4-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

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23rd March, 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrobenzoic acid

EC Number:

200-526-2

EC Name:

4-nitrobenzoic acid

Cas Number:

62-23-7

Molecular formula:

C7H5NO4

IUPAC Name:

4-nitrobenzoic acid

Test material form:

solid: crystalline

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

yes

Remarks:

water

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata
(formerly known as Pseudokirchneriella subcapitata)
Strain number: 61.81 SAG
(identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for four days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

na ( rconstituted algae growth medium)

Test temperature:

Culture temperature was checked at the beginning of the study and each day
thereafter in a flask filled with water, in the climatic chamber. In addition temperature was
continuously measured (with a min/max thermometer) within the climate chamber. The
temperature was between 23.0 and 23.1°C measured in the flask and in the range of
22.1 – 23.9°C measured within the climate chamber.

pH:

.
: The pH was measured in each test group at the start (before test solutions had been
distributed into the test vessels) and in each test vessel at the end of the test. The range of the
pH was 7.77 – 8.97 during the experiment. The pH of the control medium was not increased
by more than 1.5 units during the test.
The pH of the stock solution was adjusted to that of the culture medium (using 1N NaOH) in
order to determine the toxicity of the test item independent of the pH.

Dissolved oxygen:

na

Salinity:

na

Conductivity:

na

Nominal and measured concentrations:

See it in the attached file Table 5
Calculation of exposure concentrations
Nominal concentration
[mg/L]
Measured concentration [mg/L] Geometric mean
0-h 24-h 48-h 96-h [mg/L]
1.1 0.97 0.96 0.93 0.85 0.9
3.4 3.33 3.26 3.17 3.01 3.2
10.4 10.3 10.1 10.1 9.49 10.0
32.3 32.2 31.6 31.9 29.3 31.2
100.0 102 99.3 101 93.1 98.8

Details on test conditions:

Light Intensity:The light intensity was checked and recorded at the start of the test. The
algal culture flasks were continuously illuminated. The average light intensity measured at
the position occupied by algal culture flasks during the test was 6924 lux, which was
ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in
light intensity between the test vessels did not exceed ± 15 % and therefore provided equal
conditions for each test vessel.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 82.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 11.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results
showed that the test item 4-Nitrobenzic acid had inhibitory effects on the growth of
Raphidocelis subcapitata.
All validity criteria were met. The results are based on measured geometric mean test item
concentrations.
Biological endpoints of the study are summarised below:
Summary of the Biological Endpoints (same as Table 1)
Endpoints
(0-72 h)
Growth rate (µ)
[mg/L]
Yield (y)
[mg/L]
Calculation based on measured concentrations
EC10 6.8 1.3
95 % conf. limits 4.0 – 9.9 0.6 – 2.2
EC20 16.0 2.8
95 % conf. limits 11.2 – 22.0 1.5 – 4.3
EC50 82.4 11.7
95 % conf. limits 56.6 – 138.7 8.1 – 16.9
NOEC 3.2 3.2
LOEC 10.0 10.0

Executive summary:

Test item:	4-Nitrobenzic acid (Lot No.: H518032812)
Test species:	Raphidocelis subcapitata(formerly known asPseudokirchneriella subcapitata); Strain number: 61.81 SAG Exponentially-growing cultures; preculture was incubated for four days before the test.
Dilution water:	OECD medium, prepared in the laboratory of TOXI-COOP ZRT.
Concentrations:	Nominal concentrations of 1.1, 3.4, 10.4, 32.3 and 100.0 mg/L were investigated in the main study. Concurrent control ran. The corresponding measured geometric mean concentrations were the followings: 0.9, 3.2, 10.0, 31.2 and 98.8 mg/L. Biological results and endpoints are based on the measured geometric mean concentrations.
Test design:	Exponentially-growing cultures ofRaphidocelis subcapitatawere exposed to the test item under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 104cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test in 24-hour intervals.
Analytics:	For determination of the test item concentrations, samples were taken from each concentration level and control at each day of the test. Concentrations were determined using HPLC method with UV detection.
Endpoints:	EC10, EC20, EC50, NOEC and LOEC for both response variables (i.e. average specific growth rate and yield).
Statistics:	For the determination of the LOEC and NOEC, ANOVA and Dunnett’s test (Dunnett-t and Dunnett T3,α= 0.05) for determination of the ECx values Probit analysis was used.
Validity:	All validity criteria were met and therefore the study can be considered as valid (see section 8.1)

Results:

All biological results are based on the measured geometric mean concentrations. The biological endpoints are summarised below in Table 1.

Table 1:Summary of the biological endpoints

Endpoints (0-72 h)	Growth rate (µ) [mg/L]	Yield (y) [mg/L]
Calculation based on measured concentrations
EC10	6.8	1.3
95 % conf. limits	4.0 – 9.9	0.6 – 2.2
EC20	16.0	2.8
95 % conf. limits	11.2 – 22.0	1.5 – 4.3
EC50	82.4	11.7
95 % conf. limits	56.6 – 138.7	8.1 – 16.9
NOEC	3.2	3.2
LOEC	10.0	10.0