Butyl (S)-2-hydroxypropionate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-11-23 to 2017-03-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of the test item concentration in the test solutions was performed to determine exposure concentration and stability of the test item during exposure. Substance specific analysis was performed at the test site biochemA.
The samples were filled into 2 mL microcentrifuge tubes and stored in the freezer (≤ -18 °C). The sampling and documentation was conducted according to the following specification:
• At the start of the test (0 h), the test solutions NC, A, C and E were sampled before division to the test vessels (each group: 4 samples of 1 mL).
• After 24 h and 48 h exposure, both additional vessels of NC, A, C and E were sampled (each group: 2 samples of 1 mL).
• At the end of the test (72 h), both additional vessels and 2 replicates of the test vessels from NC, A, C and E were sampled (each group: 4 samples of 1 mL).
Of each sampled treatment, one of the samples from 0 h, 24 h, 48 h and 72 h was handed over to the analytical laboratory at the test site biochemA and analysed. The remaining samples are
stored as retain samples in the freezer until finalization of the study. Detailed information of the analytical method is given in the phase plan (Amendment No.1 to the study plan).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution was prepared by adding 216.8 mg test item to 1000 mL test medium and stirring for 35 min using a magnetic stirrer at room temperature. This stock solution was used as highest test item concentration in the test. The corresponding volume of test solution and algal inoculum (resulting in a methodical dilution by a factor of 1.11 and a nominal start concentration of 7 × 103 cells/mL) were transferred to the test vessels. Two additional vessels were prepared for treatment group A, C, E and negative control (NC).
Into each test vessel, 50 mL test item solution and test medium was transferred, respectively. The further test item concentrations were prepared by diluting the stock solution with test medium.
- Controls: yes

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettingen
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium under axenic conditions to keep it in exponential growth.

ACCLIMATION
Before the start of the test, 2.085 mL of the algae stock suspension were diluted with 47.915 mL OECD TG 201 medium to obtain a concentration of 5.0E+04 cells/mL. This preculture was incubated for 4 d at 23.0–23.3 °C and 73.2 μE/m²/s ± 5.6 % continuous lighting. For the start of the test, the cell concentration was determined using the Coulter Counter, resulting in 97.862E+05 cells/mL. To obtain a nominal inoculum concentration of 7E+04 cells/mL, 1.790 mL pre-culture were diluted into 250 mL OECD TG 201 medium.
- Acclimation period: 4 d
- Culturing media and conditions (same as test or not): 23.0–23.3 °C and 73.2 µE/m²/s ± 5.6 % continuous lighting, Holm-Hansen medium under axenic conditions
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.9–23.2 °C (required: 21–24 °C, constant within ± 2 °C)

pH:

7.6–7.8 in the control, 7.2–7.8 in the test item treatment (required: increase ≤ 1.5).

Nominal and measured concentrations:

Test item concentration nom. [mg/L]: 0, 5.0, 12.5, 31.25, 78.12, 195.31
Test item concentration measured [mg/L] at 0 h: 0, 5.45, 34.3, 211
after 72 h: 0, 4.68, 28.6, 155

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks, Schott, Mainz
- Type (delete if not applicable): open (The test vessels were sealed with cellulose stoppers)
- Material, size, headspace, fill volume: glass, 100 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no, static
- Initial cells density: 0.07 × 10^5 cells/mL
- Control end cells density: 5.056 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Medium used: Holm-Hansen medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultrapure water
- Culture medium different from test medium: OECD medium in the test, Holm-Hansen medium for culturing

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes
- Photoperiod: continuous lighting
- Light intensity and quality: 73.2 μE m^-2 s^-1 ± 5.6 %

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Coulter Counter Z2, Beckman Coulter, Krefeld
- Chlorophyll measurement: 96-well microplate, Tecan infinite F200, Tecan Group Ltd. Männedorf, Switzerland

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: yes
- Test concentrations: 1, 10 and 100 mg/L test item
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 195.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

43.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 20.84–91.96

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

78.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

78.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 15.11–384.24

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 3.30–53.22

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

78.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): Only in the highest test item concentration (195.31 mg/L), some agglutinations were visible. The morphology of the algal cells in the other test item concentrations as well as in the control showed no obvious abnormality.
- Any stimulation of growth found in any treatment: No
- Effect concentrations exceeding solubility of substance in test medium: No
The cell number over time within the different test concentrations is presented in Figure 1 below.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ExC50: 0.99 mg/L (CL 95 %: 0.89–1.10 mg/L)
- EyC50: 0.52 mg/L (CL 95 %: 0.38–0.74 mg/L)
- Other: The recent quality testing was performed in June 2016

Reported statistics and error estimates:

With the statistical software ToxRat 3.2.1 (ToxRat Solutions GmbH, Alsdorf), ECX and LOEC/NOEC were determined.

Criteria of validity

• The biomass (cell concentration) in the control increased by a factor of 72.2 and therefore ≥ 16 during the test period.  

• The mean coefficient of variation for section-by-section growth rate (day 0–1, 1–2 and 2–3) in the control is 24.2 % and therefore ≤ 35 %.

• The coefficient of variation of average specific growth rate during the test period in the control replicates is 5.3 % and therefore ≤ 7 %.

The test is valid according to OECD test guideline 201 (March 2006).

Results of chemical analysis

The samples for chemical analysis at time 0 h were taken before division of the test solutions to the test vessels and the dilution caused by addition of inoculum suspension. To take into account the dilution caused by addition of inoculum suspension, the measured test concentrations at 0 h are converted accordingly (factor of 0.9) in order to obtain the actual exposure concentrations in the test vessels. The measured test item concentrations in the test item treatments were 79.36–109.73 % of the nominal test item concentration and stable during the exposure period. As each mean of the three measured concentrations are within ± 20 % of the nominal concentrations, according to OECD 201, all results are given in relation to the nominal test item concentrations. The test item concentrations in the control were below the limit of quantification.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (growth rate) for growth inhibition of the freshwater alga Raphidocelis subcapitata by butyl-S-lactate is > 195.31 mg/L, the EC10 43.78 mg/L (95 % C.I. 20.84–91.96 mg/L), and the NOEC: 31.25 mg/L.
The measured test item concentrations in the test item treatments were within 79.36–109.73 % of the nominal test item concentration hence stable during the exposure period. Therefore, all results are given in relation to the nominal test item concentrations. The test item concentrations in the control were below the limit of quantification.

Executive summary:

In a 72 hour algal growth inhibition test, cultures of Pseudokirchneriella subcapitata (Strain No. 61.81 SAG) were exposed to butyl-(S)-lactate at nominal concentrations of 0 (control), 5.0, 12.5, 31.25, 78.12, 195.31 mg/L under static conditions in accordance with OECD guideline 201 and under GLP. The % growth inhibition in the treated algal cultures as compared to the control ranged from 3.7 % at a concentration of 5.0 mg/L to 31.1 % at a concentration of 195.31 mg/L.

The following abnormalities were noted: Only in the highest test item concentration (195.31 mg/L), some agglutinations were visible. There were no other compound related phytotoxic effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.

Results synopsis

Test organism: Pseudokirchneriella subcapitata (Strain No. 61.81 SAG)

Test Type: Static

Growth rate

72 hr EC₅₀: > 195.31mg/L

72 hr EC₂₀: 101.8 mg/L (95 % C.I.: 47.10–227.35)

72 hr EC₁₀: 43.78 mg/L (95 % C.I.: 20.84–91.96)

72 hr NOEC: 31.25 mg/L

72 hr LOEC: 78.12 mg/L

Yield

72 hr EC₅₀: 78.02 mg/L (95 % C.I.: 15.11–384. 24 mg/L)

72 hr EC₂₀: 24.35 mg/L (95 % C.I.: 6.58–91.44 mg/L)

72 hr EC₁₀: 13.25 mg/L (95 % C.I.: 3.30–53.22 mg/L)

72 hr NOEC: 31.25 mg/L

72 hr LOEC: 78.12 mg/L

Endpoint(s) effected: Growth rate, yield

Description of key information

In a growth inhibition study, Raphidocelis subcapitata were exposed to butyl-(S)-lactate over 72-h in a static study design according to OECD guideline 201 and under GLP. The following effect concentrations for growth rate were determined:

72-h E_rC₅₀: > 195.3 mg/L

72-h E_rC₂₀: 101.8 mg/L (95 % C.I.: 47.10–227.35 mg/L)

72-h E_rC₁₀: 43.78 mg/L (95 % C.I.: 20.84–91.96 mg/L)

72-h NOE_rC: 31.25 mg/L

72-h LOE_rC: 78.12 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

195.31 mg/L

EC10 or NOEC for freshwater algae:

31.25 mg/L