Sodium bis[2-chloro-4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-5-hydroxybenzenesulphonamidato(2-)]chromate(1-)

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:

2014-09-11 to 2014-11-15

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)

Version / remarks:

adopted March 23, 2006

Deviations:

yes

Remarks:

Concerning application of the Test Item and the Control

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)

Version / remarks:

August 24, 2009

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

December 15, 2000

Qualifier:

according to guideline

Guideline:

other: Residues: Guidance for generating and reporting methods of analysis in support of preregistration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Version / remarks:

SANCO/3029/99 rev.4 11/07/00

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: #119#35 (Thailand)
- Expiration date of the lot/batch: January 14, 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 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:
Filtrate of 100 mg test item/L, 1:3, 1:9, 1:27 and 1:81 dilutions, corresponding to 1.98, 0.706 and 0.241 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: T
The test item was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item was tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore, a stock suspension of 100 mg test item/L was prepared by suspending 102.1, 100.6 and 100.7 mg test item in 1021, 1006 and 1007 mL test water for preparing the test concentrations. The stock suspension was stirred for 24 to 26 h in the dark to dissolve as much test item as possible. Then, the undissolved test item was separated by filtration (0.45 µm cellulose acetate filter) and the required test concentrations were prepared from the filtrate by dilution with test water. The test concentrations were prepared just before introduction of the Lemna (= start of the test) and each test media renewal.

Appearance of the Test Item in Test Medium:
The test item caused a colouration in all freshly prepared and aged test media of the filtrate and the 1:3, 1:9 and 1:27 dilutions.

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:

Water Temperature: 24 to 25 °C; The temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.

pH:

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;
8.5 to 9.1 in the aged test media
The pH in the control increased by more than 1.5 units in the control medium. However, this does not invalidate the tests since the validity criterion was met.

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 1.98, 0.706 and 0.241 mg test item/L and a control.

Details on test conditions:

Test Environment: Controlled environment room
Light Regime: Continuous illumination
Light Intensity: 7650 Lux (mean value) with a range of 6750 to 8390 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

Remarks:

3,5 Dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL 0.926 - 1.36

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 1.98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 1.98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 1.98 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.296 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL 0.213 - 0.376

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.883 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL 0.609 - 1.10

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

1.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL 0.986 - 1.28

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

> 1.98 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.:

0.148 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL 0.091 - 0.207

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL 0.266 - 0.707

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.476 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: 95 % CL 0.365 - 0.578

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL 0.920 - 1.44

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.08 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.:

0.241 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.08 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.:

0.241 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.241 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.:

0.706 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.241 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.:

0.706 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 1.11 and >1.98 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be >1.98 and >1.98 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 0.148 and 0.510 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 0.476 and 1.22 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/L for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 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 up to and including the test concentration of 0.241 mg test item/L.
At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days; i.e. shortened and red coloured roots from the test item (0.706 and 1.98 mg test item/L) and chlorosis (1.98 mg test item/L).

Analytical Results:
In the freshly prepared test media 0.277, 0.968 and 2.493 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 0.211, 0.532 and 1.644 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 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 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 test concentrations were extrapolated by dividing the lowest measured test concentration (0.241 mg Test item/L) by the dilution factor.
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat(c) Solutions GmbH.

Influence of FAT 20028/E 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)	1.11	> 1.98	> 1.98	> 1.98
95 % conf. limits	0.926 - 1.36	n.d.	n.d.	n.d.
EC20(7-day)	0.296	1.13	0.883	> 1.98
95 % conf. limits	0.213 - 0.376	0.986 - 1.28	0.609 - 1.10	n.d.
EC10(7-day)	0.148	0.476	0.510	1.22
95 % conf. limits	0.091 - 0.207	0.365 - 0.578	0.266 - 0.707	0.920 - 1.44
7-day NOEC	0.08	0.08	0.241	0.241
7-day LOEC	0.241	0.241	0.706	0.706

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

Summary of Analytical Results

 

Sample description [mg/L]	Geometric mean [mg/L]	n
control	n.a.	12
1:81 dilution	n.a.	12
1:27 dilution	n.a.	12
1:9 dilution	0.241	12
1:3 dilution	0.706	12
Filtrate of 100 mg/L	1.98	12

 

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 20028/E TE on the growth of the freshwater plant Lemna gibba in a semi-static dose-response test are as
The 7-day EyC50 was calculated to be 1.11 and >1.98 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be >1.98 and >1.98 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/L for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/L for dry weight, respectively.

Executive summary:

The toxicity of FAT 20028/E to the aquatic plant Lemna gibba in a growth inhibition test was determined based on the OECD 221 guideline equivalent to EU C. 26 method. 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 semi-static conditions. The frond numbers were determined on day 3, 5 and 7. The dry weight of each replicate from test end was determined. The endpoints assesed were yield and growth rate based on frond number and dry weight. The test concentrations used were filtrate of 100 mg test item/L, 1:3, 1:9, 1:27 and 1:81 dilutions, corresponding to 1.98, 0.706 and 0.241 mg test item/L and a control.

Biological Results:

Growth Inhibition:

The 7-day E_yC₅₀ was calculated to be 1.11 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀ was calculated to be >1.98 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day E_yC₁₀was calculated to be 0.148 and 0.510 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₁₀was calculated to be 0.476 and 1.22 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/Lfor dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 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 up to and including the test concentration of 0.241mg test item/L.

At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days; i.e.shortened and red coloured roots from the test item (0.706 and 1.98 mg test item/L) and chlorosis (1.98 mg test item/L).

Analytical Results:

In the freshly prepared test media 0.277, 0.968 and 2.493 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 0.214, 0.532 and 1.644 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.

Conclusion:

The influence of FAT 20028/E 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 be1.11 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀ was calculated to be >1.98 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/L for dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 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

The influence of Acid Red 211 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 be1.11 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀was calculated to be >1.98 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/L for dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determinedto be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/L for dry weight, respectively.

Additional information

The toxicity of FAT 20028/E to the aquatic plant Lemna gibba in a growth inhibition test was determined based on the OECD 221 guideline equivalent to EU C. 26 method. 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 semi-static conditions. The frond numbers were determined on day 3, 5 and 7. The dry weight of each replicate from test end was determined. The endpoints assesed were yield and growth rate based on frond number and dry weight. The test concentrations used were filtrate of 100 mg test item/L, 1:3, 1:9, 1:27 and 1:81 dilutions, corresponding to 1.98, 0.706 and 0.241 mg test item/L and a control.

Biological Results:

Growth Inhibition:

The 7-day E_yC₅₀was calculated to be 1.11 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀was calculated to be >1.98 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day E_yC₁₀was calculated to be0.148 and0.510 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₁₀was calculated to be0.476 and1.22 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be0.080 and0.241 mg test item/L for frond number and0.241 and0.706 mg test item/Lfor dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determined to be0.080 and0.241 mg test item/L for frond number and0.241 and0.706 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 up to and including the test concentration of 0.241mg test item/L.

At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days;i.e.shortened and red coloured roots from the test item (0.706 and 1.98 mg test item/L) and chlorosis (1.98 mg test item/L).

Analytical Results:

In the freshly prepared test media 0.277, 0.968 and 2.493 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 0.214, 0.532 and 1.644 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.

Conclusion:

The influence of FAT 20028/E 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 be1.11 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day E_rC₅₀was calculated to be >1.98 and >1.98 mg test item/L for frond number and dry weight, respectively.

The 7-day NOE_yC and the LOE_yC were determined to be0.080 and0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/L for dry weight, respectively.

The 7-day NOE_rC and the LOE_rC were determinedto be 0.080 and 0.241 mg test item/L for frond number and 0.241 and 0.706 mg test item/L for dry weight, respectively. This study is classified acceptable and satisfies the guideline requirements for aquatic plant studies.