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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Performed in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
Identification: Linalyl isobutyrate
Chemical name: 3,7-dimethyl-1,6-octadien-3-yl 2-methylpropanoate
Empirical Formula: C14H24O2
Molecular Weight: 224.35 g/mol
EC No.: 201-108-2
CAS No.: 78-35-3

Sampling and analysis

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. Each sample bottle received 10 mL of hexane immediately after sampling and was shaken to mix. All samples were analysed on the day of sampling. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Additional samples were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24 and 48 hours. A further sample of each test concentration containing no algal cells was prepared an incubated alongside the test to provide samples for uninoculated analyses at 72 hours.

Test solutions

Vehicle:
not specified
Details on test solutions:
Range Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 4.1 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. 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. All test item preparation was performed under non-actinic lighting/shielded from the light due to the potentially light unstable nature of the test item.

A nominal amount of test item (110 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 2 hours. After 2 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore 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 further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (4.2 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 2.6, 6.4, 16, 40 and 100% v/v saturated solution.
A nominal amount of test item (110 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 2 hours. After 1 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore 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 40, 16, 6.4 and 2.6% v/v saturated solution. An aliquot (1500 mL) of each of the stock solutions was separately inoculated with 6.9 mL of algal suspension to give the required test concentrations of 2.6, 6.4, 16, 40 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none

Test conditions

Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
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 pH value of the control cultures was observed to increase from pH 8.2 at 0 hours to pH 9.8 at 72 hours. 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 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). 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 Guidelines.
Salinity:
No (freshwater)
Details on test conditions:
Range finding study:
Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Definitive study:
Nominal test concentrations of 2.6, 6.4, 16, 40 and 100% v/v saturated solution
0-Hour measured concentrations of 0.069, 0.18, 0.47, 1.3, 3.5 mg/L
Time-Weighted mean measured concentrations of 0.033, 0.086, 0.24, 0.68, 2.0 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
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:
growth rate
Remarks on result:
other: 95% CL: 1.6 - 2.4
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.77 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.47 mg/L
Nominal / measured:
meas. (initial)
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 present in the above "effects concentration" table.

In order to prevent abiotic loss due to volatilization, a closed system with no headspace was used. Under such conditions it can be considered appropriate to base the effect results on the initial measured concentrations. The key results, 72 h ErC50 and ErC10, were determined to be 2.0 and 0.77 mg/L respectively.

Analysis of test samples at 0, 24, 48 and 72 hours showed a gradual decline in measured concentrations, with the final measured concentrations being 14-25% of the initial measured concentrations. Analysis at 72 hours of samples which had been prepared at the start of the test with the omission of algal cells showed measured test concentrations that were 20-27% initial indicating that the decline in measured concentrations observed over the test duration was predominantly due to instability with possibly some adsorption of the test item to the algal cells present. GC chromatograms showed the formation of two new peaks with retention times shorter than the parent ester. The primary degradation of the test item is believed to be due to hydrolysis of the ester functional group to give the corresponding alcohol (linalool, EC 201-134-4) and carboxylic acid (2-methyl propanoic acid, EC 201-195-7). This is supported by the water solubility determination of linalyl isobutyrate where hydrolysis/degradation of the test item was also observed and where the chromatogram of a linalool marker standard gave a retention time common to one of the degradant peaks. The degradation products are expected to be less toxic than the parent ester due to their increased polarity, which is supported by available information on the ECHA websitefor linalool and 2-methylpropanoic acid. Therefore, it is appropriate to assess the toxicity of the parent substance even if exposure levels cannot be maintained to the extent necessary to comply absolutely with test guidelines. In order to give a “worse case” analysis of the data, the results were also calculated using the time-weighted mean measured test concentrations. The 72 h ErC50 and ErC10 were determined to be 1.1 and 0.24 mg/L respectively.

The effect results based on either the initial measured concentrations or time-weighted mean measured concentrations are within the same environmental classification band of ErC50 > 1 and ≤ 10 mg/L (acute classification) and ErC10 > 0.1 and ≤ 1 mg/L (chronic classification).

Results with reference substance (positive control):
Positive control studies using potassium dichromate as the reference item were conducted in a conventional test system (Harlan Study Number 41403074) and in a modified test system using completely filled and sealed vessels (Harlan Study Number 41403365).
The results from the positive control test with potassium dichromate in a conventional test system were within the normal ranges for this reference item. Additionally, the results of the positive control test with potassium dichromate in a modified test system were comparable with the conventional test system.
It is evident from these results that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system. As such it can be considered that the results obtained from the definitive test give a reliable result for the test item.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a guideline study, conducted according to GLP, Linalyl Isobutyrate was found to have an ErC50 (72 hr) (based on the initial measured test concentrations) of 2.0 mg/L, an ErC10 (72 hr) of 0.77 mg/L, a NOEC (72 hr) of 0.47mg/L and a LOEC (72 hr) of 1.3 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 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 2.0 mg/L and 0.77 mg/L respectively.