hexasodium 4-[3-(2-bromoprop-2-enamido)benzamido]-6-[(E)-2-[5-(2,3-dibromopropanamido)-2-sulfophenyl]diazen-1-yl]-5-hydroxynaphthalene-1,7-disulfonate 6-[(E)-2-[5-(2-bromoprop-2-enamido)-2-sulfophenyl]diazen-1-yl]-4-[3-(2-bromoprop-2-enamido)benzamido]-5-hydroxynaphthalene-1,7-disulfonate

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:

2014-09-26 to 2014-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Guideline followed

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

On sample from the stock solution and 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.
Additional samples of the control and the dilution solvent were taken at test start and test end without any sample preparation.

Details on test solutions:

Test Concentrations: 50, 16, 4.9, 1.5, 0.48, 0.15 and 0.047 mg test item/L and a control.

Control: In the control, test medium was used without addition of the test item.

Dosage of Test Item: A stock solution of nominal 100 mg test item/L was prepared by dissolving 62.6 mg test item into 626 mL test water by intense stirring for 60 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media. The test media were prepared just before introduction of the Lemna (= start of the test).

Appearance of the Test Item in Test Medium:
The test item caused colouration of the test media in the concentrations higher and including 0.48 mg test item/L.

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 7 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 25 °C

pH:

pH-Values: The pH-value of each treatment was measured at test start and at test end.
7.5 to 7.9 at test start;
8.6 to 9.0 at test end

Nominal and measured concentrations:

Test Concentrations: 50, 16, 4.9, 1.5, 0.48, 0.15 and 0.047 mg test item/L 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-value of each treatment was measured at test start and at test end.
7.5 to 7.9 at test start;
8.6 to 9.0 at test end
Light Regime: Continuous illumination
Light Intensity: 7727 Lux (mean value) with a range of 7100 to 8480 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.:

9.97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

nominal

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.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.734 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

8.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

19.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.248 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.104 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

2.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Biological Results
Growth Inhibition: The 7-day EyC50 was calculated to be 9.97 and 31.0 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 50 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 0.248 and 0.104 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 2.05 and 1.82 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 0.15 and 0.48 mg test item/L for frond number and 0.48 and 1.5 mg test item/L for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.15 and 0.48 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 nominal test concentration of 1.5 mg test item/L.
At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days; i.e. slightly necrosis (4.9 and 16 mg test item/L) and separated fronds (50 mg test item/L).



Analytical Results:
At the start of the test 101 % of the nominal test concentration was found (average of all test concentrations from 0.48 up to 50 mg test item/L). After 7 days test duration, 92 % of the nominal value was determined (average of all test concentrations from 0.48 up to 50 mg test item/L). During the test the Lemna were exposed to a mean of 97 % of nominal. Therefore, all reported results refer to nominal 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:

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 Bonferroni-Welch 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 Bonferroni-Welch t-test (frondnumber) and Williams t-test (dry weight).
The software used to perform the statistical analysis was ToxRatProfessional, Version 2.10.05, ToxRat Solutions GmbH.

. Influence of FAT 92348/B TE on the Growth ofLemna 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)	9.97	> 50	31.0	> 50
95 % conf. limits	7.31 - 14.1	n.d.	20.2 - > 50	n.d.
EC20(7-day)	0.880	8.02	0.734	19.9
95 % conf. limits	0.502 - 1.34	6.14 - 10.1	0.408 - 1.15	15.0 - 27.6
EC10(7-day)	0.248	2.05	0.104	1.82
95 % conf. limits	0.109 - 0.443	1.27 - 2.94	< 0.047 - 0.207	1.14 - 2.61
7-day NOEC	0.15	0.15	0.48	0.15
7-day LOEC	0.48	0.48	1.5	0.48

n.d.: not determinable
Values refer to nominal test concentrations

Summary of Analytical Results

[mg test item/L]	nominal^1	[%]	n
control	n.a.	n.a.	2
0.15*	133	9	4
0.48	101	22	3
1.5	88	10	4
4.9	96	10	4
16	101	13	4
50	99	3	4

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*NOEC was below the limit of quantification (= 0.3 mg test item/L)

Validity criteria fulfilled:

yes

Conclusions:

The 7-day ErC50 was calculated to be > 50 mg test item/L for frond number and dry weight.

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

Biological Results:

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 nominal test concentration of 1.5 mg test item/L.

At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days;i.e.slightly necrosis (4.9 and 16 mg test item/L) and separated fronds (50 mg test item/L).

Analytical Results: At the start of the test 101 % of the nominal test concentration was found (average of all test concentrations from 0.48 mg to 50 mg test item/L). After 7 days test duration, 92 % of the nominal value was determined (average of all test concentrations from 0.48 mg to 50 mg test item/L). During the test the Lemna were exposed to a mean of 97 % of nominal. Therefore, all reported results refer to nominal concentrations.

Conclusion:

The influence of FAT 92348/B 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 9.97 and 31.0 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀was calculated to be >50 mg test item/L for frond number and dry weight. The 7-day NOE_yC and the LOE_yC were determined to be 0.15 and 0.48 mg test item/L for frond number and 0.48 and 1.5 mg test item/L for dry weight, respectively

The 7-day NOE_rC and the LOE_rC were determined to be 0.15 and 0.48 mg test item/L for frond number and dry weight, respectively.

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

Description of key information

The 7-day ErC50 was calculated to be >50 mg/L for growth rate based on frond number.

Key value for chemical safety assessment

EC50 for freshwater plants:

50 mg/L

EC10 or NOEC for freshwater plants:

0.15 mg/L

Additional information

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 Method C. 26.

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 nominal test concentration of 1.5 mg test item/L.

At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days;i.e.slightly necrosis (4.9 and 16 mg test item/L) and separated fronds (50 mg test item/L).

Analytical Results: At the start of the test 101 % of the nominal test concentration was found (average of all test concentrations from 0.48 mg to 50 mg test item/L). After 7 days test duration, 92 % of the nominal value was determined (average of all test concentrations from 0.48 mg to 50 mg test item/L). During the test the Lemna were exposed to a mean of 97 % of nominal. Therefore, all reported results refer to nominal concentrations.

Conclusion:

The influence of FAT 92348/B TE on the growth of the freshwater plant Lemna gibba was assessed in a static dose-response test.

The 7-day E_yC₅₀was calculated to be 9.97 and 31.0 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀was calculated to be >50 mg test item/L for frond number and dry weight. The 7-day NOE_yC and the LOE_yC were determined to be 0.15 and 0.48 mg test item/L for frond number and 0.48 and 1.5 mg test item/L for dry weight, respectively

The 7-day NOE_rC and the LOE_rC were determined to be 0.15 and 0.48 mg test item/L for frond number and dry weight, respectively.