2-octyldodecyl 5-oxo-L-prolinate

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:

From January 13, 2017 to January 31, 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch no.: CH 192955/01
Purity/composition: 88.88%
Appearance: clear yellow liquid

Analytical monitoring:

yes

Remarks:

UPLC/MS

Details on sampling:

- Samples (2 mL) taken from the highest test concentration were analysed. At the start of the test, the actual test concentration was 22 mg/L in the undiluted saturated solution (SS). At the end of the exposure period, the measured concentration had decreased to 28% of the initial concentration. Based on these results, the Time Weighted Average (TWA) concentration was calculated and used to determine the effect parameters. The TWA concentrations was estimated to be 12 mg/L.
- Single samples (2 mL) for possible analysis were taken from all test concentrations and the control at t= 0, 24 and 72 h.. In addition, the glass wool containing undissolved residue was kept for possible analysis.

Vehicle:

no

Details on test solutions:

- Preparation of test solutions started with a loading rate of 113 mg/L applying a two-day period of magnetic stirring after which the resulting mixture was allowed to stabilize overnight. Subsequently, the SS was siphoned off over glass wool and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. The intermediate test concentrations were clear and colourless at the start of the test, while the undiluted SS was hazy.
- After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10E04 cells/mL.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

23°C

pH:

7.6-8.1

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

0, 1.0, 10 and 100% of the SS prepared at 113 mg/L

Details on test conditions:

- A combined limit/range-finding test was performed, exposing six replicates of exponentially growing algal cultures to a control and to 1.0, 10 and 100% of the SS. The initial algal cell density was 10E04 cells/mL and the total exposure period was 72 hours. (Replicates: 6 replicates each of the control and the highest test concentration, 3 replicates of each intermediate test concentration and 1 extra replicate of each test concentration for sampling purposes after 24 hours).
1 or 2 replicates of each test concentration without algae.
- Test procedures and conditions:
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Illumination: Continuously using TLD-lamps with a light intensity within the range of 86 to 87 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L

Duration:

72 h

Dose descriptor:

other: EC 10 and 20

Effect conc.:

> 12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L

Details on results:

- No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to all test concentrations when compared to the control.
- Both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) were beyond the range tested, i.e. exceeded 12 mg/L being the TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L. The 72h-NOEC for both growth rate inhibition and yield inhibition was 12 mg/L being the TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L.

Results with reference substance (positive control):

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.32 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95% confidence interval ranging from 1.2 to 1.4 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.45 mg/L with a 95% confidence interval ranging from 0.39 to 0.53 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

- For determination of the NOEC/NOEL and the EC50/ EL50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, one-sided, smaller).
- No EC50/EL50 -values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested / EL50 > maximum loading rate tested). The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) for Pseudokirchneriella subcapitata were beyond the range tested, i.e. exceeded 12 mg/L being the TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L. The 72h-NOEC for both growth rate inhibition and yield inhibition was 12 mg/L being the TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L.

Executive summary:

A study was conducted to determine the toxicity of the test substance to aquatic algae and cyanobacteria according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. A combined limit/range-finding test was performed. Six replicates of exponentially growing algal cultures (Pseudokirchneriella subcapitata) were exposed to an untreated control and to the undiluted SS prepared at 113 mg/L. In addition, three replicates per group were exposed to 1.0 and 10% of the SS. A positive control (potassium dichromate) was included as well. The initial algal cell density was 10E04 cells/mL and the total exposure period was 72 hours under statc conditions. Samples for analytical confirmation (with UPLC/MS) of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to all test concentrations when compared to the control. The values obtained following incubation with the positive control substance were within the expected range. The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the study conditions, both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) for Pseudokirchneriella subcapitata were beyond the range tested, i.e. exceeded 12 mg/L being the TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L. The 72h-NOEC for both growth rate inhibition and yield inhibition was 12 mg/L being the TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L (Augusiak, 2017).

Key value for chemical safety assessment

EC50 for freshwater algae:

12 mg/L

EC10 or NOEC for freshwater algae:

12 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to aquatic algae and cyanobacteria according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. A combined limit/range-finding test was performed. Six replicates of exponentially growing algal cultures (Pseudokirchneriella subcapitata) were exposed to an untreated control and to the undiluted SS prepared at 113 mg/L. In addition, three replicates per group were exposed to 1.0 and 10% of the SS. A positive control (potassium dichromate) was included as well. The initial algal cell density was 10E04 cells/mL and the total exposure period was 72 hours under statc conditions. Samples for analytical confirmation (with UPLC/MS) of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to all test concentrations when compared to the control. The values obtained following incubation with the positive control substance were within the expected range. The study met the acceptability criteria prescribed by the study plan and was considered valid. Under the study conditions, both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) for Pseudokirchneriella subcapitata were beyond the range tested, i.e. exceeded 12 mg/L being the TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L. The 72h-NOEC for both growth rate inhibition and yield inhibition was 12 mg/L being the TWA concentration of the test substance in a solution prepared at a loading rate of 113 mg/L (Augusiak, 2017).