3-phenyl-1H-pyrazole-4-carbaldehyde

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:

16 NOV 2020 - 22 JAN 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-phenyl-1H-pyrazole-4-carbaldehyde

EC Number:

632-673-7

Cas Number:

26033-20-5

Molecular formula:

C10H8N2O

IUPAC Name:

3-phenyl-1H-pyrazole-4-carbaldehyde

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: M19LD3723
- Purity, including information on contaminants, isomers, etc.: 103.6%
- Expiration date of the lot/batch: 2021-11-11 (retest date)
- Purity test date: 2020-05-04 (certificate of analysis release date)
- Physical appearance: Light yellow powder

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
- Frequency: at t=0 h, t=24 h and t=72 h.
- Volume: 1.8 mL from the approximate center of the test solutions.
- Storage: samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test solution at 10 mg/L but without algae (abiotic control) and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period. Additionally, reserve samples of 1.8 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of JNJ-39125190-AAA (T003897) tested was a light-yellow powder with a purity of 103.6% and was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.

For the range-finding test, preparation of test solutions started with a loading rate of 100 mg/L applying a 30-minute period of ultrasonic treatment followed by an additional 30 minutes of magnetic stirring to ensure maximum dissolution of the test item in medium. Since undissolved material was observed the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS intest medium. All test solutions were clear and colorless at the end of the preparation procedure.

For the final test, preparation of test solutions started with the highest concentration of 100 mg/L applying a one-hour period of ultrasonic treatment to accelerate dissolution of the test item in medium. The test item was completely dissolved in test medium. The obtained solution was allowed to cool down from 27°C to 24°C for half an hour at room temperature. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

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 1E+04 cells/mL.

Any residual volumes were discarded.

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 immediately 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:

between 21 and 23°C.

pH:

start: 8.0-8.1, end: 8.0-8.3

Nominal and measured concentrations:

nominal (mg/L): 0, 1.0, 3.2, 10, 32, 100, measured (mg/L; 0h): nd, 0.999, 3.29, 9.94, 10.1, 31.3, 99.3, measured (mg/L, 24h): nd, 0.986, 3.23, 9.76, 10.3, 31.3, 99.9, measured (mg/L, 48h): nd, 0.206, 0.708, 3.04, 10.5, 17.1, 95.4

The measured concentrations at the start of the test were in agreement with the nominal concentrations, i.e. were in the range of 98 to 103% relative to the nominal levels. At the end of the test, the measured concentrations varied between 21 and 96% of the initial concentrations whereby the higher concentrations remained more stable than the lower ones. The concentration measured at the end of the test in the sample taken from the solution without algae (abiotic control) was considerably higher than in the solution incubated with algae. This indicates that the presence of the algae affected the concentration of the test item in test medium throughout the test. Based on these results, the time weighted average (TWA) concentrations were calculated and used to express effect parameters: 0.63, 2.1, 6.9, 10, 26, 98 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels with aluminium caps, perforated for ventilatoin
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 4.03E+06 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes

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: no
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 82 to 85 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm, using TLD-lamps.

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.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

- Range finding study
- Test concentrations range finding: 0.10 to 100% of the SS prepared at 100 mg/L increasing by a factor of 10 and a control.
- 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.:

> 98 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: NOEC of 6.9 mg/L based on biological relevance (i.e. >10% effect on algal growth relative to control values.)

Details on results:

- NOEC yield: 2.1 mg/L, EC50 yield: 24 mg/L
- Statistically significant inhibition of growth rate and of yield was found at test concentrations of 6.9 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at 6.9 mg/L, where the observed inhibition was below 10%. The according NOEC based on biological relevance was thus set at 6.9 mg/L.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.
- Validity: .
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 403).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 24%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 1.1%).

Results with reference substance (positive control):

The batch of Raphidocelis subcapitata tested showed expected sensitivity to Potassium dichromate, based on the historical range of reference tests performed by the Test Facility in the last ten years.

Reported statistics and error estimates:

- Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.
- An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s test) and a test for homogeneity of the variances (Levene’s test). 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 TWA concentrations of the test item.The EC50-value for growth rate inhibition could not be determined because the observed effects were below 50%.ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The objective of the study was to evaluate JNJ-39125190-AAA (T003897) for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield. A full test was performed based on the results of a preceding range-finding test. The study met the validity criteria prescribed by the study plan and was considered valid. In conclusion, under the conditions of the present study with Raphidocelis subcapitata, JNJ-39125190-AAA (T003897) inhibited growth rate and yield of this freshwater algae species significantly at TWA concentrations of 6.9 mg/L and higher, i.e. the NOEC being 2.1 mg/L. The EC50 for growth rate was > 98 mg/L, the EC50 for yield was 24 mg/L. The results of the test can be considered reliable without restriction.