Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

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

Study period:

2018

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)

Deviations:

no

Principles of method if other than guideline:

Duckweed is a model organism for aquatic fresh water plants. The purpose of this test is to determine the effects of a test item on the growth of Lemna minor during a test period of 7 days. The test endpoint is inhibition of growth, expressed as logarithmic increase in measurement variable (average specific growth rate of frond number and frond area) during the exposure period. 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 ECx values are determined.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Water solubility: 240 g/L at 20°C
Arrival: 12.10.2017
Stability: The test item was found to be stable and homogeneously prepared.
Volatility: negligible
Appearance: red brown powder
Storage conditions: Room temperature, closed container
Expiration date: 17 June 2018
Safety instructions: Not dangerous

Analytical monitoring:

yes

Details on sampling:

Storage conditions: Room temperature, closed container

Vehicle:

no

Details on test solutions:

Preparation of the test, positive and negative control vessels
According to the results obtained in a range-finding pre-test the study was performed as a static test in five test concentrations with a spacing factor of 3.2.
Based on the analytical report the purity of the test item was determined to be 86.3%.
Therefore a stock solution with 115.9 mg/L test item was prepared to achieve a test concentration of 100 mg/L Bayscript Gelbkomponente in the highest test concentration.
The stock solution was prepared by adding 115.9 mg test item to 1000 mL test medium (Steinberg medium) and stirring for 12 min. using a magnetic stirrer at room temperature (25.8°C).
Five nominal test item concentrations of 115.9 mg/L, 36.2 mg/L, 11.3 mg/L, 3.5 mg/L and 1.1 mg/L (spacing factor of 3.2) was prepared by diluting the stock solution with test medium. This corresponds to an active ingredient concentration of 100 mg/L, 31.3 mg/L, 9.8 mg/L, 3.1 mg/L and 1.0 mg/L. For the control only test medium was used.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: duckweed
- Source (laboratory, culture collection): German Federal Environment Agency (UBA), Berlin-Marienfelde.

Test system and pre-culture
Tests were carried out with the duckweed Lemna minor obtained from the German Federal Environment Agency (UBA), Berlin-Marienfelde. The sterile permanent stock culture is grown on agar medium prepared according to Jungnickel & Augsten (Details see Annex I) in 100 mL Erlenmeyer flasks sealed with cellulose stoppers at room temperature. After a maximum period of 2 months the plants are transferred sterile into freshly prepared medium. The stock culture used in the test has been cultivated in the labor of Hydrotox since March 2014.
21 days before the start of the test plant colonies are transferred from the sterile agar stock culture into 150 mL beakers containing 100 mL Steinberg Medium (see 5.1.3). After 7 and 14 days they are transferred into fresh medium and grown until using for the test. This pre-culture is cultivated under test conditions (24°C ± 2°C, 85 – 135 µE m-2 s-1). At least 15 beakers were prepared to gain enough plant material for the test. Colonies consisting of 2 to 4 visible fronds are transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions.
A quality check takes place at regular intervals using a concentration range of 3,5-dichlorophenol (Sigma-Aldrich, Steinheim, Germany, Lot No. MKBS0425V, from September 2015) in order to assess the validity of the test system. The last quality check was performed in October 2017. The EC50 for the frond number was 3.0 mg/L (2.4 – 3.9 mg/L 95%-CI). This value is in-line with the requirements of the ISO 20079 (2005) [Lemna, Growth Inhibition Test] of 2.2 –3.8 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24.5 to 25.0°C

pH:

at start: 5.6 , after 7 days: 6.5 - 6.6

Nominal and measured concentrations:

115.9 mg/L, 36.2 mg/L, 11.3 mg/L, 3.5 mg/L and 1.1 mg/L. This corresponds to an active ingredient concentration of 100 mg/L, 31.3 mg/L, 9.8 mg/L, 3.1 mg/L and 1.0 mg/L.

Details on test conditions:

Conditions of exposure
The test vessels were placed in an illuminated shelf which is located in a temperature controlled laboratory. Four fluorescent tubes (Osram L36W/840, CoolWhite) were placed with a distance of 50 cm above the test vessels to obtain a light intensity of 104 µE/m² s +/- 8.4 % (PAR). Illumination took place permanently. Temperature was in a range of 24.5 to 25.0°C during exposure time. Two times during the seven-day exposure period locations of the test vessels were randomised to minimise the influence of spatial differences in light intensity or temperature.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorphenol

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

114.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 53.6 - 245.6 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

97.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Yield, Frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

28.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 15.2 - 53.5 mg/L

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

32.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidence limit: 11.3 - 91.6 mg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Details on results:

The measured test item concentrations in the test item treatments were 95.3 – 113.5 % of the nominal concentrations. Therefore the EC values are given as nominal concentrations.

Growth rate and Yield of frond number and frond area
With the five test item treatments tested between 1.1 mg/L and 115.9 mg/L (active ingredient concentration between 1.0 mg/L and 100 mg/L) inhibitions of the growth rates of the frond number could be observed with a maximum of 9.8 % in the highest test item treatment. For the growth rates of the frond area a maximum inhibition of 20.6 % could be measured also in the highest test item treatment.

Effect concentrations
The effect concentrations after 7 d exposure are calculated with the statistical software ToxRat Professional 3.2.1. As the measured concentrations are within ± 20 % of the nominal concentrations, according to OECD 221 (2006), all results are given in relation to the nominal concentrations. On behalf of the sponsor the EC values are given as the active ingredient concentration of Bayscript Gelbkomponente. Although a statistical determination of EC50 values based on growth rate was not possible, the results depicted in Table 2a and 2b clearly demonstrate that EC50 values according to frond number or frond area inevitably lie above a concentration of 100 mg/L.

Results with reference substance (positive control):

A quality check check takes place at regular intervals using a concentration range of 3.5-dichlorophenol (Sigma-Aldrich, Steinheim, Germany, Lot No. MKBS0425V, from September 2015) in order to assess the validity of the test system. The last quality check was performed in October 2017. The EC50 for the frond number was 3.0 mg/L (2.4 - 3.9 mg/L 95% Cl). This value is in-line with the requirements of the ISO 20079 (2005) [Lemna, Growth Inhibition Test] of 2.2-3.8 mg/L.

Reported statistics and error estimates:

Statistical differences between the growth rate of the test and the control vessels were checked with the software Toxrat (ToxRat Professional, version 3.2.1, ToxRat Solutions GmbH, Alsdorf).

Validity criteria fulfilled:

yes

Remarks:

Doubling time of the frond number in the controls must be less than 2.5 days (within a period of 60 hours). This corresponds approximately to a seven-fold increase within the seven-day test duration and an average specific growth rate of 0.275 per day

Conclusions:

Bayscript Gelbkomponente shows an EC50 value of > 100 mg/L (growth rate, frond number and frond area) on a freshwater aquatic plant Lemna minor.

Executive summary:

A growth inhibition test with the freshwater aquatic plant Lemna minor was conducted according to OECD 221 (23 March 2006) in order to investigate the aquatic phytotoxic effect of Bayscript Gelbkomponente.

Duckweed is a model organism for aquatic fresh water plants. The purpose of this test is to determine the effects of a test item on the growth of Lemna minor during a test period of 7 days. The test endpoint is inhibition of growth, expressed as logarithmic increase in measurement variable (average specific growth rate of frond number and frond area) during the exposure period. 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 ECx values are determined.

The EC values are given as the nominal active ingredient concentration of Bayscript Gelbkomponente. Results of frond number and frond area are:

(Lowest/ No observed) Effect Concentration	Nominal active ingredient concentration [mg/L]
	Frond number		Frond area
Cl: confidence limit	Growth rate	Yield	Growth rate	Yield
EC10 (Cl 95%)	114.7 mg/L (53.6 - 245.6 mg/L)	97.7 mg/L	28.5 mg/L (15.2 - 53.5 mg/L)	32.2 mg/L (11.3 - 91.6 mg/L)
EC50 (Cl 95 %)	> 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L
LOEC	100 mg/L	100 mg/L	9.8 mg/L	100 mg/L
NOEC	31.3 mg/L	31.3 mg/L	3.1 mg/L	31.3 mg/L

n.d.: not determined due to mathematical reason or inappropriate data.

The measured test item concentrations in the test item treatments were 95.3 - 113.5 % of the nominal concentrations. Therefore the EC values are given as nominal concentrations.

The pH-values measured at the beginning and at the end of the test. A slight pH increase of a maximum of 1.0 unit occurred which can often be observed in the Lemna Growth Inhibition Test.

The test is valid as the following criterion is fulfilled: Doubling time of the frond number in the controls must be less than 2.5 days (within a period of 60 hours). This corresponds approximately to a seven-fold increase within the seven-day test duration and an average specific growth rate of 0.275 per day.

Description of key information

A growth inhibition test with the freshwater aquatic plant Lemna minor was conducted according to OECD 221 (23 March 2006) in order to investigate the aquatic phytotoxic effect of Bayscript Gelbkomponente.

Duckweed is a model organism for aquatic fresh water plants. The purpose of this test is to determine the effects of a test item on the growth of Lemna minor during a test period of 7 days. The test endpoint is inhibition of growth, expressed as logarithmic increase in measurement variable (average specific growth rate of frond number and frond area) during the exposure period. 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 ECx values are determined.

The EC values are given as the nominal active ingredient concentration of Bayscript Gelbkomponente. Results of frond number and frond area are:

(Lowest/ No observed) Effect Concentration	Nominal active ingredient concentration [mg/L]
	Frond number		Frond area
Cl: confidence limit	Growth rate	Yield	Growth rate	Yield
EC10 (Cl 95%)	114.7 mg/L (53.6 - 245.6 mg/L)	97.7 mg/L	28.5 mg/L (15.2 - 53.5 mg/L)	32.2 mg/L (11.3 - 91.6 mg/L)
EC50 (Cl 95 %)	> 100 mg/L	104.8 mg/L (n.d.)	> 100 mg/L	232.5 mg/L (32.7 - 1510.5 mg/L)
LOEC	100 mg/L	100 mg/L	9.8 mg/L	100 mg/L
NOEC	31.3 mg/L	31.3 mg/L	3.1 mg/L	31.3 mg/L

n.d.: not determined due to mathematical reason or inappropriate data.

The measured test item concentrations in the test item treatments were 95.3 - 113.5 % of the nominal concentrations. Therefore the EC values are given as nominal concentrations.

The pH-values measured at the beginning and at the end of the test. A slight pH increase of a maximum of 1.0 unit occurred which can often be observed in the Lemna Growth Inhibition Test.

The test is valid as the following criterion is fulfilled: Doubling time of the frond number in the controls must be less than 2.5 days (within a period of 60 hours). This corresponds approximately to a seven-fold increase within the seven-day test duration and an average specific growth rate of 0.275 per day.

Key value for chemical safety assessment

EC50 for freshwater plants:

100 mg/L

EC10 or NOEC for freshwater plants:

114.7 mg/L

Additional information

EC50 result should read "> 100 mg/L" referring to growth rate, frond number