Sodium 2-[[2-amino-8-hydroxy-6-[(methylanilino)sulphonyl]-1-naphthyl]azo]-5-(chloroacetamido)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:

experimental study

Adequacy of study:

key study

Study period:

18 November 2014 to 29 November 2014

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

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009, Annex, Part C, C.26.: "Lemna sp. Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: AT-0027506800
- Expiration date of the lot/batch: January 20, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (20 ± 5 °C) under dark and dry conditions

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
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 test period, duplicate samples were taken at the end of the test from all test media and the control of the actual test by pouring together the contents of each treatment.
All samples were diluted by a factor of 2 with acetonitrile directly after sampling.

Details on test solutions:

Test Concentrations: A filtrate of nominal 100 mg test item/L and dilutions of 1:2, 1:4, 1:8 and 1:16 and a control. The geometric mean measured test item concentration in the filtrate was 0.474 mg/L.
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 149.0 mg test item in 1490 mL test water. The stock solution was heated up at 80 °C and stirred for 24 hours at room temperature in the dark to dissolve as much test item as possible The pH of the stock solution was adjusted from 7.1 to 6.3 using 1 M HCl. Then, the undissolved test item was separated by filtration (0.65 µm ± 0.45µm; Sartobra) 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 in the filtrate and the dilutions of 1:2 and 1:4.

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 plants are cultured in the laboratories of IBACON under standardised conditions according to the test guidelines.
Pre-Culture: For at least 5 days under test conditions with weekly media exchange.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

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 24 °C

pH:

pH-Values: The pH-value of each treatment was measured at test start and at test end.
6.3 at test start;
7.1 to 7.4 at test end

Nominal and measured concentrations:

Test Concentrations: A filtrate of nominal 100 mg test item/L and dilutions of 1:2, 1:4, 1:8 and 1:16 and a control. The geometric mean measured test item concentration in the filtrate was 0.474 mg/L.
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 24 °C
pH-Values: The pH-value of each treatment was measured at test start and at test end.
6.3 at test start;
7.1 to 7.4 at test end
Light Regime: Continuous illumination
Light Intensity: 7190 Lux (mean value) with a range of 6700 to 8040 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.:

> 0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.474 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.:

> 0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.474 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.:

0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.474 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.:

0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.474 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.:

> 0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.474 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.:

> 0.474 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 > 0.474 mg test item/L for frond number and dry weight.
The 7-day ErC50 was calculated to be >0.474 mg test item/L for frond number and dry weight.
The 7-day EyC10 was calculated to be >0.474 mg test item/L for frond number and dry weight.
The 7-day ErC10 was calculated to be >0.474 mg test item/L for frond number and dry weight.
The 7-day NOEyC and the LOEyC were determined to be 0.474 and >0.474 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.474 and >0.474 mg test item/L for frond number and 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 up to and including the highest concentration tested (Filtrate of nominal 100 mg test item/L).

Analytical Results:
The concentrations of the test item were analysed in the duplicate test media samples from the highest test concentration (filtrate of test item saturated stock solution) and the duplicate control samples from both sampling times. A decrease of the test item concentrations was determined during the test period. Thus all reported results refer to geometric mean measured 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 number) Growth rate
(frond number) Yield
(dry weight) Growth rate
(dry weight)
[mg test item/L] [mg test item/L] [mg test item/L] [mg test item/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:

Based on frond number and dry weight, the 7-day ErC50 and the 72-hour EyC50 (see Definitions) could not be calculated by Probit analysis due to the lack of effects.
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).
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Influence of FAT 20075/C 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)	> 0.474	> 0.474	> 0.474	> 0.474
95 % conf. limits	n.d.	n.d.	n.d.	n.d.
EC10(7-day)	> 0.474	> 0.474	> 0.474	> 0.474
95 % conf. limits	n.d.	n.d.	n.d.	n.d.
7-day NOEC	0.474	0.474	0.474	0.474
7-day LOEC	> 0.474	> 0.474	> 0.474	> 0.474

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

Summary of Analytical Results

¹

sample description	geometric mean
[mg test item/L]	concentration [mg/L]	n
Control	<LOD	4
Filtrate of 100 mg/L	0.474	4

¹The geometric mean value was calculated using the mean value of freshly prepared and aged test media
n: number of analysed samples
LOD: limit of detection = 0.005 mg test item/L

Validity criteria fulfilled:

yes

Conclusions:

The 7-day EyC50 was calculated to be >0.474 mg test item/L for frond number and dry weight.
The 7-day ErC50 was calculated to be >0.474 mg test item/L for frond number and dry weight.
The 7-day NOEyC and the LOEyC were determined to be 0.474 and > 0.474 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.474 and > 0.474 mg test item/L for frond number and dry weight, respectively.

Executive summary:

Toxicity of FAT 20075/C TE to the Aquatic Plant Lemna gibba in a Static Growth Inhibition Test was carried out according to OECD guideline 221. The purpose of this test was to determine the inhibitory effect of the test item FAT 20075/C TE on the growth of the freshwater aquatic plant Lemna gibba. Cultures of Lemna gibba were exposed in a static test to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 7 days.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and control.

At test start 12 fronds were introduced in each replicate and incubated for 7 days under static conditions. The frond numbers were determined on day 2, 5 and 7. The dry weight of each replicate from test end was determined.

Yield and growth rate based on frond number and dry weight. A filtrate of nominal 100 mg test item/L and dilutions of 1:2, 1:4, 1:8 and 1:16 and a control. The geometric mean measured test item concentration in the filtrate was 0.474 mg/L.

 

Based on the study results, 7-day EyC₅₀was calculated to be > 0.474 mg test item/L for frond number and dry weight.

The 7-day ErC₅₀was calculated to be > 0.474 mg test item/L for frond number and dry weight.

The 7-day NOEyC and the LOEyC were determined to be 0.474 and > 0.474 mg test item/L for frond number and dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be 0.474 and > 0.474 mg test item/L for frond number and dry weight, respectively.

Key value for chemical safety assessment

EC50 for freshwater plants:

0.474 mg/L

EC10 or NOEC for freshwater plants:

0.474 mg/L

Additional information

Toxicity of FAT 20075/C TE to the Aquatic Plant Lemna gibba in a Static Growth Inhibition Test was carried out according to OECD guideline 221. The purpose of this test was to determine the inhibitory effect of the test item FAT 20075/C TE on the growth of the freshwater aquatic plant Lemna gibba. Cultures of Lemna gibba were exposed in a static test to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 7 days.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and control.

At test start 12 fronds were introduced in each replicate and incubated for 7 days under static conditions. The frond numbers were determined on day 2, 5 and 7. The dry weight of each replicate from test end was determined.

Yield and growth rate based on frond number and dry weight. A filtrate of nominal 100 mg test item/L and dilutions of 1:2, 1:4, 1:8 and 1:16 and a control. The geometric mean measured test item concentration in the filtrate was 0.474 mg/L.

 

The influence of FAT 20075/C TE on the growth of the freshwater plant Lemna gibba was assessed in a static dose-response test.

The 7-day E_yC₅₀ was calculated to be >0.474 mg test item/L for frond number and dry weight.

The 7-day E_rC₅₀ was calculated to be >0.474 mg test item/L for frond number and dry weight.

The 7-day NOE_yC and the LOE_yC were determined to be 0.474 and >0.474 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be 0.474 and >0.474 mg test item/L for frond number and dry weight, respectively.