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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:
10 February 2014 to 25 February 2014
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: ISO International Standard 20079: "Water quality – Determination of the toxic effect of water constituents and waste water on duckweed (Lemna minor) – Duckweed growth inhibition test", First edition, 01 November 2005.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Sampling for analysis of test concentrations:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below:
- Frequency:
1. At the start of the test and after 5 days from the freshly prepared solutions.
2. After 2 and 7 days from the 48-hour old solutions, respectively.
- Volume 3.0 ml
- Storage Samples were stored in a freezer until analysis.

At the end of each refreshment period the replicates were pooled at each concentration before sampling, except for test concentrations of 22 mg/l and higher at day 7. Due to the presence of precipitate in the test vessels samples were taken from one replicate of each test concentration.

Additionally, reserve samples of 3.0 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Test organisms (species):
Lemna minor
Details on test organisms:
Species: Lemna minor
Source: Friedrich-Schiller-Universität Jena Dornburger Straße 159 D-07743 Jena, Germany
Reason for selection: This species has been internationally accepted as a representative for aquatic plants.


ACCLIMATION
- Culturing media and conditions (same as test or not): Modified Steinberg medium.
- Pre-culture: with 2 or 3 plants each with a maximum of 3 fronds. The total number of fronds increased by a factor of ≥ 7 in 7 days with a
doubling time of ≤ 2.5 days. The culture consisted of young, rapidly growing colonies with bright green fronds that varied in size without visible
lesions, chlorosis, gibbosity or necrosis. The pre-culture was maintained under the same conditions as used in the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
the incubator was maintained between 23.8 and 24.5 °C.
pH:
5.4-6.2
Nominal and measured concentrations:
Nominal concentrations: 10, 22, 46, 100 and 220 mg/l
Measured concentration in sampling time day 0 (fresh): 9.43, 21.2, 45.4, 101, 226 mg/l
Measured concentration in sampling time day 2 (old): 9.19, 21.4, 45.7, 102, 217 mg/l
Measured concentration in sampling time day 5 (fresh): 9.42, 21.1, 43.7, 102, 226 mg/l
Measured concentration in sampling time day 7 (old): 8.48, 20.4, 43.1, 101, 223 mg/l
Details on test conditions:
Fresh water plant culture:
- Stock culture: Axenic plant stock cultures were started by inoculating medium with fronds from a pure culture on agar. The suspensions were exposed to light in a climate room at a temperature of 22 - 26 °C.

- Light intensity: 85 to 135 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: Modified Steinberg medium; according to ISO 20079, formulated using Milli-RO water and with the following
composition:
- KNO3: 350 mg/l
- Ca(NO3)2.4H2O: 295 mg/l
- KH2PO4: 90 mg/l
- K2HPO4: 12.6 mg/l
- MgSO4.7H2O: 100 mg/l
- H3BO3: 120 μg/l
- ZnSO4.7H2O: 180 μg/l
- Na2MoO4.2H2O: 44 μg/l
- MnCl2.4H2O: 180 μg/l
- FeCl3.6H2O: 760 μg/l
- Na2EDTA.2H2O: 1500 μg/l
- pH 5.5 ± 0.2

Pre-culture: 7 days before the start of the test, pre-cultures were started with 2 or 3 plants each with a maximum of 3 fronds. The total number of
fronds increased by a factor of ≥ 7 in 7 days with a doubling time of ≤ 2.5 days. The culture consisted of young, rapidly growing colonies with bright green fronds that varied in size without visible lesions, chlorosis, gibbosity or necrosis. The pre-culture was maintained under the same conditions as used in the test.

- Test concentrations FAT 20306/B: 10, 22, 46, 100 and 220 mg/l
- Controls: Test medium without test substance or other additives
- Replicates: 3 replicates of each test concentration; 6 replicates of the control.

Test procedures and conditions
- Test duration: 7 days
- Test type: Semi-static, with renewal of test solutions on days 2 and 5
- Test vessels: 250 ml, all-glass, containing 150 ml of test solution
- Medium: Modified Steinberg medium
- Initial plant density: 3 plants, with a total of 10 fronds
- Illumination: Continuously using TLD-lamps
Intensity at the start: 86-97 μE.m-2.s-1
Intensity at the end: 85-98 μE.m-2.s-1
- Incubation: During incubation the vessels were continuously swirled.
Reference substance (positive control):
yes
Remarks:
3, 5-dichlorophenol
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
150 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
A 7-day aquatic plant toxicity test using Lemna minor with 3,5-dichlorophenol (Project 505599).
- Start of first exposure: 11 April 2014
- Completion last exposure: 18 April 2014
The study procedures described in this report were based on the OECD guideline No. 221, Adopted March 23, 2006; and ISO Standard 20079, First
edition, 01 November 2005.
This reference test was carried out to check the sensitivity of the test system used by WIL Research Europe to 3,5-dichlorophenol (Sigma-Aldrich, Art. D70600, Batch 04631AJ). Plants were exposed for a period of 7 days to 3,5-dichlorophenol concentrations of 0.56, 1.0, 1.8, 3.2 and 5.6 mg/l and to a control. The initial plant density was 3 with a total frond number of 10.

The EC50 for growth rate reduction (ERC50: 0 - 7d) was 2.5 mg/l with a 95% confidence interval ranging from 2.4 to 2.7 mg/l. According to ISO
2079 the ranges for growth rate reduction should lie between 2.2 and 3.8 mg/l. Hence, the ERC50: 0 - 7d for the present batch corresponds with this range.
The EC50 for yield inhibition (EYC50: 0 - 7d) was 2.2 mg/l with a 95% confidence interval ranging from 2.0 to 2.7 mg/l. Historical ranges are not yet available.
The protocol, raw data and report of this study are kept in the WIL Research Europe archives. The test described above was performed under GLP conditions with a QA-check.
Reported statistics and error estimates:
EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on Weibull analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test substance.
The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions ® GmbH, Germany).
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Lemna minor, FAT 20306/B inhibited growth rate and yield of this fresh water plant species significantly at 100 mg/l. The NOEC for growth rate and yield inhibition was 46 mg/l.
The 7d-EC50 for growth rate inhibition (ERC50) was similar for frond numbers and wet weight, i.e. 150 and 100 mg/l, respectively.
The 7d-EC50 for yield inhibition (EYC50) was also similar for frond numbers and wet weight, i.e. 85 and 79 mg/l.
Executive summary:

The purpose of the study was to evaluate the influence of the test substance, FAT 20306/B, on the growth of aquatic

plants (Lemna minor).

A 7-day aquatic plant toxicity test using Lemna minor with FAT 20306/B. The study procedures described in this report were based on the OECD guideline No. 221, Adopted March 23, 2006 and the ISO Standard 20079, First edition, 01 November 2005.

The batch of FAT 20306/B tested was a red powder with a purity of 93.6% and the substance was completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test. Preparation of test solutions started with a nominal concentration of 220 mg/l applying a short period of vigorous shaking to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 220 mg/l concentration in test medium.

Six replicates of Lemna minor colonies were exposed to a control, whereas three replicates per group were exposed to nominal FAT 20306/B concentrations of 10, 22, 46, 100 and 220 mg/l. The initial plant density per replicate was 3 with a total frond number of 10 per replicate. Test solutions were renewed on days 2 and 5. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the first and the last renewal period.

The analytical results of the samples taken at the start of the first and the last refreshment period showed that actual test concentrations were in agreement with nominal (94 - 103%). These concentrations remained stable during both 48-hour refreshment periods (90 - 101% of initial).

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Effect parameters obtained in this study are summarized in table below:

   Parameter (mg/l) NOEC   EC10  EC50
Growth rate Frond number  Value  46  36  150
Growth rate Wet weight  Value  46  35  100
Yield Frond number  Value  46  56  85
Yield Wet weight  Value  46  51  79

Description of key information

Under the conditions of a study with Lemna minor, FAT 20306/B inhibited growth rate of this fresh water plant species. The NOEC for growth rate and yield inhibition was 46 mg/l. The EC50 for growth rate was found being 150 mg/l, based on frond number and 100 mg/l based on wet weight.

Key value for chemical safety assessment

EC50 for freshwater plants:
100 mg/L
EC10 or NOEC for freshwater plants:
46 mg/L

Additional information

The influence of FAT 20306/B, on the growth of aquatic plants (Lemna minor) was investigated in a 7-day aquatic plant toxicity test based on the OECD guideline No. 221, Adopted March 23, 2006.

The batch of FAT 20306/B tested was a red powder with a purity of 93.6% and the substance was completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test. Preparation of test solutions started with a nominal concentration of 220 mg/l; the lower test concentrations were prepared by subsequent dilutions of the 220 mg/l concentration in test medium.

Six replicates of Lemna minor colonies were exposed to a control, whereas three replicates per group were exposed to nominal FAT 20306/B concentrations of 10, 22, 46, 100 and 220 mg/l. The initial plant density per replicate was 3 with a total frond number of 10 per replicate. Test solutions were renewed on days 2 and 5. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the first and the last renewal period. The analytical results of the samples taken at the start of the first and the last refreshment period showed that actual test concentrations were in agreement with nominal (94 - 103%). The study met the acceptability criteria prescribed by the protocol and was considered valid.

Effect parameters obtained in this study are summarized in table below:

   Parameter (mg/l) NOEC   EC10  EC50
Growth rate Frond number  Value  46  36  150
Growth rate Wet weight  Value  46  35  100
Yield Frond number  Value  46  56  85
Yield Wet weight  Value  46  51  79