5-amino-2-nitrobenzoic acid

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:

20 August - 02 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

yes

Remarks:

The pH in the highest test concentration was 5.9 at the beginning of the test and not in the recommended range of 6 to 9. However, this deviation seems negligible regarding that no immobility was observed up to the highest test concentration.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

30 May 2008

Deviations:

yes

Remarks:

The pH in the highest test concentration was 5.9 at the beginning of the test and not in the recommended range of 6 to 9. However, this deviation seems negligible regarding that no immobility was observed up to the highest test concentration.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-2-nitrobenzoic acid

EC Number:

236-283-4

EC Name:

5-amino-2-nitrobenzoic acid

Cas Number:

13280-60-9

Molecular formula:

C7H6N2O4

IUPAC Name:

5-amino-2-nitrobenzoic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

Test material form:

solid

Details on test material:

Batch / Lot No. 10044431

Specific details on test material used for the study:

Batch no.: 10044431
Storage: At room temperature, protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test media at the start of the test as well as after 24 and 48 hours of exposure. At the start of the test, the samples were taken from the freshly prepared test solutions as well as from the pure test water serving as control. For all subsequent sampling time points, the replicates of the test media were pooled before sampling.

Immediately after sampling, acetonitrile was added to each sample to obtain a composition of sample:acetonitrile (75:25; v:v). Thereafter, all samples were stored deep-frozen (at about -20 °C). In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test vessels were set up:
- Test solution: containing test water with test item (four replicates per concentration with five daphnids each)
- Control: containing pure test water (four replicates with five daphnids each)
Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution (100 mg test item/l dissolved in test water with five minutes of stirring and five minutes of ultrasonication) with test water, which were used as the test solutions in the toxicity test. Pure test water served as control.
The test vessels were filled with 50 ml of the respective test medium. Daphnia, aged less than 24 hours and already acclimatized to the test water, were introduced into the test media and the vessels were covered with a glass plate. The Daphnia were not fed during the test and the test vessels were not aerated.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISMS
- Test organism: Daphnia magna Straus, supplied by IES Ltd., 4108 Witterswil, Switzerland
- Breeding: The daphnids are cultivated in Elendt M4 medium at 18–22 °C, controlled at ±1 °C
- Illumination: 16 h per day, about 550-1100 lux, source: overhead white fluorescent tubes
- Medium: Reconstituted water (Elendt M4 medium)
- Feed: Suspension of Desmodesmus subspicatus in Elendt M4 medium with an optical density OD680 of about 12 units. This results in a final concentration of about 0.1 units from Monday to Thursday and 0.4 units on Fridays in the culture vessel.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

2.5 mM (based on the sum of Ca2+ and Mg2+), Carbonate Alkalinity: 0.8 mM

Test temperature:

18–22°C, controlled at ±1 °C

pH:

5.9 - 7.8

Dissolved oxygen:

>=3 mg O2/l (required >=3 mg O2/l)

Salinity:

not reported

Conductivity:

629 µS cm-1

Nominal and measured concentrations:

NOMINAL CONCENTRATIONS
The following test item concentrations were selected for the definitive test: 100, 50.0, 25.0, 12.5 and 6.25 mg/l.

MEASURED CONCENTRATIONS
The concentrations of 5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) in the test media at 0, 24 and 48 hours of exposure were determined by HPLC analysis. These analyses showed that the test item was correctly dosed and that the concentrations remained stable within 80-120% of the nominal concentrations over the duration of the test. Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item, which were 100, 50.0, 25.0, 12.5 and 6.25 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml glass beaker containing 50 ml of test medium, covered with a glass plate to avoid evaporation and contamination of the test solutions with dust.
- Test medium: Elendt M4 medium
- Number of Daphnia: 20 individuals per test concentration and for the control divided in four groups of five individuals.
- Age: Less than 24 hours
- Light: 16 hours photoperiod per day, about 550–1100 lux, source: overhead white fluorescent tubes.
- Temperature: 18–22 °C, controlled at ±1 °C
- pH: The pH should not vary by more than 1.5 units during the exposure.
- Feed: The daphnids are not fed during the test
- Test design: Static exposure conditions
- Test duration: 48 hours

RANGE-FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of 5-Amino-2-Nitrobenzoic Acid was performed (two replicates for each test concentration and the control; five daphnids per replicate).

EFFECT PARAMETERS MEASURED
Observations of immobilised daphnids were made after 24 and 48 hours of exposure. Any abnormal behaviour or appearance was reported.

Reference substance (positive control):

yes

Remarks:

Acute reference test with potassium dichromate conducted twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 24 and 48 hours of exposure, no immobilised test organism were observed in the control and in the test concentrations up to a nominal concentration of 100 mg/l (Table 5). Thus, the 24- and 48-hour EC50 was greater than 100 mg/l.

The no observed effect concentration (NOEC) after 24 and 48 hours was determined to be 100 mg/l.

Results with reference substance (positive control):

The result of the latest positive control test performed in March 2019 showed that the sensitivity of the test system (24-hour EC50 = 0.88 mg/l) lies within the recommended range of 0.6–2.1 mg/l according to ISO 6341:2012: Water quality – determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) – Acute toxicity test.

Reported statistics and error estimates:

The effective concentration EC50 for 24 and 48 hours was estimated based on the concentration tested and immobilisation observed.

The no observed effect concentration (NOEC) is the highest test concentration for which the test item is observed to have an immobilisation rate not exceeding 10%. Thus, immobilisation rates >10% are considered to be significant.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria i.e. immobilization in the controls <=10% and O2 concentrations at the end of the test >=3 mg/L were fulfilled.

Conclusions:

In conclusion, 5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) had no acute toxic effects on Daphnia magna up to the nominal concentration of 100 mg/l under the conditions of the test.

Executive summary:

The acute toxicity of 5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) to Daphnia magna was investigated according to the OECD guideline 202 under static exposure conditions over a period of 48 hours.

The test item 5-Amino-2-Nitrobenzoic Acid is solid, 99.4% (m/m) pure and soluble in test water up to at least 100 mg/l. Consequently, the test solutions were prepared by respective dilutions of a stock solution. The nominal test item concentrations were 100, 50.0, 25.0, 12.5 and 6.25 mg/l. Four replicates with five daphnids each were applied for every test concentration as well as for the control.

The concentrations of 5-Amino-2-Nitrobenzoic Acid in the test media at 0, 24 and 48 hours of exposure were determined by HPLC with UV detection. These analyses showed that the test item was correctly dosed and that the concentrations remained stable within 80-120% of the nominal concentrations over the duration of the test. Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item.

In the control and up to a nominal concentration of 100 mg/l no immobilised test organisms were observed during the test period of 48 hours.

The biological test results of 5-Amino-2-Nitrobenzoic Acid to Daphnia magna after 24 and 48 hours of exposure are summarised in the following table based on the nominal concentrations of the test item:

Parameter		Immobilisation (0-24 h) [mg/l]		Immobilisation (0-48 h) [mg/l]
EC50		>100		>100
NOEC		100		100

In conclusion, 5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) had no acute toxic effects on Daphnia magna up to the nominal concentration of 100 mg/l under the conditions of the test.

All validity criteria were fulfilled.