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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:
2019-06-28 to 2019-07-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
March 2006
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 761/2009, Annex VI, C.26: "Lemna sp. Growth Inhibition Test"
Version / remarks:
August 24, 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance Document on Aqueous-Phase Toxicity Testing of Difficult Test Chemicals
Version / remarks:
February 2019
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentration: 5 mg/L
- Sampling method: For determination of the test item concentration, samples (50 mL / replicate) were taken from the testing concentration and from the control in duplicates at the start (prior to distributing of the test solution to the test vessels), and at the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). A test item suspension was prepared by adding an amount of 0.0080 g test item to 1600 mL test medium (20X AAP medium) in the first and second renewal periods, 0.0083 g test item to 1660 mL test medium (20X AAP medium) in the third renewal period in order to give the nominal loading rate of 5 mg test item/L. This test item solution was handled by ultrasonic bath for approximately 10 minutes thereafter stirred for a period of approximately 24 hours to achieve equilibrated concentration. The solution was then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. The test solution was freshly prepared in the testing laboratory just before the start of each renewal period.
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: Duckweed
- Strain: G3
- Source: In-house culture kept under axenic conditions; preculture was prepared at least 7 days before the tests; healthy colonies with 3-4 fronds. Originally, the culture was obtained from Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie, Jena, Germany.
- Preculture: 7 - 10 days (7 days in this study) before testing, sufficient colonies are transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
7 d
Test temperature:
22.8 – 24.8 °C in the climate chamber
23.9 – 24.7 °C in the flask
pH:
7.85 - 8.88
Nominal and measured concentrations:
Nominal: 5 mg/L
Measured concentrations: 0.097 mg/L. Not within ± 20 % of the nominal during the test period.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beaker
- Material, size, fill volume: Glass, total capacity of 400 mL, filled with 160 mL test solution
- Type of cover: Covered by glass petri dishes
- Aeration: No
- Renewal rate of test solution: The test solution (as well control solution) was renewed twice during the test (on days 3 and 5).
- Initial frond number: The initial frond number in the test cultures was 11. The number of colonies and fronds was identical in each test vessel.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (20 X AAP Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes, the pH was adjusted to 7.5 ± 0.1 with 1 M HCl
- Photoperiod: Continuous illumination
- Light intensity and quality: 6500-10000 lux using fluorescent light tubes (with a spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED
At the start of the test, frond numbers in the test vessels were recorded. The number and appearance of fronds of Lemna gibba were determined in each testing vessel during the 168-hour test on the 3rd, 5th and 7th days. No change in plant development, frond size or appearance was noted as well as additional observations of root length, significant features of the test media or other abnormalities. In addition to determinations of frond number during the test, effects of the test item on final biomass were also assessed based on determination of dry weight at the beginning and at the end of the study.

RANGE-FINDING STUDY
In the semi-static preliminary range-finding test a test item solution was prepared by adding 0.0051 g test item into 1020 mL dilution water (20X AAP Medium) in the first renewal period. 0.0051 g test item into 1000 mL dilution water (20X AAP Medium) in the second renewal period and 0.0053 g test item into 1060 mL dilution water (20X AAP Medium) in the third renewal period to get the nominal concentration of 5 mg test substance/L. The test solution was handled in ultrasonic bath for 10 minutes, stirred rigorously for 24 hours then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. Untreated control ran parallel in the test. The pre-test was performed with three replicates per test item treated group (containing 11 fronds in total per test vessel) for a period of 7 days. A concurrent control was run (with three replicates). The results are shown in the section "Any other information on results incl. tables"
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.097 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
based on frond number and on dry weight
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.097 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
yield
Remarks:
based on frond number and based on dry weight
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 0.097 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
based on frond number and on dry weight
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 0.097 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
yield
Remarks:
based on frond number and on dry weight
Details on results:
- Change in plant development during the test: No, the plants were healthy, there were no symptoms observed at the 3rd, 5th and 7th day of the experiment.
Results with reference substance (positive control):
-Results with reference substance valid? Yes
The date of the last study with reference item 3,5-Diclilorophenol was: 11 - 18 October 2019.
Endpoints of this study were: EyfnC50 (7 day, yield based on frond numbers): 5.187 mg/L, ErfnC50 (7 day, growth rate based on frond numbers): 7.624 mg/L, EydwC50 (7 day, yield based on dry weight): 5.136 mg/L, ErdwC50 (7 day, growth rate based on dry weight): 6.497 mg/L.
Reported statistics and error estimates:
Mean values and standard deviations were calculated for each treatment and at each replicate at the start, on the 3rd, 5th days and at the end of the test.
The doubling time of frond number in the control was calculated.
The average specific growth rates were calculated for the entire test period based on frond numbers and dry weights for each treatment and each parallel.
The yield was calculated based on frond numbers and dry weights for each treatment and each parallel.
Mean value of the dry weight and standard deviation were calculated for three independent frond number samples at the start and for Lemna colonies used in the test in each replicate at the end of the test.
For the determination of the LOEC and NOEC, the calculated growth rate and yield at the test concentration were tested on significant differences to the control value using Independent Samples T-Test (α = 0.05) by SPSS PC+ software program.

Table 1: Results of the Preliminary Range-finding Test

Nominal concentrations
(mg/L)

Untreated control

WAF* (5 mg/L)

Average number of fronds
(day 7)

198.00

188.33

Growth Rates (µ)
(0-7d)

0.413

0.406

% Inhibition of µ
(0-7d)

-

1.73

* WAF = water accommodated fraction (according to OECD No. 23)

 

Based on the results of the non-GLP Preliminary Range-Finding Test (see above) and since the analytical method validation showed that the test item is poorly soluble, the main test was performed as a limit test using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 5 mg/L and a concurrent control group.

 

Analytical Results

For determination of the test item concentrations, samples were taken from the test concentration level and from the control at the start and at the end of the test. The measured concentration values were not within ± 20 % of the nominal during the experiment. Therefore, test item concentration was calculated as the time-weighted mean of the measured concentrations (according to OECD No. 23). Calculations of the test concentration are given in the table below.

 

Table 2: Calculation of Test Concentration

Nominal
concentration
(mg/L)

Measured concentration (mg/L)

Start

End

Sample measured

Mean of measured

Sample measured

Mean of measured

Control (n=6)

< LOQ

< LOQ

5 (WAF*)

0.507

0.500

0.100**

0.050

0.492

0.000***

0.316

0.324

0.000***

0.000

0.331

0.000***

0.100**

0.223

0.000***

0.050

0.345

0.100**

0.434

0.353

0.000***

0.000

0.272

0.000***

0.100**

0.179

0.000***

0.000

0.257

0.000***

0.316

0.208

0.000***

0.000

0.100**

0.000***

Mean

 

0.298

 

0.017

Time-weighted mean

0.097

 

*WAF= water accommodated fraction(OECD No. 23.); loading rate: 5 mg/L nominal

**LOQ= limit of quantification (0.2µg/mL), measured as<LOQ, therefore test concentration calculated as LOQ/2 (OECD No. 23.)

***not detected, thereforetest concentration calculated as 0.000 mg/L

 

Growth rate

No significant inhibition of the average specific growth rate based on frond number and dry weight in comparison to that of the control was observed in the test item treated group. The NOEC and LOEC values were determined using Independent Samples T-Test (α = 0.05). Accordingly, the NOEC based on growth rate was determined to be 0.097 mg/L, while the LOEC based on growth rate was determined to be >0.097 mg/L.

 

Yield

No significant inhibition of the yield based on frond number and dry weight in comparison to that of the control was observed in the test item treated group. NOEC and LOEC values were determined using Independent Samples T-Test (α = 0.05). Accordingly, the NOEC based on yield was determined to be 0.097 mg/L, while the LOEC based on yield was determined to be> 0.097 mg/L.

 

Validity of the Test

The doubling time of frond number in the control was 1.95 days (less than 2.5 days). The validity criterion was within acceptable limit and therefore the study can be considered as valid.

 

 

Validity criteria fulfilled:
yes
Conclusions:
In this 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item had no toxic effect on the test system. The overall 7-day NOEC was 0.097 mg/L (nominal 5 mg/L), the overall 7-day LOEC > 0.097 mg/L (nominal > 5 mg/L). All validity criteria were met. The results are based on the time-weighted mean of the measured test item concentrations. Test solution was prepared by utilizing the water accommodated fraction (WAF) approach
Executive summary:

The phytotoxicity of the test item C.I. Leuco Sulfur Green 2 on the freshwater aquatic plant Lemna gibba was assessed in a study according to OECD guideline 221. Exponentially growing cultures of Lemna gibba were exposed to the test item over a period of 7 days in a semi-static system under defined conditions. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach (according to OECD No. 23). Growth and inhibition of growth in the test solution of the nominal test item concentration of 5 mg/L were compared with that in the control in a limit test. The effects of C.I. Leuco Sulfur Green 2 compared to the control plant development were demonstrated by the changes of average specific growth rates and yield (both calculated on the basis of frond number and dry weight). The analytically measured test item concentration was not within ± 20 %of the nominal during the test period therefore all biological results are based on the time-weighted mean of the measured test item concentrations. In the 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item C.I. Leuco Sulfur Green 2 had no toxic effect on the test system. The overall 7-day NOEC was determined to be 0.097 mg/L (nominal 5 mg/L). The overall 7-day LOEC was determined to be >0.097 mg/L (nominal > 5 mg/L). All validity criteria were met.

Description of key information

In the 7-day growth inhibition study on lemna sp. (Lemna gibba) the obtained results showed that the test item C.I. Leuco Sulfur Green 2 had no toxic effects on the test system at its solubility limit in the test medium.

The overall 7-day NOEC was determined to be 0.097 mg/L (nominal 5 mg/L).

The overall 7-day LOEC was determined to be >0.097 mg/L (nominal 5 mg/L).

All validity criteria were met. The results are based on the time-weighted mean of the measured test item concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:
0.097 mg/L

Additional information

The phytotoxicity of the test item C.I. Leuco Sulfur Green 2 on the fresh water aquatic plant Lemna gibba was assessed in a study acording to OECD guideline 221. Exponentially growing cultures of Lemna gibba were exposed to the test item over a period of 7 days in a semi-static sytem under defined conditions. Because of low solubility of the test system, the test solution was prepared by utilizing the water accomodated fraction (WAF) approach (according to OECD No. 23). Growth and inhibition of growth in the test solution of the nominal test item concentration of 5 mg/L were compared with that in the control in a limit test. The effcets of C.I. Leuco Sulfur green 2 compared to the control plant development were demonstrated by the changes of average specific growth rates and yield (both calculated on the basis of frond number and dry weight). The analytically measured test item concentration was not within ± 20 % of the nominal during the test period therefore all biological results are based on the time-weighted mean of the measured test item concentrations.

In this 7-day growth inhibition study on lemna sp. (Lemna gibba) the obtained results showed that the test item C.I. Leuco Sulfur Green 2 had no toxic effects on the test system. The overall 7-day NOEC was determined to be 0.097 mg/L (nominal 5 mg/L). The overall 7-day LOEC was determined to be >0.097 mg/L (nominal 5 mg/L). All validity criteria were met.