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EC number: 219-145-8 | CAS number: 2372-82-9
- 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:
- 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Deviations:
- yes
- Remarks:
- ; analytical quantification of the test concentrations was not possible due to the absence of a suitable analytical method. Therefore only the stock solution used to prepare the test concentrations were analyzed.
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Qualifier:
- according to guideline
- Guideline:
- ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- - Lot/Batch number: FP 020010155
- Triamine content: 85.3%; Total amine content (commercial grade): 100% - Analytical monitoring:
- yes
- Details on sampling:
- Cell concentrations were determined photometrically with a UV/VIS Spectrophotometer (Standard Operation Procedure K 3 (9.11)). Measurements were carried out at 436 nm in a cuvette with a light path of 4 cm. To establish the relation between extinction and cell number/algal biomass, a calibration curve was made which is checked yearly. The procedure of constructing the calibration curve as well as the control procedure are described in Standard Operation Procedure E 3 (9.10).
From the relation between extinction (E) and counted cell number (N) the following calibration curve was determined using linear regression: N = 3.2034 * 10^6 * Extinction - 3.0 * 10^5 (r" = 0.9864).
The available analytical method was not suitable to quantify concentrations below 10 mg/L. Therefore only the stock solution used to prepare the test concentrations was analyzed. The measured concentration of the undiluted stock solution (1080 mg/L) differs only slightly from the nominal concentration of the stock solution (1066 mg/L). These results show that the stock solution was stable even after storage for almost 7 months in the refrigerator. All concentrations mentioned in this report are therefore based on nominal concentrations. - Vehicle:
- no
- Details on test solutions:
- STOCK AND TEST SOLUTION AND THEIR PREPARATION:
- A stock solution of approximately 1 g/L of test substance was prepared as follows: To an accurately measured amount of 0.1066 g. of test substance some OECD medium of 51.3°C was added. The test substance dissolved well under stirring. After cooling, OECD medium was added to a final volume of 100 mL. A homogeneous, clear stock solution was obtained. The pH was 8.4. It was kept stirring at room temperature before being used for the preparation of the test solutions at the start of the test.
- The test solutions were prepared by addition of the required amounts of a 1:100 diluted stock solution to the test vessels to obtain the following final nominal test concentrations: 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L. - Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISMS (Fresh water algae):
- Strain: Pseudokirchneriella subscapitata (was formally called Selenastrum capricornutum)
- Source: Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, The Windermere Laboratory, Cumbria, Ambleside, United Kingdom.
- Laboratory culture: Yes
- Method of cultivation: According to OECD 201
- Pretreatment: None
- Initial cell concentration: 1*E04 cells/mL
- Other: The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing.
The extinction of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1*E04 cells/mL in the test medium. - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
- Test temperature:
- 23.4-23.6 °C
- pH:
- 8.2-8.7
- Salinity:
- Standard OECD 201 medium
- Nominal and measured concentrations:
- Nominal test concentrations: 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L.
- Details on test conditions:
- TEST SYSTEM:
- Test vessel: 100 mL erlenmeyers containing 40 mL medium
- Renewal of test solution: No
- Number of replicates: 6 control vessels and 3 vessels per test substance concentration
- Test type: growth inhibition, endpoint biomass
TEST MEDIUM / WATER PARAMETERS:
- Test medium: The test medium (OECD medium) was prepared from concentrated solutions of the mineral salts prepared in deionized water. 3 L was prepared for the test and sterilized by filter sterilization (0.2 um).
- Macro nutrients (mg/L): NH4Cl; 15 KH2PO4; 1.6 CaCl2(H2O)2; 18 MgSO4(H2O)7; 15 MgCl2(H2O)6; 12 Fe-EDTA FeCl2(H2O)6; 0.08 Na2H2EDTA(H2O)2; 0.1 - Trace elements: H3BO3; 0.185 ZnCl2; 0.003 MnCl2(H2O)4; 0.415 CoCl2(H2O)6; 0.0015 CuCl2(H2O)2; 1*10-5 Na2MoO4(H2O)2; 0.007 NaHCO3 NaHCO3 150
- For the test an adequate amount of test medium was prepared in a 3-L vessel. This medium was sterilized by filter sterilization (0.2 μm). Adequate amounts of stock solution were added to the test vessels (100 mL erlenmeyers). The test vessels were filled with medium up to a total volume of 40 mL using a sterilized dispenser. For the concentrations 0.01 - 0.04 and 0.16 mg/L; 1L of test solution was prepared from which three times 40 mL was divided in the 100 mL test vessels. The remaining 880 mL was divided up in two portions of 440 mL from which one was stored for chemical analyses and one was transferred into a 1 L erlenmeyer. The inoculum was added to all 100 mL test vessels from an exponentially growing culture with a pipette. In addition six control replicates were included. The extinction in each 100 mL erlenmeyer was measured after 0, 24, 48 and 72 h. Algal medium was used as a blank in the spectrophotometer. The 1 L erlenmeyers with 440 mL 0.01, 0.04 and 0.16 mg/L were incubated together with the 100 mL test vessels in the orbital incubator.
- The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing. The extinction of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1*E04 cells/mL in the test medium.
TEST SOLUTION, CONTROLS AND TEST CONDITIONS:
- Dilution water: The deionized water used in the study contained not more than 0.01 mg/L of copper and had a TOC content of not more than 2.0 mg/L and a conductivity of less than 5 uS/cm. This water was produced from tap water in a water purification system.
- The test solutions were prepared by addition of the required amounts of a 1:100 diluted stock solution to the test vessels to obtain the following final nominal test concentrations: 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L (nominal).
- Controls containing only test medium were included in the test.
- The vessels were incubated in an illuminated and temperature controlled (23°C ± 2 °C) orbital incubator.
- Aeration: No
- The test vessels were rotated continuously at a speed sufficient to prevent sedimentation of the algae. The extinction in each 100 mL erlenmeyer was measured after 0, 24, 48 and 72 h. Algal medium was used as a blank in the spectrophotometer. At the end of the test five random samples were microscopically checked for purity of the algal culture.
- Intensity of irradiation: 88-89 umol/m-2s-1
- Photoperiod: Continuous
- The pH of all samples and controls were measured at the beginning (t=0 h) and at the end (t=72 h) of the test. The temperature in the culturing apparatus was continuously measured and read out at the end of the test. The light intensity was measured at the beginning and at the end of the test. At the end of the test five random samples were microscopically checked for purity of the algal culture.
- Cell concentrations were determined photometrically with a UV/VlS Spectrophotometer. Measurements were carried out at 436 nm in a cuvette with a light path of 4 cm. To establish the relation between extinction and cell density, a calibration curve was made which was checked yearly. From the relation between extinction (E) and counted cell number (N) the following calibration curve was determined using linear regression: N= 3.2034*106 E- 3.0*105 (r2 = 0.9864).
STABILITY OF THE TEST CHEMICAL SOLUTIONS:
- Stock solution were stable after 7 months in refrigerator - Reference substance (positive control):
- yes
- Remarks:
- potassium dichromate
- Duration:
- 72 h
- Dose descriptor:
- other: EbC50
- Effect conc.:
- 0.01 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Based on total amine content
- Basis for effect:
- biomass
- Remarks on result:
- other: (0.012-0.020 95% C.I.)
- Key result
- Duration:
- 72 h
- Dose descriptor:
- other: ErC50
- Effect conc.:
- 0.015 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Based on total amine content
- Basis for effect:
- growth rate
- Remarks on result:
- other: (0.012-0.020 95% C.I.)
- Key result
- Duration:
- 72 h
- Dose descriptor:
- other: ErC10
- Effect conc.:
- 0.009 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Based on total amine content
- Basis for effect:
- growth rate
- Remarks on result:
- other: (0.0045-0.0121 95% C.I.)
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- < 0.01 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Remarks:
- Based on total amine content
- Basis for effect:
- biomass
- Remarks on result:
- other:
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.01 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- biomass
- Results with reference substance (positive control):
- The sensitivity of the algae is checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year, when the cultures are grafted from the sloped agar tubes. The sensitivity was tested for compliance with the Guidelines, which means that the EC50 values are between 0.25 and 2.0 mg/L.
- Reported statistics and error estimates:
- The mean values of the extinction’s for each test substance concentration were used to calculate the growth and specific growth rate.
Where possible, the EC20,50,80 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50 (0 72 h), whereas the EC50 value calculated for the specific growth rate is termed ErC50. The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using threshold values from the William’s test. The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no significant inhibition relative to the control values. Confidence limits were computed on the basis of Fieller's theorem. All computations were performed using the TOXCALC version 5.0 program - Validity criteria fulfilled:
- yes
- Conclusions:
- Under the study conditions, the 72 h EbC50 and ErC50, based on nominal concentrations were 0.010 and 0.015 mg/L, respectively. The LOEC was determined to be at the lowest tested concentration of 0.010 mg/L. The ErC10 of 0.0095 mg/L will be used as long term endpoint.
- Executive summary:
A study was conducted to determine the acute toxicity of the test substance to the freshwater algae Pseudokirchneriella subcapitata according to OECD Guideline 201, EU Method C.3 and ISO Method 8692, in compliance with GLP. In this study, triplicate Erlenmeyers containing the freshwater algae were exposed to 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L nominal concentrations of the test substance for 72 h under static conditions. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Although quantification of the applied concentrations was not possible due to the absence of a suitable analytical method it was possible to analyze the two stock solutions used to prepare the test concentrations. These analyses showed that the stock solutions were stable even after storage for almost 7 months in the refrigerator. Considering the high water solubility and stability of the test substance, it was considered reasonable to assume that the actual concentrations would be identical to the nominal concentrations during the entire test. All the validity criteria were considered to be fulfilled. Under the study conditions, the 72 h EbC50 and ErC50, based on nominal concentrations were 0.010 and 0.015 mg/L, respectively. The LOEC was determined to be at the lowest tested concentration of 0.010 mg/L. The ErC10 of 0.0095 mg/L will be used as long term endpoint (Geurts, 2002).
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Deviations:
- no
- GLP compliance:
- yes
- Analytical monitoring:
- yes
- Vehicle:
- no
- Details on test solutions:
- For the purpose of the definitive study, the test material was dissolved directly in culture medium.
An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication (approximately 5 minutes) and the volume adjusted to 500 mL to give a 200 mg/L
stock solution. A series of dilutions were made from this stock solution to give further stock solutions of 20, 2.0, 0.20, 0.10, 0.050, 0.025 and 0.0125 mg/L. An aliquot (500 mL) of the 0.0125, 0.025, 0.050, 0.10 and 0.20 mg/L stock solutions was separately mixed with algal suspension (500 mL) to give the required test concentrations of 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours.
Pre-study recovery analysis conducted indicated that the test material adsorbed to algal cells and hence it was considered that chemical analysis of the O-Hour test samples would show relatively low measured test concentrations. Therefore, in order to confirm the correct preparation of the initial stock solution and subsequent dosing of the test preparations, samples were prepared in culture medium alone (no algal cells) at 0 hours and taken for chemical analysis.
Analysis of the test solutions containing no algal cells showed measured test concentrations to be in the range of 87% to 127% of nominal thereby indicating that the correct dosing procedures were followed.
Chemical analysis of the test solutions containing algal cells at 0 hours showed measured test concentrations to be in the range of 58% to 81 % of nominal. Analysis of the test solutions at 72 hours showed a marked decline in measured test concentrations to below the limit of quantitation (LOQ) which was assessed down to 0.0035 mg/L and 4% of nominal. These results were inline with the preliminary recovery analysis conducted, which indicated that the test material adsorbed to algal cells.
The pre-study stability analysis conducted indicated that the test material was stable in culture medium over the study period. Adsorption was not a factor in the pre-study stability analysis since no algal cells were present.
In order to give a worst case analysis of the data, it was considered justifiable to base the results on the time-weighted mean measured test concentrations. Following the recommendations of the competent authorities in the EU, in cases where the measured test concentrations were less than the LOQ, a value of half the LOQ was used for calculating the time-weighted mean measured test concentrations.
The use of the time-weighted mean measured test concentrations in the calculation of the EC50 and NOEC values had a significant effect on the results of the study. Care should be taken in the interpretation of the results based on the time-weighted mean measured test concentrations as the test material has been shown to adsorb to algal cells. - Test organisms (species):
- Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
- Details on test organisms:
- The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria.
Cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The culture was maintained in the laboratory at a temperature of 21 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration. - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
- Test temperature:
- 24 ± 1°C.
- pH:
- 7.3 - 10.5
- Salinity:
- Standard OECD 201 test medium
- Nominal and measured concentrations:
- See table below
- Details on test conditions:
- - Scenedesmus subspicatus was exposed to a geometric series of five test concentrations and a negative control. The study was conducted in glass conical flasks plugged with polyurethane foam bungs to reduce evaporation.
- Three replicate flasks were prepared for each test concentration and control. The nominal cell density in flasks at the initiation of the study was l0 cells per ml. Flasks were incubated under continuous light and constant shaking for 72 h.
- Samples were taken at 0, 24, 48 and 72 h and cell densities determined using a particle counter.
- pH was recorded at initiation and termination of the study.
- Temperature was recorded daily.
- Duplicate samples for verification of test concentrations were taken at 0 and 72 h and stored frozen.
- Inhibition of growth was calculated from comparison of area under the growth curves of the test concentration versus the control. - Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 0.012 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- act. ingr.
- Remarks:
- Based on total amine content
- Basis for effect:
- growth rate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 0.02 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- act. ingr.
- Remarks:
- Based on total amine content
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.007 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- act. ingr.
- Remarks:
- Based on total amine content
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 0.012 mg/L
- Nominal / measured:
- meas. (TWA)
- Conc. based on:
- act. ingr.
- Remarks:
- Based on total amine content
- Basis for effect:
- biomass
- Details on results:
- - At test initiation the control and test cultures were clear colourless solutions.
- At the 72 h observation, the control, 0.00625, 0.0125, 0.025 and 0.050 mg/L test cultures were bright green dispersions and the 0.1 mg/L test culture was a very pale green dispersion.
- Analysis of the test concentrations showed a decline in test concentration in solutions containing algal cells. At 0 h concentrations ranged from 58 to 81 % of nominal and at 72 h test concentrations were below the limit of quantification to 4 % of nominal. Adsorption to algal cells was considered to be the cause of the decline in test substance concentration. This was confirmed by measuring the test concentrations in test vessels not containing algae (measured test concentrations were within 87 to 127 % of the nominal concentrations). - Validity criteria fulfilled:
- yes
- Conclusions:
- Under the study conditions, the 72 h EbC50 and ErC50 of the test substance, based on measured concentrations, were 0.012 and 0.02 mg/L, respectively. The NOEC and ErC10 were determined at 0.0069 and 0.012 mg/L, respectively.
- Executive summary:
A study was conducted to determine the acute toxicity of the test substance to the freshwater algae Scenedesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Based on the results of two range finding studies, the algae were exposed to 0.00625, 0.0 125, 0.025, 0.050, 0.10 mg/L nominal concentrations of the test substance under static conditions for 72 h. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Analysis of the test concentrations showed a decline due to adsorption to the algal cells. At 0 h, concentrations ranged from 58 to 81% of nominal values; at 72 h, the concentrations ranged from below the limit of quantification to 4% of nominal. Under the study conditions, the 72 h EbC50 and ErC50 of the test substance, based on measured concentrations, were 0.012 and 0.02 mg/L, respectively. The NOEC and ErC10 were determined at 0.0069 and 0.012 mg/L, respectively (Mead, 2001, 2007).
Referenceopen allclose all
RESULTS: EXPOSED- Nominal/measured concentrations: 0.01, 0.02, 0.04, 0.08, 0.16 mg/L (nominal).
- Effect data/Element values:
Table 1: Result details: Test-Substance
Concentration
(nominal) Inhibition%
[mg/L] compared to
0 h 24 h 48 h 72 h control
0.00 0.008 0.038 0.157 0.654 0.0
0.01 0.008 0.018 0.075 0.346 12.7
0.02* 0.008 0.004 0.008 0.022 73.9
0.04 0.007 0.002 0.001 0.003 114.3
0.08 0.007 0.001 0.002 0.001 125.0
0.16 0.007 0.001 0.001 0.001 132.5
*One value was excluded from the calculations because it was an outlier (Grubbs outlier test). Validity criteria:
Validity criteria were considered as fulfilled.
- The pH measurements a maximum increase of 0.5 pH units.
- The temperature varied from 23.4 to 23.6°C during the test, and the light intensity varied between approximately 88 and 89 umol/m-2s-1, which are both in accordance with the required conditions described in the study plan.
- The five random samples taken at the end of the test were all pure and not contaminated with bacteria.
- The increase factor of cell growth was 87 over 72 h for the controls.
- A dose-response related increase in growth inhibition observed.
The pH measurements a maximum increase of 0.5 pH units. The temperature varied from 23.4 to 23.6°C during the test, and the light intensity varied between approximately 88 and 89 μmol/m-2s-1, which are both in accordance with the required conditions described in the study plan. The five random samples were all pure and not contaminated with bacteria.
ALGAL INHIBITION TEST
PROGRAM: ALGAL2A
Project:T01014AL Y12D
Concentration Time (hours) Area Inhibition Specific Inhibition
(mg/L) 0 24 48 72 (%) Growth rate (%)
0.000 0.008 0.039 0.185 0.717 13.500 0.063
0.000 0.007 0.039 0.173 0.657 12.552 0.063
0.000 0.008 0.039 0.167 0.676 12.576 0.062
0.000 0.007 0.039 0.159 0.690 12.612 0.063
0.000 0.008 0.036 0.131 0.604 10.776 0.059
0.000 0.008 0.034 0.124 0.579 10.260 0.059
Mean 0.008 0.038 0.157 0.654 12.046 0.062 0.0
0.010 0.008 0.021 0.083 0.409 6.924 0.055
0.010 0.008 0.016 0.066 0.337 5.532 0.053
0.010 0.008 0.016 0.076 0.292 5.232 0.052
Mean 0.008 0.018 0.075 0.346 5.896 51.1 0.054 12.7
0.020 0.007*0.036* 0.140*0.594* Outlier Outlier
0.020 0.008 0.003 0.009 0.027 0.132 0.020
0.020 0.008 0.005 0.007 0.017 0.012 0.011
Mean 0.008 0.004 0.008 0.022 0.072 99.4 0.016 73.9
0.040 0.007 0.001 0.001 0.002 -0.348 -0.013
0.040 0.007 0.001 0.001 0.003 -0.336 -0.009
0.040 0.007 0.003 0.002 0.005 -0.240 -0.005
Mean 0.007 0.002 0.001 0.003 -0.308 102.6 -0.009 114.3
0.080 0.007 0.001 0.002 0.002 -0.324 -0.011
0.080 0.007 0.001 0.001 0.001 -0.360 -0.019
0.080 0.007 0.001 0.002 0.001 -0.336 -0.017
Mean 0.007 0.001 0.002 0.001 -0.340 102.8 -0.015 125.0
0.160 0.007 0.001 0.001 0.000 -0.372 -0.031
0.160 0.007 0.002 0.000 0.002 -0.348 -0.019
0.160 0.007 0.001 0.001 0.001 -0.360 -0.019
Mean 0.007 0.001 0.001 0.001 -0.360 103.0 -0.021 133.8
The coefficient of average specific growth rates (72 hours test) |
|
|||||
Time (hours) |
|
|
mean |
SD ± |
%CV |
Mean |
0-24 |
24-48 |
48-72 |
72 hours test |
|
|
%CV |
0.066 |
0.065 |
0.056 |
0.0624 |
0.00522 |
8.36 |
9.35 |
0.072 |
0.062 |
0.056 |
0.0631 |
0.00803 |
12.73 |
|
0.066 |
0.061 |
0.058 |
0.0616 |
0.00397 |
6.45 |
|
0.072 |
0.059 |
0.061 |
0.0638 |
0.00689 |
10.80 |
|
0.063 |
0.054 |
0.064 |
0.0601 |
0.00543 |
9.03 |
|
0.060 |
0.054 |
0.064 |
0.0595 |
0.00520 |
8.74 |
|
|
|
mean |
0.0617 |
|
|
|
|
|
stdev |
0.0017 |
|
|
|
|
|
CV% |
2.7455 |
|
|
|
- The pH values of the control cultures were observed to pH 7.3 at 0 h to pH 10.4 -10.5 at 72 h. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution, which results in an increase in the pH of the culture. The increase in pH after 72 h was in excess of that recommended in the EEC Guidelines (1.5 pH units after 72 h). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the test guideline.
- Analysis of test solutions showed measured test concentrations to be in the range of 58 % to 81 % of nominal at 0 h and to below the LOQ at 72 h. To confirm the loss was due to adsorption to algal cells, samples were prepared in culture medium alone. Analysis of these solutions showed test concentrations in the range of 87 to 127 % of nominal. Pre-stability analysis showed that the test material was stable in the culture medium over the study period.
These factors are not considered to have affected the validity of the study.
Description of key information
Key value for chemical safety assessment
- EC50 for freshwater algae:
- 0.015 mg/L
- EC10 or NOEC for freshwater algae:
- 0.009 mg/L
Additional information
A study was conducted to determine the acute toxicity of the test substance to the freshwater algae Pseudokirchneriella subcapitata according to OECD Guideline 201, EU Method C.3 and ISO Method 8692, in compliance with GLP. In this study, triplicate Erlenmeyers containing the freshwater algae were exposed to 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L nominal concentrations of the test substance for 72 h under static conditions. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Although quantification of the applied concentrations was not possible due to the absence of a suitable analytical method it was possible to analyze the two stock solutions used to prepare the test concentrations. These analyses showed that the stock solutions were stable even after storage for almost 7 months in the refrigerator. Considering the high water solubility and stability of the test substance, it was considered reasonable to assume that the actual concentrations would be identical to the nominal concentrations during the entire test. All the validity criteria were considered to be fulfilled. Under the study conditions, the 72 h EbC50 and ErC50, based on nominal concentrations were 0.010 and 0.015 mg a.i./L, respectively. The LOEC was determined to be at the lowest tested concentration of 0.010 mg a.i./L. The ErC10 of 0.0095 mg a.i./L will be used as long term endpoint(Geurts, 2002).
A study was conducted to determine the acute toxicity of the test substance to the freshwater algae Scenedesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Based on the results of two range finding studies, the algae were exposed to 0.00625, 0.0 125, 0.025, 0.050, 0.10 mg/L nominal concentrations of the test substance under static conditions for 72 h. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Analysis of the test concentrations showed a decline due to adsorption to the algal cells. At 0 h, concentrations ranged from 58 to 81% of nominal values; at 72 h, the concentrations ranged from below the limit of quantification to 4% of nominal. Under the study conditions, the 72 h EbC50 and ErC50 of the test substance, based on measured concentrations, were 0.012 and 0.02 mg a.i./L, respectively. The NOEC and ErC10 were determined at 0.0069 and 0.012 mg a.i./L, respectively (Mead, 2001, 2007).
A study was conducted to determine effects of the test substance on the freshwater algae Pseudokirchneriella subcapitata according to EPA OTS Guideline 797.1050, in compliance with GLP. No analytical dose verification was conducted. The effects on algal growth were algistatic rather than algicidal. Under the study conditions, the nominal 96 h EC50 and NOEC values for growth rate were determined to be 0.054 and 0.012 mg/L, respectively (Thompson, 1992).
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