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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:
25 - 29 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006, Annex 5 corrected 28 July 2011
Deviations:
yes
Remarks:
The pH value in the control vessels changed by 2.5 units over the course of the exposure, instead of maximum 1.5.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266
Deviations:
yes
Remarks:
The pH value in the control vessels changed by 2.5 units over the course of the exposure, instead of maximum 1.5.
GLP compliance:
yes (incl. certificate)
Remarks:
chemical analyses were performed non-GLP

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid: volatile
Details on test material:
Batch No: 57161235
Storage: Room temperature (approx. 20°C)

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test medium, test item and algae (three replicates per concentration)
- Blank control (Bn); containing test medium and algae (six replicates)
- Since the test item was volatile, the test solutions were prepared, separately for each test concentration, by direct addition (pipetting, considering a density of 0.904 g/ml) of the respective quantities of test item to sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware. When the addition of test item was not feasible through pipetting because of a too small volume, the corresponding test concentrations were prepared by dilution of the next higher test concentration. These mixtures were gently mixed. 150 ml of each test solution was added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
Pure Algal medium, also sterile filtered into sterile test flasks and inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls. For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
- Evidence of undissolved material: Not relevant. Since the test item was volatile, the test solutions were prepared, separately for each test concentration, by direct addition (pipetting, considering a density of 0.904 g/ml) of the respective quantities of test item to sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from an axenic slope culture
- Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- Control of sensitivity: twice per year, with potassium dichromate

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
not reported
Test temperature:
Min: 23.3; Max: 24.8; Mean: 24.0
pH:
7.0 - 10.4
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
The concentrations of Vinyl propionate in the test media were measured by by purge and trap GCMS analysis (LOQ = 0.02 mg/L) at the beginning and after 24, 48 h and 72 h of exposure. At the start of the exposure 53–88% of the nominal concentrations were found. Over the course of the test the test concentrations decreased to 28–64% of nominal except in the lowest test concentration of 0.316 mg/l were the measured concentration was below LOQ (0.02 mg/L).
Therefore, the effective concentrations ErCx/EyCx were assessed based on the geometric mean of the measured concentrations of the test item over the whole 72 h test period.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 150 ml of test medium, shaken (about 130 rpm), closed with glass stoppers to avoid any loss of test item
- Test medium: Recommended OECD medium
- Preculture: Exponentially growing culture of Desmodesmus subspicatus
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity: the light intensity amounts to about 3000 lux which corresponds to about 40 µE•m-2•s-2
- Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of Vinyl propionate was performed. The results indicated that the test item is well soluble and that the test solutions can be prepared by respective dilutions of a stock solution. It also showed, that at a concentration of 100 mg/l the test item is rather stable in algal medium over 72 h. Based on the results of this non-GLP range finding test, the definitive test was performed as a limit test at the nominal concentration of 100 mg/l.

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.


DEVIATIONS FROM GUIDELINES
The pH value in the control vessels changed by 2.5 units over the course of the exposure, instead of maximum 1.5. However, since all other validity criteria were met, the test is considered to be valid. These deviations have no impact on the outcome of the study.
Reference substance (positive control):
not required
Remarks:
Control of sensitivity: None – the culture is re-purchased annually

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.94 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.:
5 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.:
< 0.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.065 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: yes (growth rate and yield)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

With respect to the endpoint growth rate, no significant effects were observed as compared to the blank controls.
Based on these effects and the measured concentrations of the test item, the median effect concentration with respect to growth rate (ErC50/0–3 days) of Vinyl propionate to Desmodesmus subspicatus was calculated to be 5.0 mg/l (95% confidence limits: 4.4 – 5.6 mg/l).
The low effect concentration with respect to growth rate ErC10 was calculated to be 0.94 mg/l (95% confidence limits: 0.65 – 1.2 mg/l).
The no observed effect concentration (NOErC) with respect to growth rate could not be determined as all test concentrations caused significant effects on the growth rate, NOErC < 0.37 mg/l.

With respect to the endpoint yield (biomass production), no significant effects were observed as compared to the blank controls.
Based on these effects and the measured concentrations of the test item, the median effect concentration with respect to yield (EyC50/0–3 days) of Vinyl propionate to Desmodesmus subspicatus was calculated to be 1.1 mg/l (95% confidence limits: 0.98 – 1.3 mg/l).
The low effect concentration with respect to yield EyC10 was calculated to be 0.065 mg/l (95% confidence limits: 0.042 – 0.092 mg/l).
The no observed effect concentration (NOEyC) with respect to yield was determined to be smaller than 0.37 mg/l.
Reported statistics and error estimates:
The effect concentrations (ErC10, ErC50 and EyC10, EyC50) were calculated with ToxRat® Standard Version 3.2.1 (ToxRat® Solutions GmbH, Alsdorf, Germany). For the calculation of the ECx and associated confidence intervals, inhibitions below 0% and inhibitions above 100% were set to 0.1% and 100%, respectively.

The statistical significance of differences between effects observed for control and test item treatments with respect to the no observed effect concentrations was determined by Welsh-t test using the ToxRat® Standard Version 3.2.1 (ToxRat® Solutions GmbH, Alsdorf, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
In the control cultures: average specific growth rate of 1.21 (>0.92)/ coefficient of variation of average specific growth rates: 2.8% (<=7%)/ mean coefficient of variation for section-by-section specific growth rates 25.4% (<=35%).
Conclusions:
The acute toxicity of Vinyl propionate (CAS no. 105-38-4) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of Vinyl propionate to Desmodesmus subspicatus was estimated to be 5.0 mg/l. The no observed effect concentration (NOErC) with respect to growth rate was determined to be <0.37 mg/l. The low effect concentration with respect to growth rate ErC10 was calculated to be 0.94 mg/l. The results of the test can be considered reliable without restriction.
Executive summary:

The growth inhibitory effects of Vinyl propionate (CAS no. 105-38-4) to the green algae Desmodesmus subspicatus were investigated according to test guideline OECD 201, over a period of 72 h.

The test item Vinyl propionate is liquid, has a high solubility (7.52 g/l, pH of 3.5–3.9 and ca. 20 °C) and is 99.88% pure. Since the test item is volatile, the test solutions were prepared, separately for each test concentration, by direct addition (pipetting) of the respective quantities of test item to test medium.

The nominal concentrations tested were 3.16, 10.0, 31.6, 100 and 316 mg/l.

Three parallel test vessels were used for each test concentration and six vessels for the blank controls.

The concentrations of Vinyl propionate in the test media were measured by by purge and trap GCMS analysis (LOQ = 0.02 mg/L) at the beginning and after 24, 48 h and 72 h of exposure. At the start of the exposure 53–88% of the nominal concentrations were found. Over the course of the test, the test concentrations decreased to 28–64% of nominal except in the lowest test concentration of 0.316 mg/l were the measured concentration was below LOQ (0.02 mg/L). Therefore, the effective concentrations ErCx/EyCx were assessed based on the geometric mean of the measured concentrations of the test item over the whole 72 h test period.

The results of the growth rate (r) and yield (y) inhibition of Vinyl propionate to the green algae Desmodesmus subspicatus are summarized in the following table showing EC values and 95% confidence limits (cl) based on the geometric mean of the measured concentrations:

 Parameter, expressed as Measured concentration (mg/l)

 ErC10

 ErC50

 NOErC

 Estimate

 0.94

 5.0

 <0.37

 Lower 95%-cl

 0.65

 4.4

 

 Upper 95%-cl

 1.2

 5.6

 

 Parameter, expressed as Measured concentration (mg/l)

 EyC10

 EyC50

 NOEyC

 Estimate

 0.065

 1.1

 <0.37

 Lower 95%-cl

0.042

 0.98

 

 Upper 95%-cl

 0.092

 1.3

 

cl: confidence limit

The 72 h ErC50 value of Vinyl propionate (CAS no. 105-38-4) on the green algae Desmodesmus subspicatus was 5.0 mg/l. This value is based on the measured concentrations.

All validity criteria were fulfilled.