Ethyl 4-oxopiperidine-1-carboxylate

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:

2016-09-26 to 2016-10-25

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 4-oxopiperidine-1-carboxylate

EC Number:

249-984-5

EC Name:

Ethyl 4-oxopiperidine-1-carboxylate

Cas Number:

29976-53-2

Molecular formula:

C8H13NO3

IUPAC Name:

ethyl 4-oxopiperidine-1-carboxylate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study reports): JNJ-126971-AAA (T000509)
- Physical state: liquid
- Appearance: clear colourless to light yellow liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15HB3826
- Manufacuring date: 2015-08-01
- Expiration date of the lot/batch: 2017-01-13 (retest date)
- Purity test date: 2016-10-28

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

OTHER SPECIFICS:
- purity: 98.5%
- appearance: clear colourless to light yellow liquid
- Water solubility: fully soluble in test medium at the concetrations tested

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken before the start of the test, after 24 hours and after 72 hours from each test medium and from the control. 2.0 mL of volume was taken. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: stored in a freezer until analysis. Additionally, reserve samples of 2.0 mL were taken for possible analysis

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L. A magnetic stirring period of 16 minutes was applied to advance complete dissolution. Lower test concentrations were prepared by subsequent dilutions in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All final test solutions were clear and colourless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal 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. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21-23°C

pH:

t=0h: 7.9 - 8.1
t=72h: 7.9 - 8.0

Dissolved oxygen:

not reported

Salinity:

not relevant

Conductivity:

not applicable

Nominal and measured concentrations:

Nominal concentrations (mg/L): 0.10, 1.0, 10, and 100 mg/L
Measured concentrations (mg/L):
after 24h: nm, nm, nm, 100 mg/L
after 72h: nm, nm, nm, 95 mg/L

nm = not measured

The measured concentrations were in agreement with the nominal concentrations (95-100%) during the entire exposure period. Therefore, the effect parameters were expressed in terms of analytically confirmed nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: 100 mL, all-glass, containing 50 mL of test solution
- Initial cells density: 1x10^4 cells/mL
- No. of vessels per concentration and per control (replicates):
* 6 replicates each for the control and the highest test concentration;
* 3 replicates for each intermediate concentrations;
* 1 extra replicate of the control and each test concentration for sampling purposes after 24 hours of exposure;
* 1 or 2 replicates of each concentration without algae.

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water
- Culture medium different from test medium: Yes. Three days before the start of the test the algal stock culture were inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test (control and 100% filtrate). Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- illumination: TLD-lamps with a light intensity within the range of 81 to 86 µE/m²/s.
- Adjustment of pH: no
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth inhibition
- Determination of cell concentrations: At the beginning, cells were counted using a microscope and a counting chamber. thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength = 20mm).
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: combined limit/range finding study was performed
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no definitive study necessary

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 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): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72h-ErC50 was 1.3 mg/L (95% confidence interval ranging from 1.2 to 1.4 mg/L)
- Other: the historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range

Reported statistics and error estimates:

Note that only the highest test concentration was statistically tested against the control group.
The differences between growth rates in the control and the 100 mg/L treatment were statistically significant but biologically not relevant (i.e. <10%).
No inhibition of yield was observed at nominal 0.10 and 1.0 mg/L. Slight inhibition of yield was observed at 10 mg/L (9%). The differences between yields in the control group and the 100 mg/L
treatment were statistically significant (15%).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular algal species Pseudokirchneriella subcapitata was performed with the test substance JNJ-126971-AAA (T000509) according to the OECD guideline 201 (GLP conditions). The test item had no inhibitory effect on the growth of Pseudokirchneriella subcapitata after the test period of 72 hours at a nominal concentration of 100 mg/L, being the regulatory limit concentration. Hence, the 72-hour NOEC for growth rate was determined to be 100 mg/L. No inhibition of yield was observed at nominal 0.10 and 1.0 mg/L. Slilght inhibition of yield was observed at 10 mg/L. The EC50 for growth rate and yield inhibition exceeded 100 mg/L.
The results of the test can be considered reliable without restrictions.