N-[4-(aminocarbonyl)phenyl]-4-nitrobenzamide

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:

2009-10-09 to 2009-10-16

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[4-(aminocarbonyl)phenyl]-4-nitrobenzamide

EC Number:

298-791-2

EC Name:

N-[4-(aminocarbonyl)phenyl]-4-nitrobenzamide

Cas Number:

93839-21-5

Molecular formula:

C14H11N3O4

IUPAC Name:

N-(4-carbamoylphenyl)-4-nitrobenzamide

Details on test material:

- Name of test material (as cited in study report): p-Nitrobenzoylaminobenzamid trocken
- Molecular formula: C14H11N3O4
- Molecular weight: 285.26 g/mol
- Physical state: Solid, white powder
- Expiration date of the lot/batch: 2010-10-01
- Storage condition of test material: Room temperature, protected from moisture and light.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Samples at start and end of the test were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution was prepared. A dispersion of 66 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor 2: 6.25 - 12.5 - 25 - 50 and 100 % of the saturated solution corresponding to initial measured concentrations of 0.0157 – 0.0314 – 0.0712 – 0.153 – 0.304 mg/L.
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22.5 °C, Min: 22 °C, Max: 23 °C

pH:

Initial measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
0.304 8.09 8.72
0.153 8.17 8.34
0.0712 8.13 8.36
0.0314 8.07 8.23
0.0157 8.16 8.43
Control 7.97 8.49

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 873651 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 h)
Results of the Range Finding Test (0-72 h)

Dilution level of the saturated solution
[%] Rate Related Inhibition[%] Yield Inhibition[%]
100 29 68
10 -2 -8
1 1 3

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.304 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and inhibition of yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.

Any other information on results incl. tables

NOEC, LOEC and EC - values of p-Nitrobenzoylaminobenzamid trocken (0-72 h)

                   based on initial measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	0.304
LOEC	> 0.304
ErC10	> 0.304
ErC20	> 0.304
ErC50	> 0.304
	Inhibition of Yield
NOEC	0.304
LOEC	> 0.304
EyC10	> 0.304
EyC20	> 0.304
EyC50	> 0.304

Cell Densities

	Initial measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
0.304		1		5074		28858	207909	925614
		2		5074		21790	170427	897132
		3		5074		23864	195671	1091558
		Mean		5074		24837	191336	971435
0.153		1		5074		28444	223420	948956
		2		5074		28952	179422	937676
		3		5074		30617	197827	1029516
		Mean		5074		29338	200223	972049
0.0712		1		5074		22860	186282	896820
		2		5074		26201	181313	921387
		3		5074		31031	210714	1085682
		Mean		5074		26697	192770	967963
0.0314		1		5074		24754	145192	837169
		2		5074		27341	172916	940592
		3		5074		28526	182383	935893
		Mean		5074		26874	166830	904551
0.0157		1		5074		27522	150722	870014
		2		5074		25480	167327	976901
		3		5074		32232	216585	1077290
		Mean		5074		28411	178211	974735
Control		1		5074		23532	184998	824193
		2		5074		26312	182768	824656
		3		5074		27788	204904	1002731
		4		5074		24615	171239	781946
		5		5074		30383	181964	843692
		6		5074		23942	183375	964687
		Mean		5074		26095	184875	873651

Evaluation after 72 h

                              

Initial measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
0.304	1		1.74	-1.20		920540	-5.98
	2		1.73	-0.60		892058	-2.70
	3		1.79	-4.41		1086484	-25.1
	Mean		1.75	-2.07		966361	-11.3
0.153	1		1.74	-1.69		943882	-8.67
	2		1.74	-1.46		932602	-7.37
	3		1.77	-3.27		1024442	-17.9
	Mean		1.75	-2.14		966975	-11.3
0.0712	1		1.73	-0.59		891746	-2.67
	2		1.73	-1.11		916313	-5.50
	3		1.79	-4.30		1080608	-24.4
	Mean		1.75	-2.00		962889	-10.9
0.0314	1		1.70	0.75		832095	4.20
	2		1.74	-1.52		935518	-7.71
	3		1.74	-1.42		930819	-7.17
	Mean		1.73	-0.73		899477	-3.56
0.0157	1		1.72	0.00		864940	0.42
	2		1.75	-2.25		971827	-11.9
	3		1.79	-4.15		1072216	-23.5
	Mean		1.75	-2.13		969661	-11.6
Control	1		1.70			819119
	2		1.70			819582
	3		1.76			997657
	4		1.68			776872
	5		1.71			838618
	6		1.75			959613
	Mean		1.72			868577

Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.53	2.06	1.49	1.70	0.317	18.7	13.4
	2	1.65	1.94	1.51	1.70	0.220	13.0
	3	1.70	2.00	1.59	1.76	0.212	12.0
	4	1.58	1.94	1.52	1.68	0.228	13.6
	5	1.79	1.79	1.53	1.71	0.148	8.67
	6	1.55	2.04	1.66	1.75	0.254	14.5
				Mean	1.72
				SD ±	0.03
				CV [%]	1.92

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study p-Nitrobenzoylaminobenzamid trocken was found not to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were > 0.304 mg/L, respectively. All effect levels are given based on initial measured concentrations of p-Nitrobenzoylaminobenzamid trocken.

Executive summary:

The toxicity of p-Nitrobenzoylaminobenzamid trocken to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL. The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2, nominal: 6.25 - 12.5 - 25 - 50 - 100 % of the saturated solution corresponding to initial measured test item concentrations of 0.0157 – 0.0314 – 0.0712 – 0.153 – 0.304 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of p-Nitrobenzoylaminobenzamid trocken were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of p-Nitrobenzoylaminobenzamid trocken at test end were in the range of 99 – 120 % of the initial measured values. The concentration of the lowest dilution level was below the LOQ of 0.03 mg/L. The lowest concentration was calculated from the second lowest test item concentration with the dilution factor of 2. All effect values are given based on initial measured concentrations of the test item.

NOEC, LOEC and EC - values of p-Nitrobenzoylaminobenzamid trocken (0-72 h)

                   based on initial measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	0.304
LOEC	> 0.304
ErC10	> 0.304
ErC20	> 0.304
ErC50	> 0.304
	Inhibition of Yield
NOEC	0.304
LOEC	> 0.304
EyC10	> 0.304
EyC20	> 0.304
EyC50	> 0.304