Hydrogen [4-[4-(p-ethoxyanilino)-4'-[ethyl(m-sulphonatobenzyl)amino]-2'-methylbenzhydrylene]-3-methylcyclohexa-2,5-dien-1-ylidene](ethyl)(m-sulphonatobenzyl)ammonium, monosodium 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:

experimental study

Adequacy of study:

key study

Study period:

2014-09-18 to 2014-11-14

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples were taken at the start and at each test medium renewal from the freshly prepared test media of all test concentrations and from the control.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the renewal periods, duplicate samples of all aged test media and control were sampled at each test medium renewal at the end of the test by pouring together the contents of each treatment.

Details on test solutions:

- Pre-Experiments: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
- Test Concentrations: Filtrate of 100 mg test item/L, 1:3, 1:9, 1:27 and 1:81 dilutions, corresponding to 17.6, 5.13 and 1.74 mg test item/L for the filtrate and the two highest dilutions and a control.
- Control: In the control, test medium was used without addition of the test item.
- Dosage of Test Item: A stock suspension of 100 mg test item/L was prepared by suspending 99.6, 99.0 and 98.1 mg test item in 996, 990 and 981 mL. The stock suspension was stirred for 24 hours in the dark to - dissolve as much test item as possible. Then, the undissolved test item was separated by filtration (0.45 µm cellulose nitrate filter) and the required test concentrations were prepared. The test concentrations were prepared just before introduction of the Lemna (= start of the test).
- Appearance of the Test Item in Test Medium: The test item caused a colouration of the test media of all test concentrations and precipitated a part of the aged test media.

Test organisms (species):

Lemna gibba

Details on test organisms:

- Species: Lemna gibba G 3
- Origin: The fronds introduced in the test were taken from IBACON's in-house laboratory culture.
- Breeding Conditions: The daphnids were bred in the laboratories of IBACON under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in the same reconstituted water of a similar quality with regards to pH, the constituent salts and total hardness as the test water used in the test. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of IBACON.
- Pre-Culture: For at least 7 days under test conditions with weekly media exchange.
- Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

The temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.
23 to 25 °C

pH:

The pH-values were measured in all freshly prepared and aged test media.
7.4 to 8.0 in the freshly prepared media;
8.4 to 8.9 in the aged test media

Nominal and measured concentrations:

Test Concentrations: Filtrate of 100 mg test item/L, 1:3, 1:9, 1:27 and 1:81 dilutions, corresponding to 17.6, 5.13 and 1.74 mg test item/L for the filtrate and the two highest dilutions and a control.
Control: In the control, test medium was used without addition of the test item.

Details on test conditions:

Test Environment: Controlled environment room
Water Temperature: The temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.
23 to 25 °C
pH-Values: The pH-values were measured in all freshly prepared and aged test media.
7.4 to 8.0 in the freshly prepared media;
8.4 to 8.9 in the aged test media
Light Regime: Continuous illumination
Light Intensity: 8593 Lux (mean value) with a range of 8160 to 8960 lux
The illumination was achieved by fluorescent tubes, installed above the test vessels. Measurements were performed once during the test at 6 places distributed over the experimental area at the surface of the test media. Differences in light intensity over the test area did not exceed ± 15 %.

Reference substance (positive control):

yes

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

5.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

6.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 17.6 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.:

> 17.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

1.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

1.58 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

5.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

5.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.449 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.781 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2.74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.193 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.:

0.193 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.193 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.579 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.579 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.579 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.579 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1.74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Biological Results
Growth Inhibition: The 7-day EyC50 was calculated to be 5.67 and 6.05 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 17.6 and > 17.6 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 0.449 and 0.781 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 2.03 and 2.74 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 0.193 and 0.579 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.193 and 0.579 mg test item/L for frond number and 0.579 and 1.74 mg test item/L for dry weight, respectively

Shape of Fronds: The shape of fronds and colonies after the test period of 7 days was not different to those in the control in the lowest concentration. At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days; i.e. coloured roots (0.579, 1.74, 5.13 and 17.6 mg test item/L), overgrowing fronds and chlorosis (5.13 mg test item/L) gibbous growth, smaller fronds and coloured fronds (17.6 mg test item/L).

Analytical Results:
In the freshly prepared test media 2.216, 5.751 and 19.050 mg test item/L were found (average of the three highest test concentrations, all other values were below the Limit of Quantification or Detection). In the aged test media 1.257, 4.843 and 16.721 mg test item/L were found (average of the three highest test concentrations, all other values were below the Limit of Quantification or Detection).
Since the test item was not well soluble in test water and a decrease of test item concentration was determined during the exposure period in all test media, the biological results are related to geometric mean concentrations.

Results with reference substance (positive control):

Results of the most recent Test with the Reference Item 3,5 Dichlorophenol performed in August to September 2014 (IBACON Project No.: 88302240)
Parameter Yield (frond #) Growth rate (frond #) Yield (dry wt) Growth rate (dry wt)
[mg/L] [mg/L] [mg/L] [mg/L]
EC50 (7-day) 6.24 10.0 6.67 9.37
95 % conf. limits 4.87 - 7.99 7.82 - 12.8 5.55 - 8.06 8.43 - 10.4

EC20 (7-day) 3.43 6.68 4.48 6.46
95 % conf. limits 1.96 - 4.47 3.21 - 8.35 3.02 - 5.41 5.05 - 7.38

EC10 (7-day) 2.50 5.41 3.64 5.32
95 % conf. limits 1.15 - 3.51 1.88 - 7.18 2.10 - 4.60 3.77 - 6.34

7-day NOEC 0.625 1.25 2.5 2.5
7-day LOEC 1.25 2.5 5.0 5.0
Values refer to nominal test concentrations

Reported statistics and error estimates:

Statistical Analysis:
The ErC50 and the EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 7-day LOEyC and NOEyC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (frond number and dry weight).
For the determination of the 7-day LOErC and NOErC values significant differences at the test concentrations compared to the control values were tested by the Williams t-test (frond number and dry weight).
For the statistical evaluation, the two lowest concentrations were extrapolated by dividing the lowest measured concentration (0.174 mg test item/L) by the appropriate dilution factor.
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat(C) Solutions GmbH.

Influence of FAT 20085/B TE on the Growth of Lemna gibba

Parameter	Yield (frond number) [mg test item/L]	Growth rate (frond number) [mg test item/L]	Yield (dry weight) [mg test item/L]	Growth rate (dry weight) [mg test item/L]
EC50 (7-day)	5.67	> 17.6	6.05	> 17.6
95 % conf. limits	4.59 - 7.17	n.d.	5.10 - 7.26	n.d.
EC20 (7-day)	1.07	5.28	1.58	5.69
95 % conf. limits	0.734 - 1.43	4.34 - 6.22	1.17 - 1.99	4.85 - 6.49
EC10 (7-day)	0.449	2.03	0.781	2.74
95 % conf. limits	0.260 - 0.665	1.44 - 2.63	0.510 - 1.07	2.11 - 3.36
7-day NOEC	0.193	0.193	0.193	0.579
7-day LOEC	0.579	0.579	0.579	1.74

n.d.: not determinable
Values refer to geometric mean measured test concentrations

Summary of Analytical Results

sample description	geometric mean
	[mg/L]^1	n
control	n.a.	12
1:81 dilution	< LOQ	12
1:27 dilution	< LOQ	12
1:9 dilution	1.74	12
1:3 dilution	5.13	12
Filtrate of 100 mg/L	17.6	12

¹geometric mean value of all arithmetic mean values per treatment group and sampling time
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable

¹mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable

Validity criteria fulfilled:

yes

Conclusions:

The influence of FAT 20085/B TE on the growth of the freshwater plant Lemna gibba was assessed in a semi-static dose-response test.
The 7-day EyC50 was calculated to be 5.67 and 6.05 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 17.6 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 0.193 and 0.579 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.193 and 0.579 mg test item/L for frond number and 0.579 and 1.74 mg test item/L for dry weight, respectively.

Executive summary:

The toxicity of the test item to the aquatic plant Lemna gibba in a growth inhibition test was determined based on the procedures indicated by the following internationally accepted methods:

- OECD 221 equivalent to EU C. 26

20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in 60 mL test medium were exposed to filtrate of 100 mg test item/L, 1:2, 1:4, 1:8 and 1:16 dilutions,

corresponding to 3.82, 1.65 and 0.829 mg test item/L for the three highest test concentrations and a control.

Biological Results:

Growth Inhibition:

The 7-day E_yC₅₀ was calculated to be5.67 and 6.05 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀ was calculated to be> 17.6 and >17.6 mg test item/L for frond number and dry weight, respectively.

The 7-day E_yC₁₀ was calculated to be 0.449 and 0.781 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₁₀ was calculated to be 2.03 and 2.74 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be 0.193 and 0.579 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be 0.193 and 0.579 mg test item/L for frond number and 0.579 and 1.74 mg test item/L for dry weight, respectively.

Shape of Fronds:

The shape of fronds and colonies after the test period of 7 days was not different to those in the control in the lowest concentration.

At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days;i.e.coloured roots (0.579, 1.74, 5.13 and 17.6 mg test item/L), overgrowing fronds and chlorosis (5.13 mg test item/L) gibbous growth, smaller fronds and coloured fronds (17.6mg test item/L).

Analytical Results: In the freshly prepared test media 2.216, 5.751 and 19.050 mg test item/L were found (average of the three highest test concentrations, all other values were below the Limit of Quantification or Detection). In the aged test media 1.257, 4.843 and 16.721 mgtest item/L were found (average of the three highest test concentrations, all other values were below the Limit of Quantification or Detection). Since the test item was not well soluble in test water and a decrease of test item concentration was determined during the exposure period in all test media, the biological results are related to geometric mean concentrations.

Conclusion:

The influence of FAT 20085/B TE on the growth of the freshwater plant Lemna gibba was assessed in a semi-static dose-response test.

The 7-day E_yC₅₀ was calculated to be 5.67 and 6.05 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀ was calculated to be >17.6 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be0.193 and 0.579 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be 0.193 and 0.579 mg test item/L for frond number and 0.579 and 1.74 mg test item/L for dry weight, respectively.

This study is classified acceptable and satisfies the guideline requirements for aquatic plant studies.

Description of key information

Toxicity of FAT 20085/B TE to the Aquatic Plant Lemna gibba in a Semi-Static Growth Inhibition Test.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

0.193 mg/L

Additional information

The influence of FAT 20085/B TE on the growth of the freshwater plant Lemna gibba was assessed in a semi-static dose-response test.

The 7-day E_yC₅₀ was calculated to be 5.67 and 6.05 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀ was calculated to be >17.6 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be 0.193 and 0.579 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be 0.193 and 0.579 mg test item/L for frond number and0.579 and 1.74 mg test item/L for dry weight, respectively.