Reaction mass of Chromate(2-), [3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)] [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]]-, lithium sodium and Chromate(2-), [3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)][N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)]-, lithium sodium

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 November 25 to December 05, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

The light homogeneity variation was between 15.1 and 15.8 % therefore not in the 15% range. The temperature ranged from 20.6 to 23.5 °C (i.e. 2.9 °C variation), which is out of the required range of 24± 2 °C

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

EC Number:

274-487-5

EC Name:

Disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Cas Number:

70236-55-4

Molecular formula:

C36H18CrN7O13S . 2 Na

IUPAC Name:

disodium [1-[(2-hydroxy-3,5-dinitrophenyl)azo]-2-naphtholato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 230, 72.8, 23.0, 7.28 and 2.30 mg/l nominal concentration
- Sampling method: Since the test item is soluble in SIS medium, the test solutions were prepared by respective dilutions of a stock solution (test item dissolved in SIS Medium) with SIS Medium.
- Sample storage conditions before analysis:

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Lemna minor
- Source (laboratory, culture collection): Umweltbundesamt, FGIII 2.5, berwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Test temperature:

from 20.6 to 23.5 °C

pH:

6.5 at the beginning of the test

Nominal and measured concentrations:

230, 72.8, 23.0, 7.28 and 2.30 mg/l nominal concentration, corresponding to 150, 47.4, 15.0, 4.74 and 1.50 mg/l of the active ingredient.

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: beakers
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 400 ml beakers, all-glass
- Type of cover: black paper up until the 200 ml
- Agitation: yes/no
- No. of colonies per vessel: 9-12 fronds
- No. of fronds per colony: 2-4 fronds
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjusted to 6.5 ± 0.2 with either 0.1 or 1 M HCl or NaOH
- Light intensity and quality: continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Lighting F18T8/6500K EUP (CH Lighting CO., Ltd., China). Homogeneity: ± 15 % (target; see section 1.10.2 Deviations)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: [manual counting / computerized image analysis]
- Determination of biomass: dry weight

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: at 1 mg/l 6 % of immobilisation based on growth rate, at 10 mg/l 19 % of immobilisation, at 100 mg/l 27 % of immobilisation.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

27.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: based on dry weight

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 192 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: based on dry weight

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

12

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: based on dry weight

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 63.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: based on dry weight

Any other information on results incl. tables

Test concentrations

The test concentrations of the test substance during the 7 day test period were determined by photometry 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 remained stable over the whole 7 day test period. The measured concentrations of the active ingredient at the beginning of the test were 150, 46.0, 15.2, 4.50 and 1.12 mg/l and 136, 44.0, 13.7, 3.77 and 0.943 mg/l after 7 days (i.e. 90, 96, 90, 84 and 85%, respectively, of the initial value). However, the lowest concentrations did not remain in the 80-120 % range of the nominal concentration. Therefore, the effective concentrations (ErCx and EyCx) were assessed based on the geometric mean (GM) of the measured concentrations of the active ingredient, which were 144, 43.7, 14.4, 4.15 and 1.00 mg/l .

Environmental conditions

The pH value in the control drifted by 0.4 units during the whole test period, which is therefore in the range allowed by the guideline (required: not more than 1.5).

The light intensity was 6930-8550 lux (mean 7382 lux; max. variation ± 15.8 %) at the start of the test and 7000-8500 lux (mean 7388 lux, max. variation ± 15.1 %) at the end of the exposure period.

The light homogeneity was therefore not in the 15 % range.

The temperature ranged from 20.6 to 23.5 °C (i.e. 2.9 °C variation), which is out of the required range of 24 ± 2 °C.

Since the test vessels were randomly re-placed and since the growth criterion in the control was fulfilled, these deviations are not expected to have had any significant impact on the outcome of the study.

Appearance of the plants at the end of the test

In the blank control the plants were very healthy with dark green fronds and roots reaching the

bottom of the test vessel.

At 1.50 and 4.74 mg/l, the plants looked like the blanks.

At 15.0 mg/l, the plants looked like the blanks but were slightly smaller.

At 47.4 mg/l, the plants looked like the blanks but were only half as big as the blanks.

At 150 mg/l, the plants had very small dark green fronds and very short roots

Validity of the test and remarks

The validity criterion was met.

At the nominal concentration of 1.5 mg/l an inhibitory effect was always detected while at the next upper concentration, i.e. 4.74 (and sometimes also for 15 mg/l) a stimulating effect was observed.

Moreover, the inhibitory effects observed at 1.5 mg/l are higher than those recorded for 15 mg/l.

While this seems like a discrepancy, it is not more than natural biological variation, which is observed when plants are exposed to concentrations below the NOEC.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

ErC10 = 27.3 mg/L based on dry weight (measured)
ErC50 = 192 mg/L based on dry weight (measured)

Executive summary:

Method

The inhibitory effects of the test 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 solutions were prepared by respective dilutions of a stock solution in SIS Medium. The test was performed at 230, 72.8, 23.0, 7.28 and 2.30 mg/l nominal concentration, corresponding to 150, 47.4, 15.0, 4.74, 1.50, and 0.474 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 photometry 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 remained stable over the whole 7 day test period. However, the lowest concentrations did not remain in the 80-120 % range of the nominal concentration. Therefore, the effective concentrations (ErCx and EyCx) 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.

Results

ECr50 = 192 mg/l based on frond number

ECr50 = 192 mg/l based on dry weight