m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

UV-VIS spectrometer

Details on sampling:

concentration based on active ingredient: 6.25 mg/L, 25 mg/L, 100 mg/L and control were sampled at 0 d, 2 d, 5 d and 7 d

Vehicle:

no

Remarks:

auxiliary: magnetic stirrer

Details on test solutions:

- a stock solution was prepared by adding 111.6 mg test item to 1000 mL test medium (Steinberg medium) and stirring for 10 min. using a magnetic stirrer at room temperature (24.8°C)
- five nominal test item concentrations of 111.6 mg/L, 55.8 mg/L, 27.9 mg/L, 13.95 mg/L and 6.98 mg/L (spacing factor of 2) was prepared by diluting the stock solution with test medium
- test solutions were prepared with 3 replicates, negative control (NC) was prepared with 6 replicates
- test volume was 100 mL
- two additional replicates of NC, 6.98 mg/L, 27.9 mg/L and 111.6 mg/L were prepared without test organism for the chemical analysis

Test organisms (species):

Lemna minor

Details on test organisms:

Tests were 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 medium prepared according to Jungnickel & Augsten (Details see Annex I) in 100 mL Erlenmeyer flasks sealed with cellulose stoppers 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 in the labor of Hydrotox since March 2014.
14 days before the start of the test plant colonies are transferred from the sterile agar stock culture into 150 mL beakers containing 100 mL Steinberg Medium. After 7 they are transferred into fresh medium and grown until using for the test. The pre-culture is cultivated under test conditions (24°C ± 2°C, 85 – 135 µE m-2 s-1). At least 15 beakers were prepared to gain enough plant material for the test. Colonies consisting of 2 to 4 visible fronds are transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24.6 - 25.3 °C

pH:

5.5 - 6.8

Nominal and measured concentrations:

Nominal (based on active ingredient) [mg/L]: 6.25, 12.5, 25.0, 50.0, 100.0
As the measured test item concentrations are within ± 20 % of the nominal concentrations, according to OECD 221 (2006), all results are given in relation to the nominal test item concentrations.

Details on test conditions:

Conditions of exposure
3-4 Lemna plant colonies consisting of 9 - 10 fronds were transferred from the pre-culture 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. The test vessels were placed in an illuminated shelf which is located in a temperature controlled laboratory. Four fluorescent tubes (Osram L36W/840, CoolWhite) were placed with a distance of 50 cm above the test vessels to obtain a light intensity of 105 µE/m² s +/- 6.7 % (PAR). Illumination took place permanently. Temperature was in a range of 24.6 to 25.3°C during exposure time. Two times during the seven-day exposure period locations of the test vessels were randomised to minimise the influence of spatial differences in light intensity or temperature.
5 test concentrations (6.25, 12.5, 25.0, 50.0, 100.0 mg/L; 3 replicates each), one negative control (NC, 6 replicates) and 3 quality control samples were prepared. Two additional replicates of negative control, 6.25 mg/L, 25 mg/L and 100 mg/L were prepared without test organism for the chemical analysis.

Measurement of frond area and frond number
Frond number and frond area were determined in each test and control vessel at the start of the experiment, at day 2, at day 5 and at the end (day 7). For this the digital image evaluation system Scanalyzer from LemnaTec was applied. Image recognition was manually checked for every picture on the screen and if necessary misreads were corrected manually.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (Sigma-Aldrich, Lot No. MKBS0425V)

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: frond area

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

81.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: frond area

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: frond area

Details on results:

The measured test item concentrations in the test item treatments were 87.0 – 113.5 % of the nominal concentrations. Therefore the EC values are given as nominal concentrations.
The effect concentrations after 7 d exposure are calculated with the statistical software ToxRat Professional 3.2.1 (Tab. 3 a/b; Appendix III).

Results with reference substance (positive control):

The last quality check was performed in October 2017. The EC50(growth rate, frond number) for the reference substance 3,5-Dichlorophenol was 3.0 mg/L (2.4 – 3.9 mg/L 95%-CI) which is in-line with the requirements of the ISO 20079 (2005) [Lemna, Growth Inhibition Test] of 2.2 – 3.8 mg/L.

Reported statistics and error estimates:

At the start of the test and at day 2, day 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 effect concentrations after 7 d exposure are calculated with the statistical software ToxRat Professional 3.2.1 (Tab. 3 a/b; Appendix III).

Validity criteria fulfilled:

yes

Remarks:

Doubling time of the frond number in the controls must be less than 2.5 days (within a period of 60 hours). This corresponds approximately to a seven-fold increase within the seven-day test duration and an average specific growth rate of 0.275 per day.

Conclusions:

After 7 d an EC50 > 100 mg/L was determined for frond area (both growth rate) of Lemna minor under exposure conditions. The lowest NOEC and lowest EC10 were determined for frond area (growth rate) to be 25 mg/L and 81.2 mg/L, respectively.

Executive summary:

A study was conducted to assess the toxicity of Bayscript gelb GGN against Lemna minor according to OECD 221 (23 March 2006) 'Lemna, Growth Inhibition Test' to investigate the aquatic phytotoxic effect of Bayscript gelb GGN. To achieve this, a series of test item concentrations in aqueous solutions was prepared (nominal base on active ingredient 6.25, 12.5, 25.0, 50.0, 100 mg/L), and the plants were allowed to grow in these concentrations, as well as in controls without the test item, for a test period of 7 days under controlled conditions.

3-4 Lemna plant colonies consisting of 9 - 10 fronds were transferred from the pre-culture into the test and control vessels at the start of the test under aseptic conditions. Test solutions were prepared with 3 replicates, negative control was prepared with 6 replicates. The test volume was 100 mL.

At the start of the test and at day 2, day 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 data obtained were analysed using a regression model in order to estimate the concentration that would cause a growth inhibition of frond number and frond area. From the inhibition of the average specific growth rates recorded in a series of dilutions of the test solution compared to the control, dose-response curves and the corresponding ECx values (EC10 and EC50) and NOEC were determined.

After 7 d an EC 50 > 100 mg/L was determined for frond number and frond area (both growth rate) of Lemna minor under exposure conditions. The lowest NOEC and lowest EC10 were determined for frond area (growth rate) to be 25 mg/L and 81.2 mg/L, respectively, and >100 mg/L each for frond number (growth rate).

It is stated in OECD 221 that for Lemna "toxicity estimates should be based on frond number and one additional measurement variable (total frond area, dry weight or fresh weight), because some substances may affect other measurement variables much more than the frond number". Thus the frond area (growth rate) was taken as the main endpoint of this study, as this is the more sensitive endpoint.

Description of key information

A study was conducted to assess the toxicity of Bayscript gelb GGN against Lemna minor according to OECD 221 (23 March 2006) 'Lemna, Growth Inhibition Test' to investigate the aquatic phytotoxic effect of Bayscript gelb GGN. To achieve this, a series of test item concentrations in aqueous solutions was prepared (nominal base on active ingredient 6.25, 12.5, 25.0, 50.0, 100 mg/L), and the plants were allowed to grow in these concentrations, as well as in controls without the test item, for a test period of 7 days under controlled conditions.

3-4 Lemna plant colonies consisting of 9 - 10 fronds were transferred from the pre-culture into the test and control vessels at the start of the test under aseptic conditions. Test solutions were prepared with 3 replicates, negative control was prepared with 6 replicates. The test volume was 100 mL.

After 7 d an EC 50 > 100 mg/L was determined for frond number and frond area (both growth rate) of Lemna minor under exposure conditions. The lowest NOEC and lowest EC10 were determined for frond area (growth rate) to be 25 mg/L and 81.2 mg/L, respectively, and >100 mg/L each for frond number (growth rate).

It is stated in OECD 221 that for Lemna "toxicity estimates should be based on frond number and one additional measurement variable (total frond area, dry weight or fresh weight), because some substances may affect other measurement variables much more than the frond number". Thus the frond area (growth rate) was taken as the main endpoint of this study, as this is the more sensitive endpoint.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

25 mg/L

Additional information

Should read: EC50 > 100 mg/L, NOEC >= 25 mg/L