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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:
January 02 - 05, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
- Concentrations: 0 (control), 6.25, 12.5, 25, 50 and 100mg/l
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions were prepared for both the range-finding and definitive tests by addition of the test sample directly to nutrient growth medium in a volumetric flask of adequate volume. Calculated volumes of the stock solution were added to nutrient growth medium and made to volume to prepare the test concentrations and provide enough solution for testing and subsequent water quality measurements. The test concentrations for both the range-finding test and definitive test were prepared separately by the addition of the test sample to nutrient growth medium in volumetric flasks of adequate volume to provide enough solution for testing and subsequent water quality measurements.
- Controls: A 0 mg/l control that contained only nutrient growth medium (nutrient stock in deionised water) was included in the preliminary (range finding) test and in the main test.
- Other: Information provided by the Sponsor indicated that the sample had a solubility of 395.7mg/l (EPI Suite prediction) in water.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata.
- Strain: CCAP 278/4 (received April 22, 2016).
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Ltd, Scottish Marine Institute, OBAN, Argyll, PA37 1QA, Scotland, United Kingdom
- Method of cultivation: Grown in the exponential phase under the following conditions: temperature: 20.1 - 21.4 °C; illumination: 6140 - 7910 lux continuous white light; orbiting: set to 200 rpm; culture medium: deionised water with added nutrients according to OECD Guideline 201. Inoculum level was adjusted to give an initial cell density of 1 x 104 cells/ml.

ACCLIMATION
- Culturing media and conditions (same as test or not): Yes.
- Any deformed or abnormal cells observed: No.
Test type:
static
Water media type:
freshwater
Remarks:
Deionised water (sterilised by autoclaving at 120 °C for 15 minutes)
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Cell density measurements were taken at 24, 48, and 72 hours
Test temperature:
Temperature range within incubator throughout the test: 20.9 - 21.9 °C (required: 21 - 24 ± 2 °C)
pH:
pH range in control and test concentrations throughout the test: 7.80 – 8.09
Nominal and measured concentrations:
Preliminary range finding test: 0 (control), 0.1, 1.0, 10, 100 mg/l (nominal)
Main test: 0 (control), 6.25, 12.5, 25, 50 and 100 mg/l (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask.
- Material, size, headspace, fill volume: 250ml.
- Initial cells density: Inoculum level adjusted to give an initial cell density of 1 x 104 cells/ml.
- No. of organisms per vessel: 1 x 104 cells/ml.
- No. of vessels per concentration (replicates): For the preliminary test method, a single replicate at each concentration was prepared. In the main test, three replicates at each test concentration were prepared.
- No. of vessels per control (replicates): For the preliminary test method, a single replicate for the control (0 mg/l) was prepared. In the main test, six control flasks were made-up.

GROWTH MEDIUM
- Standard medium used: Yes.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: De-ionised; sterilised by autoclaving at 120 °C for 15 minutes.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: The temperature (to 0.1 °C) and light intensity (lux) within the incubator was recorded at the beginning of the study, after 24, 48 hours and at the end of the 72-hour test period. The pH (to 0.01) and temperature (to 0.1 °C) were recorded for each test and control solution at the beginning of the test and on the pooled replicates at the end of the 72-hour test period.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes, sterile nutrient stock solutions were prepared.
- Adjustment of pH: The test was carried out without adjustment of the pH after addition of the test substance to the media, however, pH was adjusted to 8.09 from 8.23 during preparation of the nutrient growth medium.
- Photoperiod: Continuous white light.
- Light intensity and quality: 6140 - 7910 lux.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell densities were measured microscopically by direct cell counts on each test and control replicate, in sextuplicate at 24 hours for the control and triplicate thereafter, and in triplicate at 24, 48 and 72 hours (± 2 h) for the test concentrations. The cell counts were made using a haemocytometer and microscope.

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 0 (control), 0.1, 1.0, 10, 100 mg/l.
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K12.5r2O7) was used as part of a separate reference study (ENV 11396) to check test procedure and sensitivity of the species.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): Logarithmic growth for the duration of the study.
- Observation of abnormalities (for algal test): All cells within the control and all test concentrations appeared normal with no morphological abnormalities.
- Colour differences: When observed visually, the all conical flasks appeared clear and colourless after 24 hours. At 48 hours the control and 6.25mg/l concentrations appeared slightly green with the rest clear and colourless. At 72 hours the control was slightly green, 6.25mg/l was green, 12.5 and 25mg/l were slightly green with 50 and 100mg/l remaining clear and colourless.
- Any stimulation of growth found in any treatment: By 72 hours, cell density was on average lower in all treated media relative to the control.
- Other: The validity criteria of the study were fulfilled.
Results with reference substance (positive control):
- Results with reference substance valid: Yes.
- EC50: 1.06 mg/l.
Reported statistics and error estimates:
Determined by Bonferroni t Test, 1-tail, P = 0.05, following tests for normality of distribution and equal variances, using ToxCalc v5.0.

Results Summary

Exposure Period (hours) EyCx value (mg/l) (95% confidence limits) ErCx value (mg/l) (95% confidence limits)
EyC10 EyC20 EyC50 ErC10 ErC20 ErC50
0 to 48 6.75 (3.82 - 8.67) 8.83 (6.00 - 10.7) 14.0 (11.9 - 16.7) 5.76(1) (1.23 - 10.5) 10.6 (3.79 - 16.5) 30.1 (20.4 - 45.1)
0 to 72 4.81(1) (2.55 - 6.34) 6.39 (4.12 - 7.87) 10.4 (8.63 - 12.4) 8.06 (4.69 - 10.4) 10.7 (7.37 - 13.1) 17.5 (14.7 - 20.9)
NOEC (0-72h) 6.25mg/l (Determined by Bonferroni t Test)$* 6.25mg/l (Determined by Bonferroni t Test) $^

Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Logit.

(1) Extrapolated value – value below lowest concentration tested which was 6.25mg/l.

$ Shapiro-Wilk’s Test indicates normal distribution

^ Bartlett’s Test indicates equal variances.

* Bartlett’s Test indicates unequal variances.

All concentrations of the test substance are reported as nominal as received.

Validity criteria fulfilled:
yes
Conclusions:
2-Butoxyethyl methacrylate was considered to be non-toxic to Pseudokirchneriella subcapitata and, therefore, aquatic algae and cyanobacteria. This conclusion was based on a Growth rate EC50 value for of 17.5 mg/l at 72 hours, which is greater than the ≤1 mg/l threshold provided by CLP Regulation (EC) No 1272/2008, as well as a 72-hour NOEC and LOEC value of 6.25 and 12.5 mg/l, respectively.
Executive summary:

The toxicity of 2-butoxyethyl methacrylate to aquatic algae and cyanobacteria was determined in line with Good Laboratory Practise (GLP) and OECD Guideline 201 (Alga, Growth Inhibition Test) without deviation. Pseudokirchneriella subcapitata (strain CCAP 278/4) was selected as the test species and growth inhibition was measured over a 72-hour period as part of an initial preliminary (range-finding) test using concentrations of 0 (control), 0.1, 1.0, 10, 100mg/l and as part of a main test using concentrations of 0 (control), 6.25, 12.5, 25, 50 and 100 mg/l. The tests were performed in a nutrient growth medium consisting of sterilised deionised water and a specified amount of nutrient stock within 250 ml conical flasks. Cell densities were measured microscopically by direct cell counts at 24, 48, and 72 hours using a haemocytometer and microscope. Water quality measurements, including pH, temperature (to 0.1 °C), and light intensity (lux) were measured at specific time-points. A reference substance, potassium dichromate, was assessed in a separate study in order to verify the test procedure and sensitivity of the test species.

All study validity criteria were fulfilled. The EC50 value, which is the concentration that inhibits the growth of the algae by 50% after a period of exposure, was found to be 10.4 mg/l (EC(y)50) and 17.5 mg/l (EC(r)50) at 72 hours (determined by Maximum Likelihood-Logit). The 72-hour E(y)C50 and E(r)C50 were 10.4 mg/l and 17.5 mg/l respectively. The 72-hour NOEC and LOEC value were calculated to be 6.25 and 12.5 mg/l, respectively (determined by Bonferroni t-Test). No abnormal cells were identified. As the calculated EC50 values at 72 hours were greater than the recommended threshold of ≤1 mg/l, it can be concluded that 2-butoxyethyl methacrylate was not toxic to Pseudokirchneriella subcapitata. The registered substance is not expected to be toxic to aquatic algae and cyanobacteria based on the findings of this study.

Description of key information

The concentration of 2-butoxyethyl methacrylate that elicits a 50% response (EC50) from the freshwater algae Pseudokirchneriella subcapitata over 72-hour period was determined to be 17.5 mg/l based on growth rate. The registered substance can subsequently be regarded as not acutely toxic to aquatic algae and cyanobacteria under the conditions of the study given that this value is >1 mg/l (CLP Regulation (EC) No 1272/2008.

Key value for chemical safety assessment

EC50 for freshwater algae:
17.5 mg/L

Additional information