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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
Reliability:
1 (reliable without restriction)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
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 and stored frozen for further analysis if necessary. Only samples of the No Observed Effect Concentration (NOEC) and
above were analyzed.
Vehicle:
no
Details on test solutions:
Culture Medium
NaNO3
25.5 mg/L
MgCl2.6H2O
12.16 mg/L
CaCl2.2H2O
4.41 mg/L
MgSO4.7H2O
14.6 mg/L
K2HPO4
1.044 mg/L
NaHCO3
15.0 mg/L
H3BO3
0.186 mg/L
MnCl2.4H2O
0.415 mg/L
ZnCl2
0.00327 mg/L
FeCl3.6H2O
0.160 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

The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
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. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
See section Details on test solutions
Test temperature:
24 C
pH:
7.5
Details on test conditions:
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 Pseudokirchneriella subcapitata 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.
A nominal 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 (450 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.
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 ± 1 ºC 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.
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. All samples were stored frozen prior to analysis.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.
Experimental Preparation
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 6.25, 12.5, 25 and 50 mg/L. An aliquot (500 mL) ofeach of the stock solutions was separately inoculated with algal suspension (5.6 mL) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L.
Each of the prepared concentrations 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.
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 4.44 x 105 cells per mL. Inoculation of 500 mL of test medium with 5.6 mL of this
algal suspension gave an initial nominal cell density of 5.00 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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
47 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
54 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
68 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
47 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Range Finding test
The results showed no effect on growth 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 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.
Chemical analysis of the 10 and 100 mg/L test preparations at 0 hours and 72 hours showed that measured concentrations of near nominal were obtained, indicating that
the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the 25, 50 and 100 mg/L test preparations at 0 and 72 hours showed measured test concentrations be near nominal. As a result, the results were based on
the nominal concentrations.

Growth Data
From the data it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) 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) : 47 mg/L
ErC20 (0 - 72 h) : 54 mg/L
ErC50 (0 - 72 h) : 68 mg/L; 95% confidence limits 63 - 74 mg/L
where ErCx is the test concentration that reduced growth rate by x%.
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 (P≥0.05), between the control and the 6.25, 12.5 and
25 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth
rate was 25 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 50 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h) : 25 mg/L
EyC20 (0 - 72 h) : 31 mg/L
EyC50 (0 - 72 h) : 47 mg/L; 95% confidence limits 41 - 54 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 6.2.3. There were no statistically significant differences (P≥0.05), between the control and the 6.25, 12.5 and
25 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was
25 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 50 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 6.25, 12.5, 25 and 50 mg/L,
however no intact cells were observed to be present in the test cultures at 100 mg/L.

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.
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.7 at 0 hours to pH 8.8 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.

Observations on Test Item Solubility
At the start of the test, the control and all test cultures were observed to be clear colorless solution. After 72 hours exposure, the control cultures, along with replicate 1 and 2 of the
6.25 mg/L test culture group were observed to be green dispersions whilst replicate 3 was observed to be a bright green dispersion. The 12.5, 25 and 50 mg/L test cultures were
observed to be green dispersions and the 100 mg/L test cultures were observed to be clear colorless solutions.
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. The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Validity criteria fulfilled:
yes
Conclusions:
The effect of the 6-chloro-1-hexanol on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the nominal test concentrations gave the
following results - ErC50 (0 - 72 h) is 68 mg/L based on growth rate and EyC50 (0 - 72 h) is 47 mg/L based on yield.
Executive summary:

The effect of the 6-chloro-1-hexanol on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the nominal test concentrations gave the

following results - ErC50 (0 - 72 h) is  68 mg/L based on growth rate and EyC50 (0 - 72 h) is  47 mg/L based on yield.

Description of key information

The effect of the 6-chloro-1-hexanol on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the nominal test concentrations gave the


following results - ErC50 (0 - 72 h) is  68 mg/L based on growth rate and ErC50 (0 - 72 h) is  47 mg/L based on yield.


The effect of the 6-chloro-1-hexanol on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the nominal test concentrations gave thefollowing results - ErC50 (0 - 72 h) is  68 mg/L based on growth rate and EyC50 (0 - 72 h) is  47 mg/L based on yield.

Key value for chemical safety assessment

EC50 for freshwater algae:
68 mg/L
EC10 or NOEC for freshwater algae:
47 mg/L

Additional information