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Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Remarks:
Toxicity to the Aquatic Plant Lemna gibba
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-13 to 2017-07-03
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)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 761/2009, C.26.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
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.
The samples were diluted by a factor of two with acetonitrile.
Additional samples of the control blank and the dilution solvent were taken at test start and test end without any sample treatment.
Vehicle:
no
Details on test solutions:
Test Concentrations: 158.7, 49.6, 15.5, 4.8, 1.5 and 0.5 mg test item/L, corresponding to 100, 31.3, 9.8, 3.1, 1.0 and 0.3 mg a.i./L and a control.
Control: In the control, test medium was used without addition of the test item.
Dosage of Test Item: A stock solution of 158.7 mg test item/L was prepared by dissolving 161.3 mg test item into 1016.4 mL test water by intense stirring for 30 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
The test media were prepared just before introduction of the Lemna (= start of the test).
Appearance of the Test Item in Test Medium:
The test item caused a blue colouration of the test medium in all test concentration and over the whole test period.
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:
23.6 – 26.3 °C
pH:
7.6 – 7.7 at test start;
8.5 – 8.7 at test end
Nominal and measured concentrations:
nominal: 158.7, 49.6, 15.5, 4.8, 1.5 and 0.5 mg test item/L, corresponding to 100, 31.3, 9.8, 3.1, 1.0 and 0.3 mg a.i./L and a control.
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.
23.6 – 26.3 °C
pH-Values: The pH-value of each treatment was measured at test start and at test end.
7.6 – 7.7 at test start;
8.5 – 8.7 at test end
Light Regime: Continuous illumination
Light Intensity: 7067 Lux (mean value) with a range of 6780 to 7410 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:
For the evaluation of the quality of the Lemna gibba and the experimental conditions the substance 3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
30.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
82.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 158.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 158.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
4.33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
3.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
19.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
41.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
4.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
4.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
4.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
19.5 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
52.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
2.73 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
2.15 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
26.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
1.55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
6.02 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.642 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
5.37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Biological Results
Growth Inhibition: The 7-day EyC50 was calculated to be 30.9 (19.5)* and 82.9 (52.2)* mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 158.7 (> 100)* mg test item/L for frond number and dry weight, respectively.
The 7-day EyC10 was calculated to be 1.55 (0.977)* and 0.642 (0.404)* mg test item/L for frond number and dry weight, respectively.
The 7-day ErC10 was calculated to be 6.02 (3.79)* and 5.37 (3.38)* mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC for frond number were determined to be 0.5 (0.3)* and 1.5 (1.0)* mg test item/L.
The 7-day NOEyC and the LOEyC for dry weight were determined to be 1.5 (1.0)* and 4.8 (3.1)* mg test item/L.
The 7-day NOErC and the LOErC were determined to be 1.5 (1.0)* and 4.8 (3.1)* mg test item/L for frond number and dry weight, respectively.
* = values in brackets refer to test concentrations based on the active ingredient
Results with reference substance (positive control):
Results of the most recent Test with the Reference Item 3,5 Dichlorophenol performed in January 2017
Parameter
Yield Growth rate Yield Yield
(frond number) (frond number) (dry weight) (dry weight)
(frond number) [mg test item/L] [mg test item/L] [mg test item/L]

EC50 (7-day) 4.63 6.24 4.77 6
95 % conf. limits 4.30 - 4.98 6.07 - 6.42 4.47 - 5.08 5.77 - 6.36

EC20 (7-day) 3.11 4.79 3.41 4.92
95 % conf. limits 2.68 - 3.45 4.64 - 4.93 2.19 - 3.76 4.79 - 5.02

EC10 (7-day) 2.53 4.17 2.87 4.43
95 % conf. limits 2.06 - 2.89 4.00 - 4.32 2.28 - 3.27 4.22 - 4.57

7-day NOEC 2.5 2.5 2.5 2.5
7-day LOEC 5 5 5 5
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 LOEC and NOEC values based on frond number and biomass 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 3.2.1, ToxRat Solutions GmbH.
Validity criteria fulfilled:
yes
Remarks:
Doubling Time of Frond Number in Control: 2.0 days, validity criterion was met.
Conclusions:
The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a static concentration-response test.
The 7-day EyC50 was calculated to be 30.9 (19.5)* and 82.9 (52.2)* mg test item/L for frond number and dry weight, respectively.
The 7-day ErC50 was calculated to be > 158.7 (> 100)* mg test item/L for frond number and dry weight, respectively.
The 7-day NOEyC and the LOEyC for frond number were determined to be 0.5 (0.3)* and 1.5 (1.0)* mg test item/L.
The 7-day NOEyC and the LOEyC for dry weight were determined to be 1.5 (1.0)* and 4.8 (3.1)* mg test item/L.
The 7-day NOErC and the LOErC were determined to be 1.5 (1.0)* and 4.8 (3.1)* mg test item/L for frond number and dry weight, respectively.
The quantification of the test item in the test samples was performed using liquid chromatography with UV detection.
At the start of the test 112% of the nominal test concentrations were found (average of all test concentrations). After 7 days test duration, 107% of the nominal value was determined (average of all test concentrations). During the test the Lemna were exposed to a mean of 110% of nominal. Therefore, all reported results refer to nominal concentrations.
Executive summary:

Title: Test item: Toxicity to the Aquatic Plant Lemna gibba in a Static Growth Inhibition Test

Purpose: The purpose of this study was to determine the inhibitory effect of the test item on the growth of the freshwater aquatic plant Lemna gibba.

For this purpose, cultures of Lemna gibba were exposed in a static test to various concentrations under defined conditions. The inhibition of growth in relation to control cultures were determined over a test period of 7 days.

The test method of application and the test system are recommended by the test guidelines and Lemna gibba is the recommended test species.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines/Recommendations:

Commission Regulation (EC) No 761/2009, Annex, Part C, C.26.: “Lemna sp. Growth Inhibition Test”, Official Journal of the European Union (EN), dated August 24, 2009

OECD Guideline 221: “Lemna sp. Growth Inhibition Test”, adopted March 23, 2006

OECD Series on Testing and Assessment, No. 23, “Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”, December 15, 2000

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Material and Methods

Test Item; batch no.: CHA0 122490; content of a.i.: 63.0 % (w/w), according to certificate of analysis.

Test Species:Lemna gibba G 3

Test Design: 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 static conditions. The frond numbers were determined on day 3, 5 and 7. The dry weight of each replicate was determined at test termination.

Endpoints: Yield and growth rate based on frond number and dry weight.

Test Concentrations: 158.7, 49.6, 15.5, 4.8, 1.5 and 0.5 mg test item/L, corresponding to 100, 31.3, 9.8, 3.1, 1.0 and 0.3 mg a.i./L and a control.

Test Conditions: Water temperature: 23.6 – 26.3 °C; pH values at test start 7.6 – 7.7, at the end of the test 8.5 – 8.7; continuous illumination; mean light intensity: 7067 lux (6780 to 7410 lux).

Conclusion: The influence of the test item on the growth of the freshwater plant Lemna gibba was assessed in a static concentration-response test.

The 7-day EyC50 was calculated to be 30.9 (19.5)* and 82.9 (52.2)* mg test item/L for frond number and dry weight, respectively.

The 7-day ErC50 was calculated to be > 158.7 (> 100)* mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC for frond number were determined to be 0.5 (0.3)* and 1.5 (1.0)* mg test item/L.

The 7-day NOEyC and the LOEyC for dry weight were determined to be 1.5 (1.0)* and 4.8 (3.1)* mg test item/L.

The 7-day NOErC and the LOErC were determined to be 1.5 (1.0)* and 4.8 (3.1)* mg test item/L for frond number and dry weight, respectively. The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. At the start of the test 112% of the nominal test concentrations were found (average of all test concentrations). After 7 days test duration, 107% of the nominal value was determined (average of all test concentrations). During the test the Lemna were exposed to a mean of 110% of nominal. Therefore, all reported results refer to nominal concentrations.

* = values in brackets refer to test concentrations based on the active ingredient

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

Description of key information

The purpose of this study was to determine the inhibitory effect of the test item on the growth of the freshwater aquatic plant Lemna gibba.

The 7-day ErC50 was calculated to be > 158.7 mg test item/L for frond number and dry weight, respectively.

The 7-day NOEyC and the LOEyC for dry weight were determined to be 1.5 and 4.8 mg test item/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:
1.5 mg/L

Additional information