Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage conditions: Cool, dry, well ventilated, protected from light; in tightly closed container

Analytical monitoring:

yes

Remarks:

HPLC and UV/VIS-detection

Details on sampling:

I. Sampling Schedule:
- Samples of the stock solution and the test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations
- At the beginning of the test (day0), duplicate samples of the stock solution, the control and the test item concentrations were taken from the volumetric flasks
- At the end of the exposure period (day 7), immediately after determination of the biological and physico-chemical parameters, the replicates of each separate test solution were combined. Duplicate samples were taken of aged solutions from the control and all test item concentrations
- The volume of each sample was recorded

II. Sampling Conditions:
- Samples of a defined volume (10 mL) were taken from the designated test solution
- After sampling and before shipment, the samples were stabilised by adding acetonitrile directly after sampling (acetonitrile volume: 50% of the total sample volume; 10 mL of sample was mixedwith 10 mL of acetonitrile)

Vehicle:

no

Details on test solutions:

TEST MEDIUM:
Modified Steinberg medium was prepared according to: OECD Guideline No. 221, Annex 4. The medium will be prepared in deionised water. The pH was adjusted to 5.5±0.2. The medium was sterilised by autoclaving the medium for at least 20 minutes at set temperature of 121°C or by sterile filtration (pore size 0.2 µm).

PREPARATION OF THE STOCK SOLUTION:
At the day of the test start a stock solution (S1) was prepared by dissolving 2588 mg of the test item in 2000 mL of growth medium, resulting in a nominal concentration of 129.0 mg test item/L. This stock solution was stirred at ambient temperature for 10 minutes at 500 rpm using a magnetic stirrer. Thereafter this stock solution was left to settle for 10 minutes. The stock solution was visually examined for undissolved/particulate matter in the free water column. No non-dissolved test material was observed
The test solutions were then prepared by diluting the stock solution S1 with culture medium. All test solutions were stirred for 5 minutes using a magnetic stirrer.
The volume of the stock solution S1 was large enough to prepare all replicates of the test concentrations and all analytical samples at once. The macro- and microelement stock solutions for the test medium as well as the dilution water were sterilised by sterile filtration (pore size 0.2 µm) before use. All glassware and materials used for testing purposes were sterilised for at least 3 hours at ≥150°C using a heating furnace.

Test organisms (species):

Lemna minor

Details on test organisms:

-cultured at ECT Oekotoxikologie GmbH since May 07, 2014.
-The organisms were supplied by Friedrich-Schiller-Universität, Jena.
-The stock culture has been maintained at ECT under recommended culture conditions, thereafter.
-To adapt the plants to the test conditions, a pre-culture was inoculated by a liquid culture and incubated under test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

No data

Test temperature:

24±2°C

pH:

5.5-6.7

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

nominal concentrations: 0, 8.07, 16.1, 32.3, 64.5, 129 mg test item/L, corresponding to 6.25, 12.5, 25.0, 50.0, 100 mg Reactive Red 141

Details on test conditions:

TEST SYSTEM:
-Test organism: Lemna minor
-Test medium: Modified Steinberg medium
-Endpoints: ECx (e.g. EC50, EC20, EC10), NOEC/LOEC
-Biological parameters: Inhibition of growth in relation to control (frond number & dry weight; growth rate & yield)
-Test duration: 7 days
-Temperature (target): 24±2°C
-Light regime: Permanent
-Light intensity (target): 85–135 µE m-2 s-1, within ±15% over the incubation area
-Test units: 300 mL crystallising dishes covered by watch glasses
-Test item concentrations (nominal): 0 (control), 8.07, 16.1, 32.3, 64.5, 129 mg test item/L
-No. of replicates in the control: 6
-No. of replicates per test concentration: 3
-Renewal of test solution during exposure: None (static system)
-Chemical analysis of test concentrations: At start and at end of exposure

PRE-CULTURE CONDITIONS:
-Species: Lemna minor
-Supplier: Friedrich-Schiller-Universität, Jena, Germany
-Date of receipt: May 07, 2014
-Charge No. (ECT): Lm/070514/A
-pH-value of the culture medium: 5.6
-Culture medium: Modified Steinberg medium
-Amount of liquid stock culture per pre-culture vessel: 200±5 mL
-Pre-culture vessels: 250 mL crystallising dishes covered by watch glasses
-Number of replicates:6
-Light: Light/dark (24/0 h); type: Osram L 18W/840 (cool white)
-Light intensity: 85–135 µE m-2 s-1
-Temperature in the culture room: 24±2°C

EXPOSURE CONDITIONS:
-Age of the pre-culture: 10 days
-Number of colonies per test vessel at the beginning of the test: 4
-Number of fronds per test vessel at the beginning of the test: 12
-Number of replicates per test item concentration: 3
-Number of replicates in the control:6
-Test medium: Modified Steinberg medium
-pH of untreated test medium at test start: 5.3
-pH in test solutions during exposure: 5.2–6.7
-Light regime: 24 h light/0 h dark
-Type of light: Fluorescent tubes of universal white type (Osram L 18W/840, cool white)
-Light intensity: Mean 98.5 µE m-2 s-1;
-Temperature: Mean 23.41°C; range: 22.8–24.4°C;
-Test duration (exposure): 7 days
-Determination of frond number: Days 0, 2, 4 and 7
-Determination of dry weight: Days 0 and 7

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol, Result:Growth rate (frond number): ErC50 (0–7 d): 2.95 mg/L. This result is in accordance with the range given in the International Standard ISO 20079 (2005) for Lemna minor (ErC50 should be in the range between 2.2 mg/L and 3.8 mg/L).

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

3.52 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

50.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

3.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

56.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Samples from the test solutions were analysed to determine actual levels of the test item at start of exposure period (time 0 days) and following 7 days of exposure. At time 0 days the measured concentrations range between 94.1-106.5 % and decrease after 7 days exposure at between 58.8-112.1 % of the nominal concentrations. Therefore, the test item concentration based on nominal concentration was not stable as it declined more than 20% throughout the
exposure period.

Summary of biological results [mg test item/L] based on measured concentrations:

Parameter	EC10	EC20	EC50	NOEC	LOEC
Front number (Yield)	1.54	3.28	13.8	<4.65	≤4.65
Front number (Growth rate)	3.52	8.80	50.8	<4.65	≤4.65
Biomass (Yield)	1.45	3.07	12.9	<4.65	≤4.65
Biomass (Growth rate)	3.90	9.78	56.8	<4.65	≤4.65

Summary of biological results [mg test item/L] based on nominal concentrations:

Parameter	EC10	EC20	EC50	NOEC	LOEC
Front number (Yield)	2.75	5.31	18.7	<8.07	≤8.07
Front number (Growth rate)	5.08	12.0	61.6	<8.07	≤8.07
Biomass (Yield)	2.63	5.06	17.7	<8.07	≤8.07
Biomass (Growth rate)	5.57	13.2	68.4	<8.07	≤8.07

According to the guideline OECD 221 and REACH Guidance Document R.7b (2017), frond number is the primary measurement variable. Thus the endpoints EC10 3.52 mg/L and EC50 50.8 mg/L for the primary biological measurement variable "frond number growth rate", based on measured concentrations, can be regarded as the main endpoints of this study.

Validity criteria fulfilled:

yes

Remarks:

Doubling time of the frond number in the control (exposure period): (required: less than 2.5 days (60 h), corresponding to an approximately 7-fold increase in 7 days and an average specific growth rate of 0.275/d)

Conclusions:

In this toxicity test to aquatic plants (Lemna minor) according to OECD 221 an 7d-EC 50 value of 50.8 mg/L and an 7d-EC 10 value of 3.52 mg/L for the primary biological measurement variable "frond number growth rate" was obtained for Reactive Red 141. The results are based to measured concentrations.

Executive summary:

A growth inhibition test with the freshwater aquatic plant Lemna minor was conducted according to OECD 221 (March 2006) in order to investigate the aquatic phytotoxic effect of Reactive Red 141. Lemna minor is a model organism for aquatic fresh water plants. The aim of the study was to determine the effects of Reactive Red 141 on the growth of the freshwater aquatic plant Lemna minor. To achieve this, a series of test item concentrations in aqueous solutions was prepared (nominal 8.07, 16.1, 32.3, 64.5, 129 mg/L), and the plants were allowed to grow in these concentrations, as well as in controls without the test item, for a test period of 7 days under controlled conditions. The data obtained were analysed using a regression model in order to estimate the concentration that would cause a x% growth inhibition of front number and biomass. From the inhibition of the average specific growth rates recorded in a series of dilutions of the test solution compared to the control, dose-response curves and the corresponding ECxvalues (EC10, EC20 and EC50) were determined. A clear concentration-response relationship was observed for both biological parameters frond number and biomass evaluating growth rate and yield during the exposure period. Samples from the test solutions were analysed to determine actual levels of the test item at start of exposure period (time 0 days) and following 7 days of exposure. At time 0 days the measured concentrations ranged between 94.1-106.5 % and decreased after 7 days exposure at between 58.8-112.1 % of the nominal concentrations.

According to the guideline OECD 221 and REACH Guidance R.7b (2017), frond number is the primary measurement variable. Thus the endpoints 7d-EC50 50.8 mg Reactive Red 141/L and 7d-EC10 3.52 mg Reactive Red 141/L for the primary biological measurement variable "frond number growth rate" can be regarded as the main endpoints of this study.

All validity criteria are met. This toxicity study is classified as acceptable and satisfies the guideline requirements for the growth inhibition test with Lemna minor.

Description of key information

In this toxicity test to aquatic plants (Lemna minor) according to OECD 221 an 7d-EC 50 value of 50.8 mg/L and an 7d-EC 10 value of 3.52 mg/L for the primary biological measurement variable "frond number growth rate" was obtained for Reactive Red 141. The results are based to measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater plants:

50.8 mg/L

EC10 or NOEC for freshwater plants:

3.52 mg/L

Additional information

A growth inhibition test with the freshwater aquatic plant Lemna minor was conducted according to OECD 221 (March 2006) in order to investigate the aquatic phytotoxic effect of Reactive Red 141. Lemna minor is a model organism for aquatic fresh water plants. The aim of the study was to determine the effects of Reactive Red 141 on the growth of the freshwater aquatic plant Lemna minor. To achieve this, a series of test item concentrations in aqueous solutions was prepared (nominal 8.07, 16.1, 32.3, 64.5, 129 mg/L), and the plants were allowed to grow in these concentrations, as well as in controls without the test item, for a test period of 7 days under controlled conditions. The data obtained were analysed using a regression model in order to estimate the concentration that would cause a x% growth inhibition of front number and biomass. From the inhibition of the average specific growth rates recorded in a series of dilutions of the test solution compared to the control, dose-response curves and the corresponding ECxvalues (EC10, EC20 and EC50) were determined. A clear concentration-response relationship was observed for both biological parameters frond number and biomass evaluating growth rate and yield during the exposure period. According to the guideline OECD 221 and REACH Guidance R.7b (2017), frond number is the primary measurement variable. Thus the endpoints 7d-EC50 50.8 mg Reactive Red 141/L and 7d-EC10 3.52 mg Reactive Red 141/L for the primary biological measurement variable "frond number growth rate" can be regarded as the main endpoints of this study. All validity criteria are met.This toxicity study is classified as acceptable and satisfies the guideline requirements for the growth inhibition test with Lemna minor.