mg/L loading rate WAF showed micro-dispersions of test item to be present whilst visual inspection of the 32 and 100 mg/L loading rate WAFs showed dispersed test item remained. It was considered that further filtration at this point would not have removed any more of the dispersed test item present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.3 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
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 in the laboratory under constant agitation by orbital shaker (100 to 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 (100 to 150 rpm) and constant illumination at 24 ±1°C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
No remarks
Post exposure observation period:
NA
Hardness:
NA
Test temperature:
24 ±1°C
pH:
7.1 - 8.2
Dissolved oxygen:
NA
Salinity:
NA
Conductivity:
NA
Nominal and measured concentrations:
Nominal loading rates: 1.0, 3.2, 10, 32 and 100 mg/L
Details on test conditions:
Exposure Conditions:
Test vessels were 250 mL glass conical flasks containing 100 mL of test preparation. Six flasks each containing 100 mL of test preparation were used for the control and 3 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 4.00 x 10^5 cells per mL. Inoculation of 500 mL of test medium with 6.3 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 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.


Test condition measurements:
The pH of the control and each test concentration 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. The vortex depth was recorded at the start and end of the mixing period.


Test Organism Observations:
Samples were taken at 24, 46 and 72 hours and the cell densities determined using a haemocytometer and light microscope. Three determinations were made for each sample. The nominally 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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Limits (mg/L Loading Rate WAF) = 25 - 40 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
5.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Remarks on result:
other: 95% Confidence Limits (mg/L Loading Rate WAF) = 4.6 - 7.0 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Details on results:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.70 to 60 mg/L. Measured test concentrations in the range of 0.35 to 59 mg/L were obtained at 72 hours. 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.
Results with reference substance (positive control):
The 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 and exposure conditions 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): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 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 loading rates 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).

For growth rate, there were no statistically significant differences between the control, 1.0 and 3.2 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on growth rate was 3.2 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 10 mg/L loading rate WAF.

For yield, there were no statistically significant differences between the control and 1.0 mg/L loading rate WAF (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on yield was 1.0 mg/L loading rate WAF. Correspondingly the LOEL based on yield was 3.2 mg/L loading rate WAF.

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

 

Nominal Loading Rate (mg/L)

Cell Densities* (cells per mL)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

7.42E+03

1.40E+06

-

-

R2

7.69E+03

1.52E+06

Mean

7.55E+03

1.46E+06

1

R1

6.34E+03

1.11E+06

1

27

R2

5.22E+03

1.02E+06

Mean

5.78E+03

1.06E+06

10

R1

7.92E+03

6.94E+05

14

54

R2

6.94E+05

6.72E+05

Mean

7.57E+03

6.83E+05

100

R1

6.78E+03

2.08E+05

37

90

R2

4.72E+03

1.01E+05

Mean

5.75E+03

1.55E+05

 

* 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

  

 

Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/L)

pH

Cell Densities* (cells per mL)

pH

0 Hour

24 Hours

46 Hours

72 Hours

72 Hours

Control

R1

7.2

1.84E+04

5.84E+04

7.50E+05

7.7

R2

1.50E+04

9.34E+04

7.78E+05

R3

1.50E+04

8.34E+04

7.72E+05

R4

1.50E+04

9.67E+04

9.72E+05

R5

1.17E+04

8.34E+04

8.00E+05

R6

1.50E+04

8.50E+04

7.95E+05

Mean

1.50E+04

8.34E+04

8.11E+05

1

R1

7.2

1.84E+04

8.34E+04

7.28E+05

7.9

R2

2.00E+04

8.84E+04

7.50E+05

R3

2.17E+04

1.00E+05

6.45E+05

Mean

2.00E+04

9.06E+04

7.08E+05

3.2

R1

7.2

1.50E+04

7.67E+04

6.89E+05

8.2

R2

1.34E+04

9.50E+04

5.78E+05

R3

1.50E+04

8.00E+04

5.72E+05

Mean

1.45E+04

8.39E+04

6.13E+05

10

R1

7.2

8.35E+03

8.00E+04

1.80E+05

8.1

R2

1.34E+04

7.17E+04

2.53E+05

R3

8.35E+03

5.50E+04

2.17E+05

Mean

1.00E+04

6.89E+04

2.17E+05

32

R1

7.1

8.35E+03

1.84E+04

9.17E+04

8

R2

1.00E+04

1.34E+04

6.50E+04

R3

3.35E+03

1.50E+04

9.00E+04

Mean

7.23E+03

1.56E+04

8.22E+04

100

R1

7.1

5.00E+03

5.00E+03

1.34E+04

7.9

R2

6.65E+03

1.17E+04

1.67E+04

R3

6.65E+03

8.35E+03

1.00E+04

Mean

6.10E+03

8.35E+03

1.34E+04

  

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

R = Replicate

  

 

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

0.07

-

7.45E+05

-

R2

0.07

7.73E+05

R3

0.07

7.67E+05

R4

0.073

9.67E+05

R5

0.07

7.95E+05

R6

0.07

7.90E+05

Mean

0.071

8.06E+05

SD

0.001

8.08E+04

1

R1

0.069

3

7.23E+05

R2

0.07

1

7.45E+05

R3

0.067

6

6.40E+05

Mean

0.069

3

7.03E+05

13

SD

0.002

5.54E+04

3.2

R1

0.068

4

6.84E+05

R2

0.066

7

5.73E+05

R3

0.066

7

5.67E+05

Mean

0.067

6

6.08E+05

25

SD

0.001

6.59E+04

10

R1

0.05

30

1.75E+05

R2

0.054

24

2.48E+05

R3

0.052

27

2.12E+05

Mean

0.052

27

2.12E+05

74

SD

0.002

3.65E+04

32

R1

0.04

44

8.67E+04

R2

0.036

49

6.00E+04

R3

0.04

44

8.50E+04

Mean

0.039

46

7.72E+04

90

SD

0.002

1.49E+04

100

R1

0.014

80

8.40E+03

R2

0.017

76

1.17E+04

R3

0.01

86

5.00E+03

Mean

0.014

81

8.37E+03

99

SD

0.004

3.35E+03

  

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

R = Replicate

SD = Standard Deviation

- = Not applicable

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test item 'Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives' to the green alga Pseudokirchneriella subcapitata resulted in the 72-hour EL50, NOELR and LOELR values of 31, 3.2 and 10 mg/L, respectively based on growth rate and 72-hour EL50, NOELR and LOELR values of 5.7, 1 and 3.2 mg/L, respectively based on yield.
Executive summary:

The toxicity of 'Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives' to the freshwater green alga Pseudokirchneriella subcapitata was assessed according to the OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009). The study was conducted at a GLP accredited laboratory and met the validity criteria described in the Guideline and is therefore considered to be reliable.

 

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (3 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 haemocytometer and light microscope.

 

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.70 to 60 mg/L. Measured test concentrations in the range of 0.35 to 59 mg/L were obtained at 72 hours. The dissolved test item may have been one or several components of the test item. 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. The study resulted in 72-hour EL50, NOELR and LOELR values of 31, 3.2 and 10 mg/L, respectively based on growth rate and 72-hour EL50, NOELR and LOELR values of 5.7, 1 and 3.2 mg/L, respectively based on yield.

Description of key information

The toxicity of 'Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives' to the freshwater green alga Pseudokirchneriella subcapitata was assessed according to the OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009). The study was conducted at a GLP accredited laboratory and met the validity criteria described in the Guideline and is therefore considered to be reliable.

 

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (3 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 haemocytometer and light microscope.

 

The study resulted in 72-hour EL50, NOELR and LOELR values of 31, 3.2 and 10 mg/L, respectively based on growth rate and 72-hour EL50, NOELR and LOELR values of 5.7, 1 and 3.2 mg/L, respectively based on yield.

Key value for chemical safety assessment

EC50 for freshwater algae:
5.7 mg/L
EC10 or NOEC for freshwater algae:
1 mg/L

Additional information

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 0.70 to 60 mg/L. Measured test concentrations in the range of 0.35 to 59 mg/L were obtained at 72 hours. The dissolved test item may have been one or several components of the test item. 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.