4-Bromo-2-fluoro-N-methylbenzamide

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:

11 Sep 2020 to 22 Oct 2020

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD series on testing and assessment number 23 (2nd edition),

Version / remarks:

February 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-Bromo-2-fluoro-N-methylbenzamide

EC Number:

808-257-8

Cas Number:

749927-69-3

Molecular formula:

C8H7BrFNO

IUPAC Name:

4-Bromo-2-fluoro-N-methylbenzamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: FFP-106-200011
- Expiry date: 17 March 2021 (retest date)
- Physical Description: Almost white powder
- Purity: 99.0%
- Purity correction factor: not required
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 material in the solvent/vehicle and the exposure medium: not indicated

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: 1.5 mL from the approximate centre of the test vessels.
- 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 vessel containing 10% of the SS, but without algae and samples for analysis were taken at the start and at the end of the test period. Additionally, reserve samples of 1.5 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:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of test solutions started with a loading rate of 100 mg/L applying a fifteen- minute period of ultrasonic waves followed by a two-hour period of magnetic stirring. Against expectation, undissolved material was observed after this treatment. Accordingly, an additional twenty-minute period of ultrasonic waves was applied, followed by a two-hour period of magnetic stirring was needed. Undissolved particulate material was still observed at this point. Consequently, 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 saturated solution 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.
- 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:

Between 23-24 °C

pH:

At t=0: 7.8-7.9
At t= 72: 7.8-8.1

Dissolved oxygen:

Not reported

Salinity:

Not relevant

Conductivity:

Not relevant

Nominal and measured concentrations:

nominal: Solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading of 100 mg/L.

The concentrations measured at the start of the test were 0.99, 3.2, 9.7, 30 and 96 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. The concentrations remained stable during the exposure period, i.e. were at 99-103% of the initial values at the end of the test.

The measured concentrations were consequently at 93-102% relative to the theoretically possible nominal levels, indicating that the undissolved fraction that was removed during the preparation of test solutions was not related to the quantified test item fraction.

Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations.

The concentrations measured in the samples taken from solution without algae were comparable with the concentrations measured in the samples with algae. It was thus concluded that the presence of the algae did not affect the concentrations of the test item in test medium throughout the test.

It should be noted that a small test item response was detected in the sample taken from the control at the end of the test. The exact origin of this response could not be identified. This response was only present in the sample taken at the end of the test, and therefore, test item was probably introduced during sampling or sample preparation procedures. This response was considered negligible. Quantification was possible only by extrapolation of the calibration curve and the contribution to the lowest test concentration was estimated to be 0.22%

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels, perforated for ventilation
- 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: 211.1 x 10,000 cells/mL
- 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 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 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 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 water quality measurement: temperature measured continuously, pH at the beginning an d the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- illumination: TLD-lamps with a light intensity within the range of 84 to 88 μE/m²/s.
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 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)
- Range finding study test concentrations: 0.10, 1.00, 10 and 100 % of the SS at a loading rate of 100 mg/L
- 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.:

> 100 mg/L

Nominal / measured:

nominal

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Basis for effect:

growth rate

Remarks on result:

other: based on biological relevance

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 NOEC (growth rate): 3.2 mg/L, based on statistical significance
- 72-h EC10 (growth rate): 53 mg/L
- 72-h EC10 (yield): 15 mg/L
- 72-h EC50 (yield): 68 mg/L
- 72-h NOEC (yield): 10 mg/L, based on biological relevance
- 72-h NOEC (yield): 3.2 mg/L, based on statistical significance
- biological relevance vs. statistical significant: effects were considered not biologically relevant when the effect was lower than 10%

Results with reference substance (positive control):

Experimental Start Date: 21 Sep 2020
Experimental Completion Date: 24 Sep 2020
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.
The raw data from this study are kept in the Charles River Den Bosch archives. The test described above was performed non-GLP.

Reported statistics and error estimates:

For determination of the NOEC and the ECx 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 control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on a 3-parameter normal cumulative distribution function (CDF) using non-linear regression analysis with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the correspondingconcentrations 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:

Toxicity of T003669 to Raphidocelis subcapitata was tested during 72 hour exposure at increasing concentrations, according to OECD 201 test guideline.
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, JNJ-61789975-AAA (T003669) inhibited both growth rate and yield of this freshwater algae species significantly at the analytically confirmed nominal concentration of 10 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 68 mg/L with a 95% confidence interval ranging from 48 to 95 mg/L.
The 72h-NOEC for growth rate inhibition was 3.2 mg/L based on statistical significance and 32 mg/L based on biological relevance.
The 72h-NOEC for yield inhibition was 3.2 mg/L based on statistical significance and 10 mg/L based on biological relevance.