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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-03 to 2017-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name (as stated in the report): Silvial
Batch No.: SC00017272
Expiration date: 2017-10-13
Chemical name: 2-METHYL-3-(4-(2-METHYLPROPYL)PHENYL)PROPANAL
Purity: 99%
Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via HPLC-DAD after 0 (start of exposure), 24, 48 and 72 hours (end of the exposure). Separate replicates for each test item concentration and control for the test item analysis at the beginning of the exposure were prepared with algae. The samples for the test item analysis after 24, 48 and 72 hours were prepared with algae and incubated under test conditions.
The samples of all tested concentrations were stabilized factor 2 with acetonitrile containing 0.2% H3PO4 after sampling. The control and all samples were analyzed without further dilution. The highest sample was additionally diluted factor 2 (total dilution factor 4) with acetonitrile : HPLC water ( 50 : 50) containing 0.1% H3PO4.
All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
The test substance was found to be rapidly degraded. Therefore, a number of non-GLP preliminary range-finder tests were conducted using different methods of preparation of the test item in efforts to obtain as much of the parent substance in the test medium at the start of the test. The saturated solution approach and water-miscible solvent method were both explored. The use of solvent resulted in dispersions containing droplets of undissolved test item and gave measured concentrations that were higher in the 50 mg/L nominal test solution than in the 100 mg/L nominal test solution, which was possibly a result of the fact that the substance didn’t stay in solution after application of the stock solution in acetone in algal medium. In contrast, the saturated solution approach gave a series of test solutions with the expected increasing trend in measured concentrations and was therefore considered to be the most appropriate method of preparation of the test substance.

A saturated solution with a nominal loading of 100 mg/L test item was prepared once with demineralized water 24 ± 1 hour prior to the start of the exposure. The test item was placed on to the surface of the demineralized water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 24 ± 1 hour. After a separation phase of at least 1 hour at room temperature the saturated solution was taken from the bottom and was used for testing. The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion), which was negative. Then the components of the dilution water were added to the saturated solution.
The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25 - 50 - 100 % of the saturated solution. The dilution levels are based on the results of three preliminary range finding tests.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata HINDAK, SAG 61.81
Origin: Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen
Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day.
Culture medium: Nutrient medium Z according to LOttge et al, (1994)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 - 24°C, controlled at ± 2°C
pH:
7.88 - 9.48
Nominal and measured concentrations:
The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25 - 50 - 100 % of the saturated solution. The dilution levels are based on the results of three preliminary range finding tests.
The geometric mean measured concentration of the test item was calculated. These were determined to be 0.600 - 1.17 - 3.90 - 7.81 - 15.6 mg/L. Values < LOQ were taken with a half LOQ = 0.600 mg/L into calculation of the geometric mean measured concentration.
Details on test conditions:
The study was conducted under static conditions with an initial cell density of 6839 cells/mL. With regard to the volatility of the test item glass flasks without headspace were used to reduce losses of test item.
Three replicates per concentration level and six for the control Separate replicates for each measuring time were prepared. Test container : Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals.
Test volume: 59 mL ; Preculture: A four days old preculture, prepared in dilution water, was used as inoculum. Application was carried out by adding appropriate volumes of the test solutions to the replicate test vessels. Incubation: The flasks were positioned randomly and repositioned daily.Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (Sigma; purity 99%) was tested as a reference item
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Due to the rapid degradation of the test item and significant presence of the metabolite at the start of the test, the biological effects observed are produced by the parent substance and metabolite. Therefore, it is considered justifiable to base all effect values on the geometric mean measured test item concentrations of Silvial (parent substance and the calculated concentration of parent based on the measured concentration of the metabolite).

Effects based on yield were also reported (see attached full study report). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table. Furthermore, the preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus, only the EC10 is presented in the above "effects concentration" table. The NOEC values are available in the full study report.
Validity criteria fulfilled:
yes
Remarks:
The study meets the validity criteria of the guideline.
Conclusions:
Based on the geometric mean measured concentrations of the test item Silvial, the 72 hour-ErC50 for Algae was 1.44 mg/L (95% confidence limits: 1.17 - 3.16 mg/L) based on growth rate effects.
The 72 hour-ErC10 for Algae was 1.16 mg/L (95% confidence limits: (0.629 - 2.97 mg/L) based on growth rate effects.
Executive summary:

The toxicity of Silvial (Batch no.: SC00017272) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3 from 2017-04-03 to 2017-04-07, with the definitive exposure phase from 2017-04-04 to 2017-04-07 at the test facility. The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and ECso-values of growth rate and yield over a period of 72 hours.

The test substance was found to be rapidly degraded. Therefore, a number of non-GLP preliminary range-finder tests were conducted using different methods of preparation of the test item in efforts to obtain as much of the parent substance in the test medium at the start of the test. The saturated solution approach and water-miscible solvent method were both explored. The use of solvent resulted in dispersions containing droplets of undissolved test item and gave measured concentrations that were higher in the 50 mg/L nominal test solution than in the 100 mg/L nominal test solution, which was possibly a result of the fact that the substance didn’t stay in solution after application of the stock solution in acetone in algal medium. In contrast, the saturated solution approach gave a series of test solutions with the expected increasing trend in measured concentrations and was therefore considered to be the most appropriate method of preparation of the test substance. One metabolite of the test item was determined in all samples. The degradation product was confirmed to be 3-(4-isobutylphenyl)-2-methylpropanoic acid by comparison of the peaks observed in the chromatograms from the preliminary range-finder tests with a reference standard provided by the sponsor.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. Based on the results of the range-finder tests, a saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution. A saturated solution with a nominal loading of 100 mg test item/L was prepared with demineralized water 24 ± 1 hour prior to the start of exposure. The test item was placed on to the surface of the demineralized water with a pipette. A slow stirring procedure was applied. Gentle stirring was carried out with a magnetic stirrer at room temperature for 24 ± 1 hour. After a separation phase of at least 1 hour at room temperature the saturated solution was taken from the bottom and used for testing. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item, which was negative. Then the components of the dilution water were added to the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The concentrations of Silvial were determined at the start of the exposure (0 hours) and at the end of the exposure (72 hours) of all tested dilutions of the saturated solution and the control via HPLC- DAD. Additional replicates were analysed after 24 hours and 48 hours of the exposure due to instability of the test item. One metabolite of Silvial was determined in all samples. The concentrations of the degradation product were analytically verified in addition to the concentrations of the parent substance.

All measured concentrations (0, 24, 48 and 72 hours) were < LOQ at the highest dilution level (6.35% of the saturated solution). The measured concentrations of the test item (sum of the parent substance and the calculated concentration converted to the metabolite) in the lower dilution levels of 12.5 - 25.0 - 50.0 - 100 % of the saturated solution after 24 hours ranged from 89 to 99% of the initially measured values at the start of the exposure (0 hours). After 48 hours the measured concentrations of the test item (sum of the parent substance and the calculated concentration converted to the metabolite) were between 67 and 96% and at the end of the exposure between 85 and 95% of the initially measured values.

Using the sum of the measured concentrations of the parent substance and the calculated concentration based on the measured concentration of the metabolite, the geometric mean measured concentration of the test item was calculated. These were determined to be 0.600 - 1.17 - 3.90 - 7.81 - 15.6 mg/L. Values < LOQ were taken with a half LOQ = 0.600 mg/L into calculation of the geometric mean measured concentration.

Due to the rapid degradation of the test item and significant presence of the metabolite at the start of the test, the biological effects observed are produced by the parent substance and metabolite. Therefore, it is considered justifiable to base all effect values on the geometric mean measured test item concentrations of Silvial (parent substance and the calculated concentration of parent based on the measured concentration of the metabolite).

Based on the geometric mean measured concentrations of the test item Silvial, the 72 hour-ErC50 for Algae was 1.44 mg/L (95% confidence limits: 1.17 - 3.16 mg/L) based on growth rate effects.

The 72 hour-ErC10 for Algae was 11.16 mg/L (95% confidence limits: (0.629 - 2.97 mg/L) based on growth rate effects.

Description of key information

The toxicity of Silvial (Batch no.: SC00017272) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3 from 2017-04-03 to 2017-04-07, with the definitive exposure phase from 2017-04-04 to 2017-04-07 at the test facility.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.44 mg/L
EC10 or NOEC for freshwater algae:
1.16 mg/L

Additional information

The test substance was known to be rapidly degraded. Therefore, a number of non-GLP preliminary range-finder tests were conducted using different methods of preparation of the test item in efforts to obtain as much of the parent substance in the test medium at the start of the test. The saturated solution approach and water-miscible solvent method were both explored. The use of solvent resulted in dispersions containing droplets of undissolved test item and gave measured concentrations that were higher in the 50 mg/L nominal test solution than in the 100 mg/L nominal test solution, which was possibly a result of the fact that the substance didn’t stay in solution after application of the stock solution in acetone in algal medium. In contrast, the saturated solution approach gave a series of test solutions with the expected increasing trend in measured concentrations and was therefore considered to be the most appropriate method of preparation of the test substance. One metabolite of the test item was determined in all samples. The degradation product was confirmed to be 3-(4-isobutylphenyl)-2-methylpropanoic acid by comparison of the peaks observed in the chromatograms from the preliminary range-finder tests with a reference standard of 3-(4-isobutylphenyl)-2-methylpropanoic acid provided by the sponsor.

The definitive test was conducted under static conditions with an initial cell density of 6839 cells/mL. Based on the results of the range-finder tests, a saturated solution and further four dilution levels of the saturated solution were tested in a geometrical series with a factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution. A saturated solution with a nominal loading of 100 mg test item/L was prepared with demineralized water 24 ± 1 hour prior to the start of exposure. The test item was placed on to the surface of the demineralized water with a pipette. A slow stirring procedure was applied. Gentle stirring was carried out with a magnetic stirrer at room temperature for 24 ± 1 hour. After a separation phase of at least 1 hour at room temperature the saturated solution was taken from the bottom and used for testing. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item, which was negative. Then the components of the dilution water were added to the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The concentrations of Silvial were determined at the start of the exposure (0 hours) and at the end of the exposure (72 hours) of all tested dilutions of the saturated solution and the control via HPLC- DAD. Additional replicates were analysed after 24 hours and 48 hours of the exposure due to instability of the test item. One metabolite of Silvial was determined in all samples. The concentrations of the degradation product were analytically verified in addition to the concentrations of the parent substance.

All measured concentrations (0, 24, 48 and 72 hours) were < LOQ at the highest dilution level (6.35% of the saturated solution). The measured concentrations of the test item (sum of the parent substance and the calculated concentration converted to the metabolite) in the lower dilution levels of 12.5 - 25.0 - 50.0 - 100 % of the saturated solution after 24 hours ranged from 89 to 99% of the initially measured values at the start of the exposure (0 hours). After 48 hours the measured concentrations of the test item (sum of the parent substance and the calculated concentration converted to the metabolite) were between 67 and 96% and at the end of the exposure between 85 and 95% of the initially measured values.

Using the sum of the measured concentrations of the parent substance and the calculated concentration based on the measured concentration of the metabolite, the geometric mean measured concentration of the test item was calculated. These were determined to be 0.600 - 1.17 - 3.90 - 7.81 - 15.6 mg/L. Values < LOQ were taken with a half LOQ = 0.600 mg/L into calculation of the geometric mean measured concentration.

Due to the rapid degradation of the test item and significant presence of the metabolite at the start of the test, the biological effects observed are produced by the parent substance and metabolite. Therefore, it is considered justifiable to base all effect values on the geometric mean measured test item concentrations of Silvial (parent substance and the calculated concentration of parent based on the measured concentration of the metabolite).

Based on the geometric mean measured concentrations of the test item Silvial, the 72 hour-ErC50 for Algae was 1.44 mg/L (95% confidence limits: 1.17 - 3.16 mg/L) based on growth rate effects. The 72 hour-ErC10 for Algae was 11.16 mg/L (95% confidence limits: (0.629 - 2.97 mg/L) based on growth rate effects.