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Ecotoxicological information

Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Jun 2017 to 11 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 208 (Terrestrial Plants, Growth Test)
Version / remarks:
July, 2006
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): ZDEC
- Batch No.: 70100804
- Purity: 99.2%
- Zn. content: 18.1%
- Date of analysis: January 20, 2017
- Expiry date: January 20, 2019
- Appearance: Solid white to off white powder or Granules
- Solubility (water): 1.06 mg/L
- Storage conditions: at 20 ± 5 °C, in the dark
Analytical monitoring:
no
Vehicle:
yes
Remarks:
quartz sand
Details on preparation and application of test substrate:
The test item was weighed separately for each concentration using an analytical balance. The test item was mixed with fine quartz sand by a blender for around 5 minutes and was then applied with this mixture to the soil. The test item treated fine quartz sand was mixed with the soil by using a laboratory mixer for around 5 minutes. The amount of quartz sand depended on the water content of the used charge of soil and was the same in all test item treatment groups (40.3 g quartz sand per kg soil). For the control the same amount of untreated quartz sand per kg soil as in the test item treated groups was added to the soil. The application was done with a laboratory mixer. The soil for one treatment was treated in one to two batches and then split into the replicates. As the test item is insoluble in the used concentration no further dose verification is performed. The application order was as follows: 1. Control, 2. test item (increasing concentrations).
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Oilseed rape
- Plant family: Brassicaceae
- Variety: Smart
- Source of seed: Syngenta seeds
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Soybean
- Plant family: Fabaceae
- Variety: Cardiff
- Source of seed: Saatzucht Donau
Species:
Cucumis sativus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Cucumber
- Plant family: Cucurbitaceae
- Variety: Zircon, F1
- Source of seed: Hild Samen GmbH
Species:
other: Solanum lycopersicum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Tomato
- Plant family: Solanaceae
- Variety: Hilmar
- Source of seed: Bruno Nebelung GmbH
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Perennial ryegrass
- Plant family: Poaceae
- Variety: Deutsches Weidelgras ZS
- Source of seed: Sämereien Jehle
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Onion
- Plant family: Amaryllidaceae
- Variety: Sturon
- Source of seed: Bingenheimer Saatgut
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
21 d
Remarks:
14 to 21 days after 50% germination in the control depending on the growth of the seedlings.
Test temperature:
15.4 - 27.4 °C (mean value of 21.5 °C)
Moisture:
A spot check on soil moisture was carried out daily.
Details on test conditions:
TEST SYSTEM
- Test container: Commercial plastic flower pots (diameter of 15 cm)
- Growth chamber: Yes
- Method of seeding: Only untreated seeds were used. The seeds were introduced manually into the soil. After sowing the pots were placed on saucers and watered.
- No. of seeds / replicates / sowing details: See 'Any other information on materials and methods incl. tables'.

SOURCE AND PROPERTIES OF SUBSTRATE
The soil was steam sterilized, delivered and analysed by the LUFA Speyer, Germany.
- Soil type: LUFA 2.3 (USDA: sandy loam)
- Particle Size: All particles ≤ 0.2 cm
- Corg: 0.68 ± 0.04%
- pH: 5.84 ± 0.6
- Pesticides: No pesticide treatments were performed in the year of sampling and for at least the two previous years.

GROWTH CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- Light intensity and quality: Minimum: 201 μE/m2/s; Maximum: 381 μE/m2/s; Mean: 282 μE/m2/s
- Relative humidity: Minimum: 52%; Maximum: 96%; Mean: 69%
- Watering regime and schedules: Bottom watering (through saucers) was done where necessary. Water was given in order to assure optimal water supply of the plants.
- Fertilization: After development of the first true leaves, Ferty® 9 “Hydro” (Planta-Düngemittel GmbH) at 3 g/L and Terraflor®-AZ (Terraflor GmbH) at 0.4 g/L were added to the water up to three times a week, depending on the development of the plants.

EFFECT PARAMETERS MEASURED: Fresh weight, germination and mortality of the plants treated with the tested concentrations were compared with the results of the control plants.
- Fresh weight: The fresh weight of the above ground part of all survived plants of a pot (each pot is considered as a replicate) was determined 14 or 21 days after 50% seedling emergence in the control. If all plants of a pot were dead the value for this replicate was assessed as “0”.
- Germination: Germination was checked daily on weekdays (except weekends) until 50% of the control plants had emerged. Further checks were done weekly.
- Mortality: The number of living and dead plants was recorded 7 and 14 days or 7, 14 and 21 days after 50% seedling emergence in the control. A plant was considered dead if no living tissue could be found on the leaves or shoots. All other plants were considered living.
- Phytotoxicity: Visual phytotoxicity (e.g. chlorosis, necrosis, abnormal growth) was recorded weekly according to EPPO Standard PP 1/135 (3) after 50% seedling emergence in the control.
- Growth Stages: Growth stages at day 14 or 21 after 50% seedling emergence in the control were reported according to BBCH-Monograph - Growth stages.

VEHICLE CONTROL PERFORMED: yes (the same amount of untreated quartz sand as in the test item treated groups was added to the soil)
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 12.3, 37.0, 111, 192, 333, 577 and 1000 mg test item/kg dry soil (based on a range-finding study).
- The applied concentrations differed per plant species, see 'Any other information on materials and methods incl. tables'.
Reference substance (positive control):
no
Key result
Remarks on result:
other: see 'Any other information on results incl. tables'
Details on results:
SPECIES: BRASSICA NAPUS
- Fresh weight (14 days): The fresh weight was statistically significantly reduced at 192 mg test item/kg soil dry weight and at higher concentrations (-51.1% to -96.6%). The plant development was delayed at 192 mg test item/kg soil dry weight and at higher concentrations (growth stages at test end: BBCH 13-15 in the control, BBCH 13-14 at 192 mg/kg, BBCH 12 at 333 mg/kg, BBCH 12-13 at 577 mg/kg and BBCH 11-12 at 1000 mg/kg).
- Germination: The germination rate ranged between 90% and 95% (control: 86%) There was no statistically significant difference to the control.
- Mortality (14 days): No mortality occurred.
- Phytotoxicity (14 days): Phytotoxic effects were chlorosis and growth reduction. Clear effects were observed at 192 mg test item/kg soil dry weight and at higher concentrations (27% to 97%).
For details on the observed effects, see 'Any other information on results incl. tables'.

SPECIES: GLYCINE MAX
- Fresh weight (14 days): The fresh weight was statistically significantly reduced at all tested concentrations (-21.4% to -59.6%). The plant development was delayed at 577 and 1000 mg/kg (growth stages at test end: BBCH 13/22 in the control and BBCH 12 at 577 and 1000 mg/kg).
- Germination: The germination rate ranged between 85% and 100% (control: 95%) There was no statistically significant difference to the control.
- Mortality (14 days): No mortality occurred.
- Phytotoxicity (14 days): The only phytotoxic effect was growth reduction. Clear effects were observed at 1000 mg test item/kg soil dry weight (32%).

SPECIES: CUCUMIS SATIVUS
- Fresh weight (14 days): The fresh weight was reduced at all tested concentrations (-5.7% to -91.0%). This reduction was statistically significant at 192 mg test item/kg soil dry weight and at higher concentrations (-47.7% to -91.0%). The plant development was not clearly delayed compared to the control (growth stages at test end: BBCH 12 in the control and BBCH 12 and BBCH 11-12 in the test item treatment groups).
- Germination: The germination rate ranged between 95% and 100% (control: 100%) There was no statistically significant difference to the control.
- Mortality (14 days): No mortality occurred.
- Phytotoxicity (14 days): The only phytotoxic effect was growth reduction. Clear effects were observed at 192 mg test item/kg soil dry weight and at higher concentrations (26% to 93%).

SPECIES: SOLANUM LYCOPERSICUM
- Fresh weight (14 days): The fresh weight was statistically significantly reduced at 333 and 1000 mg test item/kg soil dry weight (-90.8% and -93.9%). The plant development was delayed compared to the control at 333 and 1000 mg ZDEC/kg soil dry weight (growth stages at test end: BBCH 12-14 in the control and BBCH 10-12 at 333 and 1000 mg/kg).
- Germination: The germination rate ranged between 85% and 100% (control: 95%) There was no statistically significant difference to the control.
- Mortality (14 days): No mortality occurred.
- Phytotoxicity (14 days): The only phytotoxic effect was growth reduction. Clear effects were observed at 333 and 1000 mg test item/kg soil dry weight (85% and 95%).

SPECIES: LOLIUM PERENNE
- Fresh weight (21 days): The fresh weight was statistically significantly reduced at 192 mg test item/kg soil dry weight and at higher concentrations (-70.0% to -93.0%). The plant development was delayed compared to the control at 577 and 1000 mg test item/kg soil dry weight (growth stages at test end: BBCH 21-22 in the control and BBCH 12-14 at 577 and 1000 mg/kg).
- Germination: The germination rate ranged between 70% and 100% (control: 90%). There was no statistically significant difference to the control.
- Mortality (21 days): No mortality occurred.
- Phytotoxicity (21 days): Phytotoxic effects were chlorosis, necrosis (two replicates) and growth reduction. One pot at 1000 mg test item/kg soil dry weight showed discolouration of the plants. Clear effects were observed at 192 mg test item/kg soil dry weight and at higher concentrations (55% to 78%).

SPECIES: ALLIUM CEPA
- Fresh weight (21 days): The fresh weight was statistically significantly reduced at 192 mg test item/kg soil dry weight and at higher concentrations (-55.4% to -87.7%). The plant development was delayed compared to the control at 333 mg test item/kg soil dry weight and at higher concentrations (growth stages at test end: BBCH 12 in the control and BBCH 11 at 333, 577 and 1000 mg/kg).
- Germination: The germination rate ranged between 95% and 100% (control: 100%) There was no statistically significant difference to the control.
- Mortality (21 days): No mortality occurred.
- Phytotoxicity (21 days): Phytotoxic effects were chlorosis and growth reduction. Clear effects were observed at 192 mg ZDEC/kg soil dry weight and at higher concentrations (40% to 92%).
Reported statistics and error estimates:
Fresh weight data were tested for normal distribution and homogeneity of variance using the Shapiro-Wilk’s test (α = 0.05) and the Levene’s test (α = 0.05). If the data were normally distributed, homogeneous and showed a monotonic dose response the Williams t-test (multiple comparison, one-sided smaller, α = 0.05) was used for comparing treatment groups and control. If the data were normally distributed and not homogeneous the Bonferroni-Welch t-test (multiple comparison, one-sided smaller, α = 0.05) was used. If the data were not normally distributed the Bonferroni-Holm U-test (multiple comparison, one-sided smaller, α = 0.05) was used. In order to determine the EC10, EC20 and EC50values, a regression analysis was performed (Probit-analysis). In the case that no significant dose response relation on the mean values for each treatment group was found (p(F) > 0.05) the regression analysis was performed using all replicates for fitting. For the mortality and germination data Fisher’s Exact Binomial Test (with Bonferroni Correction, multiple comparison, one-sided greater, α = 0.05) was used. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ® ToxRat Solutions GmbH.

Table: Summarised results of fresh weight, germination, mortality, phytotoxicity and plant growth

Time of Evaluation**

Treatment group

Germination (1) [%]

Mortality (1) [#]

Fresh weight [g]

Standard deviation

Effect* [%]

Fresh weight statistics

Phytotoxicity [%]

Growth Stage (BBCH)

Species

(mg test item/kg dry soil)

Final check

Final check

Day 7

Day 14

Day 21

Final check

Brassica napus

Control

86

0

9.68

± 2.96

-

 

0

0

-

13-15

111

90

0

10.56

± 2.70

9

n.s.2

6

7

-

13-15

192

95

0

4.74

± 1.35

-51.1

s.2

19

27

-

13-14

333

90

0

0.63

± 0.28

-93.5

s.2

61

89

-

12

577

95

0

0.82

± 0.46

-91.5

s.2

41

88

-

12-13

1000

95

0

0.3

± 0.08

-96.9

s.2

85

97

-

11-12

Glycine max

Control

95

0

7.14

± 1.63

-

 

0

0

-

13/22

111

85

0

5.61

± 1.96

-21.4

s.3

6

11

-

12-13/22

192

100

0

5.13

± 1.05

-28.1

s.3

8

11

-

12-13/22

333

100

0

4.43

± 0.89

-38

s.3

5

11

-

12-13/22

577

90

0

4.41

± 1.32

-38.3

s.3

3

9

-

12

1000

95

0

2.88

± 0.88

-59.6

s.3

16

32

-

12

Cucumis sativus

Control

100

0

10.17

± 0.75

-

 

0

0

-

12

111

100

0

9.59

± 0.93

-5.7

n.s.2

1

2

-

12

192

100

0

5.32

± 0.43

-47.7

s.2

32

26

-

12

333

100

0

1.66

± 0.39

-83.7

s.2

59

77

-

11-12

577

100

0

2.88

± 0.38

-71.6

s.2

36

59

-

12

1000

95

0

0.91

± 0.18

-91

s.2

71

93

-

11-12

Solanum lycopersicum

Control

95

0

2.31

± 1.51

-

 

0

0

-

12-13

12.3

95

0

3.18

± 1.36

37.5

n.s.2

4

5

-

13-14

37

95

0

3.85

± 2.05

66.4

n.s.2

0

0

-

12-14

111

100

0

3.71

± 0.89

60.3

n.s.2

4

5

-

13-14

333

85

0

0.21

± 0.23

-90.8

s.2

36

85

-

10-12

1000

90

0

0.14

± 0.09

-93.9

s.2

54

95

-

10-12

Lolium perenne

Control

90

0

6.88

± 1.74

 

 

0

0

0

21-22

111

100

0

7.91

± 1.12

14.9

n.s.4

0

0

0

21-22

192

95

0

1.67

± 0.45

-75.7

s.4

8

28

55

21-22

333

90

0

2.07

± 1.43

-70

s.4

24

38

46

21

577

100

0

0.96

± 0.69

-86

s.4

15

51

68

12-14

1000

70

0

0.48

± 0.24

-93

s.4

51

74

78

12-14

Allium cepa

Control

100

0

2.37

± 0.39

 

 

0

0

0

12

111

95

0

3.01

± 0.37

27

n.s.3

10

6

4

12

192

100

0

1.06

± 0.31

-55.4

s.3

0

3

40

11-12

333

100

0

0.52

± 0.26

-77.9

s.3

38

44

73

11

577

95

0

0.54

± 0.15

-77.4

s.3

13

28

69

11

1000

95

0

0.29

± 0.06

-87.7

s.3

48

53

92

11

* : negative values indicate reduction compared to control

**: day after 50% seedling emergence in the control

s.: significant

n.s.: not significant

1 no effect or no statistically significance at all tested concentrations on all six plant species, multiple comparison Fisher's exact Test after Bonferroni-Holm, α = 0.05

2 multiple comparison Bonferroni-Holm U-test, α = 0.05n.

3 multiple comparison Williams t-test, α = 0.05

4 multiple comparison Bonferroni-Welch t-test, α = 0.05

Table: summary of effect concentrations, based on fresh weight

Species

NOEC

LOEC

Statistical Analysis

 

EC10

EC20

EC50

Statistical Analysis

[mg test item/kg dry soil]

[mg test item/kg dry soil]

Brassica napus

111

192

1

 

131

149

192

4

 

lower 95%-cl

n.d.

n.d.

n.d.

upper 95%-cl

n.d.

n.d.

n.d.

r2 = 0.780

 

 

 

Glycine max

< 111

111

2

 

39.2*

108

746

4

 

lower 95%-cl

n.d.

10.3

464

upper 95%-cl

n.d.

202

2804

r2 = 0.894

 

 

 

Cucumis sativus

111

192

1

 

85.9*#

119#

222#

4

 

lower 95%-cl

n.d.

n.d.

n.d.

upper 95%-cl

n.d.

n.d.

n.d.

r2 = 0.688

 

 

 

Cucumis sativus

-

-

-

 

83.4*§

117§

222§

4

 

lower 95%-cl

62.8

94.7

199

upper 95%-cl

102

135

245

r2 = 0.668

 

 

 

Solanum lycopersicum

111

333

1

 

174#

194#

239#

4

 

lower 95%-cl

n.d.

n.d.

n.d.

upper 95%-cl

n.d.

n.d.

n.d.

r2 = 0.002

 

 

 

Solanum lycopersicum

-

-

-

 

155§

177§

226§

4

 

lower 95%-cl

3.4

7.45

33.3

upper 95%-cl

220

237

273

r2 = 0.107

 

 

 

Lolium perenne

111

192

3

 

90.3*#

115#

184#

4

 

lower 95%-cl

n.d.

n.d.

n.d.

upper 95%-cl

n.d.

n.d.

n.d.

r2 = 0.495

 

 

 

Lolium perenne

-

-

-

 

87.5*§

113§

184§

4

 

lower 95%-cl

30.4

53.6

140

upper 95%-cl

122

147

238

r2 = 0.433

 

 

 

Allium cepa

111

192

2

 

91.2*#

123#

217#

4

 

lower 95%-cl

n.d.

n.d.

n.d.

upper 95%-cl

n.d.

n.d.

n.d.

r2 = 0.658

 

 

 

Allium cepa

-

-

-

 

91.2*§

123§

217§

4

 

lower 95%-cl

46.9

76.3

177

upper 95%-cl

124

156

265

r2 = 0.593

 

 

 

n.d.: not determined due to mathematical reasons

- not relevant

* the ECx-value is extrapolated

# the ECx-value is with reservation (p(F) > α = 0.05)

§ the ECx-values are calculated on each replicate per concentration

1 multiple comparison Bonferroni-Holm U-test, α = 0.05

2 multiple comparison Williams t-test, α = 0.05

3 multiple comparison Bonferroni-Welch t-test, α = 0.05

4 Probit Analysis, cl = confidence limits

Validity criteria fulfilled:
yes
Remarks:
see 'Any other information on materials and methods incl. tables'

Description of key information

The lowest determined 21-d EC50-value is based on fresh weight of six plant species (four dicotyledonous and two monocotyledonous species, representing six plant families) and was 184 mg/kg dry soil in Deutsche Weidelgras ZS (Lolium perenne).

Key value for chemical safety assessment

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

Additional information

The toxicity to terrestrial plants was determined in a Seedling Emergence and Seedling Growth Test according OECD guideline No 208 and in compliance with GLP criteria (Ibacon GmbH, 2017). In this study, six plant species (four dicotyledonous and two monocotyledonous species, representing six plant families) were exposed to nominal test concentrations of 0 (control), 12.3, 37.0, 111, 192, 333, 577 and 1000 mg test item/kg dry soil (based on a range-finding study). The test item was added to artificial soil, by first adding the test item to quartz sand, followed by mixing with the soil (LUFA 2.3 (USDA: sandy loam)). Test concentrations were not analytically verified and thus test concentrations are given as nominal. The exposure time was 14 to 21 days after 50% germination in the control, depending on the growth of the seedlings. Only untreated seeds were used. The seeds were introduced manually into the soil. Fresh weight (at test end), germination (daily), mortality (after 7 days and at test end), phytotoxicity (weekly) and growth stages (at test end) were determined. Five of the tested plant species showed an equal sensitivity in terms of fresh weight. These plant species were Brassica napus, Cucumis sativus, Solanum lycopersicum, Lolium perenne and Allium cepa with EC50 values of 192, 222, 239, 184 and 217 mg test item/kg soil dry weight, respectively. As these values are with reservation (p(F)> α = 0.05) for Cucumis sativus, Solanum lycopersicum, Lolium perenne and Allium cepa for these plant species the EC50 values were also calculated per replicate, which are nearly identical (222, 226, 184 and 217 mg test item/kg soil dry weight, respectively). The least sensitive species was Glycine max which showed an EC50 value of 746 mg test item/kg soil dry weight. The germination rate was not statistically significantly reduced for any species tested. No mortality was observed for any species tested. Main phytotoxic effect observed for all plant species was growth reduction. Additionally Brassica napus, Lolium perenne and Allium cepa showed chlorosis and two replicates of Lolium perenne showed necrosis. Plants of one pot at 1000 mg test item/kg soil dry weight of Lolium perenne showed discolouration. Overall, the lowest determined (21-d) EC50-value based on fresh weight was 184 mg/kg dry soil in species Lolium perenne .