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EC number: 919-898-9 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
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- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
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- Endpoint summary
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
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- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data
Toxicity to aquatic algae and cyanobacteria
Administrative data
Link to relevant study record(s)
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- This study was conducted between 20 July 2017 and 26 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Reliability 1 is assigned because the study conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
- Version / remarks:
- 2006
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Version / remarks:
- EC No. 761/2009
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- Information as provided by the Sponsor.
Identification: Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and glycidyl tolyl ether
Batch: Ei 4015
Purity: 100% UVCB
Physical state/Appearance: Dark yellow viscous liquid
Expiry Date: 15 November 2021
Storage Conditions: Room temperature in the dark - Analytical monitoring:
- yes
- Details on sampling:
- Range-Finding Test
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. Only concentrations within the range to be used for the definitive test were analyzed.
Definitive Test
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours. - Vehicle:
- no
- Details on test solutions:
- Range-Finding Test
The loading rates to be used in the definitive test were determined by preliminary range finding tests.
Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. 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 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). The aqueous phase was then passed through two sheets of filter paper to remove as much undissolved test item as possible. Microscopic observations of the WAFs were performed after filtering. No micro-dispersions of test item were observed in the 10 mg/L loading rate WAF, the 100 mg/L loading rate was observed to be a slightly cloudy white dispersion with micro-dispersions of test item visible. It was considered that further filtration at this point would not have removed anymore of the undissolved test item present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.0 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF
The results of the initial range-finding test showed significant inhibition of growth occurred at both 10 and 100 mg/L loading rate WAF and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.10, 1.0 and 10 mg/L for a period of 72 hours.
Nominal amounts of test item (5.0 and 50 mg) were each separately added to the surface of 5 liters of culture medium to give the 1.0 and 10 mg/L loading rates respectively. 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 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). Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present. A dilution was made from the 1.0 mg/L loading rate WAF to give a 0.10 mg/L loading rate WAF stock solution.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.4 mL) to give the required test concentrations of 0.10, 1.0 and 10 mg/L loading rate WAF.
Significant inhibition of yield was observed to have occurred at all loading rates employed and so a third range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.0010, 0.010, 0.10 and 1.0 mg/L for a period of 72 hours.
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 mg/L loading rate. 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 mixture allowed to stand for 1-Hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.10, 0.010 and 0.0010 mg/L loading rate WAF.
An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.9 mL) to give the required test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L loading rate WAF.
Definitive Test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Due to the need to test at relatively low levels, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give the remaining test concentrations.
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 mg/L loading rate. 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 mixture allowed to stand for 1-Hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.32, 0.10, 0.032 and 0010 mg/L loading rate WAF.
An aliquot (900 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (7.9 mL) to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF. - 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 (below). The master cultures were maintained under constant agitation by orbital shaker (approximately 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 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 C until the algal cell density was approximately 104 to 105 cells/mL.
Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
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 type:
- semi-static
- Water media type:
- freshwater
- Limit test:
- yes
- Total exposure duration:
- 72 h
- Hardness:
- not reported
- Test temperature:
- Temperature was maintained at 24 ± 1 °C
- pH:
- The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 7.9 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.
- Dissolved oxygen:
- Not reported
- Salinity:
- Not reported
- Conductivity:
- Not reported
- Nominal and measured concentrations:
- Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.034 mg/L, to 0.19 mg/L. A decline in measured test concentrations to less than the LOQ in all test preparations was observed at 72 hours.
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only. - Details on test conditions:
- Experimental Design and Study Conduct
Range-Finding Test
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.
Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.
Range-Finding Tests
The loading rates 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 nominal loading rates of 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 prepared 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 haemocytometer and light microscope. 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 to 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 haemocytometer and light microscope. It was not possible to monitor algal growth using a Coulter® Multisizer Particle Counter due to the presence of dispersed test item.
The results of the initial range-finding test showed significant inhibition of growth occurred at both 10 and 100 mg/L loading rate WAF and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.10, 1.0 and 10 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 prepared for each control and test concentration.
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. 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 to 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.
Significant inhibition of yield was observed to have occurred at all loading rates employed and so a third range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.0010, 0.010, 0.10 and 1.0 mg/L for a period of 72 hours.
The control group was maintained under identical conditions but not exposed to the test item.
Exposure conditions in the third range-finding test were the same as those in the second test.
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. 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 loading rates were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Due to the need to test at relatively low levels, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give the remaining test concentrations.
Exposure Conditions
As in the range-finding tests 250 mL glass conical flasks were used. 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 5.73 x 105 cells per mL. Inoculation of 900 mL of test medium with 7.9 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 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Assessments
Test Organism Observations
Samples were taken at 24, 52 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 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.
Water Quality Criteria
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.
Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.
Chemical Analysis of Test Loading Rates
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 0 hours and stored frozen for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EL50
- Effect conc.:
- 0.25 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat. (dissolved fraction)
- Remarks:
- Loading Rate WAF
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOELR
- Effect conc.:
- 0.01 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat. (dissolved fraction)
- Remarks:
- Loading Rate WAF
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- LOELR
- Effect conc.:
- 10 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat. (dissolved fraction)
- Remarks:
- Loading Rate WAF
- Basis for effect:
- growth rate
- Details on results:
- alidation of Mixing Period
Preliminary investigational work indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.
Range-finding Test
The results of the third range-finding test showed no effect on growth at 0.0010 and 0.010 mg/L loading rate WAF. However, growth was observed to be reduced at 0.10 and 1.0 mg/L loading rate WAF.
Based on this information loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were selected for the definitive test.
Chemical analysis of the 0.010, 0.10 and 1.0 mg/L loading rate WAF test preparations taken from the third range-finding test at 0 hours showed measured test concentrations of less than the LOQ, determined to be 0.034 mg/L, 0.041 and 0.61 mg/L respectively were obtained. A decline in measured test concentrations was observed at 72 hours to less than the LOQ in all test preparations indicating that the test item was unstable under the conditions of the test.
Definitive Test
Chemical Analysis of Test Loading Rates
Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.034 mg/L, to 0.19 mg/L. A decline in measured test concentrations to less than the LOQ in all test preparations was observed at 72 hours.
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
Growth Data
Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 2.
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
ErL10 (0 to 72 hour) : 0.068 mg/L loading rate WAF
ErL20 (0 to 72 hour) : 0.11 mg/L loading rate WAF
ErL50 (0 to 72 hour) : 0.25 mg/L loading rate WAF; 95% confidence limits 0.21 to 0.31 mg/L loading rate WAF
Where ErLx is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control, 0.010, 0.032, 0.10 and 0.32 mg/L loading rate WAF test groups 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 and 0.010 mg/L loading rate WAF (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on growth rate was 0.010 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 0.032 mg/L loading rate WAF.
Inhibition of Yield
EyL10 (0 to 72 hour) : 0.014 mg/L loading rate WAF
EyL20 (0 to 72 hour) : 0.023 mg/L loading rate WAF
EyL50 (0 to 72 hour) : 0.055 mg/L loading rate WAF; 95% confidence limits 0.044 to 0.067 mg/L loading rate WAF
Where EyLx is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out as above. There were no statistically significant differences between the control and 0.010 mg/L loading rate WAF (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on yield was 0.010 mg/L loading rate WAF. Correspondingly the LOEL based on yield was 0.032 mg/L loading rate WAF.
Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 259 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.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 1.30 x 10^6 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 13% 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 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
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 0.010, 0.032, 0.10 and 0.32 mg/L loading rate WAF, however cell debris and no intact cells were observed to be present in the test cultures at 1.0 mg/L loading rate WAF.
Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.3 at 0 hours to pH 7.9 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.
Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.
Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless media column with a single globule of test item settled on the bottom of the mixing vessel. After stirring the WAF had formed a clear colorless media column with many small globules of test item on the bottom of the mixing vessel and settling towards the bottom of the mixing vessel. After a 1-Hour standing period a clear colorless media column with small globules of test item settled on the bottom of the mixing vessel. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period, all control, 0.010 and 0.032 mg/L loading rate WAF test cultures were observed to be green dispersions. The 0.10 mg/L loading rate WAF test cultures were observed to be pale green dispersions whilst the 0.32 and 1.0 mg/L loading rate WAF test cultures were observed to be clear colorless solutions. - Results with reference substance (positive control):
- A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 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. - Validity criteria fulfilled:
- yes
- Remarks:
- The data satisfied the validation criterion given in the OECD Guideline
- Conclusions:
- The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 Hour period and gave the following results:
Response Variable EL10 (mg/L LR WAF) EL20(mg/L LLR WAF) EL50 (mg/L LR WAF)
Growth Rate 0.068 0.11 0.25
Response Variable 95% Confidence Limits (mg/L LR WAF) No Observed Effect LR (NOEL) (mg/L) Lowest Observed Effect
Growth Rate 0.21 - 0.31 0.010 0.032 - Executive summary:
A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.
Methods
Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
Following preliminary range-finding tests,Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 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 Coulter®Multisizer Particle.
Results
Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.034 mg/L, to 0.19 mg/L. A decline in measured test concentrations to less than the LOQ in all test preparations was observed 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.
Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:
Response Variable
EL10
(mg/L Loading Rate WAF)EL20
(mg/L Loading Rate WAF)EL50
(mg/L Loading Rate WAF)95% Confidence Limits
(mg/L Loading Rate WAF)
No Observed Effect Loading Rate (NOEL)
(mg/L)Lowest Observed Effect Loading Rate (LOEL)
(mg/L)Growth Rate
0.068
0.11
0.25
0.21
-
0.31
0.010
0.032
Reference
Table 2 Inhibition of Growth Rate and Yield in the Definitive Test
Nominal Loading Rate |
Growth Rate (cells/mL/hour) |
Yield (cells/mL) |
|||
0 to 72 Hour |
% Inhibition |
0 to 72 Hour |
% Inhibition* |
||
Control |
R1 |
0.077 |
|
1.29E+06 |
|
R2 |
0.076 |
|
1.18E+06 |
|
|
R3 |
0.078 |
|
1.38E+06 |
|
|
R4 |
0.079 |
- |
1.50E+06 |
- |
|
R5 |
0.074 |
|
1.05E+06 |
|
|
R6 |
0.078 |
|
1.34E+06 |
|
|
Mean |
0.077 |
|
1.29E+06 |
|
|
SD |
0.002 |
|
1.58E+05 |
|
|
0.010 |
R1 |
0.077 |
0 |
1.26E+06 |
|
R2 |
0.076 |
1 |
1.20E+06 |
|
|
R3 |
0.075 |
3 |
1.11E+06 |
|
|
Mean |
0.076 |
1 |
1.19E+06 |
8 |
|
SD |
0.001 |
|
7.65E+04 |
|
|
0.032 |
R1 |
0.073 |
5 |
9.81E+05 |
|
R2 |
0.071 |
8 |
8.00E+05 |
|
|
R3 |
0.073 |
5 |
9.76E+05 |
|
|
Mean |
0.072 |
6 |
9.19E+05 |
29 |
|
SD |
0.001 |
|
1.03E+05 |
|
|
0.10 |
R1 |
0.059 |
23 |
3.51E+05 |
|
R2 |
0.060 |
22 |
3.75E+05 |
|
|
R3 |
0.058 |
25 |
3.28E+05 |
|
|
Mean |
0.059 |
23 |
3.51E+05 |
73 |
|
SD |
0.001 |
|
2.37E+04 |
|
|
0.32 |
R1 |
0.035 |
55 |
5.85E+04 |
|
R2 |
0.037 |
52 |
6.75E+04 |
|
|
R3 |
0.041 |
47 |
9.23E+04 |
|
|
Mean |
0.038 |
51 |
7.28E+04 |
94 |
|
SD |
0.003 |
|
1.75E+04 |
|
|
1.0 |
R1 |
0.003 |
96 |
1.26E+03 |
|
R2 |
-0.005 |
106 |
-1.61E+03 |
|
|
R3 |
-0.007 |
109 |
-2.02E+03 |
|
|
Mean |
-0.003 |
104 |
-7.92E+02 |
100 |
|
SD |
0.005 |
|
1.79E+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
Description of key information
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 Hour period and gave the following results:
Response Variable |
EL10 |
EL20 |
EL50 |
95% Confidence Limits (mg/L Loading Rate WAF) |
No Observed Effect Loading Rate (NOEL) |
Lowest Observed Effect Loading Rate (LOEL) |
||
Growth Rate |
0.068 |
0.11 |
0.25 |
0.21 |
- |
0.31 |
0.010 |
0.032 |
Key value for chemical safety assessment
- EC50 for freshwater algae:
- 0.25 mg/L
Additional information
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