Reaction product of diazotized 3-aminobenzenesulfonic acid coupled with naphthalen-1-amine, subsequently diazotized and coupled with 3-((ethyl(phenyl)amino)methyl)benzenesulfonic acid or 4-((ethyl(phenyl)amino)methyl)benzenesulfonic acid, sodium salt

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

other: read across on similar substance

Adequacy of study:

key study

Study period:

From the 19th of October to the 3th 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 light intensity was 6890-9310 lux (mean 7712 lux; max. variation ±21%) at the start of the test and 6780-9150 lux (mean 7656 lux, max. variation ±20%) at the end of the exposure period. The light homogeneity was therefore not in the 15% range

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:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

EC Number:

274-403-7

EC Name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Cas Number:

70210-06-9

Molecular formula:

C31H25N5Na2O6S2

IUPAC Name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Test material form:

solid: particulate/powder

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:

Since the test item is soluble in Swedish standard-Medium (SIS Medium), the test concentrations were prepared by dilution of a stock solution.
At the nominal concentrations of 31.6 mg/L the test item showed minimal and at 100 mg/L major precipitation, respectively.

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:

137, 43.2, 13.7, 4.32 and 1.37 mg/L nominal concentration of test item, corresponding to
100, 31.6, 10.0, 3.16 and 1.00 mg/L 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 6890-9310 lux (mean 7712 lux; max. variation ± 21%) at the start of the test and 6780-9150 lux (mean 7656 lux, max. variation ± 20%) at the end of the exposure period.
The light homogeneity was therefore not in the 15% range. Since the test vessels were randomly replaced and since the growth criterion in the blank control was fulfilled without a too large variation, this deviation is not expected to have any significant impact on the outcome of the study.
Measured at the beginning and at the end of the test over the whole test area at points the same distance from the light source as the Lemna fronds.
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.:

> 78.4 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.:

91.7 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.:

> 78.4 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.:

123 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.:

25.1 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: 35% at 100 mg/L nominal concentration (93.4 mg/L measured at the beginning, 55.8% mg/L measured at the end of the test)
% inhibition growth rate of Frond Number: 26% at 100 mg/L nominal concentration (93.4 mg/L measured at the beginning, 55.8% 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 that the test item was fully dissolved (82, 85, 89, 92 and 95% of the 1.00, 3.16, 10.0, 31.6 and 100 mg/l nominal concentrations, respectively), and showed that the concentrations decreased over the whole 7-day test period (57, 66, 66, 71, 67% of initial value, respectively). The initial measured test concentrations were 0.818, 2.67, 8.90, 29.1 and 95.4 mg/l and the measured geometric mean concentrations over the whole 7-day test period were 0.662, 2.35, 7.18, 25.1 and 78.4 mg/l, respectively. Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the measured concentrations of the active ingredient.

The test concentrations during the 7-day test period were determined by HPLC analysis at the beginning of the test, as well as after 2, 5 and 7 days of exposure. These analyses confirmed that the test item was fully dissolved (82, 85, 89, 92 and 95% of the 1.00, 3.16, 10.0, 31.6 and 100 mg/L nominal concentrations, respectively), and showed that the concentrations decreased over the whole 7-day test period (57, 66, 66, 71, 67% of initial value, respectively).
The initial measured test concentrations were 0.818, 2.67, 8.90, 29.1 and 95.4 mg/L and the measured geometric mean concentrations over the whole 7-day test period were 0.662, 2.35, 7.18, 25.1 and 78.4 mg/L, respectively.
Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the measured concentrations of the active ingredient.

With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 18% at 100 mg/L. No significant effects were observed at the nominal concentrations of the active ingredient of 31.6, 10.0, 3.16 and 1.00 mg/L. With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 45% at 100 mg/L. No significant effects were observed at the nominal concentrations of the active ingredient of 31.6, 10.0, 3.16 and 1.00 mg/L.

FROND NUMBER:
50% effects on the growth rate of the frond number could not be attained with the maximal concentration tested here (100 mg/l active ingredient). However, the limited effects observed at this concentration as well as the flat dose-response curve allow to estimate that the 7-day ErC50 on the duckweed Lemna minor for the endpoint frond number is >100 mg/l active ingredient.

Growth rate inhibition:
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 16% at 100 mg/L. No significant effects were observed at the nominal concentrations of the active ingredient of 31.6, 10.0, 3.16 and 1.00 mg/L.
Yield:
With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 38% at 100 mg/L. No significant effects were observed at the nominal concentrations of the active ingredient of 31.6, 10.0, 3.16 and 1.00 mg/L.

DRY WEIGHT
50% effects on the growth rate of the dry weight could not be attained with the maximal concentration tested here (100 mg/L active ingredient). However, the limited effects observed at this concentration as well as the flat dose-response curve allow to estimate that the 7-day ErC50 on the duckweed Lemna minor for the endpoint dry weight is >100 mg/l active ingredients.

APPEARANCE OF THE PLANTS
In the blank control and at the nominal concentrations of 1.00 and 10.0 mg/L, the plants were healthy with green fronds and roots reaching to the bottom of the test vessel.
At the nominal concentration of 3.16 mg/l, the plants did not appear any different from the blank controls, except for 4 fronds, which appeared yellow. This discoloration was only observed in 4 out of almost 600 fronds, and therefore, can be considered insignificant. At the nominal concentration of 31.6 mg/L, the plants were healthy with green fronds.
Nevertheless, they were slightly smaller and the roots were 5 mm shorter as compared to the blank controls. At the nominal concentration of 100 mg/L, the plants were healthy with green fronds. They were more compact and smaller and the roots were 5 mm shorter as compared to the blank controls.

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, on duckweed Lemna minor (7 days) >100 mg/L active ingredient
ErC50 dry weight, on duckweed Lemna minor (7 days) >100 mg/L active ingredient
NOEC frond number, on duckweed Lemna minor (7 days) 25.1 mg/L 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. Since the test item is soluble in Swedish standard-Medium (SIS Medium), the test concentrations were prepared by respective dilutions of a stock solution. The test was performed at 137, 43.2, 13.7, 4.32 and 1.37 mg/L nominal concentration of the test item, corresponding to 100, 31.6, 10.0, 3.16 and 1.00 mg/L, respectively, 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. These analyses confirmed that the test item was fully dissolved and showed that the concentrations decreased over the whole 7-day test period. Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on 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.

Observations

Even though 50% effects on the growth rate of the frond number could not be attained with the maximal concentration tested here (100 mg/L active ingredient), the limited effects observed at this concentration as well as the flat dose-response curve allow to estimate that the 7-day ErC50 of the substance on the duckweed Lemna minor for the endpoint frond number is >100 mg/L active ingredient. The validity criterion was fulfilled.

Conclusion

The HPLC measurements indicated that the test item concentrations decrease and do not remain in the recommended 80-120% range of the nominal concentration. Even though 50% effects on the growth rate of the frond number could not be attained with the maximal concentration tested here (100 mg/L active ingredient), the limited effects observed at this concentration as well as the flat dose-response curve allow to estimate that the 7-day ErC50 on the duckweed Lemna minor for the endpoint frond number is >100 mg/L active ingredient.

50% effects on the growth rate of the dry weight could not be attained with the maximal concentration tested here (100 mg/L active ingredient). However, the limited effects observed at this concentration as well as the flat dose-response curve allow to estimate that the 7-day ErC50 on the duckweed Lemna minor for the endpoint dry weight is >100 mg/l active ingredients. The NOEC frond number, on was found 25.1 mg/L of active ingredient.