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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Sep - 29 Sep 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom, date of inspection: 10 July 2012
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples were taken from the solvent control and the 100% v/v test group (replicates R1 – R6 pooled) at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: All 0-Hour samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 ºC for further analysis if necessary.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100% v/v to confirm that at the highest attainable concentration, no effect on algal growth was observed. For the purpose of the definitive test, the test item was prepared using a preliminary solution in dimethylformamide. An amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (200 µL) of this solvent stock solution was dispersed in 2 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes. After stirring any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v solution of test item. An aliquot (1 liter) of the stock solution was inoculated with algal suspension (10.9 mL) to give the required test concentration of 100% v/v (equivalent to a 0-Hour measured test concentration of 0.018 mg/L). The stock solutions and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the start of the test all control, solvent control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, solvent control and test cultures were observed to be pale green dispersions.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: 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 ± 1 °C.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
7.5 - 7.9
Nominal and measured concentrations:
100% v/v (nominal)
0.018 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks
- Initial cells density: 5.52 x 10^3 cells/mL (mean)
- Control end cells density: 8.11 x 10^5 cells/mL (mean)
- Mean cell density of solvent control at 0 hours: 5.51 x 10^3 cells/mL
- Mean cell density of solvent control at 72 hours: 1.00 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to OECD guideline 201

OTHER TEST CONDITIONS
- Adjustment of pH: yes, adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux, provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer Particle Counter

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10 an 100% v/v (nominal)
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 10 and 100% v/v.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test mat. WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test mat. WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test mat. WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test mat. WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test mat. WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: Test mat. WAF
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h): 1.1 mg/L; 95% confidence limits 1.0 – 1.3 mg/L
EyC50 (0 – 72 h): 0.70 mg/L
NOEC) based on growth rate: 0.50 mg/L
NOEC) based on yield: 0.25 mg/L
LOEC) based on growth rate: 1.0 mg/L
LOEC) based on yield: 0.50 mg/L
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981, Biometry. New York: W H Freeman and Company) was carried out on the growth rate and yield data after 72 hours for the solvent control and the 100% v/v test concentration to determine any statistically significant differences between the test and solvent control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001, SAS/STAT Proprietary Software Release 8.02, SAS Institute Inc, Cary, NC, USA).

Range-finding Test:

The results showed no effect on growth at the test concentrations of 10 and 100% v/v. Based on this information a single test concentration of six replicates, of 100% v/v was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration, no effect on growth was observed. Chemical analysis of the 100% v/v test preparation at 0 hours showed a measured test concentration of 0.029 mg/L was obtained. A significant decline in measured test concentration was observed at 72 hours to less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.0045 mg/L. These results indicated that the test item was unstable over the test duration.

Table: Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)

pH

Cell densities (cells/mL) (mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

R2

R3

R4

R5

R6

mean

7.5

4.93E+03

2.85E+04

1.66E+05

8.08E+05

7.9

5.17E+03

2.98E+04

1.75E+05

8.18E+05

5.57E+03

3.19E+04

1.77E+05

9.25E+05

5.92E+03

2.76E+04

1.47E+05

7.29E+05

5.78E+03

3.09E+04

1.41E+05

8.16E+05

5.76E+03

2.59E+04

1.50E+05

7.69E+05

5.52E+03

2.91E+04

1.59E+05

8.11E+05

Solvent control

R1

R2

R3

R4

R5

R6

mean

7.5

5.50E+03

3.64E+04

2.20E+05

1.16E+06

7.9

5.42E+03

3.22E+04

1.70E+05

9.84E+05

5.68E+03

2.68E+04

1.54E+05

7.72E+05

5.18E+03

3.23E+04

1.94E+05

1.11E+06

5.46E+03

2.77E+04

1.65E+05

9.21E+05

5.79E+03

2.76E+04

2.04E+05

1.07E+06

5.51E+03

3.05E+04

1.84E+05

1.00E+06

0.0065

R1

R2

R3

R4

R5

R6

mean

7.5

5.84E+03

4.32E+04

2.93E+05

1.44E+06

7.9

5.70E+03

2.92E+04

1.73E+05

1.02E+06

4.67E+03

2.77E+04

1.91E+05

1.04E+06

5.31E+03

2.95E+04

1.85E+05

1.41E+06

5.42E+03

3.02E+04

1.79E+05

9.67E+05

5.22E+03

2.52E+04

1.79E+05

9.35E+05

5.36E+03

3.08E+04

2.00E+05

1.13E+06

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

Geometric Mean Measured Test Concentration (mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition

Control

R1

R2

R3

R4

R5

R6

Mean

SD

0.071

-

8.03E+05

-

0.071

8.13E+05

0.073

9.20E+05

0.069

7.23E+05

0.071

8.11E+05

0.070

7.63E+05

0.071

8.06E+05

0.001

6.59E+04

Solvent control

R1

R2

R3

R4

R5

R6

Mean

SD

0.076

-

1.16E+06

-

0.073

9.78E+05

0.070

7.67E+05

0.075

1.11E+06

0.072

9.15E+05

0.075

1.07E+06

0.074

9.99E+05

0.002

1.43E+05

0.0065

R1

R2

R3

R4

R5

R6

Mean

SD

0.079

(7)

1.43E+06

 

0.074

0

1.01E+06

 

0.074

0

1.04E+06

 

0.078

(5)

1.40E+06

 

0.073

1

9.61E+05

 

0.073

1

9.30E+05

 

0.075

(2)

1.13E+06

(13)

0.003

 

2.27E+05

 

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 - 15 Dec 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no purity, no analytical monitoring)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Water Accomodated Fraction (WAF): The test substance (100 mg/L) was moderately stirred in algal medium for 24 h at room temperature. After this incubation time, the undissolved materials were removed by sterile filtration (0.45 µm) to obtain a sterile test substance solution. The resulting WAF was used in the test.
- Controls: yes water control
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, Germany
- Method of cultivation: Cultivated in 250 mL flasks containing 100 mL of sterile OECD medium inoculated with cell material from axenic slope culture at continuous illumination from warm white fluorescent tubes (8500-9000 lux) and 22 ± 0.2 °C (thermocontrolled room).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22 ± 0.2 °C
pH:
at the beginning of incubation: 8.0 ± 0.2
Nominal and measured concentrations:
Nominal: 0 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250 mL flaks, all-glass, with 100 mL of test medium
- Initial cells density: 10000- 20000 / mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 5
- Other: Test flasks were placed on an orbital shaker at a nominal shaking speed of 140 RPM.

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was determined in a control flask at the start and at the end of the test. pH was determined in 1 control flask after sterilisation before the test and in all test vessels at the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuous
- Light intensity and quality: From warm white fluorescent tubes (8500-9000 lux)

EFFECT PARAMETERS MEASURED: Determination of cell concentration and observations of mis-shapen cells and cell-debris were made after 24, 48 and 72 h of exposure.
- Determination of cell concentrations: Cell concentration was determined every 24 h using a spectrophotometer (680 nm wavelength). Aliquots of 5 mL were removed from each test substance served as controls. The starting cell density was determined using a counting chamber.
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Remarks on result:
other: EL50 (24 - 72 h)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
biomass
Remarks on result:
other: EL50 (24 - 72 h)
Duration:
72 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Remarks on result:
other: EL0 (24 - 72 h)
Duration:
72 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
biomass
Remarks on result:
other: EL0 (24 - 72 h)
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: 1.1% stimulation of growth rates at 100 mg/L; 20.9% stimulation of biomass at 100 mg/L

Table 1: Cell concentration (A680) in individual test cultures after 24, 48 and 72 h of exposure to the test material ((b) = Mean absorbance (A680))

Nominal concentration [mg/l]

Code

Cell concentration (A680)

0 h (b)

24 h

48 h

72 h

Control

A

B

C

D

E

0.005

0.005

0.005

0.005

0.005

0.019

0.020

0.017

0.017

0.018

0.116

0.150

0.122

0.137

0.131

0.549

0.667

0.543

0.556

0.573

100

A

B

C

0.005

0.005

0.005

0.023

0.021

0.021

0.149

0.111

0.189

0.776

0.649

0.720

 

Table 2: Average specific growth rates between 24 and 72 h

Nominal concentration [mg/l]

Code

Specific growth rate (x 0.01)

Coefficient of correlation (R)

Inhibition (%)

Mean value (%)

Standard deviation (%)

Control

A

B

C

D

E

7.01

7.31

7.22

7.27

7.21

0.999

0.996

0.997

0.994

0.996

2.7

-1.5

-0.2

-0.9

-0.1

0.0

1.6

100

A

B

C

7.33

7.15

7.36

0.999

1.000

0.990

-1.8

0.7

-2.3

-1.1

1.6

 

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
Refer to analogue justification document provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
source, CAS 85186-86-3, Oleon, 2007, D. subspicatus 72 h, RL2
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
source, CAS 85186-86-3, Oleon, 2007, D. subspicatus 72 h, RL2
Duration:
72 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
source, CAS 85186-86-3, Oleon, 2007, D. subspicatus 72 h, RL2
Duration:
72 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
source, CAS 85186-86-3, Oleon, 2007, D. subspicatus 72 h, RL2
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
source, CAS 68855-18-5, Croda, 2012, Pseudokirchneriella subcapitata, RL1
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
source, CAS 68855-18-5, Croda, 2012, Pseudokirchneriella subcapitata, RL1
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
source, CAS 68855-18-5, Croda, 2012, Pseudokirchneriella subcapitata, RL1
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
source, CAS 68855-18-5, Croda, 2012, Pseudokirchneriella subcapitata, RL1
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Remarks on result:
other: source, CAS 68855-18-5, Croda, 2012, Pseudokirchneriella subcapitata, RL1
Duration:
72
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Remarks on result:
other: source, CAS 68855-18-5, Croda, 2012, Pseudokirchneriella subcapitata, RL1
Conclusions:
No effects were observed up to the limit of water solubility.

Description of key information

EL50 (72 h) > 0.0065 mg/L (measured, geometric mean; Pseudokirchneriella subcapitata); no effects up to the limit of water solubility; read-across

EL50 (72 h) > 100 mg/L (nominal; Desmodesmus subspicatus); no effects up to the limit of water solubility; read-across

NOELR (72 h)0.0065 mg/L (measured, geometric mean; Pseudokirchneriella subcapitata); no effects up to the limit of water solubility; read-across

EL0 (72 h)100 mg/L (nominal; Desmodesmus subspicatus); no effects up to the limit of water solubility; read-across

Key value for chemical safety assessment

Additional information

There is no study available in which the toxicity of Neopentyl glycol dicaprate (CAS No. 27841-06-1) to algae and cyanobacteria was assessed. Therefore, read-across to the structurally and chemically closely related source substances Fatty acids, C8-18 and C18-unsatd., esters with neopentyl glycol (CAS 85186-86-3) and Heptanoic acid, ester with 2,2-dimethyl-1,3-propanediol (CAS 68855-18-5) was performed in accordance with Regulation (EC) No 1907/2006 Annex XI, section 1.5 in order to fulfill the data requirements for this substance. The source substances are characterized by similar chemical structure and are therefore considered suitable representatives for the assessment of the toxicity of the target substance to algae. A detailed read-across justification in provided in IUCLID section 13.

The study conducted with the source substance Fatty acids, C8-18 and C18-unsatd., esters with neopentyl glycol (CAS 85186-86-3) was performed under static conditions and according to OECD guideline 201, usingDesmodesmus subspicatusas test organism. A 100 mg/L loading rate prepared as Water Accommodated Fraction (WAF) was applied in a limit test. No growth rate inhibition was observed in the treatment or control. Hence, based on growth rate, the EL50 (72 h) was determined as > 100 mg/L loading rate (nominal, WAF) and the EL0 (72 h) was determined as100 mg/L loading rate (nominal, WAF).

The study conducted with the source substance Heptanoic acid, ester with 2,2-dimethyl-1,3-propanediol (CAS 68855-18-5) to freshwater algae is available. The study was performed under static conditions and according to OECD guideline 201, usingPseudokirchneriella subcapitataas test organism. A 100% v/v (nominal) test solution was prepared as Water Accommodated Fraction (WAF) and applied in a limit test. The measured concentration (geometric mean) at test start was 0.018 mg/L. No growth rate inhibition was observed in the treatment or control during the exposure period of 72 h. Based on growth rate, the EC50 (72 h) and EC10 (72 h) were both determined as > 0.0065 mg/L (measured, geometric mean, WAF). The NOEC (72 h) was determined as0.0065 mg/L (measured, geometric mean, WAF).

Based on the available results from structurally related read-across substances (in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5) which are characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that the target substance Neopentyl glycol dicaprate (CAS No. 27841-06-1) is not toxic to aquatic algae.