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Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-01 to 2014-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AT-29381400
- Expiration date of the lot/batch: May 12, 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:
One sample from the freshly prepared 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.
Details on test solutions:
Test Concentrations: A filtrate of nominal 100 mg/L, dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate and a control. The filtrate and its dilutions correspond to mean measured test concentrations of 11.2, 3.08, 0.913, 0.362 and 0.121 mg test item/L.
Control: In the control, test medium was used without addition of the test item.
Dosage of Test Item: The test medium of the highest test concentration of nominal 100 mg/L was prepared by dissolving 113.9 mg test item into 1139 mL test water by intense stirring for 24 hours. Then, the stock suspensions were filtered through a cellulose-nitrate filter (pore size 0.45 µm). The filtrates were diluted in a series of sequential dilutions with test water to prepare the test media. Adequate volumes of this test medium 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: There were no remarkable observations.
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 26 °C

pH:
pH-Values: The pH-value of each treatment was measured at test start and at test end.
7.6 – 8.0 at test start; 8.6 – 8.9 at test end

Nominal and measured concentrations:
A filtrate of nominal 100 mg/L, dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate and a control.
The filtrate and its dilutions correspond to mean measured test concentrations of 11.2, 3.08, 0.913, 0.362 and 0.121 mg test item/L.
Details on test conditions:
Test Environment: Controlled environment room
Light Regime: Continuous illumination
Light Intensity: 8813 Lux (mean value) with a range of 8040 to 9700 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 %.

Test Units
Type and Size: Glass vessels of 250 mL volume with approximately 200 mL test medium, covered with glass dishes.
Identification: Each test unit was uniquely identified with the serial number, treatment and replicate number.
Reference substance (positive control):
yes
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
2.67 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
6.06 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 11.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 11.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
0.497 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
1.67 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
2.3 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.:
5.93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.207 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.848 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.802 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.:
2.42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate
Remarks on result:
other: 95 % CL
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.121 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.913 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.121 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.913 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
0.362 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
3.08 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
0.362 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
3.08 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate
Details on results:
Biological Results
Growth Inhibition: The 7-day EyC50 was calculated to be 2.67 and 6.06 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 11.2 and > 11.2 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 0.207 and 0.848 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 0.802 and 2.42 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 0.121 and 0.362 mg test item/L for frond number and 0.913 and 3.08 mg test item/L for dry weight.
The 7-day NOErC and the LOErC were determined to be 0.121 and 0.362 mg test item/L for frond number and 0.913 and 3.08 mg test item/L for dry weight.

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 0.121 mg test item/L.
At the higher test item concentrations the fronds showed deviations from the control replicates after 7 days; i.e., shortened roots, smaller fronds and gibbous growth.

Analytical Results:
The concentrations of the test item were analysed in all duplicate test media samples from the freshly prepared and aged test media, from each test concentration (filtrate and dilutions of 1:81, 1:27, 1:9, 1:3 of the filtrate of a suspension of the test item in test water) and of the controls. The recovery rates of the test media samples of the lowest test concentration (dilution of filtrate 1:81) are slightly below the limit of quantification (LOQ) of the analytical method. The limit of quantification was determined to be the fortification level of nominal 0.2 mg/L (corresponding to nominal 0.1 mg/L after dilution by factor two). The results of the different test concentration level show that the determined concentrations are in good agreement with the corresponding dilution factors. Therefore the concentration of the lowest test concentration was calculated using the dilution factor and result of the next higher concentration level. The reported biological results refer to mean measured concentrations.
Results with reference substance (positive control):
Results of the most recent Test with the Reference Item 3,5 Dichlorophenol performed in January 2014 (IBACON Project No.: 88301240)
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) 5.28 9.66 6.16 9.34
95 % conf. limits 3.87 - 7.22 7.51 - 12.4 4.65 - 8.16 8.12 - 10.7

EC20 (7-day) 2.53 6.17 3.74 6.32
95 % conf. limits 1.24 - 3.53 3.15 - 7.83 1.84 - 4.88 4.53 - 7.43

EC10 (7-day) 1.73 4.87 2.88 5.16
95 % conf. limits 0.632 - 2.63 1.87 - 6.59 1.05 - 4.02 3.23 - 6.35

7-day NOEC 0.625 1.25 1.25 1.25
7-day LOEC 1.25 2.5 2.5 2.5
Values refer to nominal test 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 and the Welch t-test. 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. The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Influence of FAT 20200/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)

2.67

> 11.2

6.06

> 11.2

95 % conf. limits

1.79 - 4.29

n.d.

4.72 - 8.17

n.d.

 

 

 

 

 

EC20(7-day)

0.497

2.30

1.67

5.93

95 % conf. limits

0.217 - 0.817

1.64 - 2.98

1.03 - 2.28

4.88 - 7.04

 

 

 

 

 

EC10(7-day)

0.207

0.802

0.848

2.42

95 % conf. limits

< 0.121 - 0.400

0.447 - 1.19

0.421 - 1.30

1.66 - 3.13

 

 

 

 

 

7-day NOEC

0.121

0.121

0.913

0.913

7-day LOEC

0.362

0.362

3.08

3.08

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

Summary of Analytical Results
mean measured
sample description concentration1
[mg/L] n
control n.a. 4
dilution of filtrate 1:81 0.121 4
dilution of filtrate 1:27 0.362 4
dilution of filtrate 1:9 0.913 4
dilution of filtrate 1:3 3.08 4
filtrate of 100 mg/L 11.2 4

1mean value of all measured samples per treatment group
italic values: calculated value, since measured values below LOQ
n: number of analysed samples
n.a.: not applicable

Validity criteria fulfilled:
yes
Conclusions:
The 7-day EyC50 was calculated to be 2.67 and 6.06 mg test item/L for frond number and dry weight, respectively. The 7-day ErC50 was calculated to be >11.2 and >11.2 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 0.121 and 0.362 mg test item/L for frond number and 0.913 and 3.08 mg test item/L for dry weight.
The 7-day NOErC and the LOErC were determined to be 0.121 and 0.362 mg test item/L for frond number and 0.913 and 3.08 mg test item/L for dry weight.
Executive summary:

The toxicity of the FAT 20200/C to the aquatic plant Lemna gibba in a growth inhibition test was determined based on the procedures indicated by the following OECD 221 and EU C. 26 guidelines.

 

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 3, 5 and 7. The dry weight of each replicate from test end was determined.

A filtrate of nominal 100 mg/L, dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate and a control. The filtrate and its dilutions correspond to mean measured test

concentrations of 11.2, 3.08, 0.913, 0.362 and 0.121 mg test item/L.

Conclusion:

The 7-day EyC50 was calculated to be 2.67 and 6.06 mg test item/L for frond number and dry weight, respectively. The 7-day ErC50 was calculated to be >11.2 and >11.2 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC were determined to be 0.121 and 0.362 mg test item/L for frond number and 0.913 and 3.08 mg test item/L for dry weight.

The 7-day NOErC and the LOErC were determined to be 0.121 and 0.362 mg test item/L for frond number and 0.913 and 3.08 mg test item/L for dry weight.

Description of key information

The toxicity of FAT 20200/C to the aquatic plantLemna gibbain a growth inhibition test was determined based on the procedures indicated by the following OECD 221 and EU C. 26 guidelines. The following results were obtained:

The 7-day EyC50 was calculated to be 2.67 and 6.06 mg test item/L for frond number and dry weight, respectively. The 7-day ErC50 was calculated to be > 11.2 and > 11.2 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC were determined to be 0.121 and 0.362 mg test item/L for frond number and 0.913 and 3.08 mg test item/L for dry weight.

The 7-day NOErC and the LOErC were determined to be 0.121 and 0.362 mg test item/L for frond number and 0.913 and 3.08 mg test item/L for dry weight.

Key value for chemical safety assessment

EC50 for freshwater plants:
11.2 mg/L
EC10 or NOEC for freshwater plants:
0.121 mg/L

Additional information