1'-(phenylmethyl)-[1,4'-bipiperidine]-4'-carbonitrile

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:

2017-04-07 to 2017-06-16

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult subsances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1'-(phenylmethyl)-[1,4'-bipiperidine]-4'-carbonitrile

EC Number:

282-544-0

EC Name:

1'-(phenylmethyl)-[1,4'-bipiperidine]-4'-carbonitrile

Cas Number:

84254-97-7

Molecular formula:

C18H25N3

IUPAC Name:

1'-benzyl-[1,4'-bipiperidine]-4'-carbonitrile

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15IB3393
- Expiration date of the lot/batch: 2017-08-19 (retest date)
- Purity test date: 2017-06-01 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: 2 g/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples were taken before the start of the test and after 72 hours from each test medium and from the control. In addition, the filter containing undissolved residue was kept for possible analysis. Additionally, reserve samples of 2.0 mL were taken for possible analysis. 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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Due to an expected low water solubility of the test item, saturated solutions (SS) were prepared. The preparation of test solutions started with a loading rate of 50 mg/L applying 3 days of magnetic stirring at room temperature to ensure maximum dissolution of the test item in test medium. This resulted in a clear and colourless dispersion that contained undissolved floating material and precipitate. The obtained mixture was filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove the undissolved fraction. The filter was pre-conditioned with a small volume of test solution that was discarded. For the combined limit/range-finding test, the pH of the resulting SS was adjusted from 9.4 to 8.5 with 1.0 M HCl (Merck, Darmstadt, Germany). The obtained solution was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. 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 10^4 cells/mL.
- Controls: yes
- 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:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

22-23 °C

pH:

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

Dissolved oxygen:

not applicable

Salinity:

not relevant

Conductivity:

not relevant

Nominal and measured concentrations:

Range-finder:
nominal test concentrations: 1.0, 10 and 100% of a SS prepared at 100 mg/L (only 10% SS analysed)
measured test concentration t = 0 h: 10.0 mg/L
measured test concentration t = 72 h: 9.90 mg/L

Final test:
nominal test concentrations: 0, 1.0, 3.2, 10, 32 and 100% of a SS prepared at 50 mg/L.
measured test concentration t = 0 h: n.d., 0.522, 1.73, 5.46, 5.52, 16.1, 52.0 mg/L
measured test concentration t = 72 h: n.d., 0.512, 1.69, 5.43, 5.31, 16.6, 57.0 mg/L

n.d. = not detected

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Initial cells density: 10,000 cells/mL
- Control end cells density: 22.04 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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 (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was 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. 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
- Sterile test conditions: no information
- Adjustment of pH: the pH of the resulting SS was adjusted from 9.4 to 8.5 with 1.0 M HCl (Merck, Darmstadt, Germany)
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 600 to 120 µE.m-2.s-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: 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: x3.2
- Range finding study: yes
- Test concentrations: 1.0, 10 and 100% of the SS (setup as combined range finder/limit test)
- Results used to determine the conditions for the definitive study: yes

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.:

21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L. 13-38 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (initial)

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
- 72-h EC50 (yield) = 4.1 mg T000745/L (95% C.L. 3.3-5.1 mg/L)
- 72-h EC10 (growth rate) = 1.9 mg T000745/L (95% C.L. 00.39-3.9 mg/L)
- 72-h EC10 (yield) = 1.3 mg T000745/L (95% C.L. 0.82-2.0 mg/L)
- 72-h NOEC (yield) = 1.7 mg T000745/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 1.2 mg/L (95% C.L. 1.1 to 1.2 mg/L)
- 72-h EC50 (yield) = 0.43 mg/L (95% C.L. 0.42 to 0.44 mg/L)

Reported statistics and error estimates:

For determination of the NOEC and the EC50 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 test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller)or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding measured exposure concentrations of the test item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The final test was inoculated using algal cells from the stock culture instead of the pre-culture because the pre-culture had not grown sufficiently. Evaluation: The validity criteria were met, indicating that this had no effect on the test results

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T000745 according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had a statistically significant inhibitory effect on the growth rate of Pseudokirchneriella subcapitata at a measured concentration of 5.5 mg/L and higher during the 72-hour test period, i.e. the 72-hour NOEC for growth rate inhibition was determined to be 1.7 mg/L. The 72-hour NOEC for yield inhibition was determined to be 1.7 mg/L. The results of the test can be considered reliable without restrictions.