Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

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:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The read across approach has been detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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, the control and the test item concentrations 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 and all test item concentrations.
- Volume of sample: samples of a defined volume (10 ml) were taken from the designated test solution.
- Stabilization of samples: 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).
- storage: 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.

Vehicle:

no

Details on test solutions:

Preparation of the Stock and Test Solutions
At the day of the test start a stock solution 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 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:

TEST ORGANISM
- Species: Lemna minor.
- Source: cultured at ECT Oekotoxikologie GmbH since May 07, 2014. The organisms were supplied by Friedrich-Schiller-Universität, Jena.

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.
- 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: 24 h light / 0 h 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

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:

0, 4.95, 10.9, 24.0, 52.7, 116 mg/l, nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 ml crystallising dishes covered by watch glasses.
- No. of colonies per test vessel at the beginning of the test: 4
- No. of fronds per test vessel at the beginning of the test: 12
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates.

GROWTH MEDIUM
- Standard medium used: yes; Modified Steinberg medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Modified Steinberg medium.
- pH: pH of untreated test medium at test start 5.6.

OTHER TEST CONDITIONS
- Adjustment of pH: none.
- Photoperiod: permanet light, fluorescent tubes of universal white type (Osram L 18W/840, cool white).
- Light intensity: mean 103.3 µE m-2 s-1.

EFFECT PARAMETERS MEASURED
- Biological parameters: inhibition of growth in relation to control (frond number and dry weight; growth rate and yield).
- Determination of frond number: the frond number per replicate was counted and recorded on days 0, 3 and 5 and at the end of the test. Any changes in plant development in comparison to the control (frond size, appearance, necrosis, chlorosis, gibbosity, colony break up or loss of buoyancy, root length, morphology) were recorded. Additionally, any significant features of the test medium were recorded.
- Determination of dry weight: the dry weight of the plants was determined at the beginning of the test for a representative sample of the plants from the pre-culture vessels and at the end of the test for each replicate. For this, the colonies were collected using soft steel forceps and rinsed with demineralised water. The plants were placed in pre-weighed containers (made of stainless steel) and dried for at least 12 hours at a setting of 60 °C in a drying cabinet. The dry weight was measured after cooling in a desiccator to an accuracy of 0.1 mg.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

NOErC (7d): 7.3 mg/l (meas. geom. mean), based on frond number
NOErC (7d) < 2.73 mg/l (meas. geom. mean), based on biomass
ErC10 (7d): 6.55 mg/l (meas. geom. mean), based on frond number
ErC10 (7d): 1.01 mg/l (meas. geom. mean), based on biomass

MEASURED CONCENTRATIONS
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:

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.

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:

ErC50 (7d) > 100 mg/l (meas. geom. mean), based on the frond number
ErC50 (7d) > 100 mg/l (meas. geom. mean), based on the biomass

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 test item. Lemna minor is a model organism for aquatic fresh water plants.

The aim of the study was to determine the effects of the substance on the growth of the freshwater aquatic plant. 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).

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) frond number is the primary measurement variable. For the measured variable 'front number growth rate' the endpoints vales resulted to be ErC50 > 100 mg/l, ErC10 equal to 6.55 mg/l and NOErC equal to 7.3 mg/l were obtained based on geometric mean measured concentrations. For the measured variable biomass growth rate the endpoints vales resulted to be ErC50 >100 mg/l, ErC10 equal to 1.01 mg/l and NOErC < 2.73 mg/l were obtained based on geometric mean measured concentrations.

All validity criteria are met, thus, the study can be considered as acceptable and satisfying the guideline requirements.

Conclusion

ErC50 (7d) > 100 mg/l (meas. geom. mean), based on the frond number

ErC50 (7d) > 100 mg/l (meas. geom. mean), based on the biomass

Description of key information

Non harmful/toxic to aquatic plants other than algae (ErC50 (7d) > 100 mg/l (meas. geom. mean)).

Additional information

There is no information about the potential toxicity to aquatic plants other than algae of Direct Yellow 142, thus the available data on structural analogous Similar Substance 02 has been taken into account. The read across approach can be considered as reliable and adequate for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.

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 test item. 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).

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, all concentration levels were measured and the biological results are additionally calculated and reported based on geometric mean measured concentrations.

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. For the measured variable 'front number growth rate' the endpoints vales resulted to be ErC50 > 100 mg/l, ErC10 equal to 6.55 mg/l and NOErC equal to 7.3 mg/l were obtained based on geometric mean measured concentrations. For the measured variable biomass growth rate the endpoints vales resulted to be ErC50 >100 mg/l, ErC10 equal to 1.01 mg/l and NOErC < 2.73 mg/l were obtained based on geometric mean measured concentrations.

All validity criteria are met, thus, the study can be considered as acceptable and satisfying the guideline requirements.