Reaction products of diazotised 5,5’-methylenedianthranilic acid, coupled with resorcinol, subsequently coupled with diazotised 4-aminobenzene-1,3-disulfonic acid, chelated with copper sulphate, sodium salt

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From August 26 to December 19, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability of the source study is 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

Temperature range 21.6 to 25.2°C, slightly out of required range of 24 ± 2 °C.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
non-GLP range finding test 1, 10 and 100 mg/l;
definitive test 100, 50.0, 25.0, 12.5 and 6.25 mg/l nominal concentration

- Sampling method: the test solutions were prepared by respective dilutions of a stock solution (test item dissolved in STEINBERG Medium)

Vehicle:

no

Test organisms (species):

Lemna minor

Details on test organisms:

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

ACCLIMATION
- Acclimation period: 7-10 days
- Culturing media and conditions: same as 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)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

21.6 to 25.2 °C

pH:

5.5 at the beginning of the test

Nominal and measured concentrations:

100, 50.0, 25.0, 12.5 and 6.25 mg/l nominal concentration, corresponding to 61.9, 31.0, 15.5, 7.74 and 3.87 mg/l of the active ingredient.

Measured concentrations of the active ingredient at the beginning of the test were 65.2, 29.5, 15.3, 7.92 and 3.89 mg/l; and 40.6, 25.4, 13.8, 6.73 and 2.62 mg/l after 7 days (i.e. 62, 86, 90, 85 and 67 %, respectively, of the initial value)

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes, beakers were incubated on a black non-reflecting surface.
- Test vessel: beakers
- Material, size, headspace, fill volume: 400 ml, all-glass
- Type of cover: black paper up until the 200 ml mark
- No. of fronds per plant: 2-4 fronds
- No. of fronds per vessel: 9-12 fronds per replicate
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (STEINBERG Medium )

OTHER TEST CONDITIONS
- IlluminationIntensity: 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%

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of biomass: dry weight. Determined from duckweed at day 0 in six additional blank replicates (similar to those used in the test), and at day 7 in all blanks and all test vessels, after drying at 60 °C at least overnight.
- Determination of frond area: fronds was counted on day 0, 3, 5 and 7.
- Other: changes in plant development, e.g. in frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up, loss of buoyancy, root length and appearance were noted.

RANGE-FINDING STUDY
- Test concentrations: nominal concentrations of 1, 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: at 1 mg/l 0 % of inhibition growth rate (IGR) and 0 % of inhibition yield (IY) ; at 10 mg/l, 2 % IGR and 5 % IY; at 100 mg/l 92 % IGR and 97 IY.- Replicates: 3 for the blank (0 mg/l) and 2 at 1, 10, 100 mg/l.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 17.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: effects on frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 10.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: effects on dry weights

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 8.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: effects on frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

ca. 3.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: effects on dry weights

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 3.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: effects on frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

< 3.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: effects on dry weights

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

ca. 3.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: effects on frond number

Environmental conditions

The pH value in the control drifted by 1.5 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 7360-8930 lux (mean 8308 lux; max. variation ± 11.4 %) at the start of the test and 7400-9000 lux (mean 8359 lux, max. variation ± 11.5 %) at the end of the exposure period. The light homogeneity was therefore in the required 15 % range.

The temperature ranged from 21.6 to 25.2 °C, which is very slightly 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 is 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 3.87 mg/l, the plants were dark green, the roots reached the bottom of the test vessel, but the fronds were smaller than in the blanks.

At 7.74 mg/ and 15.5 mg/l, the plants were light green, significantly smaller than the blanks and the 3.87 mg/l concentration, and the roots partially fell off.

At 31.0 mg/l, the plants were light green and very small with only 1 or 2 fronds; the roots were hardly visible.

At 61.9 mg/l, the plants all looked dead (all fronds showed signs of chlorosis); the roots were hardly visible. Also the test item showed some signs of precipitation

Validity criteria fulfilled:

yes

Conclusions:

ErC50 = 10.6 mg/l (effects on dry weight)
NOErC < 3.36 mg/l (effects on dry weight)

Executive summary:

Method

The inhibitory effects of Similar Substance 01 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 Acid Red 310 is solid, well soluble, and has a purity of 61.9% (active ingredient). The test solutions were prepared by respective dilutions of a stock solution in STEINBERG Medium.

The test was performed at100, 50.0, 25.0, 12.5 and 6.25 mg/l nominal concentration of Acid Red 310, corresponding to 61.9, 31.0, 15.5, 7.74 and 3.87 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 of Acid Red 310 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 slightly decreased over the whole 7-day test period. Therefore, the effective concentrations (E_rC_xand E_yC_x) 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

Parameter, expressed as Measured conc. Of A.I. (mg/l)	EC50	EC10	NOEC^1
Frond Number, Growth Rate	17.5	3.40	3.36
Frond Number, Yield	8.80	n.d.	3.36
Dry Weight, Growth Rate	10.6	n.d.	< 3.36
Dry Weight, Yield	3.80	n.d.	< 3.36

¹Determined by Dunnett’s Test

Description of key information

ErC50 = 10.6 mg/l (effects on dry weight)

ErC50 = 17.5 mg/l (effects on frond number)

Key value for chemical safety assessment

EC50 for freshwater plants:

10.6 mg/L

EC10 or NOEC for freshwater plants:

3.36 mg/L