Acetamide, N-[5-[bis(2-methoxyethyl)amino]-2-[2-(2,6-dicyano-4-nitrophenyl)diazenyl]phenyl]-

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 February 2020 - 31 March 2020

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

23 March 2006

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

437-930-5

EC Name:

-

Cas Number:

51553-03-8

Molecular formula:

Hill formula: C22H23N7O5 CAS formula: C22H23N7O5

IUPAC Name:

N-{5-[bis(2-methoxyethyl)amino]-2-[2-(2,6-dicyano-4-nitrophenyl)diazen-1-yl]phenyl}acetamide

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the start of the test and at day 2, 5 and at the end of the test (day 7) the frond number and the frond area are determined using the digital image evaluation system Scanalyzer from LemnaTec (Würselen). The test endpoint is inhibition of the average specific growth rates and the yield of frond number and frond area during the exposure period. A static test design was applied with an accompanying substance specific analysis. A nominal loading rate of 100 mg/L test item was used to prepare a saturated solution of the test item. The saturated solution was filtered with a 0,45 μm aPES membrane filter to remove undissolved particles and diluted with test medium to obtain the following concentrations: 6.25 %, 12.5 %, 25 %, 50 % and 100 % (v/v) of the saturated solution. A preliminary range finding test without GLP was performed before the start of this GLP-study. Nominal loading rates of 10 mg/L and 100 mg/L test item were prepared, shaken for 24 h and filtered with a 0,45 μm a PES membrane filter. The filtered saturated solution with nominal loading rates of 10 mg/L and 100 mg/L resulted in 0 % effect.

Test solutions

Vehicle:

yes

Details on test solutions:

The test was performed with Steinberg medium.

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

Test was carried out with the duckweed Lemna minor obtained from the German Federal Environment Agency (UBA), Berlin-Marienfelde. The sterile permanent stock culture is grown on agar 2 medium prepared according to Jungnickel & Augsten (Details see Appendix I) in 100 mL sealed Erlenmeyer flasks at room temperature. After a maximum period of 2 months the plants are transferred sterile into freshly prepared medium. The stock culture used in the test has been cultivated at Hydrotox since March 2014. 14 days before the start of the test, plant colonies were transferred from the sterile agar stock culture into 150 mL beakers containing 100 mL Steinberg Medium. After 7 days they were transferred into fresh medium and grown until being used for the test. Colonies consisting of 2 to 4 visible fronds were transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions. A quality check takes place at regular intervals using a concentration range of 3,5-dichlorophenol (Sigma-Aldrich, Steinheim, Germany, Lot No. MKBS0425V, from September 2015) in order to assess the validity of the test organism. The last quality check was performed in November 2019. The EC50 for the frond number was 2.96 mg/L (2.46 – 3.54 mg/L 95%-CI). This value is in-line with the requirements of the ISO 20079 (2005) (Lemna, Growth Inhibition Test] of 2.2 –3.8 mg/L).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test conditions

Test temperature:

24 - 25 °C

pH:

5,3 - 5,8

Conductivity:

99 μE/m² s

Nominal and measured concentrations:

100 mg/L nominal loading rate to prepare a saturated solution,
tested concentrations: 6.25 %, 12.5 %, 25 %, 50 % and 100 %
(v/v) of the saturated solution

Details on test conditions:

100 mL from the test and negative control solutions were dispensed into 150 mL beakers. Test solutions were prepared with 3 replicates, negative control was prepared with 6 replicates. Two additional replicates of each treatment group were prepared without test organism for the chemical analysis. Lemna plant colonies with a total number of 9 – 10 fronds were transferred from the preculture into the test and control vessels at the start of the test. Test vessels were covered with watch glasses to minimize evaporation from the test vessels.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: v/v %

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

growth rate

Effect concentrations 2

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 100 other: v/v %

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

growth rate

Details on results:

Since test item concentrations were below the limit of calibration, the results are given in relation to the concentration of the saturated solution that was prepared by shaking a loading rate of 100 mg/L for 24 h and removing undissolved particles by filtration.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes