Sodium 2,4-diamino-5-(2-hydroxy-5-nitrophenylazo)benzenesulphonate

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:

From the 11th of November to the 29th of November, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted according to an internationally accepted test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

yes

Remarks:

The temperature ranged from 21.8 to 30.1 °C, due to technical issues. Out of the required range of 24± 2 °C, this deviation is not expected to have any significant influence on the outcome of the study, the growth of the plants was optimal.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU C.26 Commission Regulation (EU) 2016/266 of 7 December 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Mordant Brown 033

IUPAC Name:

Mordant Brown 033

Test material form:

other: solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the beginning of the test, as well as after 2, 5 and 7 days

Test solutions

Vehicle:

yes

Details on test solutions:

To prepare the standard solutions, a stock solution (test item dissolved in water:methanol (1:1, v:v)) was diluted with SIS medium:methanol (9:1, v:v) containing 10 mM ammonium acetate.
Test samples were diluted with methanol containing 100 mM ammonium acetate to obtain a final sample composition of sample:methanol (9:1, v:v) containing 10 mM ammonium acetate before HPLC analysis. If necessary, the samples were further diluted into the calibration range using SIS medium:methanol (9:1, v:v) containing 10 mM ammonium acetate.

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISMS
Exponential growing plant monoculture of Lemna minor (Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin)
TEST SYSTEM
Culture: All-glass vessel containing sterile Swedish standard-Medium (SIS Medium) and the exponential growing plant monoculture
Cultivation: At least seven days before testing, sufficient colonies are transferred aseptically into fresh sterile medium and cultured for 7 - 10 days under the conditions of the test
Illumination: Continuous (6500–10000 lux)
Temperature: 24 ± 2 °C

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Recommended Swedish standard-Medium (SIS Medium)

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

The temperature ranged between 23.4 and 25.5°C, which is within the required range (24 ± 2°C).
Measured in an additional glass vessel containing SIS medium and Lemna, at least daily.

pH:

6.5 - 7.2
Determined in the combined replicate test solutions at the beginning and at the end of the test.
The pH value in the control drifted by 0.6 units during the whole test period, which is therefore in the range as required by the guideline (required: not more than 1.5).

Nominal and measured concentrations:

268, 84.6, 26.8, 8.46 and 2.68 mg/L
nominal concentration of the test item, corresponding to
200, 63.2, 20.0, 6.32 and 2.00 mg/L of the active ingredient.

Details on test conditions:

Test vessels
400 ml beakers, all-glass, with 200 mL of test medium. The beakers are covered with black paper up until the 200 ml mark to ensure that illumination comes only from above and not from the sides.
Illumination: Intensity: continuous (6500–10000 lux), Homogeneity: ±15%.
The light intensity was 6500-8130 lux (mean 7132 lux; max. variation ±14.0%) at the start of the test and 6500-8000 lux (mean 7100 lux, max. variation ±12.7%) at the end of the exposure period. The light homogeneity was therefore in the range allowed by the guideline (±15%).
Incubation: Beakers will be incubated on a black non-reflecting surface

Reference substance (positive control):

yes

Remarks:

Yearly, with 3,5-dichlorophenol. According to guideline ISO/CD 20079, the ErC50 value based on the frond number should lie in the range 1.8–3.6 mg/l; this level of sensitivity was attained (data from 29 July 2016)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

134 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

43.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

192 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

dry weight

Basis for effect:

other:

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

44.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

dry weight

Basis for effect:

other:

Effect concentrations 5

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

14.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Frond Number: growth rate and yield Dry Weight: growth rate and yield

Details on results:

PRELIMINARY NON-GLP TEST
% inhibition yield of Frond Number: 366% at 100 mg/L nominal concentration (103 mg/L measured at the beginning, 90.1% mg/L measured at the end of the test)
% inhibition growth rate of Frond Number: 39% at 100 mg/L nominal concentration (103 mg/L measured at the beginning, 90.1 % mg/L measured at the end of the test)
The HPLC measurements indicated that the test item concentrations decrease and do not remain in the recommended 80-120% range of the nominal concentration. Nonetheless, the definitive test was performed under static conditions in accordance with the sponsor.

DEFINITIVE TEST
The test concentrations of the substance during the 7-day test period were determined by HPLC analysis. These analyses confirmed the right dosage of the test item, and showed that the concentrations of the test item decreased over the whole 7-day test period. The measured concentrations of the active ingredient at the beginning of the test were 195, 60.0, 18.9, 5.52 and 1.73 mg/L, and 176, 39.2, 10.5, 2.85 mg/L and Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

FROND NUMBER:
Growth rate inhibition: the following effects as compared to the untreated controls were observed: 60% at 200 mg/L and 28% at 63.2 mg/L. No significant effects were observed at the 20.0, 6.32 and 2.00 mg/L concentrations-
Yield inhibition:the following effects as compared to the untreated controls were observed: 87% at 200 mg/L and 59% at 63.2 mg/l. No significant effects were observed at the 20.0, 6.32 and 2.00 mg/L concentrations

DRY WEIGHT
Growth rate inhibition: the following effects as compared to the untreated controls were observed: 50% at 200 mg/l and 23% at 63.2 mg/l. No significant effects were observed at the 20.0, 6.32 and 2.00 mg/l concentrations.
Yield inhibition: the following effects as compared to the untreated controls were observed: 83% at 200 mg/L and 53% at 63.2 mg/L. No significant effects were observed at the 20.0, 6.32 and 2.00 mg/l concentrations.

APPEARANCE OF THE PLANTS
In the blank control the plants were very healthy with dark green fronds and roots reaching the bottom of the test vessel.
At 2.00, 6.32 and 20.0 mg/L, the plants looked like the blanks, with dark green fronds and roots reaching the bottom of the test vessel.
At 63.2 mg/L, the fronds were dark green but the roots were only about half of the blanks.
At 200 mg/L, the plants had small pale green fronds (sometimes only one frond), and the roots fell off.

Any other information on results incl. tables

The effective concentration ErCx with respect to growth rate, and EyCx with respect to yield, is the concentration of test item which causes an x % reduction in growth rate or yield, respectively.

These values can be determined for any of the variables considered (frond number and one other measurement variable e.g. total frond area, dry weight or fresh weight). The effect concentrations ErCx and EyCx wre determined with ToxRat® Standard Version 2.10 (ToxRat® Solutions GmbH, Alsdorf, Germany). For the calculation of the ECx and associated confidence intervals, inhibitions below 0% and inhibitions above 100% were set to 0.1% and 100%, respectively.

The no observed effect concentration (NOErC or NOEyC) is the highest concentration tested in which the test item is observed to have no statistically significant reducing effect on growth when compared with the control. The statistical significance of differences between effects observed for control and test item treatments were determined by Dunnett’s test using the ToxRat® Standard Version 2.10 (ToxRat® Solutions GmbH, Alsdorf, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1

Conclusions:

ErC50 frond number, Growth rate on duckweed Lemna minor (7 days) 134 mg/L
NOEC frond number, on duckweed Lemna minor (7 days) 14.9 mg/L
geometric mean of the measured concentrations of active ingredient

Executive summary:

Method

The inhibitory effects of the substance to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and biomass (dry weight), following the guideline OECD 221.

The test item is solid, well soluble (about 65 g/L in water). The test solutions were prepared by respective dilutions of a stock solutionin SIS medium.

The test was performed at 268, 84.6, 26.8, 8.46 and 2.68 mg/L nominal concentration of the test item, corresponding to 200, 63.2, 20.0, 6.32 and 2.00 mg/L of the active ingredient.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

The test concentrations during the 7 day test period were determined by HPLC analysis at the beginning of the test, as well as after 3, 5 and 7 days of exposure.

Observations

The analyses confirmed the right dosage of the test item, and showed that the concentrations of the test item decreased over the whole 7-day test period (90, 65, 55 and 52%, respectively, of the initial value; not assessable for the lowest test concentration). Therefore, the effective concentrations (E_rC₅₀and E_yC₅₀) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient.

The two endpoints frond number and biomass (dry weight) were investigated at days 3, 5 and 7, and each of them were assessed as growth rate and yield.

Conclusion

The 7 day E_rC₅₀ (based on frond number) on the duckweed Lemna minoris 134 mg/L. This value is based on the measured concentrations of the active ingredient. The validity criterion was fulfilled.