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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:
8 - 19 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
The test guideline was followed with the following modification:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, Cis-3-Hexenyl Salicylate falls into this catergory, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/l) of test material in culture medium for a period of 24 hours prior to removing any undissolved test material present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test material.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Cis-3-hexenyl salicylate is being used as a read across for hexyl salicylate.
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group at 0 and 72 (replicates pooled) hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored at approximately 20ºC for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.
Vehicle:
no
Details on test solutions:
The test material solutions were prepared by stirring an excess (50 mg/l) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): not reported
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C with periodic renewal of the test media.

ACCLIMATION
- Acclimation period: not reported
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Exposure was based on the initial measured concentrations.
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperature was maintianed between 24 ± 1°C
pH:
pHat the start of the exposure ranged between 7.0 and 7.2, after 72 hours of exposure values ranged between 7.1 and 7.6.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Test media were prepared by dilution of a saturated solution. The initial measured concentration of the saturated solution was 3.6 mg/L. Dilutions of this saturated solution were used to prepare four additional concentrations. Measured initial concentrations were 3.64, 1.52, 0.737, 0.353 and 0.151 mg/L. After 72 hours measured concentrations were less than the limit of quantification (0.017 mg/L) except of the two highest concentrations were measured concentrations were 0.060 and 0.091 mg/L. Overall geometric means were calculated to be 0.036, 0.055, 0.079, 0.30 and 0.58 mg/L.
Details on test conditions:
250 ml glass conical flasks were used. Six flasks each containing 100 ml of solution 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 material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 8.91 x 105cells per ml. Inoculation of 1 litre of test medium with 4.5 ml of this algal suspension gave an initial nominal cell density of 4 x 103cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS MultitronâVersion 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:
EC50
Effect conc.:
0.61 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.51 - 0.73 mg/L
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.25 - 0.30 mg/L
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
0.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Validity criteria

The following data show that the cell concentration of the control cultures increased by a factor of 56 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 : 4.04 x 103 cells per ml
Mean cell density of control at 72 hours : 2.25 x 105 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 34% 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 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd Project No: 0039/1088) used potassium dichromate as the reference material 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 material gave the following results:

ErC50 (0 – 72 h) : 0.79 mg/l
EyC50 (0 – 72 h) : 0.30 mg/l, 95% confidence limits 0.27 – 0.34 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 material.
Reported statistics and error estimates:
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).

Analytical results

Nominal conc. (mg/L)

Measured concentration (mg/L)

Geometric mean measured (mg/L)

% of the 0-Hour Measured Test Concentration

0 hours

72 hours

Control

<LOQ

<LOQ

-

-

0.3

0.151

<LOQ

0.036

24

0.6

0.353

<LOQ

0.055

16

1.2

0.737

<LOQ

0.079

11

2.4

1.52

0.0603

0.30

20

4.8

3.64

0.0910

0.58

16

LOQ – 0.017 mg/L

Given this decline in measured test concentrations it was considered appropriate to calculate the geometric mean measured test concentrations. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0085 mg/l) was used to enable calculation of the geometric mean measured concentration. 

As the test material was known to be stable in aqueous test medium and the decline observed attributed to adsorption to algal cells it may be considered that the algal cells present were exposed to nominal test concentrations of the test material throughout the test period. The results of the study are based on the initial measured concentrations.

 

 Algal cell densities

Initial measured conc. (mg/L)

Mean algal cell density (cell/mL)

0 hours

24 hours

48 hours

72 hours

Control

4.04E+03

1.17E+04

3.45E+04

2.25E+05

0.15

3.93E+03

1.16E+04

5.79E+04

2.67E+05

0.35

4.20E+03

7.11E+03

2.63E+04

5.59E+04

0.74

4.18E+03

6.56E+03

8.42E+03

2.44E+04

1.5

4.11E+03

6.44E+03

8.21E+03

8.17E+03

3.6

4.32E+03

2.97E+03

3.25E+03

3.77E+03

 

Inhibition of growth rate and yield

 

Initial measured conc. (mg/L)

Growth rate (cells/mL/hour)
(0 – 72 hours)

% inhibition

Yield (cells/mL)
 (0 – 72 hours)

% inhibition

Control

0.056

-

2.20E+05

-

0.15

0.058

-4

2.63E+05

-19

0.35

0.037

35

5.17E+04

77

0.74

0.025

55

2.03E+04

91

1.5

0.010

82

4.06E+03

98

3.6

-0.001

101

-5.55E+02

100

Validity criteria fulfilled:
yes
Conclusions:
A 72 hour algal inhibition study was performed in which Desmodesmus subspicatus cultures were exposed to a range of concentrations of Cis-3-Hexenyl Salicylate. Based on initial measured concentrations the 72 hour EyC50 was calculated to be 0.28 mg/L and the 72 hour ErC50 was 0.61 mg/L. The corresponding no-observed effect concentrations were 0.15 mg/L.

Due to the similar structural, physical and chemical properties of Cis-3-Hexenyl Salicylate, it was considered appropriatefor the purposes of read-across to Hexyl Salicylate.
Executive summary:

A 72 hour algal inhibition study was performed in which Desmodesmus subspicatus cultures were exposed to nominal concentrations of Cis-3-Hexenyl Salicylate of 0.3, 0.6, 1.2, 2.4 and 4.8 mg/L, and was used for read-across to Hexyl Salicylate. Test media were prepared by dilution of a stock solution of nominally 4.8 mg/L produced by 24 hours of stirring prior to settling and filtration. The initial measured concentrations were 0.151, 0.353, 0.737, 1.52 and 3.64 mg/L, after 72 hours exposure measured concentrations were <LOQ, except at the two highest concentrations, values were 0.06 and 0.09 mg/L.  As the test material was known to be stable in aqueous test medium and the decline observed attributed to adsorption to algal cells it may be considered that the algal cells present were exposed to nominal test concentrations of the test material throughout the test period. The results of the study were therefore based on the initial measured concentrations. The 72 hour EyC50 was calculated to be 0.28 mg/L and the 72 hour ErC50 was 0.61 mg/L. The corresponding no-observed effect concentrations were both 0.15 mg/L.

Due to the similar structural, physical and chemical properties of Cis-3-Hexenyl Salicylate, it was considered appropriate for the purposes of read-across to Hexyl Salicylate.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
It is considered appropriate to address the data requirements for hexyl salicylate by read-across to the available studies on structurally similar substances.
Hexyl salicylate and cis-3-hexenyl salicylate are both composed of the salicylate group and a carbon chain of similar length. The substances are therefore sufficiently similar in terms of chemical structure to support a read-across approach.
Reason / purpose:
read-across source
Specific details on test material used for the study:
Cis-3-hexenyl salicylate is being used as a read across for hexyl salicylate.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.61 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.51 - 0.73 mg/L
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.25 - 0.30 mg/L
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
0.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Validity criteria

The following data show that the cell concentration of the control cultures increased by a factor of 56 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 : 4.04 x 103 cells per ml
Mean cell density of control at 72 hours : 2.25 x 105 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 34% 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 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd Project No: 0039/1088) used potassium dichromate as the reference material 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 material gave the following results:

ErC50 (0 – 72 h) : 0.79 mg/l
EyC50 (0 – 72 h) : 0.30 mg/l, 95% confidence limits 0.27 – 0.34 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 material.
Reported statistics and error estimates:
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).

Analytical results

Nominal conc. (mg/L)

Measured concentration (mg/L)

Geometric mean measured (mg/L)

% of the 0-Hour Measured Test Concentration

0 hours

72 hours

Control

<LOQ

<LOQ

-

-

0.3

0.151

<LOQ

0.036

24

0.6

0.353

<LOQ

0.055

16

1.2

0.737

<LOQ

0.079

11

2.4

1.52

0.0603

0.30

20

4.8

3.64

0.0910

0.58

16

LOQ – 0.017 mg/L

Given this decline in measured test concentrations it was considered appropriate to calculate the geometric mean measured test concentrations. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0085 mg/l) was used to enable calculation of the geometric mean measured concentration. 

As the test material was known to be stable in aqueous test medium and the decline observed attributed to adsorption to algal cells it may be considered that the algal cells present were exposed to nominal test concentrations of the test material throughout the test period. The results of the study are based on the initial measured concentrations.

 

 Algal cell densities

Initial measured conc. (mg/L)

Mean algal cell density (cell/mL)

0 hours

24 hours

48 hours

72 hours

Control

4.04E+03

1.17E+04

3.45E+04

2.25E+05

0.15

3.93E+03

1.16E+04

5.79E+04

2.67E+05

0.35

4.20E+03

7.11E+03

2.63E+04

5.59E+04

0.74

4.18E+03

6.56E+03

8.42E+03

2.44E+04

1.5

4.11E+03

6.44E+03

8.21E+03

8.17E+03

3.6

4.32E+03

2.97E+03

3.25E+03

3.77E+03

 

Inhibition of growth rate and yield

 

Initial measured conc. (mg/L)

Growth rate (cells/mL/hour)
(0 – 72 hours)

% inhibition

Yield (cells/mL)
 (0 – 72 hours)

% inhibition

Control

0.056

-

2.20E+05

-

0.15

0.058

-4

2.63E+05

-19

0.35

0.037

35

5.17E+04

77

0.74

0.025

55

2.03E+04

91

1.5

0.010

82

4.06E+03

98

3.6

-0.001

101

-5.55E+02

100

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the target substance hexyl salicylate to aquatic algae is estimated to be a 72 h ErC50 = 0.61 mg/L and EyC50 = 0.28 mg/L, and a NOEC = 0.15 mg/L, based on read-across from a study testing Cis-3-Hexenyl Salicylate.
Executive summary:

The toxicity of the target substance hexyl salicylate to aquatic algae is estimated to be a 72 h ErC50= 0.61 mg/L and 72 h EyC50= 0.28 mg/L, based on read-across from a study testing cis-3-hexenyl salicylate.

 Due to the similar structural, physical and chemical properties of cis-3-hexenyl Salicylate, it was considered appropriate for the purposes of read-across to hexyl salicylate.

 As explained in the justification for type of information, hexyl salicylate and cis-3-hexenyl Salicylate share similar structural, physical and chemical properties. Both substances are composed of the salicylate group and a carbon chain of similar length. The substances are therefore sufficiently similar in terms of chemical structure to support a read-across approach.

Description of key information

Two algal studies were performed:
A 72 hour algal inhibition study was performed in which Pseudokirchneriella subcapitata cultures were exposed to nominal concentrations of hexyl salicylate of 1.0, 10 and 100 mg/L. As no analytical determinations were performed the results of the study are based on nominal exposure concentrations. The 72 hour EC50 was observed to be >100 mg/L (the highest concentration tested).
A 72 hour algal inhibition study was performed in which Desmodesmus subspicatus cultures were exposed to nominal concentrations of Cis-3-Hexenyl Salicylate of 0.3, 0.6, 1.2, 2.4 and 4.8 mg/L, and was used for read-across to hexyl salicylate. Test media were prepared by dilution of a stock solution of nominally 4.8 mg/L produced by 24 hours of stirring prior to settling and filtration. The initial measured concentrations were 0.151, 0.353, 0.737, 1.52 and 3.64 mg/L, after 72 hours exposure measured concentrations were <LOQ, except at the two highest concentrations, values were 0.06 and 0.09 mg/L. As the test material was known to be stable in aqueous test medium and the decline observed attributed to adsorption to algal cells it may be considered that the algal cells present were exposed to nominal test concentrations of the test material throughout the test period. The results of the study were therefore based on the initial measured concentrations. The 72 hour EyC50 was calculated to be 0.28 mg/L and the 72 hour ErC50 was 0.61 mg/L. The corresponding no-observed effect concentrations were both 0.15 mg/L. Due to the similar structural, physical and chemical properties of Cis-3 -Hexenyl Salicylate, it was considered appropriate for the purposes of read-across to Hexyl Salicylate.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.61 mg/L
EC10 or NOEC for freshwater algae:
0.15 mg/L

Additional information

Two algal studies were performed, one with hexyl salicylate and the other with Cis-3-Hexenyl Salicylate as a read-across study. The hexyl salicylate study was conducted with a wide spread of concentrations, the highest two of which were above the expected water solubility value, additionally no analytical verification was performed. The Cis-3-Hexenyl Salicylate study was conducted with a range of concentrations that were less than the limit of water solubility and with analytical verification. Therefore the results of the Cis-3-Hexenyl Salicylate study will be used to provide the appropriate endpoint for algae. Due to the similar structural, physical and chemical properties of Cis-3 -Hexenyl Salicylate, it was considered appropriate for the purposes of read-across to Hexyl Salicylate. Based on the Cis-3-Hexenyl Salicylate the 72 ErC50 of 0.61 mg/L is the appropriate endpoint.