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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:
The study was conducted between 7 September 2017 and 3 November 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
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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:
11-oxahexadecan-16-olide
EC Number:
222-225-5
EC Name:
11-oxahexadecan-16-olide
Cas Number:
3391-83-1
Molecular formula:
C15H28O3
IUPAC Name:
1,7-dioxacycloheptadecan-8-one
Test material form:
liquid
Specific details on test material used for the study:
Lot No.: VE00497116
Aspect: colourless liquid, may crystallize
Purity: 98.9%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using external calibration. The test item gave a chromatographic profile consisting of one peak.
The analytical method was developed by IES Ltd. These experiments were not performed according to the regulations of GLP (IES Study Number 20170206). The materials and methods as described in this report refer to the analysis of the samples from the main test.

Test Item
The test item described in the biological part of this study was used to prepare spiked samples for method validation work.

Analytical Standard
The test item described in the biological part of this study was also used as the analytical standard.

Analytical Procedure
Storage: The samples were stored frozen (at about -20 °C) and protected from light until analysis was performed.

Reagents and Solvents
Water prepared in-house using an ELGA water purification system
Test water as described in the biological part of this study
Acetonitrile, HPLC grade Sigma-Aldrich, no. 34998

Preparation of Calibration Solutions
The test item (23.55 mg) was dissolved in acetonitrile and made up to the mark in a 10 mL volumetric flask to prepare a stock solution with a concentration of 2355 mg/L. A stock solution aliquot of 1 mL was diluted to 11 mL with acetonitrile/water (1/1, v/v) to obtain a working solution with a concentration of 214 mg/L.
Defined volumes of this working solution were further diluted with acetonitrile/water (1/1, v/v) to obtain calibration solutions of the test item in the range of 0.321 to 15.9 mg/L. These solutions were used to calibrate the analytical system.

Preparation of Spiked Samples
To demonstrate the validity of the method, untreated test water was spiked with the test item. The test item (25.25 mg) was dissolved in acetonitrile and made up to the mark in a 10 mL volumetric flask to prepare a stock solution with a concentration of 2525 mg/L. A stock solution aliquot of 1 mL was diluted to 10 mL with acetonitrile to obtain a fortification solution with a concentration of 253 mg/L.
Defined volumes of the fortification solution and the stock solution were diluted to 10 mL with
test water to obtain spiked samples with concentrations of 1.14 mg/L (0.045 mL from fortification solution) and 25.3 mg/L (0.1 mL from stock solution). Five spiked samples were prepared per concentration level and subjected to the same treatment as a test sample but without storage. In addition, test water without the test item was analyzed after sample preparation (analytical blank).

Preparation of Test Samples
The storage stability of the test samples was demonstrated with the measurements of spiked samples (IES non-GLP study number 20170206). The % nominals found in the spiked samples stored frozen were found to be 105 and 103 % (see Table 7) following 1 day of storage at -20 °C.
Test samples and control samples were thawed at room temperature for 1 hour. The 10 mL samples were completely diluted with acetonitrile by a factor of two. The test and control samples from day 3 were centrifuged (2465 g for 5 minutes) due to the presence of algae. One spiked sample solution from each concentration level was also centrifuged.

Instrumental Setup
Autosampler: VWR-Hitachi CM5210
Pump: VWR-Hitachi CM5110
Detector: VWR-Hitachi CM5430
Column Oven: VWR-Hitachi CM5310
Column: Symmetry C18, 75 mm x 4.6 mm; 3.5 μm
Eluent A: Water
Eluent B: Acetonitrile
Injection Volume: 500 μL
Flow Rate: 1.5 mL/minute
Temperature: 40 °C in a thermostatic oven
Detection Wavelength: 210 nm
Retention Time: Approximately 5.9 minutes

Calculations
Injected samples were quantified by the peak area with reference to the respective calibration curve. The latter was obtained by correlation of the areas of the calibration solutions to their corresponding concentration in mg/L.

Test solutions

Details on test solutions:
Range Finding Test
For determination of the appropriate test concentrations for the main test, a range-finding test with the following concentrations was performed in a closed system: The undiluted filtrate with a loading rate of 100 mg/L and the dilutions 1:10 and 1:100. For this, three replicates per treatment were tested in parallel with a control.
The choice of the closed system was based on a stirring pre-experiment (IES Study 20170205) which showed that the test item was not stable over 48 hours. Even if the analytical results of this pre-experiment did not demonstrate the volatility of the test item, the range finding test and the main test were performed in a closed system as a precaution.
The highest test concentration (undiluted filtrate) was prepared by mixing 108.01 mg of the test item into 1080 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark in a closed vessel. The stirring time was based on a stirring pre-experiment (IES Study 20170205) which showed, that the maximum amount of dissolved test item was reached after this stirring time.
After stirring, the suspension was filtered through a 0.45 μm membrane filter (Whatman, NC45). The filter was saturated by 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the test item.
Main Test
Based on these results and in agreement with the Sponsor the following concentrations of MUSK R1 were selected for the main test: The undiluted filtrate with a loading rate of 100 mg/L and the dilutions 1:2, 1:4, 1:8 and 1:16. Additionally, a control (test water without test item) was tested in parallel.
The main test was performed in a closed system. The test design included three replicates per test concentration and six replicates for the control.
The test was started using a nominal algal cell density of 5000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined using an electronic particle counter (Cell Counter CASY TT, OLS, Bremen/Germany).
A static test design was applied. The duration of the test was 72 hours.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at IES Ltd Laboratories under standardized conditions according to the test guidelines.

Study design

Test type:
static
Water media type:
other: Synthetic test water (AAP Medium) according to OECD Guideline No. 20 was used in the study.
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
The water hardness (calculated) of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).
Test temperature:
The water temperature during the test was maintained at 23 °C.
pH:
The pH of the test media was in the range of 7.6 to 7.8 during the test period.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval of 1.9 - 2.0 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval of 2.0 - 2.1 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval of 2.3 - 2.4 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate

Any other information on results incl. tables

VALIDITY

The values for the validity criteria of the test were calculated by the statistical software programToxRat Professional® [7].

In the control, the biomass increased by a factor of 162 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled.

The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 18 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled.

The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 0.5 %. According to the OECD test guideline, the coefficient of variation must not be higher than 0.5 %. Thus, the validity criterion was fulfilled.

All validity criteria were thus fulfilled for the control.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:
The growth rate EC50 was 2.4 mg/L with 95 % confidence limits of 2.3 - 2.4 mg/L. The corresponding NOEC was 1.02 mg/L, LOEC 1.5 mg/L, EC20 2.1 mg/L and the EC10 was 1.9 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 2.4 mg/L and 1.9 mg/L respectively.
Executive summary:

The impact of the test item MUSK R1 on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU)No 2016/266, C.3.

The preparation of the test media was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.