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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
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)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each loading rate WAF 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 each occasion and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
The culture medium used for the range-finding tests, initial experiments and definitive test was the same as that used to maintain the stock culture.
The culture medium is

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):
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 aeration and constant 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 103 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 104 - 105 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
See test solution details
Test temperature:
24 ± 1 °C
pH:
7.5
Dissolved oxygen:
> 3 mg/l
Nominal and measured concentrations:
See section Details on results
Details on test conditions:
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
Range-Finding Tests
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 90 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by preliminary range-finding tests. The initial 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% v/v saturated solution for a period of 72 hours.
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.4 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
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 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.
The results of the initial range-finding test showed significant inhibition of growth occurred at all test concentrations and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal test concentrations of 0.0010, 0.010, 0.10 and 1.0% v/v saturated solution for a period of 72 hours.
A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0, 0.10, 0.010 and 0.0010% v/v saturated solution. An aliquot (450 mL) of each of the 0.0010, 0.010, 0.10 and 1.0% v/v saturated solution stock solutions was separately inoculated with algal suspension (2.0 mL) to give the required test concentrations of 0.0010, 0.010, 0.10 and 1.0% v/v saturated solution.
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 control group was maintained under identical conditions but not exposed to the test item.
Exposure conditions in the second range-finding test were the same as those in the initial 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. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.
Definitive Test
Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 0.0032, 0.010, 0.032, 0.10 and 0.32% v/v saturated solution.
Exposure Conditions
As in the range-finding tests 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.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.00 x 105 cells per mL. Inoculation of 450 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.:
0.046 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: value determined by extrapolation of the growth curve
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.021 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.059 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
Range-finding Tests
The results showed no effect on growth at the test concentrations of 0.0010 and 0.010% v/v saturated solution. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100% v/v saturated solution.
Based on this information test concentrations of 0.0032, 0.010, 0.032, 0.10 and 0.32% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 0.010, 0.10 and 1.0% v/v saturated solution test preparations at 0 hours (see Annex 4) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.11 mg/L, 0.13 and 1.0 mg/L respectively were obtained. A decline in measured concentration was observed at 72 hours to less than the LOQ for the 0.010 and 0.10% v/v test samples and to 0.75 mg/L for the 1.0% v/v test sample indicating that the test item was either unstable and/or was adsorbing to the algal cells present.
Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours (see Annex 4) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.11 mg/L were obtained from the 0.0032, 0.010, 0.032 and 0.10% v/v saturated solution test preparations. A measured concentration of 0.24 mg/L was obtained from the 0.32% v/v saturated solution test preparation. Measured concentrations of less than the LOQ were obtained from all test samples at 72 hours.
Given this decline in measured test concentrations, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.053 and 0.11 mg/L for the 0.10 and 0.32% v/v saturated solution test groups respectively. It was considered inappropriate to calculate the geometric mean measured concentrations for the remaining test preparations as measured concentrations of less than the LOQ were obtained at both 0 and 72 hours. In order to determine ECx values it was necessary to determine a measured concentration for the 0.10% v/v saturated solution test group. Using a value of half of the LOQ for this test level was considered to give a “worst case” result.
Care should be taken in the interpretation of the results of the study as, despite extensive method development work having been conducted to improve the sensitivity of the analytical method, it was not possible to accurately quantify the exposure concentrations for the lower test levels. Furthermore, in only being able to plot the two highest test levels, extrapolation of the growth curve was required to attain several of the required ECx values.
Growth Data
From the data 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 based on the geometric mean measured test concentrations:
Inhibition of Growth Rate
ErC10 (0 - 72 h): 0.046 mg/L*
ErC20 (0 - 72 h): 0.074 mg/L
ErC50 (0 - 72 h): 0.17 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 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 between the control, 0.0032, 0.010 and 0.032% v/v saturated solution test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.032% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.10% v/v saturated solution.
6.2.4 Inhibition of Yield
EyC10 (0 - 72 h): 0.021 mg/L*
EyC20 (0 - 72 h): 0.031 mg/L*
EyC50 (0 - 72 h): 0.059 mg/L
Where:
EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out. There were no statistically significant differences between the control, 0.0032, 0.010 and 0.032% v/v saturated solution test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.032% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.10% v/v saturated solution.
Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
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
Validity criteria fulfilled:
yes
Conclusions:
The effect of the 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results - ErC10 (0 - 72 h): 0.046 mg/L, ErC50 (0 - 72 h): 0.17 mg/L based on growth rate and EyC10 (0 - 72 h): 0.021 mg/L, EyC50 (0 - 72 h): 0.059 mg/L based on inhibition of the yield.
Executive summary:

The effect of the 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results - ErC10 (0 - 72 h): 0.046 mg/L, ErC50 (0 - 72 h): 0.17 mg/L based on growth rate and EyC10 (0 - 72 h): 0.021 mg/L, EyC50 (0 - 72 h): 0.059 mg/L based on inhibition of the yield.

Description of key information

The effect of the 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl tolyl ether on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results - ErC10 (0 - 72 h): 0.046 mg/L, ErC50 (0 - 72 h): 0.17 mg/L based on growth rate and EyC10 (0 - 72 h): 0.021 mg/L, EyC50 (0 - 72 h): 0.059 mg/L based on inhibition of the yield.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.17 mg/L

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