Disodium 5-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4-hydroxy-6-(methylamino)naphthalene-2-sulphonate

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:

26 September 2014 to 24 October 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:

yes

Remarks:

The stock solution was stirred for 2 h, 15 and 10 minutes, respectively. No impact of this deviation on study outcome

Qualifier:

equivalent or similar to guideline

Guideline:

other: equivalent to Commission Regulation (EC) No 761/2009, C.26., 2009

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD Principles of Good Laboratory Practice Germany

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SBL/036/LR5B (India)
- Expiration date of the lot/batch: May 13, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the freezer (≤ -20 °C), under dark and dry conditions

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.

Details on test solutions:

Test Concentrations: 50, 16, 5.0, 1.6, 0.5, 0.16 and 0.05 mg test item/L and a control corresponding to following geometric mean measured concentrations:
2.85, 2.67, 2.71, 1.45, 0.483 and 0.129 mg test item/L.
Control: In the control, test medium was used without addition of the test item.
Dosage of Test Item: Before test start and before each test media renewal a stock solution of nominal 50 mg test item/L was prepared by dissolving 50.6, 52.9 and 50.6 mg test item in 1012, 1058 and 1012 mL test water by intense stirring for 2 hours, 15 and 10 minutes, respectively. 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) and each test medium renewal.
Appearance of the Test Item in Test Medium:
The test item caused pink coloration of the test media in all test concentrations beside the lowest tested concentration of 0.05 mg test item/L. The coloration increase with increasing test concentration.

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:

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 7.8 in the freshly prepared media;
8.2 to 9.0 in the aged test media

Nominal and measured concentrations:

Test Concentrations: 50, 16, 5.0, 1.6, 0.5, 0.16 and 0.05 mg test item/L and a control corresponding to following geometric mean measured concentrations:
2.85, 2.67, 2.71, 1.45, 0.483 and 0.129 mg test item/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-values were measured in all freshly prepared and aged test media.
7.5 to 7.8 in the freshly prepared media; 8.2 to 9.0 in the aged test media
Light Regime: Continuous illumination
Light Intensity: 8422 Lux (mean value) with a range of 7820 to 8930 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.:

32 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.:

30.8 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.:

> 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.:

7.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

9.81 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.:

27.9 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.:

30.8 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.:

3.45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

5.4 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.:

13.7 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.:

16.2 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:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Growth Inhibition: The 7-day EyC50 was calculated to be 32.0 and 30.8 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 50 and > 50 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 3.45 and 5.40 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 13.7 and 16.2 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 1.6 and 5.0 mg test item/L for frond number and 5.0 and 16 mg test item/L for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 1.6 and 5.0 mg test item/L for frond number and 5.0 and 16 mg test item/L for dry weight, respectively
The test item concentrations decrease during the test period. The reduction was not even for all concentrations, the higher test concentrations of nominal 5, 16 and 50 mg test item/L decrease more obvious than the lower concentrations. At the same time the biological effects show a good dose response relationship. Therefore, the geometric mean measured concentrations do not reflect the biological effects properly. It seems the biological are effects by the initial test item concentrations. For this reason, all result evaluations are based on the nominal concentrations.
Based on the results of the statistical evaluation, the 7-day NOEC for yield based on frond number and dry weight, and for growth rate based on dry weight is < 0.05 mg test item/L with an associated LOEC 0.05 mg test item/L. However, according to the Guideline all concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. It was therefore necessary to determine the NOECs and LOECs by visual inspection of the raw data. Therefore, the NOEC for yield based on frond number and dry weight, and for growth rate based on dry weight was determined to be 1.6 mg/L with a LOEC of 5.0 mg test item/L.


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 16 mg test item/L.
At the highest test item concentration of 50 mg test item/L the fronds showed deviations from the control replicates after 7 days; gibbous growth, shortened roots, slightly necrosis.


Analytical Results:
At the start of the test item renewal periods 102 % of the nominal test concentration was found (average of all test concentrations from 0.16 to 50 mg test item/L). At the end of the test item renewal periods 44 % of the nominal value was determined (average of all test concentrations from 0.16 to 50 mg test item/L). During the test the Lemna were exposed to a mean of 96 % of nominal.

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)
ParameterYield
(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 Dunnett's t-test (frond number) and the Bonferroni-Welch 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 Bonferroni-Welch 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 92354/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)	32.0	> 50	30.8	> 50
95 % conf. limits	10.1 - n.d.	n.d.	15.2 - > 50	n.d.
EC20(7-day)	7.41	27.9	9.81	30.8
95 % conf. limits	n.d.	9.42 - 44.2	0.276 - 18.6	18.9 - 40.9
EC10(7-day)	3.45	13.7	5.40	16.2
95 % conf. limits	n.d.	0.741 - 23.4	< 0.05 -12.2	5.07 - 23.9
7-day NOEC	1.6	1.6	5.0	5.0
7-day LOEC	5.0	5.0	16	16

n.d.: not determinable
Values referto nominal testconcentrations

Summary of Analytical Results

sample description	% of nominal
[mg test item/L]	initial mean^1	aged mean^2	geometric mean^3
control	n.a.	n.a.	n.a.
0.16	107	61	80
0.5	106	88	97
1.6	101	82	91
5	99	30	54
16	100	3	17
50	98	0	6

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 7 day EC50, EC10 and NOEC of FAT 92354/B for yield (fond numbers), biomass and groth rate are:

The 7-day EyC50 was calculated to be 32.0 and 30.8 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50was calculated to be > 50 and > 50 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10was calculated to be 3.45 and 5.40 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10was calculated to be13.7 and 16.2 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be1.6 and 5.0 mg test item/L for frond number and 5.0 and 16 mg test item/Lfor dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be1.6 and 5.0 mg test item/L for frond number and 5.0 and 16 mg test item/L for dry weight, respectively

Executive summary:

The toxicity of the FAT 92354/B to the aquatic plant Lemna gibba in a growth inhibition test was determined according to OECD 221 equivalent to EU C. 26 guidelines. This study encompassed 8 treatment groups (7 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 3, 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. Test Concentrations used were 50, 16, 5.0, 1.6, 0.5, 0.16 and 0.05 mg test item/L and a control corresponding to following geometric mean measured concentrations: 2.85, 2.67, 2.71, 1.45, 0.483 and 0.129 mg test item/L. Test Conditions during the test was -water temperature: 23 to 24 °C; pH values in fresh media 7.5 to 7.8, in aged media 8.2 to 9.0; continuous illumination; mean light intensity: 8422 lux (7820 to 8930 lux).

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 16mg test item/L.

At the highest test item concentration of 50 mg test item/L the fronds showed deviations from the control replicates after 7 days; gibbous growth, shortened roots, slightly necrosis.

Growth Inhibition:

The 7-day E_yC₅₀was calculated to be32.0 and30.8 mg test item/Lfor frond number and dry weight, respectively.

The 7-day E_rC₅₀was calculated to be > 50 and > 50 mg test item/L for frond number and dry weight, respectively.

The 7-day E_yC₁₀was calculated to be3.45 and5.40 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₁₀was calculated to be13.7 and16.2 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be1.6 and5.0 mg test item/L for frond number and5.0 and16 mg test item/Lfor dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be1.6 and5.0 mg test item/L for frond number and5.0 and16 mg test item/L for dry weight, respectively

The test item concentrations decrease during the test period. The reduction was not even for all concentrations, the higher test concentrations of nominal 5, 16 and 50 mg test item/L decrease more obvious than the lower concentrations. At the same time the biological effects show a good dose response relationship. Therefore, the geometric mean measured concentrations do not reflect the biological effects properly. It seems the biological are effects by the initial test item concentrations. For this reason, all result evaluations are based on the nominal concentrations. Based on the results of the statistical evaluation, the 7-day NOEC for yield based on frond number and dry weight, and for growth rate based on dry weight is < 0.05 mg test item/Lwith an associated LOEC 0.05 mg test item/L. However, according to the Guideline all concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC. It was therefore necessary to determine the NOECs and LOECs by visual inspection of the raw data. Therefore, the NOEC for yield based on frond number and dry weight, and for growth rate based on dry weight was determined to be 1.6 mg/L with a LOEC of 5.0 mg test item/L. This study is classified acceptable and satisfies the guideline requirements for aquatic plant studies.

Description of key information

The 7-day E_yC₅₀ was calculated to be 32.0 and 30.8 mg test item/L for frond number and dry weight, respectively.

Key value for chemical safety assessment

EC50 for freshwater plants:

30.8 mg/L

EC10 or NOEC for freshwater plants:

1.6 mg/L

Additional information

The influence of FAT 92354/B on the growth of the freshwater plant Lemna gibba was assessed in a semi-static dose-response test. The test item concentrations decrease during the test period. The reduction was not even for all concentrations, the higher test concentrations of nominal 5, 16, and 50 mg test item/L decrease more obvious than the lower concentrations. At the same time, the biological effects show a good dose-response relationship. It seems the biological are effects by the initial test item concentrations. Therefore, the geometric mean measured concentrations do not reflect the biological effects properly. For this reason, all result evaluations are based on the nominal concentrations.

The 7-day E_yC₅₀ was calculated to be 32.0 and 30.8 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀ was 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 1.6 and 5.0 mg test item/L for frond number and 5.0 and 16 mg test item/L for dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be 1.6 and 5.0 mg test item/L for frond number and 5.0 and 16 mg test item/L for dry weight, respectively.