Disodium 3,3'-[sulphonylbis[p-phenyleneazo(5-imino-3-methyl-1H-pyrazole-4,1-diyl)]]bis(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 2nd of July to the 3rd of August, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test 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:

Temperature 23.2- 26.4°C during the whole test period; Light intensity 6620-8430 lux(variation ±16%) at the start of the test and 6530-8520 lux (max. variation ±17%) at the end of the exposure period

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 3,3'-[sulphonylbis[p-phenyleneazo(5-imino-3-methyl-1H-pyrazole-4,1-diyl)]]bis(benzenesulphonate)

EC Number:

275-148-4

EC Name:

Disodium 3,3'-[sulphonylbis[p-phenyleneazo(5-imino-3-methyl-1H-pyrazole-4,1-diyl)]]bis(benzenesulphonate)

Cas Number:

71033-19-7

Molecular formula:

C32H26N10Na2O8S3

IUPAC Name:

disodium 3,3'-[sulphonylbis[p-phenyleneazo(5-imino-3-methyl-1H-pyrazole-4,1-diyl)]]bis(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 3, 5 and 7 days

Test solutions

Vehicle:

yes

Details on test solutions:

STEINBERG Medium

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, comprising 2-5 fronds.
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:

STEINBERG Medium

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

23.2 and 26.4°C during the whole test period; which is not within the required 24±2 °C. S
ince the growth criterion in the control was fulfilled, this minimal deviation of +0.4°C does not have a significant impact on the outcome of the study.

pH:

7.0 - 8.2
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 0.7 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:

130, 50.0, 19.2, 7.40 and 2.84 mg/L nominal concentration of the test item (corresponding to 100, 38.5, 14.8, 5.69 and 2.19 mg/L active ingredient). At all concentrations, the test item was fully dissolved, no suspended matter or supernatant was visible

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.
Pre-culture: Exponentially growing plant monoculture of Lemna minor (the culture was visibly free from contamination by other organisms such as algae and protozoa).
Illumination: Intensity: continuous (6500–10000 lux), Homogeneity: ±15%.
The light intensity was 6620-8430 lux (mean 7270 lux; max. variation ±16%) at the start of the test and 6530-8520 lux (mean 7258 lux, max. variation ±17%) at the end of the exposure period. The light homogeneity was not in the 15% range, but since the test vessels were randomly re-placed and since the growth criterion in the control was fulfilled, this deviation is not expected to have a significant impact on the outcome of the study.

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.:

> 91 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 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.:

> 91 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.:

12.5 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.:

4.27 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: 19% at 100 mg/L nominal concentration (99.9 mg/L measured at the beginning, 97.7% mg/L measured at the end of the test)
% inhibition growth rate of Frond Number: 9% at 100 mg/L nominal concentration (99.9 mg/L measured at the beginning, 97.7% 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 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 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 95.6, 36.6, 13.6, 5.05 and 1.94 mg/L and 87.1, 31.4, 8.30, 3.82 and 1.18 mg/L (91, 86, 61, 76 and 61%, respectively, of the initial value) after 7 days. Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the geometric mean (GM) of the measured concentrations, which were 1.48, 4.27, 10.9, 33.1 and 91.0 mg/L.

FROND NUMBER:
Growth rate inhibition:
the following effects as compared to the untreated controls were observed: 10% at 100 mg/L, 4% at 38.5 mg/L and 4% at 14.8 mg/L. No significant effects were observed at the nominal concentrations of 5.69 and 2.19 mg/L, as determined by Dunnett’s test.
Yield:
the following effects as compared to the untreated controls were observed: 23% at 100 mg/L, 10% at 38.5 mg/L and 10% at 14.8 mg/L. No significant effects were observed at the nominal concentrations of 5.69 and 2.19 mg/L, as determined by Dunnett’s test. Based on these data the median effect concentrations to Lemna minor with respect to growth rate (ErC50) and to yield (EyC50) for the endpoint frond number were both estimated to be >91.0 mg/L measured concentration of active ingredient.
The no-observed-effect concentrations to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint frond number were both 4.27 mg/l measured concentration of active ingredient, as determined by Dunnett’s test.

DRY WEIGHT
Growth rate inhibition:
the following effects as compared to the untreated controls were observed: 33% at 100 mg/L, 27% at 38.5 mg/L, 23% at 14.8 mg/L, 16% at 5.69 mg/L and 12% at 2.19 mg/L. With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 66% at 100 mg/L, 58% at 38.5 mg/L, 51% at 14.8 mg/L, 39% at 5.69 mg/L and 32% at 2.19 mg/L.
Yield:
the following effects as compared to the untreated controls were observed: 66% at 100 mg/L, 58% at 38.5 mg/L, 51% at 14.8 mg/L, 39% at 5.69 mg/L and 32% at 2.19 mg/L.
Based on these data the median effect concentration to Lemna minor with respect to growth rate (ErC50) and to yield (EyC50) for the endpoint dry weight were estimated to be respectively >91.0 mg/L and 12.5 mg/L (95%confidence limits: 7.52-21.0 mg/L) measured concentration of active ingredient. The no-observed-effect concentrations to Lemna minor with respect to growth rate (NOErC) and yield (NOEyC) for the endpoint dry weight were both <1.48 mg/L measured concentration of active ingredient, as determined by Dunnett’s test.

APPEARANCE OF THE PLANTS
In the blank control the plants were healthy, as well as at the test concentrations of 2.19, 5.69, 14.8 and 38.5 mg/L. At 100 mg/L, the size of the plants were only about two thirds of the plants in the control. Apart from the smaller size, the plants appeared healthy.

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) >91 mg/L
ErC50 dry weight, on duckweed Lemna minor (7 days) >91 mg/L
geometric mean of the measured concentrations of the active ingredient.

Executive summary:

Method

The inhibitory effects 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 (328.5 ± 1.81 g/L at 20.0 ± 0.5°C) and has a purity of 77.1% (active ingredient). The test solutions were prepared by respective dilutions of a stock solution in STEINBERG medium. The test was performed at the nominal test item concentrations of 130, 50.0, 19.2, 7.40 and 2.84 mg/L, corresponding to 100, 38.5, 14.8, 5.69 and 2.19 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.

Observations

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 the right dosage of the test item, but showed that the concentrations of the test item decreased over the whole 7 day test period (91, 86, 61, 76 and 61%, respectively, of the initial value). Therefore, the effective concentrations (ErC50 and EyC50) 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 ErC50 of on the duckweed Lemna minor was > 91.0 mg/L. This value is based on the geometric mean of the measured concentrations of the active ingredient. The validity criterion was fulfilled.