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Toxicity to terrestrial plants

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Endpoint:
toxicity to terrestrial plants: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 September 2015 to 15 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants, Growth Test)
Deviations:
yes
Remarks:
two deviations with no impact on integrity of the study (see below)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Chemical analysis was carried out to verify the concentration of test item in the acetone solutions prepared for all rates of application.
- Aliquots (2 x 10 mL) of the treatment solutions were supplied for analysis in glass vials.
Vehicle:
yes
Remarks:
acetone
Details on preparation and application of test substrate:
TEST SOIL
- The soil used in the study was a sandy loam (batch number GIL1401).
- The percentage organic matter was 1.89%, by calculation, based on the measured organic carbon percentage of 1.1% (see Appendix 1, attached).
- The carbon content, pH and particle size of the soils were analysed and the soil was sieved prior to use to remove all particles > 2 mm.

TREATMENTS AND METHODS OF SOIL PREPARATION AND APPLICATION
- Prior to treatment the moisture content of the soil was determined to be 9.44% and the appropriate amount of moist soil was weighed out (14,118 g for each treatment of carrot and oat and 11,765 g for each treatment of tomato).
- Aliquots of sand (300 g for carrot and oat and 250 g for tomato) were also weighed out for each treatment rate, to give a total weight equivalent to 13,200 g dry substrate for carrot and oat, and 11,000 g dry substrate for tomato.
- Seven treatments were applied for each species (water and solvent controls and test item at rates of 12.35, 37.04, 111.11, 333.33 and 1000 mg a.i./kg dry soil for all species).
- Test item was prepared in acetone as shown in the table below.
- For carrot and oat, 66 mL from each of the solutions was poured over 300 g of sand. For tomato 55 mL was poured over 250 g of sand. When the acetone had evaporated, the sand was mixed into the appropriate amount of weighed moist soil. This soil was then moistened with reverse osmosis (RO) water (420 mL was added for carrot and oat and 350 mL for tomato) and mixed using a handheld mixer to ensure that the soil had a structure suitable for sowing the seeds before it was distributed among labelled replicate pots.
- The treatments were applied in the order of water and solvent controls then test item at 12.35, 37.04, 111.11, 333.33 and 1000 mg a.i./kg dry soil for all species.
- Chemical analysis was carried out to verify the concentration of test item in the acetone solutions prepared for all rates of application. Aliquots (2 x 10 mL) of the treatment solutions were supplied in glass vials. The results of the analysis are presented in Annex 2 (attached).
- The treated soil was then distributed among labelled replicate pots. For the untreated water and solvent control groups the same amounts of sand and moist soil was used as for the treatment groups. For the water control the dry sand was mixed into the moist soil and for the solvent controls the appropriate volume of acetone was added and allowed to evaporate off. Water was then added as for the treated groups. The pH of the soil was recorded after application (see Appendix 3, attached).
Species:
Daucus carota
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Apiaceae (Umbelliferae)
Species:
Lycopersicon esculentum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Family: Solanaceae
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
Family: Poaceae (Gramineae)
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
22 d
Remarks:
Total exposure period 21 days for oat and 22 days for carrot and tomato
Post exposure observation period:
Not applicable
Test temperature:
AIR TEMPERATURE
- Carrot: 17.3 to 26.8 °C
- Tomato: 17.3 to 26.1 °C
- Oat: 17.3 to 22.5 °C
pH:
- pH 6.5 to 6.7 (see Appendix 3, attached)
Moisture:
- Moisture content of soil prior to treatment: 9.44 %
Details on test conditions:
TEST SPECIES
- Seeds were purchased from commercial suppliers and stored dry in glass bottles and in the dark.
- Germination trials indicated that seed viability was > 80 % for all species.

EXPERIMENTAL DESIGN
- A total of 14 replicates, each containing three seeds were used for carrot with 20 replicates of two seeds for tomato and ten replicates of four seeds for oat.
- For all species 9.5 cm internal diameter plastic pots were used, each filled to a depth of approximately 75 mm.
- The seeds were spaced out on the soil surface and approximately 5 mm of the same soil was added to cover the seeds.
- Each pot was labelled with the study number, species name, dose group, replicate number, treatment date and the name of the Study Director.

ENVIRONMENTAL CONDITIONS
- Plants were watered as required, maintaining seedling health and growth (visually determined on a per-species and per-treatment basis).
- Plants were maintained in controlled environment rooms providing a continuous 16-hour period of light per day. - Temperatures were maintained at levels suitable for the individual species.
- Temperature, humidity and light intensity, (initially at soil level then at the top of the canopy after germination) were recorded daily throughout the exposure period.
- A summary of environmental data is given in the table below.

OBSERVATIONS AND MEASUREMENT OF GROWTH
- Each pot was inspected daily and the number of emerged plants recorded, together with any herbicidal symptoms.
- Emergence was defined as any plant material observed above the soil surface.
- After 50% emergence in the controls was achieved, observations continued every one to three days until the plants were harvested.
- Observations included visual assessments based on the overall condition of surviving seedlings. These assessments were conducted using a uniform scoring system based on plant size and vigour, where 0 indicated no
abnormal effects and 10 indicated plant death (see Appendix 5, attached).
- Plants from all treatment and control groups were harvested 14-17 days after germination of at least 50% of the control seedlings, by cutting each stem at soil level, giving a total exposure period of 21 days for oat with 22 days for carrot and tomato.
- The height of each plant was then recorded before they were placed into individually labelled paper bags and dried to a constant weight at approximately 60 °C at which time the dry weight was recorded.
- The height of dicotyledons was measured to the growing point and the length of the longest leaf was recorded for monocotyledons.

EVALUATION OF DATA
- Germination, survival, height and dry weight data were tabulated according to species, treatment level and replicate.

VALIDITY CRITERIA
- The validity criteria for this study were met.
- The protocol requirement for control seedling emergence of a minimum of 70% for all species was achieved.
- Germination in the water controls was 100% for all species.
- No visible treatment related effects were observed in the water controls.
- Control survival was 100% for all species.
- For each of the plant species tested, environmental conditions were identical, and the growing media contained the same amounts of soil matrix, support media, or substrate from the same source.
Nominal and measured concentrations:
RANGE-FINDING STUDY
- Nominal concentrations of 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry soil

DEFINITIVE TEST
- Nominal concentrations of 12.35, 37.04, 111.11, 333.33 and 1000 mg a.i./kg dry soil
Reference substance (positive control):
no
Species:
Daucus carota
Duration:
22 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
germination
Species:
Daucus carota
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
germination
Species:
Daucus carota
Duration:
22 d
Dose descriptor:
EC50
Effect conc.:
734.9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: height
Species:
Daucus carota
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
111.11 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: height
Species:
Daucus carota
Duration:
22 d
Dose descriptor:
EC50
Effect conc.:
445.67 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight
Species:
Daucus carota
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
111.11 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight
Species:
Lycopersicon esculentum
Duration:
22 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
germination
Species:
Lycopersicon esculentum
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
333.33 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
germination
Species:
Lycopersicon esculentum
Duration:
22 d
Dose descriptor:
EC50
Effect conc.:
797.47 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: height
Species:
Lycopersicon esculentum
Duration:
22 d
Dose descriptor:
LOEC
Effect conc.:
111.11 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: height
Species:
Lycopersicon esculentum
Duration:
22 d
Dose descriptor:
EC50
Effect conc.:
497.91 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight
Species:
Lycopersicon esculentum
Duration:
22 d
Dose descriptor:
NOEC
Effect conc.:
111.11 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
654 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
germination
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
333.33 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
germination
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
334.19 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: height
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
111.11 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: height
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
240.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
111.11 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight
Details on results:
MEASURED CONCENTRATIONS
- The mean concentrations of test item in test formulations prepared for application in the definitive test were within 3% of nominal concentrations, confirming accurate formulation.
- The actual concentrations ranged between 97.5 – 102.8% of nominal.
- The formulation analysis report is presented in Annex 2 (attached).

EFFECT ON GERMINATION
- Mean germination is presented in Table 1 (attached).
- The LC25, LC50 and NOEC values are presented in Table 4 (attached) and the individual replicate data are given in Appendix 4 (attached).
- There were treatment-related effects on plant germination at 1000 mg a.i./kg for all the species tested and at 333.33mg a.i./kg for oat.
- Germination in the water and solvent controls was 100% for all species. Reductions in germination of 9.5, 42.5 and 85.0% were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil. There was also a 5.0% reduction in germination in oat at 333.33 mg a.i./kg dry soil.
- The LC25 values for germination were estimated to be >1000 mg a.i./kg dry soil for carrot, and 730 and 496 mg a.i./kg dry soil for tomato and oat respectively.
- The LC50 values for germination were estimated to be >1000 mg a.i./kg dry soil for carrot and tomato and 654 mg a.i./kg dry soil for oat.
- The NOEC values were 1000 mg a.i /kg dry soil for carrot and 333.33 mg a.i./kg dry soil for tomato and oat.

EFFECT ON PLANT SURVIVAL
- The number of plants surviving at harvest and the percentage survival are given in Table 1 (attached).
- There were treatment related effects on plant survival for both carrot and oat at 1000 mg a.i./kg dry soil and for oat at 333.33 mg a.i./kg dry soil.
- There was 100% survival of all species in the water and solvent controls and at all rates of application up to and including 333.33 mg a.i./kg dry soil.
- Survival rates of 73.7, 100 and 83.3% of germinated seedlings were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil and 92.1% for oat at 333.33 mg a.i./kg dry soil.

EFFECT ON PLANT GROWTH AND HEALTH
- There were no visible effects on carrot at rates of up to 37.04 mg a.i./kg dry soil or on tomato and oat at rates of up to 111.11 mg a.i./kg dry soil.
- Very slight growth inhibition was recorded in one replicate for carrot at 111.11 mg a.i./kg dry soil with 25 - 40 and 80% growth inhibition recorded at 333.33 and 1000 mg a.i./kg dry soil respectively. Slight and slight-moderate phytotoxicity respectively was also recorded.
- For tomato slight growth inhibition (<10%) was recorded at 333.33 with ca. 80% at 1000 mg a.i./kg dry soil. Phytotoxicity was also recorded in three replicates at 1000 mg a.i./kg dry soil.
- For oat 50% and 80% growth inhibition was recorded at 333.33 and 1000 mg a.i./kg dry soil respectively.
- The mean height and dry weights per plant and percentage effect on growth in comparison to the solvent controls are given in Table 2 and Table 3 respectively (attached).
- The EC25, EC50 and NOEC values are presented in Table 4 (attached).
- The full data sets for height are given in Appendix 7 (attached) and in Appendix 8 for dry weight (attached).
- Following statistical analysis significant reductions in height and dry weight were calculated for all of the test species at 333.33 and 1000 mg a.i./kg dry soil, in comparison to the solvent controls.
- The EC25 values for height were estimated to be 409.80, 429.90 and 304.95 mg a.i./kg dry soil for carrot, tomato and oat respectively.
- The EC50 values for height were estimated to be 734.90, 797.47 and 334.19 mg a.i./kg dry soil for carrot, tomato and oat respectively.
- The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.
- The EC25 values for dry weight were estimated to be 251.62, 354.63 and 166.01 mg a.i./kg dry soil for carrot, tomato and oat respectively.
- The EC50 values for dry weight were estimated to be 445.67, 497.91 and 240.60 mg a.i./kg dry soil for carrot, tomato and oat respectively.
- The NOEC values for effects on dry weight were 111.11 mg a.i./kg dry soil for all species.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
- Statistical analyses of germination, height and dry weights in comparison to the solvent control were performed using Williams test or Dunnett’s test, with the SAS 9.1.3 programme (SAS Institute Inc. 2002).
- The water control was compared to the solvent control using the t test.
- The height and dry weight responses were assumed to be log normally distributed. Therefore the data were log transformed prior to analysis to stabilize the variance and the means were presented as back transformed geometric means.
Validity criteria fulfilled:
yes
Conclusions:
There were treatment-related effects on plant germination at 1000 mg a.i./kg dry soil for all the species tested and at 333.33 mg a.i./kg dry soil for oat.
 
The LC25 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and 730 and 496 mg a.i./kg dry soil for tomato and oat respectively. The LC50 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and tomato and 654 mg a.i./kg dry soil for oat. The NOEC values were 1000 mg a.i./kg dry soil for carrot and 333.33 mg a.i./kg dry soil for tomato and oat.
 
There was 100 % survival of carrot and tomato at all rates of application up to and including 333.33 mg a.i./kg dry soil. Survival rates of 73.7, 100 and 83.3 % of germinated seedlings were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil and 92.1 % for oat at 333.33 mg a.i./kg dry soil.
 
The EC25 values for height were estimated to be 409.80, 429.90 and 304.95 mg a.i./kg dry soil for carrot, tomato and oat respectively. The EC50 values for height were estimated to be 734.90, 797.47 and 334.19 mg a.i./kg dry soil for carrot, tomato and oat respectively. The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.
 
The EC25 values for dry weight were estimated to be 251.62, 354.63 and 166.01 mg a.i./kg dry soil for carrot, tomato and oat respectively. The EC50 values for dry weight were estimated to be 445.67, 497.91 and 240.60 mg a.i./kg dry soil for carrot, tomato and oat respectively. The NOEC values for effects on dry weight were 111.11 mg a.i./kg dry soil for all species.
 
The study was considered valid as the validation criteria were met and no visible treatment-related effects were observed in the controls.
Executive summary:

GUIDELINE

The study was conducted in accordance with OECD Guideline No 208 “Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test” adopted 19 July 2006.

 

METHODS

The study was carried out with carrot, tomato and oat to determine if the test substance exerts a detrimental effect to plants during early critical stages in their development. The investigation was a dose-response test designed to evaluate the phytotoxic effects of the test substance. Each plant species was sown into treated soil and monitored for 14 to 16 days following a minimum of 50 % germination in the controls.

 

Following initial range-finding at 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry soil, the definitive test was conducted as a dose-response test at 12.35, 37.04, 111.11, 333.33 and 1000 mg a.i./kg dry soil for all species.

 

For carrot, 14 replicate pots of three seeds were sown for each treatment rate, with 20 pots of two seeds for tomato and ten pots of four seeds for oat. An untreated water control and a solvent control were also included for all species. The soil used was sandy loam, sieved to remove all particles > 2 mm.

 

After application and sowing, the pots were checked daily and the numbers of emerged seedlings were recorded. Emergence, survival and sublethal effects were recorded daily up to a minimum of 50 % emergence of control seedlings and thereafter every one to three days. Plants were harvested 14-16 days after 50 % control germination, giving a total exposure period of 21 days for oat and 22 days for carrot and tomato. The height of each individual plant was then measured and the dry weight was recorded.

 

EFFECT ON GERMINATION AND PLANT SURVIVAL

Germination in the water and solvent controls was 100 % for all species. Reductions in germination of 9.5, 42.5 and 85.0 % were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil. There was also 5.0 % reduction in germination in oat at 333.33 mg a.i./kg dry soil.

 

There was 100 % survival of all species in the water and solvent controls and at all rates of application up to and including 333.33 mg a.i./kg dry soil for carrot and tomato. Survival rates of 73.7, 100 and 83.3 % of germinated seedlings were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil and 92.1 % for oat at 333.33 mg a.i./kg dry soil.

 

EFFECT ON PLANT GROWTH AND HEALTH

At harvest, measurement of the height and dry weight for each surviving seedling from each replicate pot was conducted. Following statistical analysis, significant reductions in height and dry weight were estimated for all species at 333.33 and 1000 mg a.i./kg dry soil in comparison to the solvent controls.

 

There were no visible effects on carrot at rates of up to 37.04 mg a.i./kg dry soil or on tomato and oat at rates of up to 111.11 mg a.i./kg dry soil.

 

Very slight growth inhibition was recorded in one replicate for carrot at 111.11 mg a.i./kg dry soil with 25-40 and 80 % growth inhibition recorded at 333.33 and 1000 mg a.i./kg dry soil respectively. Slight (333.33 mg a.i./kg dry soil) and slight-moderate (1000 mg a.i./kg dry soil) phytotoxicity was also recorded. For tomato, slight growth inhibition (< 10 %) was recorded at 333.33 mg a.i./kg dry soil with approximately 80 % inhibition at 1000 mg a.i./kg dry soil. Phytotoxicity was also recorded in three replicates at 1000 mg a.i./kg dry soil.

 

For oat, 50 % and 80 % growth inhibition was recorded at 333.33 and 1000 mg a.i./kg dry soil respectively.

 

Effects on height and dry weight in comparison to the solvent control are summarised in the table below.

 

Species

Treatment

Rate (mg a.i./kg dry soil)

Geometric mean height (cm)

% effect on mean height

p

Geometric mean dry weight (mg)

% effect on mean wet weight

p

Carrot

Water control

0

2.509

-4

0.610 (T)

2.829

+23

0.093 (T)

Carrot

Solvent control

0

2.615

0

-

2.306

0

-

Carrot

Test item

12.35

2.608

0

1.000 (W)

2.292

1

1.000 (W)

Carrot

Test item

37.04

2.542

-3

1.000 (W)

2.601

+13

1.000 (W)

Carrot

Test item

111.11

2.667

+2

1.000 (W)

2.348

+2

1.000 (W)

Carrot

Test item

333.33

2.081

-20

0.007** (W)

1.479

-36

< 0.001*** (W)

Carrot

Test item

1000

0.883

-66

< 0.001*** (W)

0.489

-79

< 0.001*** (W)

Tomato

Water control

0

5.241

+8

0.218 (T)

18.473

-6

0.575 (T)

Tomato

Solvent control

0

4.831

0

-

19.709

0

-

Tomato

Test item

12.35

4.627

4

1.000 (W)

16.969

-14

0.569 (D)

Tomato

Test item

37.04

4.943

+2

1.000 (W)

15.597

-21

0.166 (D)

Tomato

Test item

111.11

4.891

+1

1.000 (W)

21.615

+10

0893 (D)

Tomato

Test item

333.33

3.938

-18

0.003** (W)

14.036

29

0.017* (D)

Tomato

Test item

1000

1.874

-61

< 0.001*** (W)

1.920

90

< 0.001*** (D)

Oat

Water control

0

36.098

0

0.974 (T)

45.679

0

0.990 (T)

Oat

Solvent control

0

36.279

0

-

45.587

0

-

Oat

Test item

12.35

36.428

0

1.000 (W)

46.958

+3

1.000 (W)

Oat

Test item

37.04

36.511

+1

1.000 (W)

44.937

-1

1.000 (W)

Oat

Test item

111.11

37.545

+3

1.000 (W)

41.738

-8

0.742 (W)

Oat

Test item

333.33

15.276

-58

< 0.001*** (W)

10.459

-77

< 0.001*** (W)

Oat

Test item

1000

1.109

-97

< 0.001*** (W)

0.793

-98

< 0.001*** (W)

pvalues are for the comparison with the solvent control using William’s test (W), Dunnett’s test (D) and the t-test (T)

** = p< 0.01

*** = p< 0.001

- = Not applicable

 

The LC25 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot, 730 mg a.i./kg dry soil for tomato and 496 mg a.i./kg dry soil for oat.

 

The LC50 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and tomato and 654 mg a.i./kg dry soil for oat. The NOEC values were 1000 mg a.i./kg dry soil for carrot and 333.33 mg a.i./kg dry soil for tomato and oat.

 

The EC25 values for height were estimated to be 409.80 mg a.i/kg dry soil for carrot, 429.90 a.i/kg dry soil for tomato and 304.95 a.i/kg dry soil for oat. The EC50 values for height were estimated to be 734.90 mg a.i./kg dry soil for carrot, 797.47 mg a.i./kg dry soil for tomato and 334.19 mg a.i./kg dry soil for oat. The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.

 

The EC25 values for dry weight were estimated to be 251.62 mg a.i./kg dry soil for carrot, 354.63 mg a.i./kg dry soil for tomato and 166.01 mg a.i./kg dry soil for oat. The EC50 values for dry weight were estimated to be 445.67 mg a.i./kg dry soil for carrot, 497.91 mg a.i./kg dry soil for tomato and 240.60 mg a.i./kg dry soil for oat. The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.

 

CONCLUSION

There were treatment-related effects on plant germination at 1000 mg a.i./kg dry soil for all the species tested and at 333.33 mg a.i./kg dry soil for oat.

 

The LC25 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and 730 and 496 mg a.i./kg dry soil for tomato and oat respectively. The LC50 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and tomato and 654 mg a.i./kg dry soil for oat. The NOEC values were 1000 mg a.i./kg dry soil for carrot and 333.33 mg a.i./kg dry soil for tomato and oat.

 

There was 100 % survival of carrot and tomato at all rates of application up to and including 333.33 mg a.i./kg dry soil. Survival rates of 73.7, 100 and 83.3 % of germinated seedlings were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil and 92.1 % for oat at 333.33 mg a.i./kg dry soil.

 

The EC25 values for height were estimated to be 409.80, 429.90 and 304.95 mg a.i./kg dry soil for carrot, tomato and oat respectively. The EC50 values for height were estimated to be 734.90, 797.47 and 334.19 mg a.i./kg dry soil for carrot, tomato and oat respectively. The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.

 

The EC25 values for dry weight were estimated to be 251.62, 354.63 and 166.01 mg a.i./kg dry soil for carrot, tomato and oat respectively. The EC50 values for dry weight were estimated to be 445.67, 497.91 and 240.60 mg a.i./kg dry soil for carrot, tomato and oat respectively. The NOEC values for effects on dry weight were 111.11 mg a.i./kg dry soil for all species.

 

The study was considered valid as the validation criteria were met and no visible treatment-related effects were observed in the controls.

Endpoint:
toxicity to terrestrial plants: short-term
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
30 May 2016 to 04 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
other: HJ/T 153-2004
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- The initial concentration of test item at the start of the test was dissolved in 30 mL acetone and determined by ICP-MS after 15 min ultrasonication.
- Test substrates used for initial concentration analysis were prepared as follows:
(i) Samples of blank control and solvent control: 1.00 mL extracting solution was diluted to 10 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(ii) Samples of 12.5 mg/L: 0.2 mL extracting solution were diluted to 10 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(iii) Samples of 25.0 mg/L: 0.4 mL extracting solution were diluted to 10 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(iv) Samples of 35.0 mg/L: 0.1 mL extracting solution were diluted to 25 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(v) Samples of 50.0 mg/L: 0.2 mL extracting solution were diluted to 100 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP·MS.
(vi) Samples of 70.0 mg/L, 100 mg/L: 0.1 mL extracting solution were diluted to 100 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by lCP-MS.
(vii) Samples of 140 mg/L: 0.1 mL extracting solution were diluted to 250 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(viii) Samples of 200 mg/L, 280 mg/L: 0.05 mL extracting solution were diluted to 100 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(ix) Samples of 400 mg/L: 0.025 mL extracting solution were diluted to 100 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(x) Samples of 420 mg/L, 560 mg/L: 0.1 mL extracting solution were diluted to 250 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(xi) Samples of 800 mg/L: 0.0625 mL extracting solution were diluted to 500 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
(xii) Samples of 1000 mg/L: 0.05 mL extracting solution were diluted to 500 mL with 4% nitric acid solution (v/v). Then filtrated by 0.22 μm membrane (discarding the initial 5.00 mL filtrates) and analysed by ICP-MS.
Vehicle:
yes
Remarks:
acetone
Species:
Brassica campestris var. chinensis
Details on test organisms:
Cabbage (seed lot S20160530BC)
Species:
Lactuca sativa
Details on test organisms:
Lettuce (seed lot S20160530LS)
Species:
Oryza sativa
Details on test organisms:
Rice (seed lot S201605300)
Species:
Brassica oleracea var. capitata
Details on test organisms:
Kohlrabi (seed lot S20160530BO)
Species:
Cucumis sativus
Details on test organisms:
Cucumber (seed lot S20160530C)
Species:
Zea mays
Details on test organisms:
Corn (seed lot S20160530Z)
Species:
other: Sesamum indicum
Details on test organisms:
Sesame (seed lot S20160530S)
Species:
Lycopersicon esculentum
Details on test organisms:
Tomato (seed lot S20160530LE)
Species:
Glycine max (G. soja)
Details on test organisms:
Soybean (seed lot S20160530G)
Species:
Raphanus sativus
Details on test organisms:
Radish (seed lot S20160530R)
Test type:
seed germination/root elongation toxicity test
Study type:
laboratory study
Substrate type:
artificial soil
Remarks:
quartz sand
Limit test:
no
Total exposure duration:
8 d
Remarks:
Total exposure duration was 7 d for Oryza sativa and Zea mays
Post exposure observation period:
Not applicable
Test temperature:
25 ± 1 °C
pH:
See Table 1 (attached)
Moisture:
Not reported
Details on test conditions:
TEST PURPOSE
- The test was designed to assess the phytotoxicity of the test substance on seeds of commercially important terrestrial plants. To this end, seeds were treated with prepared test medium in the dark until at least 65 % of the control seeds have germinated and developed roots that are at least 20 mm long. The number of seeds that germinate was counted, and root lengths measured. The EC10 and EC50 for seed germination and root elongation were determined and reported for each of the species tested.

SELECTION OF SPECIES
- Ten species were chosen as the test organism because of their economic or ecological importance in China.
- The seeds were held in refrigeration facilities in cold storage (5 °C) in moisture-proof containers at seed moisture contents of < 10 %.
- The seeds were brought in from Nanjing Research Institute of Vegetables and Flowers. The information on growing season collected and germination percentage was provided by the supplier of the seeds.

APPARATUS
- Water quality parameter analyzer (HQ30d, HACH Corporation, parameters including pH and Temperature)
- Climatic chambers (3MCLIMACELL, MMM Group)
- Refrigeration facilities (BCD-218, Siemens)
- Petri dishes (Diameter= 200 mm, Dahua glassware ltd., Xuzhou)
- Vernier caliper (16EX, Mahr, Germany)
- Electronic balance (MS105DU, METTLER TOLEDO, Switzerland)
- ICP-MS (Thermo Scientific X SERIES 2 ICP-MS, USA).

RANGE FINDING TEST
- Nominal concentrations of 100, 500 and 1000 mg/L.
- Blank control.
- Solvent control without test substance.
- No replicate.
- A stock solution of 100000 mg/L was prepared by dissolving 5.0103 g test substance in 50 mL acetone.
- Details of media preparation are shown in the attached table.

DEFINITIVE TEST
- Because significant phytotoxicity was showed in the range-finding test, a definitive test was performed.
- One control group without test substance was tested in parallel.
- Three replicates each with 10 seed per species for each concentration and control.
- Taking into account the numbers of plant species, the number of replicates and the number of
tested concentrations, the workload had to be spread over time. Batch 1 test started on 13 June 2016; Batch 2 test started on 08 July 2016; Batch 3 test started on 20 July 2016; Batch 4 test started on 28 July 2016.
- Batch 1: A stock solution of 5000 mg/L was prepared by dissolving 0.5015 g test substance in 100 mL acetone.
- Batch 2: A stock solution of 5000 mg/L was prepared by dissolving 0.5017 g test substance in 100 mL acetone.
- Batch 3: A stock solution of 5126 mg/L was prepared by dissolving 0.5126 g test substance in 100 mL acetone.
- Batch 4: A stock solution of 5112 mg/L was prepared by dissolving 0.5112 g test substance in 100 mL acetone.
- The test medium was prepared by adding test solution to 10 g quartz sand for each container. The
mixture was thoroughly mixed. After evaporating to dryness, 90 g quartz sand was added and the
obtained mixture was again thoroughly mixed; giving a final volume of deionized water of 30 mL.
- Details of media preparation are shown in the attached table.

TEST PERFORMANCE
- seeds were positioned on the quartz sand substrate, and the radicle end of the seed was
aligned in the direction of this growth. The dishes were sealed with preservative film with some
holes and placed in a climatic chamber.
- During the test, the following conditions were maintained:
(i) Light: complete darkness.
(ii) Temperature: 25 ± 1 °C.
(iii) Humidity: 80 ± 5 %.
(iv) Seeding densities: 15 seeds/ dish for range-finding test, 10 seeds/ dish for the definitive test.
(v) Replicates: none for range-finding test; triple replicates for the definitive test.
- The test included one run for each of the plant species.
- The test period was ended when at least 65 % of the control seeds have germinated and developed roots that are at least 20 mm long.
- When both conditions were satisfied, the mean number of germinated seeds and mean root length per treatment (and control) were determined.
- The EC50 and EC10 for values were determined for germination and root elongation for each species tested.

OBSERVATIONS AND MEASUREMENTS
- The exposure durations for each test species were shown in the table attached.
- When exposure ended, the number of seeds that germinate was counted and root lengths measured for each test species.
- All root elongation measurements for a given species were made sequentially before proceeding to the next species.
- Means and standard deviations were calculated for each treatment and control.
- The root length was measured from the transition point between the hypocotyl and root to the tip of the root.
- Temperature in the germination facility was reported at the start and the end of the test.
- The pH of the test solutions was recorded at the initiation of the test.

VALIDITY OF THE TEST
- The validity of the test was assessed with respect to the acceptability criteria prescribed in the
study protocol:
(1) Control group: Blank control and solvent control series were also run in addition to the treatment series.
(2) Validity of test result: During the whole test period, more than 65 % of the control seeds have germinated at the end of test.
- The test was considered valid.
Nominal and measured concentrations:
- Brassica chinensis L: Nominal concentrations of 35.0, 70.0, 140, 280, 420 and 560 mg/L for definitive test (Batch 1).
- Lacluca saliva: Nominal concentrations of 35.0, 70.0, 140, 280, 420 and 560 mg/L for definitive test (Batch 1).
- Oryza saliva L: Nominal concentrations of 35.0, 70.0, 140, 280, 420 and 560 mg/L for definitive test (Batch 2).
- Zea mays: Nominal concentrations of 35.0, 70.0, 140, 280, 420 and 560 mg/L for definitive test (Batch 2).
- Cucumis salivus: Nominal concentrations of 12.5, 25.0, 50.0, 100, 200 and 400 mg/L (Batch 3).
- Sesamum indicum: Nominal concentrations of 12.5, 25.0, 50.0, 100, 200 and 400 mg/L (Batch 3).
- Lycopersicon esculenlum: Nominal concentrations of 12.5, 25.0, 50.0, 100, 200 and 400 mg/L (Batch 3).
- Brassica oleracea: Nominal concentrations of 50.0, 100, 200, 400, 800 and 1000 mg/L (Batch 4).
- Glycine max: Nominal concentrations of 25.0, 50.0, 100, 200, 400 and 800 mg/L (Batch 4).
- Raphanus sativus: Nominal concentrations of 25.0, 50.0, 100, 200, 400 and 800 mg/L (Batch 4).
Reference substance (positive control):
no
Species:
Brassica campestris var. chinensis
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Lactuca sativa
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
237 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Oryza sativa
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
216 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Brassica oleracea var. capitata
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Cucumis sativus
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
202 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Zea mays
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
517 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
other: Sesamum indicum
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
159 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Lycopersicon esculentum
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
330 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Glycine max (G. soja)
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Raphanus sativus
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
587 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
germination
Species:
Brassica campestris var. chinensis
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
152 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
Lactuca sativa
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
184 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
Oryza sativa
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
187 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
Brassica oleracea var. capitata
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
398 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
Cucumis sativus
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
110 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
Zea mays
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
122 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
other: Sesamum indicum
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
241 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
Lycopersicon esculentum
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
296 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
Glycine max (G. soja)
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
348 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Species:
Raphanus sativus
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
218 other: mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
boron content
Basis for effect:
other: root elongation
Details on results:
TEST CONDITIONS
- The pH and temperature of the control and treatment groups measured during the test are shown in Tables 1 and 2, respectively (attached).
- During the whole test period, the temperature in the germination facility was maintained in the range of 24.7 °C to 25.3 °C. At the beginning of the test, the pH value of the control medium was between 7.20 and 7.28, while the pH of freshly prepared test media was between 6.17 and 7.18.

ANALYSIS OF TEST CONCENTRATIONS
- Specificity: Under the above ICP-MS conditions, 4% nitric acid solution (v/v) and 0.1 mg/L standard working solution were analysed. At mass number of 10.8, there was no interference peak for 4% nitric acid solution (v/v), and there was interference peak for standard working solution. And the analytical method had specificity.
- Calibration curve: A series of standard solutions with concentrations of 0.00, 0.02, 0.05, 0.10, 0.20 and 0.30 were measured using ICP-MS. Based on the test result, a linear regression equation was obtained between the concentration: A=2E+06c+2307.4, with good linearity of r2 - 0.9935, where A represents peak area of boron (B); and c is the concentration of the test substance (mg/L) (see Figure 1, attached). The results show that linearity for concentration range of 0.00 mg/L to 0.30 mg/L is good.
- Limit of Detection (LOD) and Limit of Quantification (LOQ): The detection limit ofICP-M8 for the test substance was 0.01 mg/L (S/N ≥ 3), and the quantitation limit was 0.02 mg/L (S/N ≥ 10).
- Analysis of test concentrations: The measured concentrations corresponding to the different test solutions are listed in Table 1 (attached).
- The analysed concentration in batch 1 of 35.0 mg/L, 70.0 mg/L, 140 mg/L, 280 mg/L, 420 mg/Land 560 mg/L were 34.5 mg/L, 73 mg/L, 157.5 mg/L, 260 mg/L, 435 mg/Land 577.5 mg/L.
- The analysed concentration in batch 2 of 35.0 mg/L, 70.0 mg/L, 140 mg/L, 280 mg/L, 420 mg/L and 560 mg/L were 32.75 mg/L, 75 mg/L, 147.5 mg/L, 268 mg/L, 427.5 mg/Land 570 mg/L.
- The analysed concentration in batch 3of12.5 mg/L, 25.0 mg/L, 50.0 mg/L, 100 mg/L, 200 mg/L and 400 mg/L were 12.15 mg/L, 27.5 mg/L, 54.5 mg/L, 107 mg/L, 220 mg/Land 412 mg/L.
- The analysed concentration in batch 4 of25.0 mg/L, 50.0 mg/L, 100 mg/L, 200 mg/L, 400 mg/L, 800 mg/Land 1000 mg/L were 26.5 mg/L, 51.5 mg/L, 103 mg/L, 204 mg/L, 404 mg/L, 896 mg/L and 115 0 mg/L.

EFFECTS ON GERMINATION RATE AND ROOT ELONGATION
- At the end of both the range-finding and the definitive tests in all tested species, 86.7 % to 100 % of the control seeds germinated and the roots developed were longer than 20 mm.
- Table 3 and Table 4 (attached) show Germination Rates and Root Lengths observed for the seeds of different species in the range-finding test. Among the ten tested species, two did not present any inhibition of their germination rates when exposed to test item: Brassica chinensis L. and Brassica oleracea. The germination rates of the 8 other species were inhibited with a maximal effect at 1000 mg/L. At this concentration, the inhibition rates ranged from approximately 66.7 to 100 % depending on the plant species.
- Regarding root length, the ten species seemed to be affected. Tables 5 to Table 24 (attached) show germination rates and root lengths observed for the seeds of different species in the definitive test. Among the ten tested species, three did not present any inhibition of their germination rates when exposed to test item: Brassica chinensis L., Brassica oleracea. and Glycine max. The germination rates of the seven other species were inhibited and the magnitude of the effect increased with the concentration of test item. At the highest concentrations tested for each species, the inhibition rates ranged from 53.3 to 85.2 % depending on the plant species. Regarding root length, the ten species seemed to be affected. Again, the effects on root length increased with the concentration of 083197 54. At the highest concentrations tested for each species, the inhibition rates> 73%. Except Brassica oleracea, 9 other seeds showed germinated but the roots were perish in the high concentrations treatments.
- The EC1o and EC5o values for germination or root elongation for 10 species are given in the attached table together with the 95% confidence limits.
Reported statistics and error estimates:
DATA PROCESSING
- The Probit Model (SPSS 17.0) was used to calculate the EC10, EC50 and the 95% confidence limits.
Validity criteria fulfilled:
yes
Conclusions:
Results of exposure to the test item for 7-8 days obtained under valid test conditions permitted EC10 and EC50 values to be determined for ten species together with 95 % confidence intervals.
Executive summary:

GUIDELINE

Under valid conditions, the phytotoxicity of the test substance was investigated on seeds of commercially impo1tant terrestrial plants according to “The guidelines for the testing of chemicals" (HJ/T 153-2004).

 

METHODS

In the test, ten plant species (see the details of species in the table below) were exposed to a series of concentrations of the test substance. A blank control without the test substance was run at the same time. During the whole test period, the temperature in the germination facility was maintained in the range of 24.7 °C to 25.3 °C. At the beginning of the test, the pH value of the blank control medium and solvent control medium were 7.20 to 7.28 and 7.18 to 7.29, while the pH of the freshly prepared test media was between 6.17 and 7.18. At the end of the test, more than 65 % of the control seeds germinated and the roots developed were longer than 20 mm. Therefore, the study met the acceptability criteria prescribed by the protocol and the test was considered valid.

 

RESULTS

 

The results showed that under valid test conditions, the EC10 and EC50 for germination or root elongation for the ten species were as shown in the table below.

 

Species

EC10 germination (mg/L)

EC10

root elongation (mg/L)

EC50 germination (mg/L)

EC50

root elongation (mg/L)

Brassica chinensisL.

> 1000*

42.4

(95% CI = 32.8 to 51.9)

> 1000*

152

(95% CI = 134 to 170)

Lactuca sativa

59.5

(95% CI = 29.9 to 87.9)

49.3

(95% CI = 37.5 to 60.6)

237

(95% CI = 180 to 320)

184

(95% CI = 162 to 212)

Oryza sativa

50.4

(95% CI = 14.8 to 86.4)

56.8

(95% CI = 44.0 to 68.5)

216

(95% CI = 143 to 337)

187

(95% CI = 162 to 222)

Brassica oleracea

> 1000

88.6

(95% CI = 41.8 to 136)

> 1000

398

(95% CI = 292 to 565)

Cucumis sativus

70.9

(95% CI = 57.9 to 83.1)

23.4

(95% CI = 17.5 to 29.1)

202

(95% CI = 180 to 230)

110

(95% CI = 93.5 to 135)

Zea mays

230

(95% CI = 191 to 262)

44.5

(95% CI = 35.4 to 52.8)

517

(95% CI = 467 to 591)

122

(95% CI = 108 to 137)

Sesamum indicum

64.8

(95% CI = 31.9 to 92.6)

28.3

(95% CI = 19.1 to 37.3)

159

(95% CI = 117 to 222)

241

(95% CI = 176 to 388)

Lycopersicon esculentum

147

(95% CI = 122 to 169)

27.3

(95% CI = 17.3 to 37.0)

330

(95% CI = 295 to 380)

296

(95% CI = 203 to 546)

Glycine max

564

(95% CI not applicable)

54.5

(95% CI = 12.1 to 94.7)

> 1000*

348

(95% CI = 205 to 1343)

Raphanus sativus

95.4

(95% CI = 37.9 to 153)

49.3

(95% CI = 37.3 to 60.9)

587

(95% CI = 375 to 1331)

218

(95% CI = 186 to 264)

* EC10 and EC50 > 1000 mg/L reflects the results of both the range-finding and the definitive tests.

Description of key information

Key study

There were treatment-related effects on plant germination were at 1000 mg a.i./kg dry soil for all the species tested and at 333.33 mg a.i./kg dry soil for oat (OECD 208).

 

The LC25 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and 730 and 496 mg a.i./kg dry soil for tomato and oat respectively. The LC50 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and tomato and 654 mg a.i./kg dry soil for oat. The NOEC values were 1000 mg a.i./kg dry soil for carrot and 333.33 mg a.i./kg dry soil for tomato and oat.

 

There was 100 % survival of carrot and tomato at all rates of application up to and including 333.33 mg a.i./kg dry soil. Survival rates of 73.7, 100 and 83.3 % of germinated seedlings were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil and 92.1 % for oat at 333.33 mg a.i./kg dry soil.

 

The EC25 values for height were estimated to be 409.80, 429.90 and 304.95 mg a.i./kg dry soil for carrot, tomato and oat respectively. The EC50 values for height were estimated to be 734.90, 797.47 and 334.19 mg a.i./kg dry soil for carrot, tomato and oat respectively. The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.

 

The EC25 values for dry weight were estimated to be 251.62, 354.63 and 166.01 mg a.i./kg dry soil for carrot, tomato and oat respectively. The EC50 values for dry weight were estimated to be 445.67, 497.91 and 240.60 mg a.i./kg dry soil for carrot, tomato and oat respectively. The NOEC values for effects on dry weight were 111.11 mg a.i./kg dry soil for all species.

 

The study was considered valid as the validation criteria were met and no visible treatment-related effects were observed in the controls.

Supporting study

Results of exposure to test item for 7-8 days obtained under valid test conditions permitted EC10 and EC50 values to be determined for ten species together with 95 % confidence intervals ( HJ/T 153-2004).

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:
333 mg/kg soil dw

Additional information

Key study

GUIDELINE

The study was conducted in accordance with OECD Guideline No 208 “Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test” adopted 19 July 2006.

 

METHODS

The study was carried out with carrot, tomato and oat to determine if the test substance exerts a detrimental effect to plants during early critical stages in their development. The investigation was a dose-response test designed to evaluate the phytotoxic effects of the test substance. Each plant species was sown into treated soil and monitored for 14 to 16 days following a minimum of 50 % germination in the controls.

 

Following initial range-finding at 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry soil, the definitive test was conducted as a dose-response test at 12.35, 37.04, 111.11, 333.33 and 1000 mg a.i./kg dry soil for all species.

 

For carrot, 14 replicate pots of three seeds were sown for each treatment rate, with 20 pots of two seeds for tomato and ten pots of four seeds for oat. An untreated water control and a solvent control were also included for all species. The soil used was sandy loam, sieved to remove all particles > 2 mm.

 

After application and sowing, the pots were checked daily and the numbers of emerged seedlings were recorded. Emergence, survival and sublethal effects were recorded daily up to a minimum of 50 % emergence of control seedlings and thereafter every one to three days. Plants were harvested 14-16 days after 50 % control germination, giving a total exposure period of 21 days for oat and 22 days for carrot and tomato. The height of each individual plant was then measured and the dry weight was recorded.

 

EFFECT ON GERMINATION AND PLANT SURVIVAL

Germination in the water and solvent controls was 100 % for all species. Reductions in germination of 9.5, 42.5 and 85.0 % were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil. There was also 5.0 % reduction in germination in oat at 333.33 mg a.i./kg dry soil.

 

There was 100 % survival of all species in the water and solvent controls and at all rates of application up to and including 333.33 mg a.i./kg dry soil for carrot and tomato. Survival rates of 73.7, 100 and 83.3 % of germinated seedlings were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil and 92.1 % for oat at 333.33 mg a.i./kg dry soil.

 

EFFECT ON PLANT GROWTH AND HEALTH

At harvest, measurement of the height and dry weight for each surviving seedling from each replicate pot was conducted. Following statistical analysis, significant reductions in height and dry weight were estimated for all species at 333.33 and 1000 mg a.i./kg dry soil in comparison to the solvent controls.

 

There were no visible effects on carrot at rates of up to 37.04 mg a.i./kg dry soil or on tomato and oat at rates of up to 111.11 mg a.i./kg dry soil.

 

Very slight growth inhibition was recorded in one replicate for carrot at 111.11 mg a.i./kg dry soil with 25-40 and 80 % growth inhibition recorded at 333.33 and 1000 mg a.i./kg dry soil respectively. Slight (333.33 mg a.i./kg dry soil) and slight-moderate (1000 mg a.i./kg dry soil) phytotoxicity was also recorded. For tomato, slight growth inhibition (< 10 %) was recorded at 333.33 mg a.i./kg dry soil with approximately 80 % inhibition at 1000 mg a.i./kg dry soil. Phytotoxicity was also recorded in three replicates at 1000 mg a.i./kg dry soil.

 

For oat, 50 % and 80 % growth inhibition was recorded at 333.33 and 1000 mg a.i./kg dry soil respectively.

 

Effects on height and dry weight in comparison to the solvent control are summarised in the table below.

 

Species

Treatment

Rate (mg a.i./kg dry soil)

Geometric mean height (cm)

% effect on mean height

p

Geometric mean dry weight (mg)

% effect on mean wet weight

p

Carrot

Water control

0

2.509

-4

0.610 (T)

2.829

+23

0.093 (T)

Carrot

Solvent control

0

2.615

0

-

2.306

0

-

Carrot

Test item

12.35

2.608

0

1.000 (W)

2.292

1

1.000 (W)

Carrot

Test item

37.04

2.542

-3

1.000 (W)

2.601

+13

1.000 (W)

Carrot

Test item

111.11

2.667

+2

1.000 (W)

2.348

+2

1.000 (W)

Carrot

Test item

333.33

2.081

-20

0.007** (W)

1.479

-36

< 0.001*** (W)

Carrot

Test item

1000

0.883

-66

< 0.001*** (W)

0.489

-79

< 0.001*** (W)

Tomato

Water control

0

5.241

+8

0.218 (T)

18.473

-6

0.575 (T)

Tomato

Solvent control

0

4.831

0

-

19.709

0

-

Tomato

Test item

12.35

4.627

4

1.000 (W)

16.969

-14

0.569 (D)

Tomato

Test item

37.04

4.943

+2

1.000 (W)

15.597

-21

0.166 (D)

Tomato

Test item

111.11

4.891

+1

1.000 (W)

21.615

+10

0893 (D)

Tomato

Test item

333.33

3.938

-18

0.003** (W)

14.036

29

0.017* (D)

Tomato

Test item

1000

1.874

-61

< 0.001*** (W)

1.920

90

< 0.001*** (D)

Oat

Water control

0

36.098

0

0.974 (T)

45.679

0

0.990 (T)

Oat

Solvent control

0

36.279

0

-

45.587

0

-

Oat

Test item

12.35

36.428

0

1.000 (W)

46.958

+3

1.000 (W)

Oat

Test item

37.04

36.511

+1

1.000 (W)

44.937

-1

1.000 (W)

Oat

Test item

111.11

37.545

+3

1.000 (W)

41.738

-8

0.742 (W)

Oat

Test item

333.33

15.276

-58

< 0.001*** (W)

10.459

-77

< 0.001*** (W)

Oat

Test item

1000

1.109

-97

< 0.001*** (W)

0.793

-98

< 0.001*** (W)

pvalues are for the comparison with the solvent control using William’s test (W), Dunnett’s test (D) and the t-test (T)

**p< 0.01

***p< 0.001

- Not applicable

 

The LC25 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot, 730 mg a.i./kg dry soil for tomato and 496 mg a.i./kg dry soil for oat.

 

The LC50 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and tomato and 654 mg a.i./kg dry soil for oat. The NOEC values were 1000 mg a.i./kg dry soil for carrot and 333.33 mg a.i./kg dry soil for tomato and oat.

 

The EC25 values for height were estimated to be 409.80 mg a.i/kg dry soil for carrot, 429.90 a.i/kg dry soil for tomato and 304.95 a.i/kg dry soil for oat. The EC50 values for height were estimated to be 734.90 mg a.i./kg dry soil for carrot, 797.47 mg a.i./kg dry soil for tomato and 334.19 mg a.i./kg dry soil for oat. The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.

 

The EC25 values for dry weight were estimated to be 251.62 mg a.i./kg dry soil for carrot, 354.63 mg a.i./kg dry soil for tomato and 166.01 mg a.i./kg dry soil for oat. The EC50 values for dry weight were estimated to be 445.67 mg a.i./kg dry soil for carrot, 497.91 mg a.i./kg dry soil for tomato and 240.60 mg a.i./kg dry soil for oat. The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.

 

CONCLUSION

There were treatment-related effects on plant germination at 1000 mg a.i./kg dry soil for all the species tested and at 333.33 mg a.i./kg dry soil for oat.

 

The LC25 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and 730 and 496 mg a.i./kg dry soil for tomato and oat respectively. The LC50 values for germination were estimated to be > 1000 mg a.i./kg dry soil for carrot and tomato and 654 mg a.i./kg dry soil for oat. The NOEC values were 1000 mg a.i./kg dry soil for carrot and 333.33 mg a.i./kg dry soil for tomato and oat.

 

There was 100 % survival of carrot and tomato at all rates of application up to and including 333.33 mg a.i./kg dry soil. Survival rates of 73.7, 100 and 83.3 % of germinated seedlings were recorded for carrot, tomato and oat respectively at 1000 mg a.i./kg dry soil and 92.1 % for oat at 333.33 mg a.i./kg dry soil.

 

The EC25 values for height were estimated to be 409.80, 429.90 and 304.95 mg a.i./kg dry soil for carrot, tomato and oat respectively. The EC50 values for height were estimated to be 734.90, 797.47 and 334.19 mg a.i./kg dry soil for carrot, tomato and oat respectively. The NOEC values for effects on height were 111.11 mg a.i./kg dry soil for all species.

 

The EC25 values for dry weight were estimated to be 251.62, 354.63 and 166.01 mg a.i./kg dry soil for carrot, tomato and oat respectively. The EC50 values for dry weight were estimated to be 445.67, 497.91 and 240.60 mg a.i./kg dry soil for carrot, tomato and oat respectively. The NOEC values for effects on dry weight were 111.11 mg a.i./kg dry soil for all species.

 

The study was considered valid as the validation criteria were met and no visible treatment-related effects were observed in the controls.

Supporting study

GUIDELINE

Under valid conditions, the phytotoxicity of the test substance was investigated on seeds of commercially impo1tant terrestrial plants according to “The guidelines for the testing of chemicals" (HJ/T 153-2004).

 

METHODS

In the test, ten plant species (see the details of species in the table below) were exposed to a series of concentrations of the test substance. A blank control without the test substance was run at the same time. During the whole test period, the temperature in the germination facility was maintained in the range of 24.7 °C to 25.3 °C. At the beginning of the test, the pH value of the blank control medium and solvent control medium were 7.20 to 7.28 and 7.18 to 7.29, while the pH of the freshly prepared test media was between 6.17 and 7.18. At the end of the test, more than 65 % of the control seeds germinated and the roots developed were longer than 20 mm. Therefore, the study met the acceptability criteria prescribed by the protocol and the test was considered valid.

 

RESULTS

 

The results showed that under valid test conditions, the EC10 and EC50 for germination or root elongation for the ten species were as shown in the table below.

 

Species

EC10 germination (mg/L)

EC10

root elongation (mg/L)

EC50 germination (mg/L)

EC50

root elongation (mg/L)

Brassica chinensisL.

> 1000*

42.4

(95% CI = 32.8 to 51.9)

> 1000*

152

(95% CI = 134 to 170)

Lactuca sativa

59.5

(95% CI = 29.9 to 87.9)

49.3

(95% CI = 37.5 to 60.6)

237

(95% CI = 180 to 320)

184

(95% CI = 162 to 212)

Oryza sativa

50.4

(95% CI = 14.8 to 86.4)

56.8

(95% CI = 44.0 to 68.5)

216

(95% CI = 143 to 337)

187

(95% CI = 162 to 222)

Brassica oleracea

> 1000

88.6

(95% CI = 41.8 to 136)

> 1000

398

(95% CI = 292 to 565)

Cucumis sativus

70.9

(95% CI = 57.9 to 83.1)

23.4

(95% CI = 17.5 to 29.1)

202

(95% CI = 180 to 230)

110

(95% CI = 93.5 to 135)

Zea mays

230

(95% CI = 191 to 262)

44.5

(95% CI = 35.4 to 52.8)

517

(95% CI = 467 to 591)

122

(95% CI = 108 to 137)

Sesamum indicum

64.8

(95% CI = 31.9 to 92.6)

28.3

(95% CI = 19.1 to 37.3)

159

(95% CI = 117 to 222)

241

(95% CI = 176 to 388)

Lycopersicon esculentum

147

(95% CI = 122 to 169)

27.3

(95% CI = 17.3 to 37.0)

330

(95% CI = 295 to 380)

296

(95% CI = 203 to 546)

Glycine max

564

(95% CI not applicable)

54.5

(95% CI = 12.1 to 94.7)

> 1000*

348

(95% CI = 205 to 1343)

Raphanus sativus

95.4

(95% CI = 37.9 to 153)

49.3

(95% CI = 37.3 to 60.9)

587

(95% CI = 375 to 1331)

218

(95% CI = 186 to 264)

* EC10 and EC50 > 1000 mg/L reflects the results of both the range-finding and the definitive tests.