Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)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; not below 0 °C (stored at ambient temperature)

Analytical monitoring:

yes

Details on sampling:

SAMPLING FOR CHEMICAL ANALYSIS
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, duplicate samples of the stock solution (S1), the control (C0) and the test item concentrations (C1–C5) were taken from the volumetric flasks.
- At the end of the exposure period, 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 (C0) and all test item concentrations (C1–C5).
- The volume of each sample was recorded.
Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.
Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The volume of each sample was recorded. If necessary, test solutions of more than one vessel were united in order to provide sufficient sample volume for extraction.
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 mixed with 10 mL of acetonitrile). The samples were stored in amber glass bottles of sufficient volume (e.g. twice the designated sample volume) in the dark at a temperature of ≤ -18°C if not advised otherwise.

Samples were transferred frozen on dry ice to the test site for chemical analysis to the following delivery address:
Currenta Analytik Probenannahme
D-51368 Leverkusen
Germany

The samples arrived at the analytical laboratory deep frozen and in good condition. The dates of transfer of the samples from ECT to the laboratory for chemical analysis were recorded in the raw data. The study director sent a sample list to the principal investigator.
Each sample was analysed independently.
All samples taken from the test and stored at ECT (except the archived sample of the test item) were disposed upon completion of the study, unless the sponsor has requested in writing before the end of the study to arrange for longer-term storage.

Vehicle:

no

Details on test solutions:

Preparation of the Stock and Test Solutions
At the day of the test start a stock solution (S1) was prepared by dissolving 2323 mg of the test item in 2000 mL of growth medium, resulting in a nominal concentration of 116.0 mg test item/L. This stock solution was stirred at ambient temperature for 10 minutes 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 10 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:

The test system used in this study was Lemna minor, 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.
The species Lemna minor was chosen as a representative of freshwater aquatic plants. The selection of the test system is based on requirements of the test guideline.

PRE-CULTURE CONDITIONS:
To adapt the plants to the test conditions, a pre-culture was inoculated by a liquid culture and incubated under test 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: 5.5
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

Test type:

static

Water media type:

freshwater

Remarks:

Water Quality Measurements: temperature and pH in each treatment, temperature will additionally be recorded at least once per day throughout the test in a surrogate vessel held under the same conditions

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24 ± 2^C (Mean value: 23.91°C, Minimum value: 23.4°C, Maximum value: 24.0°C)

pH:

5.5 - 6.4

Nominal and measured concentrations:

Nominal: 0 (control), 4.95, 10.9, 24.0, 52.7, 116 mg/L corresponding to 4.26, 9.38, 20.7, 45.4, 100 mg (calculated by multiplying the nominal test item concentrations given in mg test item/L with 0.861, stipulated content of Direct Yellow 86)

Details on test conditions:

TEST SYSTEM
-Test system: 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), 4.95, 10.9, 24.0, 52.7, 116 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

EXPOSURE CONDITIONS
- Age of the pre-culture: 7 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: 5.6
- pH in test solutions during exposure: 5.5–6.4
- 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 103.3 µE m-2 s-1
- Temperature: Mean 23.91°C; 23.40–24.0°C;
- Test duration (exposure): 7 days
- Determination of frond number: Days 0, 3, 5 and 7
- Determination of dry weight: Days 0 and 7

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

7.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

ca. 2.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: NOEC < 2.73 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

6.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

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 97–102 % and decrease after 7 days exposure at between 40–100 % 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.
All concentration levels were measured, and the biological results are additionally calculated and reported based on geometric mean measured concentrations at each concentration level.

Results with reference substance (positive control):

Growth rate (frond number): ErC50 (0–7 d): 2.95 mg/L
These results are 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). The index 50 indicates 50% growth inhibition compared to the control.

Reported statistics and error estimates:

Data Assessment and Statistical Evaluation

Growth inhibition is expressed as the effect of the test item concentration that reduced the frond number compared to the control by a certain percentage. The biological results, i.e. average specific growth rate (µ), and yield (Y), based on fronds numbers, and dry weight, respectively, are evaluated statistically. The statistical methods chosen are recorded.
The analytically measured concentrations are assessed in relation to the nominal concentrations. The biological endpoints are expressed based on nominal concentrations. If the measured concentrations deviate from the nominal concentrations by more than 20%, the endpoints are expressed additionally based on measured concentrations; the method use to correct the biological endpoints is reported.

The data were evaluated for normal distribution by Shapiro-Wilk’s test and for homogeneity of variances by Levene's Test.
Williams multiple sequential t-test for homogeneous variances and Welch-t test for inhomogeneous variances were applied to find out whether there were significant differences between the control and the various test item concentrations with regard to frond number (yield and growth rate) and biomass (yield and growth rate). The 3-parametric logistic CDF and the 3-parametric normal CDF Probit analyses were used to determine the concentration-response function.
The statistical software package ToxRat 3.2 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.
Further software packages were used for calculations: Excel 2010, 2013 & 2016, Microsoft GmbH, Unterschleißheim, Germany

Validity criteria fulfilled:

yes

Remarks:

Doubling time of the frond number in the control (exposure period): 1.9 days (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:

An OCED 221 study was carried out to determine the effects of Direct Yellow 86 on the growth of the freshwater aquatic plant Lemna minor. After 7 days of exposure an EC50 value of > 100 mg/L (front number/biomass, growth rate), an EC10 value of 6.55 mg/L (front number, growth rate), an EC10 value of 1.01 mg/L (biomass, growth rate) and a NOEC of 7.3 (frond number, growth rate) were determined.

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 Direct Yellow 86. Lemna minor is a model organism for aquatic fresh water plants.

The aim of the study was to determine the effects of Direct Yellow 86 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 4.95, 10.9, 24.0, 52.7, 116 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 (temperature, illumination).

The data obtained were analysed using a regression model in order to estimate the concentration that would cause a x% growth inhibition, i.e. ECx (e.g. EC50, EC20 or EC10). As additional endpoints the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) will be reported if relevant.

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 97–102 % and decrease after 7 days exposure at between 40–100 % 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.

All concentration levels were measured, and the biological results are additionally calculated and reported based on geometric mean measured concentrations at each concentration level.

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) front number is the primary measurement variable. For the measured variable 'front number growth rate' the endpoints EC50 >100 mg Direct Yellow 86/L, EC10 6.55 mg Direct Yellow 86/L and NOEC 7.3 mg Direct Yellow 86/L were obtained based on geometric mean measured concentrations.

For the measured variable biomass growth rate the endpoints EC50 >100 mg Direct Yellow 86/L, EC10=1.01 mg Direct Yellow 86/L and NOEC < 2.73 mg Direct Yellow 86/L were obtained based on geometric mean measured concentrations.

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

An OCED 221 study was carried out to determine the effects of Direct Yellow 86 on the growth of the freshwater aquatic plant Lemna minor. After 7 days of exposure an EC50 value of > 100 mg/L (front number/biomass, growth rate), an EC10 value of 6.55 mg/L (front number, growth rate), an EC10 value of 1.01 mg/L (biomass, growth rate) and an a NOEC of 7.3 (frond number, growth rate) were determined.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

1.01 mg/L

Additional information

EC50 value should read "> 100 mg/L"; EC50 and NOEC refer to biomass, growth rate

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 Direct Yellow 86. Lemna minor is a model organism for aquatic fresh water plants.

The aim of the study was to determine the effects of Direct Yellow 86 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 4.95, 10.9, 24.0, 52.7, 116 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 (temperature, illumination).

The data obtained were analysed using a regression model in order to estimate the concentration that would cause a x% growth inhibition, i.e. ECx (e.g. EC50, EC20 or EC10). As additional endpoints the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) will be reported if relevant.

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 97–102 % and decrease after 7 days exposure at between 40–100 % 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.

All concentration levels were measured, and the biological results are additionally calculated and reported based on geometric mean measured concentrations at each concentration level.

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) front number is the primary measurement variable. For the measured variable 'front number growth rate' the endpoints EC50 >100 mg Direct Yellow 86/L, EC10 6.55 mg Direct Yellow 86/L and NOEC 7.3 mg Direct Yellow 86/L were obtained based on geometric mean measured concentrations.

For the measured variable biomass growth rate the endpoints EC50 >100 mg Direct Yellow 86/L, EC10 1.01 mg Direct Yellow 86/L and NOEC < 2.73 mg Direct Yellow 86/L were obtained based on geometric mean measured concentrations.

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.