Benzamide, N- [3- (diethylamino) propyl] - 4- nitro- , hydrochloride (1:1)

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:

30 Apr 2018 - 16 Aug 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

diethyl({3-[(4-nitrophenyl)formamido]propyl})azanium chloride

EC Number:

814-319-5

Cas Number:

110393-28-7

Molecular formula:

C14H22N3O3Cl

IUPAC Name:

diethyl({3-[(4-nitrophenyl)formamido]propyl})azanium chloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test item name: N-[3-(Diethylamino)propyl]-4-nitrobenzamide, hydrochloride
Batch number: Op 1/16 and Op 2/16
Other name(s): Nitrobenzamid x HCl
EAS Test item code: M-00013846
Appearance / colour: solid / off-white
Chemical name: N-[3-(Diethylamino)propyl]-4-nitrobenzamide, hydrochloride
CAS number: 110393-28-7
Purity analysed: 94.2 % w/w
Molecular weight: 315.8 g/mol
Density: not applicable
Signal word(s): none
Issue date of certificate: 18 Apr 2017
Expiry date: 18 Apr 2019
Storage conditions: ambient (5 °C - 30 °C), dark, dry

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Analytical samples were analysed from all test item concentrations and control from samples taken at test start and 48 hours from aged solutions.

Test solutions

Vehicle:

no

Details on test solutions:

The necessary amount of test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. Afterwards the solution was clear and transparent. Lower test solutions were prepared by dilution of the stock solution with test medium. ~ 50 mL of the prepared solutions were transferred to each test vessel.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus, Clone V, was used as the test organism. The animals are continuously bred in the laboratory and were originally purchased in a healthy condition from the Federal Environment Agency in Berlin/Germany.
Daphnia magna was bred as single culture (1 daphnid per 100 mL) in Elendt M4 medium. The pH-value of the aerated water was within a range of 6.0 – 9.0. The dissolved oxygen was above 60 % saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH. The animals were fed with single cell green algae (Desmodesmus subspicatus, formerly Scenedesmus subspicatus) at least three times a week.
The daphnids were reared at a temperature of 20 ± 2 °C in a climatic chamber with 16 hours of illumination and 8 hours of darkness. The medium was changed three times per week. A pipette was used to separate the young daphnids from the adults.
Freshly hatched daphnids less than 24 hours old were used for the test.

Study design

Test type:

static

Water media type:

other: Elendt M4

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

12°dH (214 mg/L as CaCO3)

Test temperature:

19.4 – 19.8 °C

pH:

7.54 – 7.89

Dissolved oxygen:

≥ 7.9 mg/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

100, 71.4, 51.0, 36.4, 26.0 mg test item/L nominal
132, 102, 75.0, 51.0, 37.2 mg test item/L measured

Details on test conditions:

The total hardness (as CaCO3) of the test medium (untreated control) was determined to be 12°dH (214 mg/L CaCO3); the mean pH-value of the untreated control was determined to be 7.73 ± 0.15 (Std. Dev.), the mean temperature of the control and all test item concentrations was measured to be 19.6 ± 0.1 °C (Std. Dev.) and the mean oxygen concentration was determined to be 8.8 ± 0.4 mg/L (Std. Dev.).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

132 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 132 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

101 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Control immobilisation: The percentage of immobilisation should be ≤ 10 %. In this study the control immobilisation was 0 %.
Oxygen concentration: The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in all test units. In this test, the dissolved oxygen concentration at the end of the test was ≥ 7.9 mg/L.

After 24 hours of exposure no immobilisation was observed in the control, up to and including 51.0 mg/L and at 100 mg/L. No immobilisation higher than the allowed control immobilisation was observed at 71.4 mg/L.
After 48 hours of exposure no immobilisation was observed in the control and 36.4 mg/L. No immobilisation higher than the allowed control immobilisation was observed at 26.0 mg/L. 30 % immobilisation was observed at 51.0 mg/L and 45 % immobilisation was observed at 71.4 mg/L. At the highest test item concentration of 100 mg/L 85 % of the daphnids were immobile.
After 24 hours the mobile daphnids in all test item concentration appeared to be partly sluggish. After 48 hours the mobile daphnids at 26.0 and 36.4 mg/L appeared to be partly sluggish. At test item cocnentrations 51.0 to 100 mg/L all mobile daphnids appeared to be very sluggish and weak.

Results with reference substance (positive control):

The results indicate an EC50 (24 h) of the reference item potassium dichromate between 1.00 and 2.00 mg/L. Since the results are in accordance with the requirements of the OECD guideline 202 and fall within the historical data generated with the reference item at the testing facility, the daphnids were suitable for the determination of the toxicological effects of the test item.

Reported statistics and error estimates:

EC50-values of daphnids exposed to the test item evaluated using measured concentrations
EC50 (24h): >132 mg/L
EC50 (48 h): 101 mg/L (95 % confidence limit: 89.2 to 112 mg/L)

Any other information on results incl. tables

EC₅₀and NOEC-values of daphnids exposed to the test item evaluated using measured concentrations

Endpoint	N-[3-(Diethylamino)propyl]-4-nitrobenzamide, hydrochloride [mg/L] (measured)^3)
	24 h	48 h
NOEC	132	51.0
EC50	> 132^1)	101^2)
95 % confidence limit of EC50	-	89.2 - 112^2)

- not applicable

¹⁾Due to an inhibition below 50% the database was weak for probit analysis which hence was not performed

²⁾Weibull analysis using linear max. likelihood regression

³⁾Based on the geometric mean of measured concentration levels

Analytical Results:

The initial measured content of N-[3-(Diethylamino)propyl]-4-nitrobenzamide, hydrochloride was between 130 and 144 % of nominal and the aged measured content was between 134 and 150 % of nominal.Therefore toxicological endpoints were evaluated using the measured concentrations (based on the geometric mean of each measured concentration level) of the test item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

According to the results of the test, the EC50 (48 h) for immobilisation was determined to be 101 mg/L (measured). The corresponding NOEC (48 h) was 51.0 mg/L (measured).

Executive summary:

Validity criteria:

Control immobilisation: The percentage of immobilisation should be ≤ 10 %. In this study the control immobilisation was 0 %.

Oxygen concentration: The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in all test units. In this test, the dissolved oxygen concentration at the end of the test was ≥ 7.9 mg/L.

Test conditions: The total hardness (as CaCO₃) of the test medium (untreated control) was determined to be 12°dH (214 mg/L CaCO₃); the mean pH-value of the untreated control was determined to be 7.73±0.15 (Std. Dev.), the mean temperature of the control and all test item concentrations was measured to be 19.6±0.1 °C (Std. Dev.) and the mean oxygen concentration was determined to be 8.8 ± 0.4 mg/L (Std. Dev.).

Analytical Results:  The initial measured content of N-[3-(Diethylamino)propyl]-4-nitrobenzamide, hydrochloride was between 130 and 144 % of nominal and the aged measured content was between 134 and 150 % of nominal.Therefore toxicological endpoints were evaluated using the measured concentrations (based on the geometric mean of each measured concentration level) of the test item.

Statistical Results:  EC₅₀and NOEC-values of daphnids exposed to the test item evaluated using measured concentrations

	N-[3-(Diethylamino)propyl]-4-nitrobenzamide, hydrochloride [mg/L] (measured)^3)
	24 h	48h
NOEC	132	51.0
EC50	> 132^1)	101^2)
95 % confidence limit of EC50	-	89.2 - 112^2)

- not applicable

¹⁾Due to an inhibition below 50% the database was weak for probit analysis which hence was not performed

²⁾Weibull analysis using linear max. likelihood regression

³⁾Based on the geometric mean of measured concentration levels