5-amino-2-nitrobenzoic acid

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:

27 August - 2 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266

Deviations:

no

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 including the control at the start of the test (without algae) and after 24, 48 and 72 hours of exposure (containing algae). For sampling 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, aliquots from the respective replicates were pooled per treatment before sampling and centrifuged at 4500 g for 10 min to separate the algae.

Immediately after sampling, acetonitrile was added to obtain a composition of sample:acetonitrile (75:25; v:v). Thereafter, all samples were stored deep-frozen (atabout -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 flasks were set up:
- Test solution: containing test water, test item and algae (three replicates per test concentration)
- Control: containing test water and algae (six replicates)

Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution, which was prepared by dissolving 100 mg test item/l in test water applying five minutes of stirring and five minutes of ultrasonication. This stock solution was subsequently diluted using test water and sterile glassware. 100 ml of each test media were added to the sterile test flasks and inoculated with an exponentially growing preculture of algae. Pure test water handled as the test solutions and inoculated with the exponentially growing preculture of algae mentioned above served as control.

For the incubation the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min. The test flasks were repositioned daily during the exposure.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Test organism: Raphidocelis subcapitata (SAG Nr. 61.81), supplied by the SAG Culture Collection of Algae at Göttingen University, 37073 Göttingen, Germany.
- Culture: 250 ml flask containing 100 ml of OECD medium inoculated with an exponentially growing preculture of Raphidocelis subcapitata.
- Illumination: Continuous, 4440 – 8880 lux ± max. 15% variation, source: Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland).
- Temperature: 21–24 °C maintained at ±2 °C in a thermo-controlled room.
- Control of sensitivity: For evaluation of the algal quality and the test procedure, potassium dichromate is tested as a positive control twice a year.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

The calculated water hardness of the test water is 24.2 mg/l as CaCO3.

Test temperature:

21–24 °C maintained at ±2 °C in a thermo-controlled room.

pH:

5.4 - 8.2

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

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, 48 and 72 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 within 80–120% of the nominal value over the duration of the test. Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml flask, all-glass, with 100 ml of test medium, shaken (130 rpm), capped with air permeable stoppers
- Test medium: Reconstituted water (OECD medium) prepared according to the test guidelines was used for algal cultivation and testing.
- Preculture: Exponentially growing liquid culture of Raphidocelis subcapitata kept under the same environmental conditions as in the test.
- Illumination: Source: Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland).
- Light intensity and homogeneity: Continuous, 4440 – 8880 lux ± max. 15% variation. The test flasks were repositioned daily during the exposure.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 7.9; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: Approximately 5’000 cells/ml; all test media contained the same initial density of algal cells.

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test (three replicates for the control and two replicates per test concentration) with nominal test item concentrations of 1, 10 and 100 mg/l was performed.

EFFECT PARAMETERS MEASURED
Biomass: Algal biomass is determined at 24, 48 and 72 hours of exposure using a spectrophotometer at 680 nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). To this end, aliquots of 5 ml are removed from each test flask. At test start (0 hours) the nominal value is applied.

Reference substance (positive control):

yes

Remarks:

Control of sensitivity: Potassium dichromate is tested as a positive control twice a year.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

61.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

48.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

86.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

AVERAGE SPECIFIC GROWTH RATE
With respect to the endpoint average specific growth rate, the following effects were observed compared to the control: 18% at 100 mg/l and 2.0% at 50.0 mg/l. No significant effects were observed at all lower test concentrations.
The 72-h EC50 based on average specific growth rate of 5-Amino-2-Nitrobenzoic Acid to Raphidocelis subcapitata was estimated to be >100 mg/. The EC10 was calculated to be 61.8 mg/l (95% confidence interval: 43.3–107 mg/l). These ECx were computed using a linear regression with a probit link function and a maximum likelihood algorithm (slope = 0.734, intercept = –2.60, degrees of freedom = 13, p(Chi²) = 1.00, r2 =0.736, p(F) <0.001).
The LOEC with respect to average specific growth rate was determined to be 50.0 mg/l according to a Dunnett’s multiple t-test (one-sided smaller, a = 0.05). The corresponding NOEC is therefore 25.0 mg/l.

YIELD
With respect to the endpoint yield, the following effects were observed compared to the control: 62% at 100 mg/l, 11% at 50.0 mg/l and 7.2% at 25.0 mg/l. No significant effects were observed at all lower test concentrations.
The 72-h EC50 based on yield of 5-Amino-2-Nitrobenzoic Acid to Raphidocelis subcapitata was calculated to be 86.6 mg/l (95% confidence interval: 80.7–93.0 mg/l). The EC10 was calculated to be 48.1 mg/l (95% confidence interval: 39.2–54.8 mg/l). These ECx were computed using a linear regression with a probit link function and a maximum likelihood algorithm (slope = 5.02, intercept = – 9.73, degrees of freedom = 13, p(Chi²) = 1.00, r2 = 0.859, p(F) < 0.001).
The LOEC with respect to yield was determined to be 25.0 mg/l according to a Dunnett’s multiple t-test (one-sided smaller, a = 0.05). The corresponding NOEC is therefore 12.5 mg/l.

OBSERVATIONS AND ENVIRONMENTAL CONDITIONS
The microscopic examination of the algal cells at the end of the test showed no abnormal appearance of the algae growing at the highest test concentration of 100 mg/l. The appearance of the algal cells was not affected by the test item up to at least this concentration.
The test media turned yellow after dissolving the test item in the test water. No other remarkable observations were made concerning the appearance of the test media.
The pH value in the control replicates changed by 0.4 units. The light intensity was maintained at 4796 lux ± 6%. The temperature ranged from 22 to 23 °C. All values were within the guideline ranges.
The pH in the test solutions showed a dependency on the test concentration, the higher the concentration, the lower the pH. This is most likely due to the test item having a carboxylic acid moiety. No pH adjustments were performed after the dosage of the test item.

Results with reference substance (positive control):

The result of the positive control test performed in October 2019 showed that the sensitivity of the test system based on average specific growth rate (72-h EC50 = 1.02 mg/l) was in line with a ring test yielding 1.19 mg/l, SD = 0.27 (ISO 8692:2012: Water quality - Fresh water algal growth inhibition test with unicellular green algae).

Reported statistics and error estimates:

Statistical analysis was performed with respect to the determination of the effective concentrations, the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC). The statistical analysis was conducted with the software ToxRat® Pro 3.2.1 (ToxRat® Solutions GmbH, Alsdorf, Germany).

Any other information on results incl. tables

Validity of the test:

Validity criterion (applies to control cultures)	Required value	Achieved value
Average specific growth rate [d^-1]	>=0.92	1.85
CV of average specific growth rate over 3 days (%)	<=7	1.0
CV of section-by-section specific growth rates (%)	<=35	4.5

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See table in section "any other information on results incl. tables".

Conclusions:

The 72-h EC50 value based on average specific growth rate of 5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) on the green alga Raphidocelis subcapitata was >100 mg/l and the EC10 was 61.8 mg/l based on nominal test item concentrations.

Executive summary:

The growth inhibitory effects of 5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) to the green alga Raphidocelis subcapitata were investigated according to OECD guideline 201 over a period of 72 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. Three parallel test vessels were used for each concentration of the test item and six vessels for the control.

The concentrations of 5-Amino-2-Nitrobenzoic Acid in the test media at 0, 24, 48 and 72 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 within 80–120% of the nominal value over the duration of the test. Therefore, the effective concentrations ECx were assessed based on the nominal concentrations of the test item.

With respect to the endpoint average specific growth rate, the following effects were observed compared to the control: 18% at 100 mg/l and 2.0% at 50.0 mg/l. No significant effects were observed at all lower test concentrations.

With respect to the endpoint yield, the following effects were observed compared to the control: 62% at 100 mg/l, 11% at 50.0 mg/l and 7.2% at 25.0 mg/l. No significant effects were observed at all lower test concentrations.

The results of the average specific growth rate and yield inhibition of 5-Amino-2-Nitrobenzoic Acid to the green alga Raphidocelis subcapitata are summarised in the following table based on the nominal concentrations of the test item:

Parameter (0 -72 h)	Growth rate (mg/L)	Yield (mg/L)
EC50	>100	86.6
EC10	61.8	48.1
LOEC	50.0	25.0
NOEC	25.0	12.5

The 72-h EC50 value based on average specific growth rate of 5-Amino-2-Nitrobenzoic Acid (CAS no. 13280-60-9) on the green alga Raphidocelis subcapitata was >100 mg/l and the EC10 was 61.8 mg/l based on nominal test item concentrations. All validity criteria were fulfilled.