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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:
2019-05-31 to 2019-06-07
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 23, 2006
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EU) No 761/2009, Annex VI, C.26: „Lemna sp. Growth Inhibition Test“. Dated 24 August 2009
Version / remarks:
August 24, 2019
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD Series on Testing and Assessment No. 23 (Second Edition), ENV/JM/MONO(2000)6/REV1, 8 February 2019
Version / remarks:
February 8, 2019
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: 106132249
- Expiration date: 29 September 2020
Analytical monitoring:
yes
Details on sampling:
- Sampling method: For determination of the test item concentration, samples (50 mL per replicate) were taken from the testing concentration and from the control at the start (prior to distributing of the test solution to the test vessels), and at the end of each renewal period.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF THE TEST SOLUTION
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 0.0360 g test item to 1200 mL test medium (20X AAP medium) in the first renewal period, 0.0451 g test item to 1503 mL test medium (20X AAP medium) in the second renewal period and 0.0450 g test item to 1500 mL test medium (20X AAP medium) in the third renewal period in order to give the nominal loading rate of 30 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 at the start of each renewal period.
Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Strain: Lemna gibba (G3)
- Source: 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:
22.4 – 24.7 °C in the climate chamber
22.9 – 23.1 °C in the flask
pH:
8.12 - 8.79
Nominal and measured concentrations:
Nominal: 30 mg/L
Measured concentration: 0.33 mg/L. Not within ± 20 % of the nominal during the experiment.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beaker
- Material, size fill volume: Glass, total capacity of 100 mL, fill with 160 mL test solution.
- Type of cover: Glass petri dishes
- Aeration: No
- Renewal rate of test solution: The test and control solution were 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 replicates per concentration: 6
- No. of replicates per control: 6

GROWTH MEDIUM
- Standard medium: yes, (20X AAP Medium)

TEST MEDIUM / WATER PARAMETERS
- Source / preparation of dilution water: According to guideline
Other testing conditions
- Sterile condition: 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)

EFFECTS 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. Colonies were located in knots in the test item treated group. 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 TEST
In the semi-static preliminary range-finding test, The test item solution was prepared by adding 0.0150 g test item into 500 mL dilution water (20X AAP Medium) in the first renewal period, 0.0150 g test item into 500 mL dilution water (20X AAP Medium) in the second renewal period and 0.0151 g test item into 503 mL dilution water (20X AAP Medium) in the third renewal period to get the nominal concentration of 30 mg test substance/L. The test solutions were 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 is tested at least twice a year
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
based on frond number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Remarks:
based on frond number
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
based on frond number
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Remarks:
based on frond number and based o 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 cultures 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.

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 each renewal period. The measured concentration values were not within ± 20 % of the nominal during the experiment. Therefore, test item concentration was calculated as the geometric mean of the measured concentrations (according to OECD No. 23). Calculation of the test concentration is given in the table below.

Table 2: Calculation of the Test Concentration

Concentration
(mg/L)

Mean of Measured concentration (mg/L) (n=6)

First renewal period

Second renewal period

Third renewal period

Start

End

Start

End

Start

End

Control

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

30(WAF*)

0.371

0.307

0.368

0.290

0.362

0.278

Geometric

mean

0.33

*WAF = water accommodated fraction (OECD No. 23.); loading rate: 30 mg/L nominal

Growth Rate

No significant inhibition of the average specific growth rate based on frond number 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 growth rate (frond number) was determined to be 0.33 mg/L (measured), while the LOEC based on growth rate (frond number) was determined to be > 0.33 mg/L (measured).

Significant inhibition of the average specific growth rate based on 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 growth rate (dry weight) was determined to be < 0.33 mg/L (measured), while the LOEC based on growth rate (dry weight) was determined to be < 0.33 mg/L (measured).

Yield

No significant inhibition of the yield based on frond number 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 (frond number) was determined to be 0.33 mg/L (measured), while the LOEC based on yield (frond number) was determined to be > 0.33 mg/L (measured).

Significant inhibition of the yield based on 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 (dry weight) was determined to be < 0.33 mg/L (measured), while the LOEC based on yield (dry weight) was determined to be > 0.33 mg/L (measured).

Validity of the Test

The doubling time of frond number in the control was 1.80 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 the 7-day growth inhibition study on Lemna sp. (Lemna gibba) the obtained results showed that the test item Black DB had no toxic effects on the growth rate and yield based on frond number. The 7-day NOEC based on frond number was determined to be 0.33 mg/L (nominal 30 mg/L). The 7-day LOEC based on frond number was determined to be > 0.33 mg/L (nominal > 30 mg/L). The 7-day NOEC based on dry weight was determined to be < 0.33 mg/L (nominal < 30 mg/L). The 7-day LOEC based on dry weight was determined to be < 0.33 mg/L (nominal < 30 mg/L). All validity criteria were met. The results are based on the nominal and on geometric mean of the measured test item concentrations. Test solution was prepared by utilizing the water accommodation fraction (WAF) approach.
Executive summary:

The phytotoxicity of the test item Black DB 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 (168 hours) 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 30 mg/L were compared with that in the control in a limit test. The effects of Black DB 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 nominal and on the geometric 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 Black DB had no toxic effects on the growth rate and yield based on frond number but had significant toxic effect on the growth rate and yield based on dry weight. The 7-day NOEC based on frond number was determined to be 0.33 mg/L (nominal 30 mg/L). The 7-day LOEC based on frond number was determined to be > 0.33 mg/L (nominal > 30 mg/L). The 7-day NOEC based on dry weight was determined to be < 0.33 mg/L (nominal < 30 mg/L). The 7-day LOEC based on dry weight was determined to be < 0.33 mg/L (nominal < 30 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 Black DB had no toxic effects on the growth rate and yield based on frond number.

The 7-day NOEC based on frond number was determined to be 0.33 mg/L (loading rate: 30 mg/L).

The 7-day LOEC based on frond number was determined to be > 0.33 mg/L (loading rate: 30 mg/L).

All validity criteria were met. The results are based on the nominal and on geometric mean of the measured test item concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:
0.33 mg/L

Additional information

The phytotoxicity of the test item Black DB 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 (168 hours) 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 30 mg/L were compared with that in the control in a limit test. The effects of Black DB 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 nominal and on the geometric 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 Black DB had no toxic effects on the growth rate and yield based on frond number but had significant toxic effect on the growth rate and yield based on dry weight. The 7-day NOEC based on frond number was determined to be 0.33 mg/L (nominal 30 mg/L). The 7-day LOEC based on frond number was determined to be > 0.33 mg/L (nominal > 30 mg/L). The 7-day NOEC based on dry weight was determined to be < 0.33 mg/L (nominal < 30 mg/L). The 7-day LOEC based on dry weight was determined to be < 0.33 mg/L (nominal < 30 mg/L). All validity criteria were met.