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EC number: 223-384-3 | CAS number: 3865-34-7
- 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
- Explosiveness
- 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
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- 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
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- 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:
- 11 December 2017 to
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: read-across target
- 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:
- 2009
- Deviations:
- no
- 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 at 0 and 72 hours and stored frozen for further analysis if necessary. - Vehicle:
- no
- Details on test solutions:
- PRELIMINARY MEDIA PREPARATION TRIAL
A nominal amount of test material (1100 mg) was dispersed, in duplicate, in 11 litres of deionised reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
- Centrifugation at 10000 g for 30 minutes
- Centrifugation at 40000 g for 30 minutes
- Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter)
- Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 litres discarded in order to pre-condition the filter)
Visual inspection of the centrifuged test samples showed that an oily film of test material was floating at the surface, as such centrifugation was considered inappropriate as a means of removing undissolved test material.
The results obtained from the filtered test samples indicated that no significant increase in the dissolved test material concentration occurred when the preparation period was increased to 48 hours.
Based on this information the test material was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test material by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 1 litre discarded) to give a nominal test concentration of approximately 49 mg/L. - Test organisms (species):
- Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Method of cultivation: The master cultures were kept under constant agitation by orbital shaker (100 to 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 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
- Test temperature:
- 24 ± 1 °C
- pH:
- 7.4 - 8.1
- Nominal and measured concentrations:
- 0 hours:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured: 0.389, 1.20, 2.95, 7.63 and 20.4 mg/L.
72 hours:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured: 0.360, 1.06, 3.38, 7.85 and 19.2 mg/L. - Details on test conditions:
- TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed- the flasks were plugged with polyurethane foam bungs
- Fill volume: 100 mL
- Initial cells density: 5.00 x10^3cells/ mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
TEST 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 and Na2EDTA.2H2O 0.30 mg/L.
- The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
OTHER TEST CONDITIONS
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm)
- Constantly shaken at approximately 150 rpm for 72 hours.
EFFECT PARAMETERS MEASURED:
- Test Organism Observations: Samples were taken at 23, 48 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 material 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 preparation 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.
- Test material concentrations were analytically verified.
TEST CONCENTRATIONS
- Range finding study: 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% v/v saturated solution for a period of 72 hours.
- Based on the results of the range-finding test, two initial experiments were conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no effect on algal growth was observed. In both instances significant inhibition of growth was observed to have occurred in the 100% v/v saturated solution test preparations after 24 hours exposure suggesting that the results obtained from the range-finding test were erroneous.
- Based on the results of the initial experiments the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 % v/v saturated solution.
- Preparation of test material solutions: A nominal amount of test material (1100 mg) was dispersed in 11 litres 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 material was removed by filtration through a 0.2 μm Sartorius Sartopre filter (first approximate 1 liter 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 stock solutions of 32, 10, 3.2 and 1.0 % v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 11.4 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 abnd 100% v/v saturated solution.
DATA EVALUATION
- Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation: µ = (ln Nn – ln N1) / (tn – t1)
Where:
µ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement
The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
Percentage inhibition of growth rate for each replicate test material vessel was calculated using the following equation:
Ir = [(µc - µt) / µc] x 100
Where:
Ir = percentage inhibition of average specific growth rate
µc = mean average specific growth rate for the control cultures
µt = average specific growth rate for the test culture
- Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:
Y = Nn – N0
Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test
For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:
Iy = [(Yc – Yt)/ Yc ] x 100
Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group
DETERMINATION OF ECx VALUES
- For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
- Where appropriate 95 % confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).
STATISTICAL ANALYSIS
- 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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met:
- The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
- The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3) must not exceed 35%.
- The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 7.6 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95 % Confidence Limits 6.5 - 8.9 mg/L
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 2 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Remarks on result:
- other: 95 % Confidence Limits 1.8 - 2.3 mg/L
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 1.2 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks:
- and yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 3 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks:
- and yield
- Details on results:
- RANGE-FINDING TEST
- The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
- Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the initial experiments. 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 saturated solution test preparations at 0 and 72 hours showed measured test concentrations of 8.1 and 7.9 mg/L respectively were obtained indicating that the test material was stable under test conditions.
INITIAL EXPERIMENTS
- Based on the results of the range-finding test, two initial experiments were conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no effect on algal growth was observed. In both instances significant inhibition of growth was observed to have occurred in the 100% v/v saturated solution test preparations after 24 hours exposure suggesting that the results obtained from the range-finding test were erroneous.
DEFINITIVE TEST
- Verification of Test Concentrations: Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.39 to 20 mg/L. There was no significant change in the measured concentrations at 72 hours and so the results are based on the 0-hour measured test concentrations 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 1. From the data given in Table 1, 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 material over the 72-hour exposure period.
- Accordingly the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 to- 72 hour): 2.0 mg/L
ErC20 (0 to 72 hour): 3.3 mg/L
ErC50 (0 to 72 hour): 7.6 mg/L; 95 % confidence limits 6.5 to 8.9 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, 0.39 and 1.2 mg/L test concentrations (P ≥ 0.05), however all other test concentrations were significantly different (P < 0.05) and, therefore the NOEC based on growth rate was 1.2 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 3.0 mg/L.
Inhibition of Yield
EyC10 (0 to 72 hour): 0.86 mg/L
EyC20 (0 to 72 hour): 1.2 mg/L
EyC50 (0 to 72 hour): 2.0 mg/L; 95 % confidence limits 1.8 to 2.3 mg/L
Where:
EyCx is the test concentration 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, 0.39 and 1.2 mg/L test concentrations (P ≥ 0.05), however all other test concentrations were significantly different (P < 0.05) and, therefore the NOEC based on yield was 1.2 mg/L. Correspondingly the LOEC based on yield was 3.0 mg/L.
- Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 293 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 : 1.46 x 10^6 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 6% 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 to 72 hours) was 3% 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 0.39, 1.2, 3.0 and 7.6 mg/L, however no intact cells were observed to be present in the test cultures at 20 mg/L.
- Water Quality Criteria: Temperature was maintained at 24 ±1°C throughout the test. The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.0 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 Material Solubility: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-hour test period all control, 0.39 and 1.2 mg/L test cultures were observed to be green dispersions. The 3.0 mg/L test cultures were pale green dispersions; the 7.6 mg/L test cultures were extremely pale green dispersions whilst the 20 mg/L test cultures were clear colourless solutions. - Results with reference substance (positive control):
- - Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference material gave the following results: ErC50 (0 to 72 h): 1.6 mg/L; 95% confidence limits 1.4 - 1.8 mg/L and EyC50 (0 to 72 h): 0.77 mg/L; 95% confidence limits 0.68 - 0.87 mg/L.
- No Observed Effect Concentration based on growth rate: 0.25 mg/L
- No Observed Effect Concentration based on yield: 0.25 mg/L
- Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
- Lowest Observed Effect Concentration 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. - Validity criteria fulfilled:
- yes
- Conclusions:
- Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the 0-hour measured test concentrations: Growth rate EC50: 7.6 mg/L with 95 % confidence limits of 6.5 to 8.9 mg/L and yield inhibition EC50: 2.0 mg/L with 95% confidence limits of 1.8 to 2.3 mg/L with a NOEC of 1.2 mg/L and LOEC of 3.0 mg/L.
- Executive summary:
The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.
A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 49 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.
Following a preliminary range-finding test and initial experiments, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solutions were prepared by stirring an excess (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.39 to 20 mg/L. There was no significant change in the measured concentrations at 72 hours and so the results are based on the 0-hour measured test concentrations only.
The validity criteria of the test were fulfilled.
Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the 0-hour measured test concentrations: Growth rate EC50: 7.6 mg/L with 95% confidence limits of 6.5 to 8.9 mg/L and yield inhibition EC50: 2.0 mg/L with 95% confidence limits of 1.8 to 2.3 mg/L with a NOEC of 1.2 mg/L and LOEC of 3.0 mg/L.
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read-across material
- Justification for type of information:
- Read-across to structurally similar substance Dimethyldineodecanoatetin (CAS 68928-76-7), see attached justification.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 7.6 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95 % Confidence Limits 6.5 - 8.9 mg/L
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 2 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Remarks on result:
- other: 95 % Confidence Limits 1.8 - 2.3 mg/L
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 1.2 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks:
- and yield
- Key result
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 3 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks:
- and yield
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Not specified
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Preferred study for this SIDS endpoint; GLP guideline study. No analytical confirmation of exposure concentrations.
- Reason / purpose for cross-reference:
- other: read-across target
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Deviations:
- no
- GLP compliance:
- yes
- Analytical monitoring:
- no
- Details on sampling:
- not applicable
- Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A single 1,000 mg/L WAF was formulated by combining 0.5 g of test material and 500 mL of dilution water in a glass mixing vessel equipped with a magnetic stirrer (the vortex extended approximately 25 % of the distance from the surface to the bottom of the mixing vessel). The mixture was stirred for approximately 24 hours and allowed to settle for approximately 1 hour. Following the settling period the water phase containing the WAF was removed from the mixing vessel with a siphon using care to exclude any material on the surface, bottom, or sides of the mixing vessel. Test media was prepared at 0.10, 1.0, 10, 100, and 1,000 mg/L by combining the appropriate volume of the 1,000 mg/L WAF and dilution water.
- Eluate: NA
- Differential loading: NA
- Controls: Dilution water control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Dilution control, 1.10, 1.0, 10, 100, and 1,000 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The 1,000 mg/L WAF was cloudy throughout the test. No other insoluble material was observed during the test. - Test organisms (species):
- Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Common name: Green algae
- Strain: Selenastrum capricornutum
- Source (laboratory, culture collection): University of Texas at Austin and delivered to T.R. Wilbury on April 11, 1995.
- Age of inoculum (at test initiation): The inoculum used for the test was from a single source that had been growing for 8 days prior to testing.
- Method of cultivation: No data
ACCLIMATION
- Acclimation period: Approximately 14 days
- Culturing media and conditions (same as test or not): Same as test media.
- Any deformed or abnormal cells observed: No - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 96 h
- Post exposure observation period:
- not applicable
- Hardness:
- no data
- Test temperature:
- 24.0 °C at test initiation to 23.9 °C at test termination
- pH:
- 7.3-10.2
- Dissolved oxygen:
- no data
- Salinity:
- no data
- Nominal and measured concentrations:
- nominal: 0.10, 1.0, 10, 100 and 1000 mg/L
- Details on test conditions:
- TEST SYSTEM
- Test vessel: 125 500 mL glass Erlenmeyer flasks
- Type (delete if not applicable): open / closed open
- Material, size, headspace, fill volume: Fill volume was less than 50 % of vessel volume.
- Aeration: Rotary shaker adjusted to 100 ppm in an incubator
- Type of flow-through (e.g. peristaltic or proportional diluter): Static
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 10, 000 cells/mL
- Control end cells density: 1,804,000 cells/mL
- No. of organisms per vessel: 10,000 cells/mL
- No. of vessels per concentration (replicates): 2 Replicates
- No. of vessels per control (replicates): 2 Replicates
- No. of vessels per vehicle control (replicates): None
GROWTH MEDIUM
- Standard medium used: yes/no Yes, sterile enriched media identical to media used for this test and maintained at test conditions for at lest 14 days before the definitive test. The inoculum used for the test was from a single source that had been growing for 8 days prior to testing.
- Detailed composition if non-standard medium was used: NA
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used for acclimation of test organisms and for all toxicity testing was sterile enriched media with a pH of 7.5. Dilution water was analysed for particulate matter at the start of the test and media from a control vessel was analysed for particulate matter at the end of the test.
- Total organic carbon: non detect
- Particulate matter: non detect at 0-hr; 33 mg/L at 96-hr
- Metals: all non detect; iron 0.03 mg/L
- Pesticides: non detect
- Chlorine: non detect
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: NA
- Culture medium different from test medium: No
- Intervals of water quality measurement: Start and end of test.
OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: 24 hour’s light
- Light intensity and quality: Approximately 400 foot-candles
- Salinity (for marine algae): NA
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth rate- see table
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: NA
- Other: Calculations and Statistical Methods – The average specific growth rate was calculated as the natural log of the number of cells/ml at 72 and 96 hours minus the natural log of the number of cells/ml at 0 hours divided by the exposure period. The percent change from the control was calculated by subtracting the treatment average specific growth rate from the control average specific growth rate, dividing the difference by the average specific growth rate in the control, and multiplying that value by 100. The EC50 and NOEC values were calculated using nominal concentrations of test substance, when warranted. The values were computed twice, once using the average number of cells/ml at each concentration expressed as a percent of the control and a second time using the average specific growth rate expressed as the percent change from the control. The binomial/nonlinear interpolation method (Stephan, 1983) was used to calculate EC50 values. The slope of the 96-hour dose response curve (cell density) was computed using the probit method. The no observed effect concentration (NOEC) is the highest concentration of test substance that allowed cell growth equal to at least 90% of the control growth.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: Each concentration approximately 10 % of the next higher concentration.
- Justification for using fewer concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: 1.10, 1.0, 10, 100, 1,000 mg/L
- Results used to determine the conditions for the definitive study: This is a range finding study. - Reference substance (positive control):
- no
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 270 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 120 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: cell density
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: cell density
- Details on results:
- - Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: After 120 hours, examination of the media from the flask containing a 0.50 ml subsample of test media from the flask containing the 1,000 mg/L Alkyltin MA WAF and 50 mL of fresh media contained 472,000 algal cells/mL, indicating that the effect of the test material at the concentration was algistatic.
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The 1,000 mg/L WAF was cloudy throughout the test. No other insoluble material was observed during the test.
- Effect concentrations exceeding solubility of substance in test medium: greater than 1,000 mg/L - Results with reference substance (positive control):
- not applicable
- Reported statistics and error estimates:
- Data were evaluated using the binomial/non-linear interpolation method (Stephan 1983). The slope of the 96-h dose response curve (cell density) was computed using the probit methd. The NOEC was defined as the highest concentration tested that allowed cell growth equal to at least 90% of the control growth.
- Validity criteria fulfilled:
- yes
- Conclusions:
- Exposure of the freshwater alga, Selenstrum capricornutum, to the test material resulted in a 72-h EC50 based on growth rate of 270 mg/L and a 72-h NOEC based on growth rate of 10 mg/L .
- Executive summary:
The toxicity of the WAF of the test material to the freshwater alga, Selenastrum capricornutum, was investigated during a range-finding study. The test was performed under static conditions with the WAF of 0.10, 1.0, 10, 100 and 1000 mg/L mixtures of test material and water. Cell counts were made at 0, 72 and 96 hours with a haemocytometer.
A 72 -h EC50 based on the growth rate was 270 mg/L with a no observed effect concentration at 72 -h of 10 mg/L based on growth rate.
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study conducted on read-across material
- Justification for type of information:
- Read-across to structurally similar substance DMTE (Dimethyltin bis (2-ethylhexyl thioglycolate) CAS 57583 -35 -4), see attached justification.
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 270 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 120 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: cell density
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 10 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: cell density
Referenceopen allclose all
Table 1: Inhibition of Growth Rate and Yield in the Definitive Test
0-Hour Measured Test Concentration |
Growth Rate (cells/mL/hour) |
Yield (cells/mL) |
|||
0 to 72 Hour |
% Inhibition |
0 to 72 Hour |
% Inhibition* |
||
Control |
R1 |
0.076 |
|
1.16E+06 |
|
R2 |
0.077 |
|
1.25E+06 |
|
|
R3 |
0.080 |
|
1.60E+06 |
|
|
R4 |
0.080 |
- |
1.57E+06 |
- |
|
R5 |
0.079 |
|
1.49E+06 |
|
|
R6 |
0.081 |
|
1.67E+06 |
|
|
Mean |
0.079 |
|
1.46E+06 |
|
|
SD |
0.002 |
|
2.05E+05 |
|
|
0.39 |
R1 |
0.081 |
[3] |
1.66E+06 |
|
R2 |
0.078 |
1 |
1.34E+06 |
|
|
R3 |
0.077 |
3 |
1.32E+06 |
|
|
Mean |
0.079 |
0 |
1.44E+06 |
1 |
|
SD |
0.002 |
|
1.92E+05 |
|
|
1.2 |
R1 |
0.076 |
4 |
1.14E+06 |
|
R2 |
0.076 |
4 |
1.19E+06 |
|
|
R3 |
0.076 |
4 |
1.16E+06 |
|
|
Mean |
0.076 |
4 |
1.16E+06 |
20 |
|
SD |
0.000 |
|
2.45E+04 |
|
|
3.0 |
R1 |
0.064 |
19 |
4.86E+05 |
|
R2 |
0.059 |
25 |
3.47E+05 |
|
|
R3 |
0.053 |
33 |
2.17E+05 |
|
|
Mean |
0.059 |
26 |
3.50E+05 |
76 |
|
SD |
0.006 |
|
1.35E+05 |
|
|
7.6 |
R1 |
0.047 |
41 |
1.39E+05 |
|
R2 |
0.047 |
41 |
1.39E+05 |
|
|
R3 |
0.046 |
42 |
1.33E+05 |
|
|
Mean |
0.047 |
41 |
1.37E+05 |
91 |
|
SD |
0.001 |
|
3.45E+03 |
|
|
20 |
R1 |
0.015 |
81 |
9.37E+03 |
|
R2 |
0.005 |
94 |
1.95E+03 |
|
|
R3 |
0.003 |
96 |
1.19E+03 |
|
|
Mean |
0.008 |
90 |
4.17E+03 |
100 |
|
SD |
0.006 |
|
4.52E+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
Mean cell growth data (number of cells/ml x 10^3) at 72 hours, by nominal concentration of the WAF tested:
Control (0 mg/L): 838 (72 h)
0.10 mg/L: 811 (72 h)
1.0 mg/L: 822 (72 h)
10 mg/L: 803 (72 h) 100 mg/L: 465 (72 h)
1000 mg/L: <13 (72 h)
Average specific growth rate at 72 hours, by nominal concentration of the WAF tested:
Control (0 mg/L): 0.062 (72 h)
0.10 mg/L: 0.061 (72 h)
1.0 mg/L: 0.061 (72 h)
10 mg/L: 0.061 (72 h)
100 mg/L: 0.053 (72 h)
1000 mg/L: 0.004 (72 h)
Description of key information
Read-across to DMT-Neodecanoate (CAS 68928 -76 -7)
Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the 0-hour measured test concentrations: Growth rate EC50: 7.6 mg/L with 95% confidence limits of 6.5 to 8.9 mg/L and yield inhibition EC50: 2.0 mg/L with 95% confidence limits of 1.8 to 2.3 mg/L with a NOEC of 1.2 mg/L and LOEC of 3.0 mg/L.
Read-across to structurally similar substance DMTE (Dimethyltin bis (2-ethylhexyl thioglycolate))
EC50 (72-h) 270 mg/L based on growth rate for Selenastrum capricornutum (OECD 201)
Key value for chemical safety assessment
- EC50 for freshwater algae:
- 7.6 mg/L
- EC10 or NOEC for freshwater algae:
- 1.2 mg/L
Additional information
Read-across to DMT-Neodecanoate (CAS 68928 -76 -7)
The effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
A preliminary media preparation trial indicated that a dissolved test material concentration of approximately 49 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.
Following a preliminary range-finding test and initial experiments, Pseudokirchneriella subcapitata was exposed to solutions of the test material at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solutions were prepared by stirring an excess (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.39 to 20 mg/L. There was no significant change in the measured concentrations at 72 hours and so the results are based on the 0-hour measured test concentrations only.
The validity criteria of the test were fulfilled.
Under the conditions of the study, exposure of Pseudokirchneriella subcapitata to the test material over 72 hours gave the following results based on the 0-hour measured test concentrations: Growth rate EC50: 7.6 mg/L with 95% confidence limits of 6.5 to 8.9 mg/L and yield inhibition EC50: 2.0 mg/L with 95% confidence limits of 1.8 to 2.3 mg/L with a NOEC of 1.2 mg/L and LOEC of 3.0 mg/L.
Read-across to structurally similar substance DMTE (Dimethyltin bis (2-ethylhexyl thioglycolate))
The toxicity of the WAF of the test material to the freshwater alga, Selenastrum capricornutum, was investigated during a range-finding study. The test was performed under static conditions with the WAF of 0.10, 1.0, 10, 100 and 1000 mg/L mixtures of test material and water. Cell counts were made at 0, 72 and 96 hours with a haemocytometer.
A 72 -h EC50 based on the growth rate was 270 mg/L with a no observed effect concentration at 72 -h of 10 mg/L based on growth rate.
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