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Ecotoxicological information

Toxicity to aquatic plants other than algae

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Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-23 to 2014-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Disperse Red 302

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test media of the only test concentration 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 sampled once undiluted and once diluted by a factor of 2 with acetonitrile.
One additional sample of the control and the dilution solvent were taken at test start and test end without any sample treatment.

Test solutions

Vehicle:
no
Details on test solutions:
Test Concentrations: Filtrate of nominal 100 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 suspension of nominal 100 mg test item/L was prepared by dissolving 138.5 mg test item into 1385 mL test water by intense stirring for 24 hours in the dark. The stock suspension was filtered through a cellulose-nitrate filter (pore size 0.45 µm). The filtrate was used as only test concentration. 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

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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d

Test conditions

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. 22 to 25 °C

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


Nominal and measured concentrations:
Test Concentrations: Filtrate of nominal 100 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.
22 to 25 °C
pH-Values: The pH-value of each treatment was measured at test start and at test end.
7.6 to 8.0 at test start;
8.8 at test end
Light Regime: Continuous illumination
Light Intensity: 7803 Lux (mean value) with a range of 7200 to 8410 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

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
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
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 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.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 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.:
> 100 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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 100 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 > 100 mg test item/L for frond number and dry weight.
The 7-day ErC50 was calculated to be > 100 mg test item/L for frond number and dry weight.
The 7-day EyC10 was calculated to be > 100 mg test item/L for frond number and dry weight.
The 7-day ErC10 was calculated to be > 100 mg test item/L for frond number and dry weight.
The 7-day NOEyC and the LOEyC were determined to be ≥ 100 and > 100 mg test item/L for frond number and dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be ≥ 100 and > 100 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 100 mg test item/L.


Analytical Results:
At the start of the test and after 7 days test duration all values were below the Limit of Detection.
Therefore, all reported results refer on 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:
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 Students 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 Students t-test (frond number and dry weight).
Since the 5th control replicate showed a contamination and reduced frond number, this replicate was excluded from all result evaluations.
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Any other information on results incl. tables

Influence of FAT 93503/B 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)

> 100

> 100

> 100

> 100

95 % conf. limits

n.d.

n.d.

n.d.

n.d.

 

 

 

 

 

EC10(7-day)

> 100

> 100

> 100

> 100

95 % conf. limits

n.d.

n.d.

n.d.

n.d.

 

 

 

 

 

7-day NOEC

≥ 100

≥ 100

≥ 100

≥ 100

7-day LOEC

> 100

> 100

> 100

> 100

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

Summary of Analytical Results

     
sample description % of
[mg/L] nominal1 n
     
control n.a. 4
Filtrate of 100 mg/L < LOD 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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The influence of FAT 93503/B TE on the growth of the freshwater plant Lemna gibba was assessed in a static dose-response test.
The 7-day EC50 and the 7-day EC10 for the parameters growth rate and yield of the endpoints frond number and dry weight could not be calculated due to the low effects of the test item. Therefore the 7-day EC50 and the 7-day EC10 was determined to be higher than the limit of water solubility of the test item, represented by the filtrate of nominal 100 mg test item/L, the highest concentration tested.
The 7-day NOEC was determined to be at least the filtrate of nominal 100 mg test item/L for the parameters growth rate and yield for frond number and dry weight and the associated 72-hour LOEC was determined to be higher than the filtrate of nominal 100 mg test item/L for both parameters and both endpoints.
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:

Growth Inhibition:

The 7-day EyC50was calculated to be >100 mg test item/Lfor frond number and dry weight.

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

The 7-day EyC10was calculated to be >100 mg test item/Lfor frond number and dry weight.

The 7-day ErC10was calculated to be >100 mg test item/Lfor frond number and dry weight.

The 7-day NOEyC and the LOEyC were determined to be ≥100 and >100 mg test item/L for frond number and dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be ≥100 and >100 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 100mg test item/L.

Analytical Results: At the start and at the end of the test all values were below the Limit of Detection.Therefore all results are reported on nominal concentrations.

Conclusion:  

The influence of FAT 93503/B TE on the growth of the freshwater plant Lemna gibba was assessed in a static dose-response test.

The 7-day EC50 and the 7-day EC10 for the parameters growth rate and yield of the endpoints frond number and dry weight could not be calculated due to the low effects of the test item. Therefore the 7-day EC50 and the 7-day EC10 was determined to be higher than the limit of water solubility of the test item, represented by the filtrate of nominal 100 mg test item/L, the highest concentration tested.

The 7-day NOEC was determined to be at least the filtrate of nominal 100 mg test item/L for the parameters growth rate and yield for frond number and dry weight and the associated 72-hour LOEC was determined to be higher than the filtrate of nominal 100 mg test item/L for both parameters and both endpoints.

   

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