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

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:

2009-11-08 to 2009-11-12

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 202 (Daphnia sp. Acute Immobilisation 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
- Analytical purity: 96.44 % (w/w)
- Purity test date: 2009-04-29
- Expiration date of the lot/batch: 2011-04-29
- Storage condition of test material: Room temperature, protected from moisture and light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The saturated solution and the control were analytically verified via HPLC after 0 hours (fresh media) and after 48 hours (old media).
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions. The recovery rates of the active ingredient should be within ± 20 % of the initially measured value.
- 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 stock solution of the twenty fold of the water solubility (3.1 mg/L) of the test item was prepared with dilution water two days prior to application. Therefore 62 mg/L of the test item was weighed out.
- Eluate: Dilution water
- Differential loading: The saturated solution* was tested as limit concentration
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water at test start: 263 (mg CaCO3/L)

Test temperature:

20.5 °C

pH:

Water Quality Parameters at Test Start (0 hours)
(measured in one additional replicate of the saturated solution and the control)
pH-Value
Saturated Solution 7.53
Control 7.98

Water Quality Parameters at the Test End (48 hours)
(measured in all replicates)
pH-Values
Replicates
1 2 3 4
Saturated Solution 7.52 7.58 7.57 7.56
Control 7.47 7.51 7.51 7.53

Dissolved oxygen:

Water Quality Parameters at Test Start (0 hours)
(measured in one additional replicate of the saturated solution and the control)
Dissolved
O2-Concentration

[mg/L]
Saturated Solution 7.27
Control 8.37

Water Quality Parameters at the Test End (48 hours)
(measured in all replicates)
Dissolved O2 –Concentration [mg/L]
Replicates
1 2 3 4
Saturated Solution 7.54 7.50 7.42 7.57
Control 7.32 7.27 7.49 7.47

Salinity:

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: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Acc. to Directive 92/69/EEC Method C.2, Annex 1
- Conductivity: Dilution water at test start: 640 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Daphnid immobility was determined in all groups after 24 hours and 48 hours. An animal was considered to be immobile, if it was not able to swim within 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:2
- Range finding study
Immobilization Rates of the Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Dilution level
of the saturated solution IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
1:1 0 0 0 0 0 0
1.10 0 10 5 0 10 5
1.100 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.22 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.67 (CI 1.63 - 1.71) mg/L

Reported statistics and error estimates:

The effects of the saturated solution were evaluated directly from the observation data.

EC50-value was only calculated for the reference item by sigmoidal dose-response regression. The confidence interval for EC50 were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.

Any other information on results incl. tables

Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure

                  (n = 20, divided into 4 replicates with 5 daphnids each)

IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
Saturated solution	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the saturated solution* of the test item p-Aminobenzoylaminobenzamid TF, corresponding to the initially measured concentration of 1.22 mg/L, no effect was observed.

Executive summary:

In the acute immobilization test to Daphnia magna (STRAUS) the effects of the saturated solution* of the test item p-Aminobenzoylaminobenzamid TF with a nominal loading of 62 mg/L, was determined according to OECD 202 (2004). The limit test was conducted under static conditions over a period of 48 hours .

20 test organisms were exposed to the saturated solution* and the control. The saturated solution* and the control were analysed via HPLC after 0 hours (fresh media) and 48 hours (old media). The final recovery rate of the test item in the saturated solution* was 98 % of the initially measured concentration. All effect values given are based on the initially measured test item concentration. Water quality parameters (pH-value and dissolved oxygen concentration), measured in the fresh (0 hours) an old media (48 hours), were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC-Values, NOEC and LOEC

(based on the initially measured test item concentration)

	Test Duration [h]	Initially Measured Test Item Concentration [mg/L]	Confidence Interval p = 95% [mg/L]
EC50	24	> 1.22	not applicable
	48	> 1.22	not applicable
EC100	24	> 1.22	---
	48	> 1.22	---
NOEC	48	1.22	---
LOEC	48	> 1.22	---

At the saturated solution* of the test item p-Aminobenzoylaminobenzamid TF,
corresponding to the initially measured concentration of 1.22 mg/L,
no effect was observed.