[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

lemna

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 December 2017 to 22 January 2018

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken at 0 hours (initial value) from fresh test solutions, after 3 and 5 days from fresh and aged test solutions and after 7 days from aged test solutions from all test concentrations and control. For each sampling also a retain sample was taken.
All samples were stored deep frozen until they were transferred to the analytical laboratory. The samples were thawed to ambient temperature, mixed with 10 mL acetonitrile and shaken well using a Vortex-Mixer. The samples were then diluted with acetonitrile/test medium (1:1, v/v) prior to analysis by HPLC-MS/MS.

Vehicle:

no

Remarks:

dissolved in test medium

Details on test solutions:

A stock solution was prepared in test medium and further diluted to achieve the required concentrations

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: duck weed
- Strain: Lemna gibba G3 (Alismatales: Araceae)
- Source: inhouse clone from primary culture by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, U.S.A.
- Method of cultivation: cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 10000 lux. The temperature was 24 ± 2 °C.

Before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test. Young, light-green plants of similar size and comprising 2 - 4 fronds were transferred onto fresh medium and cultured for 7-10 days prior to testing, with two further transfers onto fresh medium before initiating the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

no data

Test temperature:

23.4 – 23.7 °C (measured in separate vessel held under the same conditions as the test vessels)

pH:

fresh solutions: 7.27 – 7.59
aged solutions: 8.70-9.29 (at 100 mg/L 7.44-7.99)

Nominal and measured concentrations:

Nominal: 0, 0.298, 0.954, 3.05, 9.77, 31.3 and 100 mg/L
Measured (fresh): 97-106% of nominal
Measure (aged):

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL screw cap bottles each containing ~150 mL test medium
- Aeration: no
- Renewal rate of test solution: on day 3 and 5
- No. of fronds per colony: 4
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM:no data

TEST MEDIUM / WATER PARAMETERS: 20xAAP test medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: T8-fluorescent tubes, length 900 mm, LT30W/865 daylight; 7391 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting
- Determination of biomass: dry weight on day 7 (for t=0 based on 6X12 fronds from the culture); dried at 60°C for about 72 hours
- Determination of frond area: no
- Other: plant development, frond size, necrosis and additional observations

RANGE-FINDING STUDY
- Test concentrations: 0, 0.100, 1.00, 10.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: inhibition of yield at 1.00 mg/L and above; inhibition of growth rate 100 mg/L
Shortened roots, yellow fronds, smaller and singularized fronds were observed at 100 mg/L and at 10 mg/L gibbosity of fronds was reported

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.298 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: based on yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

3.05 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: based on growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

36.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: based on yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

76.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on yield and growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

53.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

69.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: based on growth rate

Details on results:

On day 3 shortened roots, gibbosity, brown and singularized fronds were observed at 100 mg/L.
On day 5 shortened roots, yellow fronds and small fronds were observed at concentrations of 31.3 and 100 mg/L. Additionally at concentrations of 31.3 mg/L gibbosity were found. At 100 mg/L fronds were singularized.
On day 7 the same observations were done as on day 5. Additionally at concentrations of 31.3 mg/L fronds were singularized.

The measured initial concentration of the substance ranged from 87 % to 106 % of nominal with a mean initial concentration of 95 % of nominal. As the pH of the test solutions (except at 100 mg/L) increases due to growth of the plants to approximately pH 9 and above, hydrolysis can be expected, which is more or less “confirmed” by the absence of an effect on pH and thus on test substance concentration in the solutions at 100 mg/L, where growth and yield are most strongly reduced. Therefore toxicological endpoints were evaluated using nominal concentrations of the test item.
See attached document.

Results with reference substance (positive control):

EC50 growth rate 3,5-dichlorophenol = 6.78 mg/L (frond numbers), 4.63 mg/L (dry weight)

 Frond numbers: mean values

	Mean of frond numbers Days				Yield based on mean frond numbers
Nominal conc. [mg/L]	0	3	5	7	7-0
Control	12	43	91	145	133
0.298	12	47	103	169	157
0.954	12	40	89	133	121*
3.05	12	39	88	136	124
9.77	12	39	88	118	106*
31.3	12	37	71	105	93*
100	12	18	26	28	16*

*Statistically significant different to the controls

Frond numbers: inhibition of yield

Nominal conc. [mg/L]	Inhibition [%]
	7-0 days
Control	0.0
0.298	-18.0^1)
0.954	9.0
3.05	6.8
9.77	20.3
31.3	30.1
100	88.0

¹⁾Value was set to zero for EC_{10, 20, 50}-calculation

Frond numbers: mean growth rates

	Days
Nominal conc. [mg/L]	3	5	7
	Mean growth rate µ [d^-1]
Control	0.4275	0.4047	0.3561
0.298	0.4527	0.4305	0.3781
0.954	0.3982	0.4013	0.3440
3.05	0.3957	0.3975	0.3471
9.77	0.3896	0.3985	0.3264*
31.3	0.3720	0.3545	0.3096*
100	0.1348	0.1568	0.1224*

* Statistically significant different to the controls

        

Frond numbers: inhibition of growth rates

	Days
Nominal conc. [mg/L]	3	5	7
	Inhibition growth rate [%]
Control	0.0	0.0	0.0
0.298	-5.9	-6.4	-6.2^1)
0.954	6.9	0.8	3.4
3.05	7.4	1.8	2.5
9.77	8.9	1.5	8.3
31.3	13.0	12.4	13.1
100	68.5	61.3	65.6

¹⁾Value was set to zero for EC_{10, 20, 50}-calculation

Frond numbers: mean doubling times (T_d)

	Days
Nominal conc. [mg/L]	3	5	7	7
	Td[days]			Td[hours]
Control	1.621	1.713	1.946	46.7
0.298	1.531	1.610	1.833	44.0
0.954	1.741	1.727	2.015	48.4
3.05	1.752	1.744	1.997	47.9
9.77	1.779	1.739	2.124	51.0
31.3	1.863	1.955	2.239	53.7
100	5.142	4.421	5.663	135.9

Dry weight: mean values

	Mean dry weight [g]		Yield based on mean dry weight [g]
Nominal conc. [mg/L]	Days
	0	7		7-0
Control	0.0027	0.0152		0.0125
0.298		0.0195		0.0168
0.954		0.0145		0.0118
3.05		0.0157		0.0130
9.77		0.0145		0.0118
31.3		0.0143		0.0116
100		0.0042		0.0015*

* Statistically significant different to the controls

Dry weight: inhibition of yield

Nominal conc. [mg/L]	Inhibition [%]
	7-0 days
Control	0
0.298	-34.4^1)
0.954	5.6^1)
3.05	-4.0^2)
9.77	5.6
31.3	7.2
100	88.0

1)Value was omitted for EC_{10, 20, 50}-calculation

2)Value was set to zero for EC_{10, 20, 50}-calculation

Dry weight: mean growth rate and inhibition of growth rate

	Day 7
Nominal conc. [mg/L]	Mean growth rate µ [d^-1]	Inhibition Growth rate [%]
Control	0.2470	0.0
0.298	0.2822	-14.3^1)
0.954	0.2397	3.0^1)
3.05	0.2517	-1.9^2)
9.77	0.2398	2.9
31.3	0.2376	3.8
100	0.0628	74.6*

1)Value was omitted for EC_{10, 20, 50}-calculation

2)Value was set to zero for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

 

Validity criteria fulfilled:

yes

Conclusions:

The EC50 based on growth rate is 69.8 mg/L based on biomass, The NOEC based on growth rate is 3.05 mg/L based on frond numbers

Executive summary:

In a study with a semi-static design (renewal on day 3 and 5) the vegative growth of Lemna gibba G3 was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. Lemna was exposed at nominal concentrations of 0, 0.298, 0.954, 3.05, 9.77, 31.3 and 100 mg/L (initial measured concentrations were within 87 % to 106 % of nominal, but due to a increase of the pH during the test the concentrations in the aged solutions were very low due to some hydrolysis of the substance).

Significant inhibitory effects were determined for yield of frond numbers at test item concentrations of 0.954 mg/L (nominal) and above. For growth rate of frond numbers significant inhibitory effects were determined at 9.77 mg/L (nominal) and above. For yield of dry weight and growth rate of dry weight significant inhibitory effects weredetermined at 100mg/L (nominal) and above. The overall LOEC was therefore determined to be 0.954 mg/L (nominal), the corresponding NOEC was set at 0.298 mg/L (nominal).

The EC50-value for yield of frond numbers was determined to be 36.9 mg/L (nominal), the EC50-value for growth rate of frond numbers was determined to be 76.7 mg/L (nominal). The EC50-value for yield of dry weight was determined to be 53.9 mg/L (nominal), the EC50-value for growth rate of dry weight was determined to be 69.8 mg/L (nominal).

Description of key information

In a study with a semi-static design (renewal on day 3 and 5) the vegative growth of Lemna gibba G3 was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. Lemna was exposed at nominal concentrations of 0, 0.298, 0.954, 3.05, 9.77, 31.3 and 100 mg/L The EC50 based on growth rate is 69.8 mg/L based on biomass, The NOEC based on growth rate is 3.05 mg/L based on frond numbers

Key value for chemical safety assessment

EC50 for freshwater plants:

69.8 mg/L

EC10 or NOEC for freshwater plants:

3.05 mg/L