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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:
from 26 jun 2017 to 29 sept 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item name: Ekaland DPTT PD
Other name(s): DPTH; DPTT
Batch number 15042440523
EAS Test item code M-00012578
Appearance / colour solid / beige to yellowish
Chemical name bis(piperidinothiocarbonyl) hexasulfide
CAS number 971-15-3
Purity analysed 93.2 % w/w
Molecular weight 448.82 g/mol
Density not applicable
Issue date of certificate 23 May 2017
Expiry date 23 May 2018
Storage conditions ambient (5 °C - 30 °C), dark, dry
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
The test item was mixed in an appropriate amount of quartz sand. This mixture was incorporated with a drilling machine into the final volume of dry soil by thorough mixing to reach the required amount of soil.
Finally, a defined amount of tap water was mixed into the soil of each treatment group to achieve a water content of 35 - 40 % of the WHCmax.
Immediately after mixing, the test soil was distributed to the replicate pots and the seeds were sown.
Species:
Lolium perenne
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
Supplier: Becker-Schöll
Lot No: 2015_LP_001
Untreated seeds from commercial supplier(s) were used in this study. Care was taken that within species only seeds of the same size were used.
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
Supplier: Hild
Lot No: 2014_Ac_001
Species:
Pisum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Seed supplier: Hild
Lot No: 14488-001
Species:
Lactuca sativa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Seed supplier: Hild
Lot No: 09517-031
Species:
Daucus carota
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Seed supplier: Hild
Lot No: 14109-423
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Seed supplier: KWS
Lot No: 2015-Bn-001
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Remarks:
The experimental phase was performed in a controlled environment greenhouse located in D-75245 Neulingen-Göbrichen, Germany near the test facility.
Substrate type:
artificial soil
Remarks:
Silty sand composed of 48.2 % sand, 42.5 % silt and 9.3 % clay, with a pH of 7.3, a total organic carbon content < 0.3 % and an electronic conductivity of 78 µS/cm.
Limit test:
no
Total exposure duration:
21 d
Remarks:
An additional concentration was tested for Brassica napusin a separate trial with 60 seeds per treatment group in order to verify the results of the first trial for this species
Test temperature:
Trial S17-01684-L1: Air temperature (min/max/mean) [°C]: 17.60 / 44.05 / 27.24
Trial S17-01684-L2: Air temperature (min/max/mean) [°C]: 13.02 / 35.56 / 22.50
pH:
7.3
Moisture:
Trial S17-01684-L1: Relative humidity (min/max/mean) [%]: 19.64 / 88.68 / 53.41
Trial S17-01684-L2: Relative humidity (min/max/mean) [%]: 26.25 / 89.85 / 58.49
Details on test conditions:
Photoperiod (light /dark ) [h]: 16 / 8
Trial S17-01684-L1: Light intensity [lux]: 12880 - 16310
Trial S17-01684-L2: Light intensity [lux]: 12890 - 14685
Nominal and measured concentrations:
Nominal concentrations:
0, 13.5, 40.3, 121, 363, 1090 mg test item/kg sdw, * (for all species in trial S17-01684-L1)
333 mg test item/kg sdw, * (only for Brassica napus in trial S17-01684-L2)

* analysed purity considered
Reference substance (positive control):
no
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
Daucus carota
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
647 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
seedling emergence
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
333 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Shoot dry weight
Species:
Daucus carota
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Shoot dry weight
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Shoot dry weight
Species:
Pisum sativum
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Shoot dry weight
Species:
Allium cepa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Shoot dry weight
Species:
Lolium perenne
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 090 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Shoot dry weight
Details on results:
1. Seedling Emergence
The sum of emerged seedlings was calculated for each treatment group at the final assessment day (21 DA50E). The percent seedling emergence was calculated in relation to the number of seeds initially sown. The inhibition of emergence was calculated based on the sum of emerged seedlings compared to the control group. Seedling emergence was not statistically significantly reduced compared to the control for all species tested in both trials. The highest inhibition of seedling emergence compared to the control was observed at 363 mg test item/kg sdw in the plant species Lolium perenne and Daucus carota with inhibitions of 29.2 % and 27.6 %, respectively. It is noted that there was no clear dose response relationship for the effects observed on seedling emergence for any of the species tested, except for Lactuca sativa, and so the effects are not considered to be treatment related.

2. Post-Emergence Mortality
The cumulative mean mortality of emerged plants was calculated for each treatment group from the number of dead seedlings in relation to the number of emerged plants for the final assessment (21 DA50E).
Post-emergence mortality occurred in Allium cepa in the course of this study. One plant died at 1090 mg test item/kg sdw resulting in a post-emergence mortality of 4.3 % (based on 23 emerged plants).

3. Phytotoxcicity
Phytotoxicity was reported as median value per treatment group for the final assessment day (21 DA50E). No symptoms of phytotoxcicity could be observed for any of the species tested in both trials. Thus, the median for all species tested was 1, indicating a normal plant appearance.

4. Shoot Dry Weight
The total shoot dry weight per replicate was divided by the number of plants survived in the respective replicate to determine the average shoot dry weight per plant. The mean value and standard deviation were determined per treatment group based on the replicate values. The inhibition on shoot dry weight compared to the control was calculated in percent for each test item group. Statistically significant effects on shoot dry weight could be detected for Brassica napus in the first trial (Dunnett`s t-test; one sided smaller, a = 0.05). A statistically significant difference compared to the control for Brassica napus at 13.5 and 40.3 mg test item /kg sdw was considered as not treatment related since no statistically significant difference to the control was detected at 111 mg test item/kg sdw and because the replicate values are partly within the control range and thus reflect the natural variability. This was veryfied by the second trial conducted only for Brassica napus with a higher number of replicates at 333 mg test tiem/kg sdw. The highest inhibition on shoot dry weight compared to the control was determined for Brassica napus with 29.2 % at 363 mg test item/kg sdw followed by Allium cepa with 23.5 % 1090 mg test item/kg sdw.


Reported statistics and error estimates:
A statistical evaluation was performed for the data of seedling emergence and shoot dry weight.
For determination of significant difference to the control the significance level was set to a = 0.05 for all tests.

Trial S17-01684-L1
The data of seedling emergence were analysed by the multiple Chi²-test with Bonferroni-Holm adjustment or the multiple Fisher`s exact Test with Bonferrroni-Holm adjustment for trial S17-01684-L1.
The data of shoot dry weight were tested for normality and homoscedasticity using Shapiro-Wilk`s Test and Levene-Test followed followed by a William`s test in case that both requirements were fulfilled and the trend analysis by contrast was significant. If the trend analysis by contrast was not significant the Dunnett`s t-test was conducted. The Welch`s t-test was conducted in case that the data were non-homogenous. The significance level was set to a = 0.05 for all tests.
The effect concentrations with their 95 % confidence limits were calculated by Probit analysis (based on mean values) using linear max. likelihood regression.

Trial S17-01684-L2
The data of seedling emergence were transformed and analysed with the Student`s t-test. The data of shoot dry weight were analysed with the Two-sample t-test.

Seedling emergence of dicotyledonous species (emerged plants per treatment group; inhibition compared to control)

Application rate
[mg test item/kg sdw]

21 DA50E

Emerged plants (based on 30 seeds)

Inhibition compared to control
[%]

Total

[%]

Brassica napus(Trial S17-01684-L1)

Control (0)

25

83

-

13.5

25

83

0.0

40.3

29

97

-16.0

121

27

90

-8.0

363

29

97

-16.0

1090

25

83

0.0

Brassica napus(Trial S17-01684-L2) (based on 60 seeds)

Control (0)

55

92

-

333

51

85

7.3

Daucus carota

Control (0)

29

97

-

13.5

26

87

10.3

40.3

25

83

13.8

121

25

83

13.8

363

21

70

27.6

1090

27

90

6.9

Lactuca sativa

Control (0)

29

97

-

13.5

30

100

-3.4

40.3

30

100

-3.4

121

30

100

-3.4

363

28

93

3.4

1090

25

83

13.8

Pisum sativum

Control (0)

22

73

-

13.5

18

60

18.2

40.3

19

63

13.6

121

21

70

4.5

363

18

60

18.2

1090

19

63

13.6

Seedling emergence of monocotyledonous species (emerged plants per treatment group; inhibition compared to control)

Application rate
[mg test item/kg sdw]

21 DA50E

Emerged plants

Inhibition compared to control
[%]

Total

[%]

Allium cepa

Control (0)

23

77

-

13.5

21

70

8.7

40.3

25

83

-8.7

121

24

80

-4.3

363

26

87

-13.0

1090

23

77

0.0

Lolium perenne

Control (0)

24

80

-

13.5

23

77

4.2

40.3

19

63

20.8

121

18

60

25.0

363

17

57

29.2

1090

19

63

20.8

LOEC, NOEC and EC10/50of DPTH for seedling emergence 21 days after at least 50 % of the seedlings in the control group had emerged


Species

DPTH [mg test item/kg sdw]

 

LOEC

NOEC

EC10

(95% confidence limits)

EC50

(95% confidence limits)

Dicotyledonous species

Brassica napus1)

-

= 1090

s.n.r.

> 1090

Daucus carota

-

= 1090

n.d.

> 1090

Lactuca sativa

-

= 1090

647

(243 – 1663)

> 1090

Pisum sativum

-

= 1090

n.d.

> 1090

Monocotyledonous species

Allium cepa

-

= 1090

> 1090

> 1090

Lolium perenne

-

= 1090

n.d.

> 1090

1)    Both trials considered.

n.d.: not determinable, due to a lack of a clear dose-response relationship

s.n.r: statistics not reliable

Shoot dry weight per living plant [g] of dicotyledonous species (1/2)

Application rate
[mg test item/kg sdw]

21 DA50E

Mean value [g]

± SD [g]

Inhibition
(% compared to control)#

Brassica napus(Trial S17-01684-L1)

Control (0)

0.120

0.018

-

13.5

0.098(*a)

0.014

18.3

40.3

0.101(*a)

0.013

15.8

121

0.111

0.018

7.5

363

0.085*a

0.007

29.2

1090

0.101*a

0.019

15.8

Brassica napus(Trial S17-01684-L2)

Control (0)

0.461

0.128

-

333

0.434

0.102

5.9

Shoot dry weight per living plant [g] of dicotyledonous species (2/2)

Application rate
[mg test item/kg sdw]

21 DA50E

 

Mean value [g]

± SD [g]

Inhibition
(% compared to control)#

 

Daucus carota

 

Control (0)

0.029

0.007

-

 

13.5

0.039

0.010

-34.5

 

40.3

0.042

0.011

-44.8

 

121

0.039

0.009

-34.5

 

363

0.041

0.008

-41.4

 

1090

0.037

0.009

-27.6

 

Lactuca sativa

 

Control (0)

0.044

0.011

-

 

13.5

0.043

0.007

2.3

 

40.3

0.043

0.003

2.3

 

121

0.041

0.004

6.8

 

363

0.043

0.003

2.3

 

1090

0.039

0.013

11.4

 

Pisum sativum

Control (0)

0.390

0.138

-

13.5

0.416

0.115

-6.7

40.3

0.334

0.145

14.4

121

0.387

0.110

0.8

363

0.423

0.183

-8.5

1090

0.351

0.074

10.0

Shoot dry weight per living plant [g] of monocotyledonous species (1/2)

Application rate
[mg test item/kg sdw]

21 DA50E

Mean value [g]

± SD [g]

Inhibition
(% compared to control)#

Allium cepa

Control (0)

0.017

0.005

-

13.5

0.017

0.005

0.0

40.3

0.016

0.005

5.9

121

0.016

0.002

5.9

363

0.014

0.004

17.6

1090

0.013

0.003

23.5

Shoot dry weight per living plant [g] of monocotyledonous species (2/2)

Application rate
[mg test item/kg sdw]

21 DA50E

Mean value [g]

± SD [g]

Inhibition
(% compared to control)#

Lolium perenne

Control (0)

0.012

0.003

-

13.5

0.012

0.002

0.0

40.3

0.019

0.009

-58.3

121

0.015

0.004

-25.0

363

0.021

0.011

-75.0

1090

0.015

0.006

-25.0

SD:  Standard Deviation

#       Negative values indicated that there was an increase in shoot dry weight compared to the control

LOEC, NOEC and EC10/EC50of DPTH for shoot dry weight at day 21 after 50 % of the seedlings in the control group had emerged


Species

DPTH [mg test item/kg sdw]

 

LOEC

NOEC

EC10

(95% confidence limits)

EC50

(95% confidence limits)

Dicotyledonous species

Brassica napus1)

363*a

333

n.d.

> 1090

Daucus carota

-

= 1090

> 1090

> 1090

Lactuca sativa

-

= 1090

s.n.r

> 1090

Pisum sativum

-

= 1090

n.d.

> 1090

Monocotyledonous species

Allium cepa

-

= 1090

s.n.r

> 1090

Lolium perenne

-

= 1090

> 1090

> 1090

1)   Both trials considered.

n.d.: not determinable, due to a lack of a clear dose-response relationship

s.n.r: statistics not reliable

LOEC Determined with:aDunnett`s t-test; one-sided smaller, a = 0.05

Validity criteria fulfilled:
yes
Conclusions:
The study was conducted to determine the effects of DPTH on the seedling emergence and early growth of four dicotyledonous and two monocotyledonous species. Despite some deviations from the study plan (air temperature and humidity outside recommended ranges), since the validity criteria were met for all species tested on day 21, the study can be regarded as valid.
The study was performed as a rate response test to determine the LOEC, NOEC, EC10 and EC50 for seedling emergence and shoot dry weight on day 21, where possible. An additional trial was conducted for Brassica napus at a single concentration to verify the results of the first trial.
No statistically significant differences could be determined for the parameter seedling emergence in both trials. An EC10 with 95 % confidence limits could be determined for Lactuca sativa as 647 (243 – 1663) mg test item/kg sdw.
A statistically significant difference compared to the control for shoot dry weight could be determined in the first trial for Brassica napus with a LOEC of 363 mg test item/kg sdw. An EC10 and EC50 could not be calculated. In the second trial no statistically significant differences compared to the control were observed. Therefore, an overall LOEC of 363 mg test item/kg sdw and NOEC of 333 mg test item/kg sdw can be considered for Brassica napus for the parameter shoot dry weight.

Validity criteria:
- Seedling emergence
The control seedling emergence was = 70 % (actually: 73 % to 97 %) 21 days after at least 50 % emergence in the control.
- Phytotoxicity
The control seedlings of each species did not exhibit visible phytotoxic effects (e.g. chlorosis, necrosis, wilting, leaf and stem deformations) and control plants exhibit only normal variation in growth and morphology for the particular species.
- Mean Survival
The mean survival of emerged control seedlings was = 90 % (actually: 100 %) 21 days after at least 50 % emergence in the control.
- Cultivation Conditions
The environmental conditions for each particular species were identical and growing media contained the same amount of soil matrix, support media, or substrate from the same source.

Executive summary:

The OECD TG 208 study objective was to determine the effects of DPTH on the seedling emergence and early growth of six non-target terrestrial plant species (Dicotyledonous species: Brassica napus, Daucus carota, Lactuca sativa, Pisum sativum; Monocotyledonous species: Allium cepa, Lolium perenne) under greenhouse conditions.

DPTH was incorporated into the soil at five application concentrations (13.5, 40.3, 121, 363, 1090 mg test item/kg sdw). Seeds were sown in pots filled with the test item treated or control soil. The anaylsed purity of 93.2 % was considered for the preparation of the test item groups. Results were compared to the tap water treated control.In each treatment group a total of 30 seeds were sown. In an additional trial S17-01684-L2, a total of 60 seeds ofBrassica napuswas treated at a single rate (333 mg test item/kg sdw) in order to verify the results of the tril S17-01684-L1. The test duration was from seeding until 21 days after at least 50 % of the seeds in the control had emerged in each species. During this period, plants were assessed for seedling emergence, post emergence mortality and phytotoxicity symptoms on day 7, 14 and 21. The effects on shoot dry weight were determined for day 21. The Lowest Observed Effect Concentration (LOEC), the No Observed Effect Concentration (NOEC) and the Effect Concentrations (EC10 and EC50) for seedling emergence and shoot dry weight were determined, where possible.

No statistically significant differences could be determined for the parameter seedling emergence in both trials. An EC10 with 95 % confidence limits could be determined for Lactuca sativa as 647 (243 – 1663) mg test item/kg sdw.

A statistically significant difference compared to the control for shoot dry weight could be determined in the first trial for Brassica napus with a LOEC of 363 mg test item/kg sdw. An EC10 and EC50 could not be calculated because this effect seemed like an artifact. In the second trial no statistically significant differences compared to the control were observed. Therefore, a NOEC of 333 mg test item/kg sdw can be considered for Brassica napus for the parameter shoot dry weight as a worst-case.

Description of key information

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:
333 mg/kg soil dw

Additional information

The OECD TG 208 study objective was to determine the effects of DPTH on the seedling emergence and early growth of six non-target terrestrial plant species (Dicotyledonous species:Brassica napus,Daucus carota,Lactuca sativa,Pisum sativum; Monocotyledonous species:Allium cepa,Lolium perenne) under greenhouse conditions.

DPTH was incorporated into the soil at five application concentrations (13.5, 40.3, 121, 363, 1090 mg test item/kg sdw). Seeds were sown in pots filled with the test item treated or control soil. The anaylsed purity of 93.2 % was considered for the preparation of the test item groups. Results were compared to the tap water treated control.In each treatment group a total of 30 seeds were sown. In an additional trial S17-01684-L2, a total of 60 seeds ofBrassica napuswas treated at a single rate (333 mg test item/kg sdw) in order to verify the results of the tril S17-01684-L1. The test duration was from seeding until 21 days after at least 50 % of the seeds in the control had emerged in each species. During this period, plants were assessed for seedling emergence, post emergence mortality and phytotoxicity symptoms on day 7, 14 and 21. The effects on shoot dry weight were determined for day 21.

No statistically significant differences could be determined for the parameter seedling emergence in both trials. An EC10 with 95 % confidence limits could be determined for Lactuca sativa as 647 (243 – 1663) mg test item/kg sdw.

A statistically significant difference compared to the control for shoot dry weight could be determined in the first trial for Brassica napus with a LOEC of 363 mg test item/kg sdw. An EC10 and EC50 could not be calculated. In the second trial no statistically significant differences compared to the control were observed. Therefore, an overall LOEC of 363 mg test item/kg sdw and NOEC of 333 mg test item/kg sdw can be considered for Brassica napus for the parameter shoot dry weight