4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

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:

2009-11-06 to 2009-11-13

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:

4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

EC Number:

277-874-7

EC Name:

4-amino-N-[4-(aminocarbonyl)phenyl]benzamide

Cas Number:

74441-06-8

Molecular formula:

C14H13N3O2

IUPAC Name:

4-amino-N-(4-carbamoylphenyl)benzamide

Details on test material:

- Name of test material (as cited in study report): p-Aminobenzoylaminobenzamid TF
- Molecular formula: C14H13N3O2
- Molecular weight: 255.28 g/mol
- Physical state: Solid, beige powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit concentration and the control were analytically verified at the beginning and the end of the test via HPLC-DAD analysis.
- Sampling method: Samples at test start and end 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 62 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 was tested in a limit test corresponding to a initially measured concentration of 1.23 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:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Initially measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
1.23 8.19 8.63
Control 8.12 8.54

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 378432 cells/mL
- No. of vessels per concentration (replicates): 6
- 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)


Saturated solution[%] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 0 1
10 0 2
1 0 - 1

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.23 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: rate-related and yield inhibition

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
- Effect concentrations exceeding solubility of substance in test medium: Yes

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:

The NOEC and LOEC were determined by calculation of statistical significance of growth rates and yield.
t-test was carried out for the determination of statistically significant differences compared to control replicates. When running a t-test a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value was a=0.05. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked biological not significant.

Any other information on results incl. tables

EC-Values of p-Aminobenzoylaminobenzamid TF (0-72 h)

                                based on the initially measured test item concentration [mg/L]

                               

EC-Values	Rate-related inhibition
NOEC	1.23
LOEC	> 1.23
ErC10	> 1.23
ErC20	> 1.23
ErC50	> 1.23
	Yield inhibition
NOEC	1.23
LOEC	> 1.23
EyC10	> 1.23
EyC20	> 1.23
EyC50	> 1.23

Cell Densities

	Initially measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
1.23		1		5074		25406	116139	466939
		2		5074		23840	106414	433827
		3		5074		19645	107324	404102
		4		5074		21548	108304	418592
		5		5074		20662	120325	449420
		6		5074		20142	113605	444483
		Mean		5074		21874	112019	436227
Control		1		5074		22860	69424	381364
		2		5074		19666	88472	396341
		3		5074		15775	87582	335850
		4		5074		16525	96311	347006
		5		5074		19891	84835	369363
		6		5074		20137	102219	440666
		Mean		5074		19142	88141	378432

Evaluation after 72 h

                            Statistically significant differences of growth rates and yield compared to

                                control values are marked (+), not significant differences are marked (-).

Initially measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.23	1		1.51	-4.97		461865	-23.7
	2		1.48	-3.26		428753	-14.8
	3		1.46	-1.62		399028	-6.88
	4		1.47	-2.43		413518	-10.8
	5		1.50	-4.08		444346	-19.0
	6		1.49	-3.83		439409	-17.7
	Mean	(+)*	1.48	-3.37	(+)*	431153	-15.5
Control	1		1.44			376290
	2		1.45			391267
	3		1.40			330776
	4		1.41			341932
	5		1.43			364289
	6		1.49			435592
	Mean		1.44			373358

Section-by-Section and Average Specific Growth Rates of the Control Group

	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	1.51	1.11	1.70	1.44	0.302	21.0	13.7
	2	1.36	1.50	1.50	1.45	0.085	5.84
	3	1.13	1.71	1.34	1.40	0.294	21.0
	4	1.18	1.76	1.28	1.41	0.311	22.1
	5	1.37	1.45	1.47	1.43	0.056	3.89
	6	1.38	1.63	1.46	1.49	0.125	8.42
				Mean	1.44
				SD ±	0.03
				CV [%]	2.27

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the effects of the test item p-Aminobenzoylaminobenzamid TF on the growth of the freshwater green alga Desmodesmus subspicatus were tested. No inhibiting effects on specific growth rate and yield were found in the limit concentration of 1.23 mg/L. The EC50-values for specific growth rate inhibition (ErC50) and inhibition of yield (EyC50) after 72 h were > 1.23 mg/L.
All effect levels are given based on the initially measured test item concentration.

Executive summary:

The toxicity of p-Aminobenzoylaminobenzamid TF to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was to assess the effects on growth rate and yield production over a period of 72 h in a limit test.
The study was conducted under static conditions with an initial cell density of nominally 2 - 5 x 10³ cells/mL. Based on a preliminary test, a definitive test with the saturated solution was tested as limit concentration corresponding to an initially measured test item concentration of  1.23 mg/L. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.
The concentration of p-Aminobenzoylaminobenzamid TF was determined at test start and end via HPLC-UV analysis. The measured concentration at test end was 99 % of the initially measured test item concentration. All effect levels are given based on the initially measured test item concentration

EC-Values of p-Aminobenzoylaminobenzamid TF (0-72 h)

                                based on the initially measured test item concentration [mg/L]

                               

EC-Values	Rate-related inhibition
NOEC	1.23
LOEC	> 1.23
ErC10	> 1.23
ErC20	> 1.23
ErC50	> 1.23
	Yield inhibition
NOEC	1.23
LOEC	> 1.23
EyC10	> 1.23
EyC20	> 1.23
EyC50	> 1.23