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Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 - 18 Mar 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The test concentration could not be measured since the measured values were below the limit of quantification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4400 (Aquatic Plant Toxicity Test using Lemna spp. Tiers I & II))

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Remarks:

National Institute of Pharmacy and Nutrition, Budapest, Hungary (date of issue: 03 August 2018)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and 100 mg/L nominal loading rate WAF
- Sampling method: Duplicate samples were taken (2 x ~15 mL from three replicates) at the beginning and at the end of the first and at the last renewal periods from the control and at the applied test concentration level
- Sample storage conditions before analysis: Samples were frozen and kept approximately at -20°C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set was retained as a back-up at the Test Facility, if required for any confirmatory analyses.

Vehicle:

no

Details on test solutions:

- The test item is poorly soluble in water, so test solutions were prepared using a saturated solution method (nominal loading rate of 100 mg/L, water accommodated fraction, WAF) according to the Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD No. 23. The saturated test item solution was prepared by dispersing/dissolving the amount of test item into the test medium (20x-AAP medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30 °C and then equilibrated for about 24 hours at approximately 20 °C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter. The maximum amount of test material in the medium was exposed applying this procedure. Prior to treatment of each renewal period, test item solution was prepared by the method described above and distributed into the appropriate test vessels just before introduction of Lemna colonies (fronds) (start of the treatments).

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: duckweed
- Strain: LBASF 01 LG 264 (strain code at the test Facility)
- Source (laboratory, culture collection): BASF AG, Agricultural Center, Limburgerhof, Germany
- Method of cultivation: Axenic stock cultures are grown in appropriate culturing vessels in the same medium and at similar climatic conditions, which are used in the experiment. The stock cultures are regularly transferred to fresh medium to act as initial test plant.

ACCLIMATION
- Acclimation period: 14 days
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells/plants observed: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

22.2 – 22.8 °C water temperature
22.2 - 24.2 °C climate chamber temperature

pH:

7.53 - 8.88

Nominal and measured concentrations:

- Nominal concentration: 100 mg/L nominal loading rate WAF
- Measured concentration 0 h (beginning): < LOQ
- Measured concentration 7 d (end of last renewal period): < LOQ

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes
- Test vessel: Sterile 400 mL glass beakers covered by glass Petri dishes
- Type (delete if not applicable): open
- Fill volume: 160 mL
- Type of cover: glass Petri dishes
- Aeration: no
- Agitation: no
- Renewal rate of test solution (frequency/flow rate): 24 hours
- No. of fronds per colony: 11
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (20x-AAP medium), but slightly modified according to the experience of the laboratory

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test medium same as growth medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: Culture temperature was checked at the start and at the end of the renewal periods in a flask filled with water, in the climatic chamber. The pH was measured at the start and at the end of the renewal periods in the control and at the test concentration replicates.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 7225 lux cool-white fluorescent light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting at the beginning of the test and on days 3, 5, and 7
- Determination of biomass: dry weight was determined at the beginning of the experiment of three parallel Lemna gibba samples containing 11 fronds (the same as used for inoculate the test vessels) were collected, entirely dried at 60oC in heating cabinet, and the dry weight measured with an accuracy of 0.1 mg. At the end of the test the colonies, fronds, parts of the plant were collected from each test vessel. These samples were entirely dried and handled as at the beginning of the experiment.
- Other: Any change in plant development, frond size, appearance, significant features of the test media and other abnormalities were noted every 24 hours

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10, 100 mg/L nominal WAF
- Results used to determine the conditions for the definitive study: no significant effects were observed at any tested concentration, NOEC = 100 mg/L nominal loading rate WAF

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Con. based on nominal loading rate WAF

Duration:

7 d

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other:

Remarks:

conc. based on nominal loading rate WAF

Duration:

7 d

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: overall

Remarks on result:

other: Conc. based on nominal loading rate WAF

Details on results:

There were no changes in plant development at the test item treated group during the experiment compared to the plant development in control groups.

Results with reference substance (positive control):

Date of the last study with the reference item 3,5-Dichlorophenol was 31 Jan. - 07 Feb 2020. The results were as follows:
The ErfnC50 : 3.59 mg/L, (95 % confidence limits: 3.21 – 4.02 mg/L)
The EyfnC50 : 2.66 mg/L, (95 % confidence limits: 2.38 – 2.98 mg/L)
The ErdwC50 : 3.73 mg/L, (95 % confidence limits: 3.38 – 4.13 mg/L)
The EydwC50 : 2.55 mg/L, (95 % confidence limits: 2.29 – 2.84 mg/L)

Table 1:Frond numbers, average specific growth rates and yield (based on frond numbers)

Nominal loading rate WAF [mg/L]	Frond numbers					Growth rates			Average specific growth rates 0-7 Days	Yield 0-7 Days
	Days					Days
		0	3	5	7	0-3	3-5	5-7
Control		11	23	55	128	0.2459	0.4359	0.4223	0.3506	117
		11	23	54	120	0.2459	0.4267	0.3993	0.3414	109
		11	23	55	126	0.2459	0.4359	0.4145	0.3483	115
		11	23	52	124	0.2459	0.4079	0.4345	0.3461	113
		11	23	52	110	0.2459	0.4079	0.3746	0.3289	99
		11	24	58	120	0.2601	0.4412	0.3635	0.3414	109
	Mean	11.00	23.17	54.33	121.33	0.2482	0.4259	0.4015	0.3428	110.33
	SD	0.00	0.41	2.25	6.41	0.0058	0.0147	0.0278	0.0077	6.41
100.0		11	23	52	122	0.2459	0.4079	0.4264	0.3437	111
		11	23	51	110	0.2459	0.3982	0.3843	0.3289	99
		11	23	53	124	0.2459	0.4174	0.4250	0.3461	113
		11	23	52	118	0.2459	0.4079	0.4097	0.3390	107
		11	23	52	117	0.2459	0.4079	0.4055	0.3378	106
		11	22	52	115	0.2310	0.4301	0.3968	0.3353	104
	Mean	11.00	22.83	52.00	117.67	0.2434	0.4115	0.4080	0.3385	106.67
	SD	0.00	0.41	0.63	5.01	0.0060	0.0109	0.0163	0.0061	5.01

 

Table 2:Growth rates (m) and yield (y) and their respective percentage inhibition during the test period based on frond number

Nominal loading rate WAF (mg/L)	Growth rate (m) and % inhibition ofm		Yield and % inhibition of yield
	0-7d		0-7d
	m	% Im	yield	% Iy
Control	0.3428	0.0	110.33	0.0
100.0	0.3385	1.26	106.67	3.32

 

Table 3:Validity criteria according to OECD 221 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 per day.	2.02 days	Yes

Validity criteria fulfilled:

yes

Remarks:

See table 3 on "any other information on results incl. tables".

Description of key information

Lemna gibba: 7-day EL50 (growth rate) > 100 mg/L nominal loading WAF (OECD 221, 2006)

Lemna gibba: 7-day NOELR (overall) >= 100 mg/L nominal loading WAF (OECD 221, 2006)

Additional information

There is one study available in which the toxicity of Glycerides, C16-18 (even numbered) mono- and di- and their citrates (EC 701-358-7)t o Lemna gibba was assessed.

The GLP study was performed according to OECD 221 (2006) using Lemna gibba as test organism. A limit test with a nominal loading rate of 100 mg/L was conducted in a semi-static exposure regime. This test solution was prepared as Water Accommodated Fraction (WAF). The test concentration was analytically determined at the start and at the end of the first and at the last renewal periods. The test item concentration could not be analytically quantified during the whole experiment, consequently, the measured concentrations were below the Limit of Quantification (LOQ = 0.038 mg/L). This may have been caused by adsorption to Lemna fronds and/or roots in test solution. Therefore, all results are expressed as the nominal loading rate WAF. No inhibition of growth was observed after 7 d for the tested concentration. Therefore, an EL50 (7 d) of > 100 mg/L (nominal) and a NOELR (7 d) of >= 100 mg/L (nominal) was derived.