1-acetyl-4-(4-hydroxyphenyl)piperazine

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:

2016-06-06 to 2016-12-12

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Except for the characterization and stability of the test item have been performed according to cGMP Guidelines.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15FB3131
- manufacture date: 2015-06-12
- Expiration date of the lot/batch: 2017-06-11 (retest date)
- Purity test date: 2015-09-04

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

OTHER SPECIFICS:
- purity: 100%
- appearance: white powder
- Water solubility: 4.3 g/L

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. 1.8 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 1.8 mL were taken for possible analysis

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 test concentration of 100 mg/L (combined limit/range-finding test) or with a stock solution of 10 mg/L (final test) applying a short period of ultrasonic treatment followed by magnetic stirring to accelerate the dissolving of the test item in the test medium. The lower test concentrations were prepared by subsequent dilutions 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 104 cells/mL.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions up to and including 10 mg/L were clear and colourless, while the concentration of 100 mg/L was slightly pink.

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: Algae stock cultures were started by inoculating growth medium (M1) 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.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2, according to OECD 201) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): yes

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

23-24*C

pH:

t=0h: 8.0-8.2
t=72h: 8.1-8.8

Dissolved oxygen:

not reported

Salinity:

not relevant

Conductivity:

not applicable

Nominal and measured concentrations:

Limit range test:
- Nominal concentrations (mg/L): 0.10, 1.0, 10, and 100 mg/L
- Measured concentrations (mg/L):
after 72h: nm, nm, nm, m
nm = not measured
m = measured
The measured concentration decreased to a concentration below the detection limit after 72 hours of exposure.

final test:
Nominal concentrations (mg/L): 0.032, 0.1, 0.32, 1.0, 3.2 mg/L
Measured concentrations (mg/L):
after 24h: 0.000148, 0.0119, 0.0836, 0.394, 1.68 mg/L
after 72h: nm, nm, 0.000056, 0.129, 0.907 mg/L

nm = not measured
At the start of the test, the actual measured concentrations were 0.0146, 0.0598, 0.237, 0.885 and 3.07 mg/L, i.e. 45% to 96% relative to the expected 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 cell density: 1 x 10^4 cells/mL
- No. of vessels per concentration and per control (replicates):
3 replicates of each test concentration;
6 replicates of the control;
1 extra replicate of each test group for sampling after 24 hours
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 the control and the test concentration closest to the EC50 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 99 to 101 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 = 10mm).
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x3.2
- Range finding study: combined limit/range finding study was performed (0.10, 1.0, 10, 100 mg/L)
- Test concentrations: 0.032, 0.10, 0.32, 1.0, 3.2 mg/L
- Results used to determine the conditions for the definitive study: the expected EC50 for growth rate inhibition approximated a nominal concentration of 1.0 mg/L. The expected EC50 for yield inhibition was between nominal concentrations of 0.1 and 1.0 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 1.1 to 1.4 mg/l

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (TWA)

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) = 0.16 mg/L (based on TWA, 95% confidence interval)
- 72-h EC10 (growth rate) = 0.16 mg/L (TWA)
- 72-h EC10 (yield) = 0.02 mg/L (TWA)
- 72-h NOEC (yield) = 0.009 mg/L (TWA)

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

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-119379-AAA (T001141) according to the OECD guideline 201 (GLP conditions). It can be concluded that the test item had a significant inhibitory effect on the growth of Pseudokirchneriella subcapitata at a TWA concentration of 0.35 mg/L and higher during the 72-hour test period, i.e. the 72-hour NOEC for growth rate was determined to be 0.048 mg/L. The NOEC for yield inhibition was determined to be 0.009 mg/L.
The results of the test can be considered reliable without restrictions.

Description of key information

Based on the guideline study (Migchielsen, 2016) it is concluded that the test item, JNJ-119379-AAA (T001141), had a significant inhibitory effect on the growth of Pseudokirchneriella subcapitata at a TWA concentration of 0.35 mg/L and higher during the 72-hour test period, i.e. the 72-hour NOEC for growth rate was determined to be 0.048 mg/L. The NOEC for yield inhibition was determined to be 0.009 mg/L. The results of the test can be considered reliable without restrictions.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.35 mg/L

EC10 or NOEC for freshwater algae:

0.009 mg/L