Benzamide, 3-amino-N-[4-(aminocarbonyl)phenyl]-4-methoxy-, hydrochloride (1:?)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

3-amino-N-(4-carbamoylphenyl)-4-methoxybenzamide hydrochloride

EC Number:

618-990-3

Cas Number:

939961-06-5

Molecular formula:

C15H15N3O3xClH

IUPAC Name:

3-amino-N-(4-carbamoylphenyl)-4-methoxybenzamide hydrochloride

Details on test material:

- Name of test material (as cited in study report): Aminoanisoylaminobenzamid Hydrochlorid
- Molecular formula: C15H16ClN3O3
- Molecular weight: 321.77 g/mol
- Analytical purity: 94.7 % (w/w)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: One loading level of 100 mg/L was tested as limit test
- Sampling method: THe limit concentration and the control were analytically verified at the beginning and the end of the test via HPLC analysis. Separate replicates for the test item analysis after 0 h were prepared. At the end of the test samples were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 +/- 2 °C if necessary

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mg/L, freshly prepared with dilution water and dispersion treatment was ultrasound for 30 minutes at 40 °C.
- Eluate: Dilution water
- Differential loading: 100 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

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

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
100 7.89 8.16
Control 7.93 8.46

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of Aminoanisoylaminobenzamid Hydrochlorid

Sampling date 2009-05-04
Test start 2009-05-07
Test end
Start of analysis 2009-05-04
2009-05-07

Nominal concentration Aminoanisoylaminobenzamid Hydrochlorid
Test item

[mg/L] Main component
[mg/L] Meas.
conc.
[mg/L] RR

[%] Meas.
conc.
[mg/L] RR

[%]
100 94.7 85.1 90 84.3 89
Control < LOQ — < LOQ —

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: 4878 cells/mL
- Control end cells density: Mean 900551 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)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition
[%] Yield Inhibition

[%]
100 -6 -31
10 -2 -7
1 -4 -19

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

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: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.320 (CI 0.294 - 0.358) mg/L

Reported statistics and error estimates:

All calculations were based on the fluorescence values of each replicate.

Any other information on results incl. tables

Effect Levels of Aminoanisoylaminobenzamid Hydrochlorid (0-72 h)

                                based on nominal concentration of the test item [mg/L]

                               

	Yield inhibition
NOEC	< 100
LOEC	100
EyC10	100
EyC20	> 100
EyC50	> 100
	Rate-related inhibition
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
EyC50	> 100

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
100		1		4878		29534	296480	790655
		2		4878		30160	273552	559950
		3		4878		31667	285337	749187
		4		4878		33180	274535	700925
		5		4878		30224	289765	760880
		6		4878		32906	297884	770990
		Mean		4878		31279	286259	722098
Control		1		4878		23274	216936	926281
		2		4878		37375	209157	905675
		3		4878		36378	248663	912077
		4		4878		38009	208145	877627
		5		4878		37788	220862	771701
		6		4878		37219	241583	1009943
		Mean		4878		35007	224224	900551

Evaluation after 72 h

                                Statistically significant differences of growth rates and yield compared to
                                control values are marked (+), not significant differences are marked (-).       

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.696	2.43		785777	12.27
	2		1.581	9.05		555072	38.03
	3		1.678	3.47		744309	16.90
	4		1.656	4.74		696047	22.29
	5		1.683	3.17		756002	15.59
	6		1.688	2.92		766112	14.47
	Mean	(+)*	1.664	4.30	(+)	717220	19.93
Control	1		1.749			921403
	2		1.741			900797
	3		1.744			907199
	4		1.731			872749
	5		1.688			766823
	6		1.778			1005065
	Mean		1.738			895673

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	1.563	2.232	1.452	1.749	0.4223	24.2	20.3
	2	2.036	1.722	1.466	1.741	0.2858	16.4
	3	2.009	1.922	1.300	1.744	0.3870	22.2
	4	2.053	1.700	1.439	1.731	0.3082	17.8
	5	2.047	1.766	1.251	1.688	0.4037	23.9
	6	2.032	1.870	1.430	1.778	0.3114	17.5
				Mean	1.738
				SD ±	0.03
				CV [%]	1.68

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the effects of the test item Aminoanisoylaminobenzamid Hydrochlorid on the growth of the freshwater green alga Desmodesmus subspicatus were tested. The EC50-values for specific growth rate inhibition (ErC50) and inhibition of yield (EyC50) after 72 h were > 100 mg/L.

Executive summary:

The toxicity of Aminoanisoylaminobenzamid Hydrochlorid to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 , from May 04 to 07, 2009. 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 approximately 2 - 5 x 10³cells/mL. One limit concentration of 100 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.
The concentration of Aminoanisoylaminobenzamid Hydrochlorid was analysed at test start and end via HPLC analysis. Measured concentrations at test start and end well met the nominal ranges.

Effect Levels of Aminoanisoylaminobenzamid Hydrochlorid (0-72 h)

                                based on nominal concentration of the test item [mg/L]

                               

	Yield inhibition
NOEC	< 100
LOEC	100
EyC10	100
EyC20	> 100
EyC50	> 100
	Rate-related inhibition
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
EyC50	> 100

Aminoanisoylaminobenzamid Hydrochlorid caused no significant inhibiting effects > 20 % on algal growth tested at the limit concentration of 100

mg/L.