Ammonium 3-nitrobenzoate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-08-22 - 2018-08-24 (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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 202, adopted 13. Apr. 2004: ”Daphnia sp., Acute Immobilisation Test“

Deviations:

yes

Remarks:

but not affecting the integrity or validity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No. 440/2008, Method C.2. “Daphnia sp. Acute Immo-bilisation Test”, adopted 30. May 2008

Deviations:

yes

Remarks:

but not affecting the integrity or 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)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 / 26 / 57 / 126 / 263 / 571 mg test item/L (nominal)
- Sample storage conditions before analysis: no

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 571.8 mg/L (= 100.14 mg/L of solid component) test item in dilution water was prepared. The lower concentrations were prepared by dilution of this stock solution with dilution water.
- Controls: medium resp. Potassium dichromate
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
Stock solution: 571.8 mg/L (= 100.14 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna STRAUS Berlin
- Source: Origin: Umweltbundesamt Berlin, In-house breeding since 27. September 2007
- Age of parental stock (mean and range, SD):
- Feeding during test: no

ACCLIMATION
Not applicable; Daphnia magna is bred in the laboratory throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical. The husbandry is performed similar to the method described in the OECD guideline
- Type and amount of food: green algae (Desmodesmus subspicatus)
- Feeding frequency: twice a week
- Health during acclimation (any mortality observed):

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
24 hours before the start of the test, the adult animals were separated from the young. 0.5 hours before test start, the adults were caught with the help of a glass tube, and the new-born daphnia, aged between 0 and 23.5 hours, were sieved from the medium and immediately placed into a beaker containing dilution water.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test conditions

Hardness:

Parameter Concentration in mg/L
CaCl2*2H2O 293.80
MgSO4*7H2O 123.30
NaHCO3 64.80
KCl 5.80
Resulting hardness in mmol/L: 2.502
Resulting hardness in mg CaCO3/L: 250

Test temperature:

23.1 – 23.8 °C

pH:

7.8 - 8.0

Dissolved oxygen:

8.7 - 9.1 mg/L

Nominal and measured concentrations:

nominal: 0 / 26 / 57 / 126 / 263 / 571 mg/L
measured, t=0h: 0.00 / 22.26 / 54.00 / 121.75 / 249.14 / 554.99 mg/L
measured, t=48h: 0.00 / 24.90 / 60.30 / 134.32 / 278.18 / 599.66 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass beakers, nominal volume 50 mL, tall shape, fill volume 20 ± 5 mL
- Aeration: After preparation, the dilution water was aerated.
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 hours
- Light intensity: neon tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mobility: 24 and 48 hours

VEHICLE CONTROL PERFORMED: n/a

RANGE-FINDING STUDY
0% immobilisation at 1 / 10 and 100 mg test item/L (nominal)

Reference substance (positive control):

yes

Remarks:

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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

solid Ammonium-3-nitrobenzoate

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

solid Ammonium-3-nitrobenzoate

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

solid Ammonium-3-nitrobenzoate

Basis for effect:

mobility

Effect concentrations 7

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 571 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 8

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

solid Ammonium-3-nitrobenzoate

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none stated
- 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
- Relevant effect levels: 24h-EC50
- Limit test: no
- Dose-response test: yes
- ECx: The 24h-EC50 value was determined as 1.9 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline. Since the fits showed sufficient statistical correspondence of the data with the dose-response-equation, this reference study can be considered valid.

Reported statistics and error estimates:

The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.2.1.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 202 and EU method C.2 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 daphnids.
The study was performed using 5 concentrations ranging from 26 to 571 mg/L (4.6 – 100 mg solid component/L). For each test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.
Four concentrations showed immobilisation between 5 and 15% immobilisation. One of the animals was immobilised in the blank control. Hence, the EC50 was determined as > 571 mg/L (test item) resp. > 100 mg/L (Ammonium-3-nitrobenzoate).
Based on the obtained results, Ammonium-3-nitrobenzoate does not need to be classified as toxic to the environment, neither acutely nor chronic, according to the Regulation (EC) No. 1272/2008.

Executive summary:

The study was conducted under GLP according to OECD guideline 202 and EU method C.2 on the registered substance itself. One valid experiment was performed.

The study was performed using 5 concentrations ranging from 26 to 571 mg/L (aqueous solution, corresponding to 4.6 – 100 mg solid component/L). For each test concentration and the blank control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.

Four concentrations showed immobilisation between 5 and 15% immobilisation. One of the animals was immobilised in the blank control.

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV determination. The concentrations determined at the start of the test were between 86 – 97 % of the nominal concentration. At the end of the test the determined concentrations were between 96 – 107 % of the nominal concentration. Slightly higher measured concentrations at the end of the test might be caused due to evaporation. Since measured concentrations stayed within the range of ± 20% of the nominal concentrations, the determination of the biological results was based on the nominal concentrations.

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

 

Parameter	Value (nominal test item concentration)	Value (nominal solid component concentration)
24h EC50	> 571 mg/L	> 100 mg/L
48h EC50	> 571 mg/L	> 100 mg/L
48h NOEC	≥ 571 mg/L	≥ 100 mg/L
48h LOEC	> 571 mg/L	> 100 mg/L

 

Ammonium-3-nitrobenzoate does not need to be classified as toxic to the environment, neither acutely nor chronic, according to the Regulation (EC) No. 1272/2008.