Ammonium 3-nitrobenzoate

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:

2018-08-21 - 2018-08-27 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

yes

Remarks:

but not affecting the integrity and validity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBITION TEST,” adopted 07. December 2015

Deviations:

yes

Remarks:

but not affecting the integrity and validity of the study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 3-nitrobenzoate

EC Number:

242-965-2

EC Name:

Ammonium 3-nitrobenzoate

Cas Number:

19328-56-4

Molecular formula:

C7H5NO4.H3N

IUPAC Name:

ammonium 3-nitrobenzoate

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Details on test material:

- Storage: Room Temperature (20 ± 5 °C)

Specific details on test material used for the study:

The test item was stored in the test facility in a closed vessel dark and dry at 20 ± 5°C.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 / 26 / 57 / 126 / 263 / 571 mg/L nominal concentration
- Sample storage conditions before analysis: yes, 2 days

Test solutions

Vehicle:

no

Remarks:

test medium was used

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution containing 571.6 mg/L in algal medium (demineralised water enriched with minerals but without algae) was prepared (= 100.0 mg/L solid component due to 82.5 % water content of test item). The lower treatments were prepared by dilution of this stock solution with algal medium.
- Controls: medium resp. Potassium dichromate
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Stock solution: 571.4 mg/L (= 100.0 mg/L of solid component)
Dilutions: 0 / 26 / 57 / 126 / 263 / 571 mg test item/L (nominal)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga.
- Strain: Desmodesmus subspicatus, SAG Strain Number 86.81
- Source (laboratory, culture collection):
Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Test temperature:

21.7 – 23.2 °C

pH:

8.1 - 9.9

Nominal and measured concentrations:

nominal: 0 / 26 / 57 / 126 / 263 / 571 mg/L concentration (= 0 / 4.6 / 10 / 22 / 46 / 100 mg/L solid component due to 82.5 % water content of test item)
measured, t=0h: 0.00 / 23.62 / 54.57 / 117.64 / 246.91 / 536.81 mg/L
measured, t=72h: 0.00 / 25.60 / 55.04 / 119.22 / 253.02 / 553.53 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: glass flasks, total volume 65 mL, 45 ±1 mL fill volume
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 2160 cells/mL
- Control end cells density: 403870 cells/mL
- No. of vessels per concentration (replicates): 3 replicates for each treatment
- No. of vessels per control (replicates): 6 replicates for the blank control

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Other: pH, appearance

TEST CONCENTRATIONS
- Range finding study
no toxicity could be observed in all treatments

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ammonium-3-nitrobenzoate (solid)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ammonium-3-nitrobenzoate (solid)

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ammonium-3-nitrobenzoate (solid)

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ammonium-3-nitrobenzoate (solid)

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ammonium-3-nitrobenzoate (solid)

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ammonium-3-nitrobenzoate (solid)

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ammonium-3-nitrobenzoate (solid)

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Ammonium-3-nitrobenzoate (solid)

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

aqueous solution of Ammonium-3-nitrobenzoate

Basis for effect:

growth rate

Effect concentrations 10

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

aqueous solution of Ammonium-3-nitrobenzoate

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 11

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

aqueous solution of Ammonium-3-nitrobenzoate

Basis for effect:

growth rate

Effect concentrations 12

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

aqueous solution of Ammonium-3-nitrobenzoate

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 13

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

aqueous solution of Ammonium-3-nitrobenzoate

Basis for effect:

growth rate

Effect concentrations 14

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

aqueous solution of Ammonium-3-nitrobenzoate

Basis for effect:

biomass

Remarks:

yield

Effect concentrations 15

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

aqueous solution of Ammonium-3-nitrobenzoate

Basis for effect:

growth rate

Effect concentrations 16

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

aqueous solution of Ammonium-3-nitrobenzoate

Basis for effect:

biomass

Remarks:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: slightly
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
Parameter Value 95% confidence interval
72h ErC50 0.79 mg/L 0.79 mg/L – 0.79 mg/L
72h EyC50 0.39 mg/L 0.35 mg/L – 0.45 mg/L

Reported statistics and error estimates:

Since no inhibition of algae growth has occurred, no statistical evaluation was performed. The results are based on the nominal concentrations of the test item and additionally on the concentration of the solid component (based on 82.5% water content of test item).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of Ammonium-3-nitrobenzoate towards algae. No inhibition of algal growth was observed in all tested concentrations up to 571 mg/L test item) resp. 100 mg/L Ammonium-3-nitrobenzoate. Hence, Ammonium-3-nitrobenzoate does not need to be classified as hazardous to the environment, neither acute nor chronic, according to Regulation 1272/2008 and amendments.

Executive summary:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself.

One valid experiment was performed.

The study was performed using 5 concentrations ranging from 26 to 571 mg/L of an aqueous solution of Ammonium-3-nitrobenzoate. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

No inhibition of algal growth was observed in all tested concentrations.

At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV.

The measured concentrations lay between 91% and 96 % of the nominal concentrations at the beginning of the test and between 95 % and 98% of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the nominal concentrations.

Since no inhibition of algae growth has occurred, no statistical evaluation was performed. The results are based on the nominal concentrations of the test item and additionally on the concentration of the solid component (based on 82.5% water content of test item).

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item Ammonium-3-nitrobenzoate (aqueous solution) were determined:

 

Table Results of the test item (based on test item)

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	≥ 571 mg/L	> 571 mg/L	> 571 mg/L	> 571 mg/L
Yield	≥ 571 mg/L	> 571 mg/L	> 571 mg/L	> 571 mg/L

 

Table Results of the test item (based on solid component Ammonium-3-nitrobenzoate, considering 82.5% water content)

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	≥ 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L
Yield	≥ 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L

 

Ammonium-3-nitrobenzoate does not need to be classified as hazardous to the environment, neither acute nor chronic, according to Regulation 1272/2008 and amendments.