Reaction mass of tetrasodium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)] and tetraammonium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)]

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

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:

Reaction mass of tetrasodium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)] and tetraammonium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)]

EC Number:

943-486-8

Molecular formula:

refer to the molecular formula of the constituent

IUPAC Name:

Reaction mass of tetrasodium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)] and tetraammonium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)]

Details on test material:

Chemical name: Reaction mass of tetrasodium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)] and tetraammonium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)]
EC: 943-486-8

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 167 mg/l nominal concentration, , corresponding to 100 mg/l of the active ingredient.
Since the test item is soluble, the test solution was prepared by directly dissolving the test item in Swedish standard-Medium (SIS Medium; Table 2). The resulting solution was used as the test solution in the toxicity test.
Pure SIS medium served as blank controls.
The pH of the SIS medium was adjusted before the test to pH 6.5 ± 0.2.

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM - Exponential growing plant monoculture of Lemna minor (Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin)
-Culture: All-glass vessel containing sterile Swedish Standard (SIS) medium (Table 2) and the exponentially growing plant monoculture. Healthy Lemna minor plants consist of colonies comprising 2-5 fronds.
-Cultivation:At least seven days before testing, sufficient colonies were transferred aseptically into fresh sterile medium and cultured for 7-10 days under the conditions of the test.
-Illumination: Continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Lighting F18T8/6500K EUP (CH Lighting CO., Ltd., China)
-Temperature: 24 ± 2 °C
-Control of sensitivity:Yearly, with 3,5-dichlorophenol

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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: based on frond number

Effect concentrations 2

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Remarks on result:

other: based on frond number

Details on results:

The test concentrations of Direct Blue 267:1 during the 7-day test period were determined by photometry at the beginning of the test, as well as after 2, 5 and 7 days of exposure (Table 3). 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 was 102 and 98.3 mg/l at the end (i.e. 96% of the initial value).
Therefore, the effective concentrations (ErC50 and EyC50) were assessed based on the nominal concentration of the active ingredient (Table 3).

EFFECTS ON FROND NUMBER
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 8% at 100 mg/l (Table 4, Figures 1 and 2).
Based on these effects and the nominal concentration of the test item, the median effect concentration with respect to the frond number’s growth rate (frond number ErC50) of Direct Blue 267:1 to Lemna minor was estimated to be >100 mg/l.
The no observed effect concentration (NOErC) with respect to the frond number’s growth rate was determined to be <100 mg/l.
With respect to yield inhibition, although an average of 23% effects were observed at 100 mg/l as compared to the untreated controls, these effects are not statistically significant, which is due to the variability of the data (Table 5, Figure 3).
Based on these effects and the nominal concentrations of the test item, the median effect concentration with respect to the frond number’s yield (frond number EyC50) of Direct Blue 267:1 to Lemna minor was estimated to be >100 mg/l.
The no observed effect concentration (NOEyC) with respect to the frond number’s yield was determined to be 100 mg/l.


EFFECTS ON DRY WEIGHT
With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 23% at 100 mg/l (Table 6, Figure 4).
Based on these effects and the nominal concentration of the test item, the median effect concentration with respect to the dry weight’s growth rate (dry weight r ErC50) of Direct Blue 267:1 to Lemna minor was estimated to be >100 mg/l.
With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 54% at 100 mg/l (Table 7, Figure 5).
Based on these effects and the nominal concentration of the test item, the median effect concentration with respect to the dry weight’s yield (dry weight EyC50) of Direct Blue 267:1 to Lemna minor was estimated to be <100 mg/l.
The no observed effect concentration with respect to the dry weight’s growth rate (NOErC) and dry weight’s yield (NOEyC) was determined to be <100 mg/l.

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 to the bottom of the test vessel.
At 100 mg/l, the plants looked healthy with dark green roots reaching to the bottom of the test vessel. The plants were slightly smaller as compared to the blank controls.

REMARKS
The 8% effects with respect to the frond number’s growth rate (frond number ErC50) are solely due to two blank replicates (A and D; see also Table 4), which are unusually high. A Student-T test without these two blanks shows that there is no significant difference between Blanks and 100 mg/l with respect to the frond number’s growth rate (frond number NOErC).

Any other information on results incl. tables

ENVIRONMENTAL CONDITIONS

The physico-chemical conditions of the test are reported in Table 8.

The pH value in the control drifted by 1.3 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 6950-8340 lux (mean 7663 lux; max. variation±9%) at the start of the test and 7240-8290 lux (mean 75813 lux, max. variation±7%) at the end of the exposure period. The light homogeneity was therefore in the 15% range.

The temperature ranged between 21.3 and 26.0 °C (i.e. 4.7°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, this deviation was not expected to have a significant impact on the outcome of the study.

Precipitation of the test item was observed after the 7-day test period.

VALIDITY OF THE TEST

The validity criterion as specified in section5.5was met (see footnote of Table 4), i.e.

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.

.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The substance was tested for toxicity to aquatic plants (Lemna minor) following OECD 221. Under the experimental conditions the ECr50> 100 mg/l based on frond number

Executive summary:

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 (16.44 g/l in water) and has a purity of 60% (active ingredient). The test solution was prepared by directly dissolving the test item in Swedish standard-Medium.

The test was performed as a limit test at 167 mg/l nominal concentration of the test item, corresponding to 100 mg/l of the active ingredient.

Six parallel test vessels were used for the test concentration and six vessels for the blank controls.

The test concentrations of the item during the 7-day test period were determined by photometry at the beginning of the test, as well as after 2, 5 and 7 days of exposure. These analyses confirmed the right dosage of the test item, and showed that the concentration of the test item could be maintained satisfactorily over the whole 7-day period. Therefore, the effective concentrations (E_rC₅₀and E_yC₅₀) were assessed based on the nominal concentration of the active ingredient.

The two endpoints frond number and biomass (dry weight) were investigated at days 2, 5 and 7, and each of them were assessed as growth rate and yield.

The results of the toxicity assessment of the substance to the duckweed Lemna minor are summarized in the following table showing EC_xvalues based on the nominal concentration of the active ingredient:

ECr50, frond number > 100 mg/l

ECy50, frond number > 100 mg/l

ECr50, biomass, > 100 mg/l

ECy50, biomass > 100 mg/l