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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-21 to 2015-11-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
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 (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
α,2,6,6-tetramethyl-2-cyclohexene-1-butyraldehyde
EC Number:
277-458-5
EC Name:
α,2,6,6-tetramethyl-2-cyclohexene-1-butyraldehyde
Cas Number:
73398-85-3
Molecular formula:
C14H24O
IUPAC Name:
2-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butanal
Constituent 2
Chemical structure
Reference substance name:
α,2,6,6-tetramethylcyclohexene-1-butyraldehyde
EC Number:
282-993-2
EC Name:
α,2,6,6-tetramethylcyclohexene-1-butyraldehyde
Cas Number:
84518-22-9
Molecular formula:
C14H24O
IUPAC Name:
2-methyl-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butanal
Specific details on test material used for the study:
Test material name (as stated in the report): Cetonal
Batch No.:SC00011028
Expiry date: 23 June, 2016

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
All concentration levels and the control were analytically verified via LC-MS/MS from freshly prepared media after 0 hours (start of exposure) and every 24 hours.
Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 24, 48 and 72 hours, separate replicates were prepared with and without algae at the beginning of exposure and incubated under test conditions.

Test solutions

Vehicle:
no
Details on test solutions:
A saturated solution with a nominal loading of 1000 mg test item/L was prepared once with dilution water one day prior to the start of the exposure. A slow stirring procedure was applied. Gentle stirring was carried out with a magnetic stirrer at room temperature for 24 hours. After a separation of the phases of 1 hour, the saturated solution was taken from the homogeneous phase and was used for testing. Five concentrations were tested in a geometrical series with a dilution factor of 1.4 (nominal): 26.0 - 36.4 - 51.0 - 71.3 - 100 % of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata HINDÄK, SAG 61.81
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 35-70 pE -nr2 S'1 for 24 hours per day.
Culture medium: Nutrient medium Z according to Lüttge et al. (1994)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol Ca+Mg/L.
Test temperature:
21.5 - 22.5 °C, mean value: 22.0 °C
pH:
8.05 - 8.56
Nominal and measured concentrations:
Test concentrations (nominal): 26.0, 36.4, 51.0, 71.3, and 100% of the saturated solution and a control
Measured concentrations: Time weighted average of the test item with Algae : 0.521, 0.788, 0.935, 1.49 and 3.29 mg test item/L
Measured concentrations: Time weighted average of the test item without Algae : 0.599, 0.793, 1.17, 1.66 and 3.47 mg test item/L
Details on test conditions:
The test media were clear and dissolved throughout the test period. A static test was carried out. With regard to the volatility of the test item tightly closed glass flasks without headspace were used to reduce losses of the test item. Three replicates per concentration and six for the control. Separate replicates for each measuring time were prepared.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr207)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 3.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.935 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Effects based on yield were also reported (see attached full study report and executive summary). 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 v1.1). 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 the 72-h EC50 and EC10 based on growth rate are selected as key results for classification purposes.
Results with reference substance (positive control):
The toxicity of potassium dichromate (Sigma, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2015- 10-13 to 2015-10-16 (with headspace) (batch number MKBQ9179V, purity 100 %) and 2015-11- 10 to 2015-11-13 (without headspace) (batch number MKBQ9179V, purity 100 %), respectively. The reference item toxicity is in the valid range following test facility SOPs.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, CETONAL was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on time weighted average mean measured test item concentrations): The EC50 values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both >3.29 mg/L, respectively. The EC10 values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.69 (2.44 - 2.90) mg/L and 1.33 (1.10 - 1.62) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were for both 0.935 mg/L.
The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. 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 the 72-h EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be > 3.29 mg/L and 2.69 mg/L respectively.
Executive summary:

The toxicity of CETONAL (Batch no.: SC00011028) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 Method C.3. The study was conducted under static conditions with an initial cell density of 6153 cells/mL. With regard to the volatility of the test item, closed bottles without headspace were used to reduce losses of the test item. A saturated solution with a nominal loading of 1000 mg test item/L was prepared once with dilution water one day prior to the start of the exposure. A slow stirring procedure was applied. Gentle stirring was carried out with a magnetic stirrer at room temperature for 24 hours. After a separation of the phases of 1 hour, the saturated solution was taken from the homogeneous phase and was used for testing. Five concentrations were tested in a geometrical series with a dilution factor of 1.4 (nominal): 26.0 - 36.4 - 51.0 - 71.3 - 100 % of 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 test media were clear and dissolved throughout the test period. The concentrations of the test item CETONAL in all test concentrations and the control were analytically verified via LC-MS after 0, 24, 48 and 72 hours of exposure. The concentrations of the test item decreased within 72 h in samples with and without algae. Therefore, time weighted average concentration of 0.521 - 0.788 - 0.935 - 1.49 - 3.29 mg/L were calculated for samples with algae and used for evaluation of affect values.

CETONAL was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on time weighted average mean measured test item concentrations): The EC50 values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both >3.29 mg/L, respectively. The EC10 values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 2.69 (2.44 - 2.90) mg/L and 1.33 (1.10 - 1.62) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were for both 0.935 mg/L.