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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:
2014-08-15 to 2014-08-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
yes
Remarks:
pH-value and appearance of the test item and light intensity
Qualifier:
equivalent or similar to
Guideline:
other: Commission Regulation (EC) No 761/2009, C.26., 2009
GLP compliance:
yes (incl. certificate)
Remarks:
German GLP
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: SBL/027/LRB (India)
- Expiration date of the lot/batch: May 05, 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 from the freshly prepared test media of all test concentrations and the control were taken at test start and at the test medium renewals of 3 and 6 days. 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 were taken at the test medium renewals at 1 and 4 days and at the end of the test from all aged test media and the control of the actual test by pouring together the contents of each treatment. All samples were diluted by a factor of 2 with acetonitrile. From all freshly prepared and aged controls during the above mentioned renewal periods and the dilution solvent, one additional sample was taken without any sample treatment.
Details on test solutions:
Test Concentrations: 80.0, 25.0, 7.81, 2.44, 0.76 and 0.24 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 each test media renewal a stock solution of nominal 80 mg test item/L was prepared by dissolving the test item in the test water by intense stirring for 10 minutes.
Day 0: 80.18 mg test item in 1002.25 ml
Day 1: 81.33 mg test item in 1016.625 ml
Day 2: 81.62 mg test item in 1020.25 ml
Day 3: 80.62 mg test item in 1007.75 ml
Day 4: 80.45 mg test item in 1005.625 ml
Day 5: 81.30 mg test item in 1016.25 ml
Day 6: 80.11 mg test item in 1001.38 ml
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 a colouration of the test water. The concentration of 0.24 mg test item /L caused a slightly pink colouration. The concentrations of 0.76 and 2.44 mg test item/L caused a pink colouration. The concentration of 7.81 mg test item/L caused a strong pink colouration. The concentration of 25.0 mg test item/L caused a red colouration. The concentration of 80.0 mg test item/L caused a strong red colouration.
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: 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-values were measured on day 0, 3 and 6 in the freshly prepared and on day 1, 4 and 7 in the aged test media.
7.6 to 7.7 in the freshly prepared media;
8.3 to 8.8 in the aged test media

Nominal and measured concentrations:
Test Concentrations: 80.0, 25.0, 7.81, 2.44, 0.76 and 0.24 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-values were measured on day 0, 3 and 6 in the freshly prepared and on day 1, 4 and 7 in the aged test media.
7.6 to 7.7 in the freshly prepared media;
8.3 to 8.8 in the aged test media
Light Regime: Continuous illumination
Light Intensity: 8550 Lux (mean value) with a range of 7040 to 9450 lux
The illumination was achieved by fluorescent tubes, installed above the test vessels. Measurements were performed once during the test at 12 places distributed over the experimental area at the surface of the test media. Differences in light intensity over the test area did exceed ± 15 %.

Reference substance (positive control):
yes
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 80 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.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 80 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.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 80 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.:
20.7 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.:
56.1 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.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 80 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.:
7.81 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.:
80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
7.81 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
80 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.:
25 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.:
> 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 80 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 > 80 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be >80 mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 20.7 and 56.1 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be >80 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 7.81 and 25 mg test item/L for frond number and 80 and >80 mg test item/L for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 7.81 and 25 mg test item/L for frond number and 80 and >80 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 nominal test concentration of 80 mg test item/L.



Analytical Results:
At the start of the test 110 % of the nominal test concentration was found (average of all test concentrations). After 7 days test duration, 100 % of the nominal value was determined (average of all test concentrations). During the test the Lemna were exposed to a mean of 105 % of nominal. Therefore, all reported results refer to 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:
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 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 Welch t-test (dry weight).
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat(R) Solutions GmbH.

Influence of FAT 40062/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)

> 80

> 80

> 80

> 80

EC20 (7-day)

> 80

> 80

> 80

> 80

EC10 (7-day)

20.7

> 80

56.1

> 80

95 % conf. limits

9.78 - 33.2

 

n.d.

n.d.

37.5 - > 80

7-day NOEC

7.81

7.81

> 80

> 80

7-day LOEC

25

25

> 80

> 80

 

n.d.: not determinable

Values refer to nominal test concentrations

Validity criteria fulfilled:
yes
Conclusions:
The influence of FAT 40062/C TE on the growth of the freshwater plant Lemna gibba was assessed in a semi-static dose-response test.
The 7-day EyC50 was calculated to be >80 mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be >80 mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC were determined to be 7.81 and 25 mg test item/L for frond number and 80 and >80 mg test item/L for dry weight, respectively.
The 7-day NOErC and the LOErC were determined to be 7.81 and 25 mg test item/L for frond number and 80 and >80 mg test item/L for dry weight, respectively.
Executive summary:

The toxicity of FAT 40062/C to the aquatic plant Lemna gibba in a growth inhibition test was determined according to OECD guideline 221 equivalent to EU C. 26. This study encompassed 7 treatment groups (6 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 were Yield and growth rate based on frond number and dry weight. The test concentrations used were 80.0, 25.0, 7.81, 2.44, 0.76 and 0.24 mg test item/L and a control. Water temperature was 22 to 25 °C; pH values at test start in the freshly prepared test media 7.6 to 7.7, in the aged test media 8.3 to 8.8; continuous illumination; mean light intensity: 8550 lux (7040 to 9450 lux).

 

Biological Results:

Growth Inhibition:

The 7-day EyC50was calculated to be >80 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC50was calculated to be >80 mg test item/L for frond number and dry weight, respectively.

The 7-day EyC10was calculated to be 20.7 and 56.1 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC10was calculated to be >80 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC were determined to be7.81 and 25 mg test item/L for frond number and 80 and >80 mg test item/L for dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be7.81 and 25 mg test item/L for frond number and 80 and >80 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 nominal test concentration of 80mg test item/L

 

Analytical Results: At the start of the test 110% of the nominal test concentration was found (average of all test concentrations). After 7 days test duration, 100 % of the nominal value was determined (average of all test concentrations). During the test the Lemna were exposed to a mean of 105 % of nominal. Therefore, all reported results refer to nominal concentrations.

 

In conclusion, the influence of FAT 40062/C 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 >80 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC50was calculated to be >80 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC were determined to be 7.81 and 25 mg test item/L for frond number and 80 and >80 mg test item/L for dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be 7.81 and 25 mg test item/L for frond number and 80 and >80 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 toxicity of FAT 40062/C to the aquatic plant Lemna gibba in a growth inhibition test was determined according to OECD guideline 221 equivalent to EU C. 26.This study encompassed 7 treatment groups (6 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 were yield and growth rate based on frond number and dry weight. The test concentrations used were 80.0, 25.0, 7.81, 2.44, 0.76 and 0.24 mg test item/L and a control. Water temperature was 22 to 25 °C; pH values at test start in the freshly prepared test media 7.6 to 7.7, in the aged test media 8.3 to 8.8; continuous illumination; mean light intensity: 8550 lux (7040 to 9450 lux).

 

Biological Results:

Growth Inhibition:

The 7-day EyC50was calculated to be> 80 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC50was calculated to be> 80 mg test item/L for frond number and dry weight, respectively.

The 7-day EyC10was calculated to be20.7 and 56.1 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC10was calculated to be> 80 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC were determined to be7.81 and 25 mg test item/L for frond number and 80 and> 80 mg test item/L for dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be7.81 and 25 mg test item/L for frond number and 80 and> 80 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 nominal test concentration of 80mg test item/L

 

Analytical Results: At the start of the test 110% of the nominal test concentration was found (average of all test concentrations). After 7 days test duration, 100 % of the nominal value was determined (average of all test concentrations). During the test the Lemna were exposed to a mean of 105 % of nominal. Therefore, all reported results refer to nominal concentrations.

 

In conclusion, the influence of FAT 40062/C TE on the growth of the freshwater plantLemna gibbawas assessed in a semi-static dose-response test.

The 7-day EyC50was calculated to be >80 mg test item/L for frond number and dry weight, respectively.

The 7-day ErC50was calculated to be >80 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC were determined to be 7.81 and 25 mg test item/L for frond number and 80 and >80 mg test item/L for dry weight, respectively.

The 7-day NOErC and the LOErC were determined to be 7.81 and 25 mg test item/L for frond number and 80 and >80 mg test item/L for dry weight, respectively.

 

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

Key value for chemical safety assessment

EC50 for freshwater plants:
80 mg/L
EC10 or NOEC for freshwater plants:
7.81 mg/L

Additional information