Benzamide, 3-amino-4-methoxy-N-methyl-

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

3-amino-4-methoxy-N-methylbenzamide

EC Number:

813-034-3

Cas Number:

878160-14-6

Molecular formula:

C9H12N2O2

IUPAC Name:

3-amino-4-methoxy-N-methylbenzamide

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure (48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of the test item concentrations and analyzed. At the end of the exposure (48 hours), samples of the old media were taken from additional replicates, which were prepared with test media, but without daphnids and incubated under test conditions until sampling.

Test solutions

Vehicle:

no

Details on test solutions:

Test concentrations
Five concentration levels of the test item in a geometric series with a separation factor of 2, prepared by dilution of the stock solution of 100 mg/L with dilution water, were tested as follows: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
The test concentrations were selected based on the results of a non-GLP preliminary range finding test and a first definitive test.

Preparation of the stock solution
The stock solution (100 mg/L of the test item were weighed out) was prepared with dilution water (Table 2)) one day prior to the start of the exposure (at -24 hours). The stock solution was treated with ultrasound for 10 minutes at room temperature and stirred thereafter with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5)

Reason for the selectionof the test system
Daphnia magna is the preferred species in accordance with the
test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

see any other information on material and methods incl. tables

Test temperature:

see any other information on material and methods incl. tables

pH:

see any other information on material and methods incl. tables

Dissolved oxygen:

see any other information on material and methods incl. tables

Conductivity:

see any other information on material and methods incl. tables

Details on test conditions:

Test method
The study was performed under static conditions, since the measured concentrations remained stable over 48 hours under test conditions.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL in each test replicate (see ‘Application’)

Dilution water
Same as culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each loading level and the control.

Age of the daphnids
Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.
 
Application
20 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 25.7 - > 100

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

90.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CI: 24.8 - > 100

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017 04-06 to 2017-04-07.

EC50-Value (with 95% confidence limits) of the Reference Item Potassium dichromate based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.72 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
0.6 - 2.1 mg/L, acc. to OECD 202 (2004)
95% confidence limits 1.34 - 1.93 mg/L

Reported statistics and error estimates:

Methods of evaluation
The EC50- and EC100-values after 24 and 48 hours were estimated directly from the observation data. The EC10-values were determined by non-linear regression analysis. The effect levels (EC10 / 50 / 100) given are based on the nominal concentrations of the test item.

EC-values and statistical analyses
The EC10-values after 24 and 48 hours were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism.
Since immobilization rates greater than 50% were not observed, the calculation of an EC50-value was not possible. The concentration-effect relationships of the test item are shown graphically.
The EC50-value for the reference item and its confidence limits were calculated by sigmoidal dose-response regression as specified above.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Rates after 24 and 48 hours of Exposure

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

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	20	40	40	40	35	20	60	40	60	45
50.0	0	0	0	0	0	0	0	0	0	0
25.0	0	0	0	0	0	0	0	0	0	0
12.5	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

 Absolute Numbers of immobile Daphnids after 24 and 48 hours ofExposure

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

Nominal test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	1 / 5	2 / 5	2 / 5	2 / 5	7 / 20	1 / 5	3 / 5	2 / 5	3 / 5	9 / 20
50.0	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
25.0	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
12.5	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
6.25	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item 3-Amino-4-methoxy-N-methylbenzamide, the 48 hour-EC50 for Daphnia magna was > 100 mg/L (95% confidence limits: not applicable).

Immobilization was observed only at the highest concentration level of 100 mg/L. No immobility or any adverse effects were observed at the nominal test concentrations 6.25 mg/L to 50.0 mg/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item 3-Amino-4-methoxy-N-methylbenzamide were determined according to OECD 202 (2004) .

The study was conducted under static conditions over a period of 48 hours with five concentration levels of the test item in the range of 6.25 to 100 mg/L in a geometric series with a separation factor of 2.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

The concentrations of the test item 3-Amino-4-methoxy-N-methylbenzamide were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure (48 hours) in all concentration levels and in the control.

The measured concentrations of the test item were in the range of 104 to 115% of the nominal values in fresh media at the start of the exposure (0 hours) and 105 to 115% in old media at the end of the exposure (48 hours).

All measured test item concentrations were within ± 20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test. The effect concentrations (EC_{10 / 50 / 100}) given are based on the nominal concentrations of the test item 3-Amino-4-methoxy-N-methylbenzamide.

The validity criteria of the test guideline were fulfilled.

 

 EC₁₀-, EC₅₀- (with 95% Confidence Limits) and EC₁₀₀-Values

(based on the nominal concentrations of the test item)

Effect concentrations	Test duration [h]	3-Amino-4-methoxy-N-methylbenzamide Nominal test item concentrations [mg/L]
EC10 (with 95% confidence limits)	24	92.0	(Cl: 25.7 – > 100)
	48	90.2	(Cl: 24.8 – > 100)
EC50 (with 95% confidence limits)	24	> 100	(Cl: Not applicable)
	48	> 100	(Cl: Not applicable)
EC100	24	> 100
	48	> 100