Pentasodium bis[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-5-nitrophenyl)azo]naphthalene-2,7-disulphonato(4-)]chromate(5-)

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:

2012-11-09 to 2012-01-02

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

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 apart from one aged replicate of 32 mg/L, which was not taken at day 5.

Details on test solutions:

Test Concentrations: 100, 32, 10, 3.2 and 1.0 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: Before test start and before the test medium renewal test medium of the highest test concentration of nominal 100 mg/L was prepared by dissolving 108.6, 104.1 and 100.8 mg test item in 1086, 1041 and 1008 mL test water by intense stirring for 10 minutes. Then, adequate volumes were mixed into test water to obtain the desired test concentrations. The test media were prepared just before introduction of the Lemna (= start of the test) and each test medium renewal.
Appearance of the Test Item in Test Medium: The appearance of the test item in the test media was visually determined on each observation day in all test concentrations.

Test organisms (species):

Lemna gibba

Details on test organisms:

Species: Lemna gibba G 3
Origin: The fronds introduced in the test were taken from our 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 14 days under test conditions with weekly media exchange.

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 24 °C

pH:

The pH-values were measured in all freshly prepared and aged test media.
7.5 to 7.6 in the freshly prepared media;
8.5 to 8.7 in the aged test media

Nominal and measured concentrations:

nominal: 100, 32, 10, 3.2 and 1.0 mg test item/L and a control.
measured: 104, 32, 10, 3.2 and 1.0 mg test item/L and < LOD for the control

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-values were measured in all freshly prepared and aged test media.
7.5 to 7.6 in the freshly prepared media;
8.5 to 8.7 in the aged test media
Light Regime: Continuous illumination
Light Intensity: 8477 lux (mean value) with a range of 7660 to 9000 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 %.

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

95.2 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.:

78.2 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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

7.33 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.:

3.68 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.:

76.6 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.:

67.3 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.:

1.92 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.:

0.744 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.:

13.1 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.:

8.08 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.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

10 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 95.2 and 78.2 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be >100 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 1.92 and 0.744 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 13.1 and 8.08 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 3.2 and 10 mg test item/L, respectively.
Based on the results of the statistical evaluation, the 7-day NOEC for yield (frond number) is <1.0 mg test item/L with an associated LOEC 1.0 mg test item/L. However, all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. Therefore the 7-day NOEC for yield (frond number) determined directly from the data is 3.2 mg test item/L, with an associated LOEC of 10 mg test item/L.
The 7-day NOErC and the LOErC were determined to be 3.2 and 10 mg test item/L, respectively.

Analytical Results:
In the freshly prepared test media 103 % of the nominal test concentration was found (average of all test concentrations of nominal 1.0 to 100 mg test item/L). In the aged test media 95 % of the nominal value was determined (average of all test concentrations of nominal 1.0 to 100 mg test item/L). During the test the lemna were exposed to a mean of 99 % of nominal. Therefore, all reported results refer to nominal concentrations.

Reported statistics and error estimates:

Statistical Analysis:
The EC50/20/10 (the concentrations of the test item corresponding to 50, 20 and 10 % inhibition of growth rate (frond number and dry weight) and yield (frond number and dry weight) compared to the control), and 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 the Dunnett's t-test (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 the Dunnett's t-test (dry weight).
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Influence of FAT 40210/F TE on the Growth of Lemna gibba

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)	95.2	>100	78.2	> 100
95 % conf. limits	64.5 - >100	n.d.	46.1 - > 100	n.d.
EC20(7-day)	7.33	76.6	3.68	67.3
95 % conf. limits	4.21-10.8	56.7 - > 100	1.40 - 6.56	44.1 - > 100
EC10(7-day)	1.92	13.1	0.744	8.08
95 % conf. limits	0.754 -3.47	8.23 - 18.4	0.144 - 1.82	3.75 - 13.1
7-day NOEC	3.2	3.2	3.2	3.2
7-day LOEC	10	10	10.0	10.0

n.d.: could not be determined
Values referto nominal testconcentrations

Validity criteria fulfilled:

yes

Conclusions:

The 7-day ErC50 was calculated to be >100 and >100 mg test item/L for frond number and dry weight, respectively, while the 7-day NOErC and the LOErC were determined to be 3.2 and 10 mg test item/L, 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

The influence of FAT 40210/F TE on the growth of the freshwater plant Lemna gibba was assessed in a semi-static dose-response test.

The 7-day E_yC₅₀was calculated to be 95.2 and 78.2 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀was calculated to be >100 and > 100 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be 3.2 and 10 mg test item/L, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be 3.2 and 10 mg test item/L, respectively. This study is classified acceptable and satisfies the guideline requirements for aquatic plant studies.

Description of key information

The influence of FAT 40210/F TE on the growth of the freshwater plant Lemna gibba was assessed in a semi-static dose-response test.

The 7-day EyC50was calculated to be 95.2 and 78.2 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC50was calculated to be >100 and > 100 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC were determined to be 3.2 and 10 mg test item/L, respectively.

The 7-day NOErC and the LOErC were determined to be 3.2 and 10 mg test item/L,

Key value for chemical safety assessment

EC50 for freshwater plants:

78.2 mg/L

EC10 or NOEC for freshwater plants:

3.2 mg/L