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Toxicity to terrestrial plants

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Reference
Endpoint:
toxicity to terrestrial plants: long-term
Remarks:
Although the test does not cover chronic effects or effects on reproduction, it covers a sensitive stage in the life-cycle of a plant and data obtained form this study are generally used as estimate of chronic toxicity.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
Test substrate:
A 2:1 mixture of natural soil LUFA 2.2 (batch number: Sp2.23618, loamy sand (DIN classification)) and quartz sand (12a) was used.
Grain size: average 2 mm
Clay (< 0.002 mm): 6.0 %
Silt (0.002 - 0.063 mm): 9.1 %
Sand (0.063 - 2 mm): 84.9 %
Carbon content: 0.986 %
pH-value: 5.36 ± 0.04

Origin:
Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer (LUFA), Obere Langgasse 40, 67346 Speyer, Germany
Dörentrup Quarz GmbH Co. KG, An der Sandgrube 1,
31089 Duingen, Germany

Mixing and Application:
The control and test item treatments were applied once at the beginning of the study. The respective test item amount was weighed out for each test item concentration and blended
thoroughly with quartz sand (1 % of the entire soil amount per concentration). Afterwards the test item - quartz sand mixture and an appropriate amount of demineralised water (adjusting the soil to a moisture of 45 % of the maximum water holding capacity (MWHC)) were added to the soil. Subsequently, the soil was mixed carefully with a mixer (3 min) to ensure a homogeneous distribution.

Controls:
Test substrate with 1 % quartz sand

Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: oat; Flämingsgold- Origin: KWS LOCHOW GMBH, Ferdinand-von-Lochow-Strafe 5, 29303 Bergen, Germany
Species:
Allium cepa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: onion; Exhibition- Origin: HILD SAMEN GMBH, Postfach 1161, 71666 Marbach, Germany
Species:
Beta vulgaris
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: sugar beet; Fiorella- Origin: KWS Saat SE. Grimsehlstrage 31, 37574 Einbeck, Germany
Species:
Brassica napus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Common name: rape, Sherlock- Origin: KWS Saat SE, Grimsehlstraße 31, 37574 Einbeck, Germany
Species:
Lactuca sativa
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Common name: lettuce, Mafalda- Origin: Hild Samen GmbH, Postfach 1161, 71666 Marbach, Germany
Species:
Glycine max (G. soja)
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
Common name: soybean, Obelix- Origin: Delley Samen und Pflanzen AG, Schloss Delley 40, route de Portalban, CH-1567 Delley, Switzerland
Test type:
seedling emergence and seedling growth test
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
14 d
Remarks:
Two test periods: Test period A: Oats, onion, sugar beet, rape, lettuce: 2019-01-09 to 2019-01-30, onion prolonged until 2019-02-06 Test period B: Soybean: 2019-01-22 to 2019-02-12
Test temperature:
- Nominal: 22 ± 10 °C
- Actual: 12.7 - 23.1 °C
pH:
5.36 ± 0.04
Moisture:
Adjusted to a moisture of 45 % of the maximum water holding capacity (WHC).
Details on test conditions:
Number of replicates: 8 replicates per test concentration and control tested in a randomised block design;
Number of seeds: 5 seeds per test container (soybean: 3 seeds);
Test container: Non-porous plastic containers (standard flower pots) with a diameter of ca. 12 cm were used. The pots were labelled with the study number and all necessary additional information to ensure unique identification;
Test substance application: The control and test item treatments were applied once at the beginning of the study. Due to large soil amounts per concentration, several batches were prepared per concentration and applied seperately.
Application technique: The respective test item amount was weighed out for each test item concentration and blended thoroughly with quartz sand (1 % of the entire soil amount per concentration). Afterwards the test item - quartz sand mixture and an appropriate amount of demineralised water (adjusting the soil to a moisture of 45 % of the maximum water holding capacity (MWHC)) were added to the soil. Subsequently, the soil was mixed carefully with a mixer (3 min) to ensure a homogeneous distribution.The treated soil was transferred to the test container;
Sowing: Test containers were filled each with the same amount of treated soil (see application technique) and seeding holes were made with a seedling pistil. One seed was given into each hole and covered with soil. During cultivation the test containers were bottom watered and fertilized as necessary.;
Room: The test was conducted in a climatic hall. The test conditions approximated those conditions necessary for normal growth or typical environmental conditions for the species and varieties tested;
Photoperiod: 16h;
llumination: nominal >3500 lux, actual 4444 ± 396 lux;
Relative humidity: nominal 70 ± 30 %, actual 33.0-100%
Watering / Fertilisation: Bottom watering of the test containers with fertilized tap water as needed.
Fertilizer: HAKAPHOS® SOFT SPEZIAL 16+8+22(+3) For watering a 0.05 ‰ solution was used. Origin: Grünes Landhaus, St. Godehard-Str. 23, 31139 Hildesheim, Germany
Effect parameters measured: During the 21 - 28 day observation period (from day of application) the plants were observed on day 7, 14 and 21, onion additionally on day 28, for number of emerged seedlings, number of dead plants and visual phytotoxic effects. The rating of the treated plants was done in relation to the untreated control plants. Observations included all variations, either inhibitory or stimulatory, between the treated replicates and the untreated controls. Such variations may be phytotoxic symptoms (e.g. chlorosis, necrosis, wilting), effects of growth
and development rates. At the end of the study the shoot heights (in cm) and the shoot fresh weights of the shoots (in g) were measured additionally, directly after cutting the plants at the soil surface. The shoot height was measured per plant. Average mean values per replicate were calculated. The shoot fresh weight was determined for all plants from the same pot and considered as one replicate.;
Physical/chemical parameters measured: The room temperature and relative humidity were recorded continuously throughout the test with a data logger and a hygrothermograph, respectively. The illumination is determined twice per year.;
Chronological test description:
- Determination of the dry weight and the MWHC of the soil
- Application and sowing
- Watering and fertilization working daily
- Observation for visual phytotoxic effects, number of emerged
seedlings and number of dead plants at least every 7 days.
- Final shoot heights and shoot fresh weight measurements
Nominal and measured concentrations:
nominal: 1000 - 500 - 250 - 125 - 62.5 mg/kg DW (factor 2)
Reference substance (positive control):
no
Remarks:
No reference item is recommended for this test according to the test guideline (OECD 208).
Key result
Species:
Avena sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot height, shoot fresh weight, number of emerged seedlings
Key result
Species:
Allium cepa
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot height, shoot fresh weight, number of emerged seedlings
Remarks on result:
other: For onion the rate of emergence of the control plants reached > 50 % within 14 days. The test with onion was prolonged until 28 days
Key result
Species:
Beta vulgaris
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot height, shoot fresh weight, number of emerged seedlings
Key result
Species:
Brassica napus
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot height, shoot fresh weight, number of emerged seedlings
Key result
Species:
Lactuca sativa
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot height, shoot fresh weight, number of emerged seedlings
Key result
Species:
Glycine max (G. soja)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: shoot height, shoot fresh weight, number of emerged seedlings
Details on results:
Potential toxic effects of the test item were assessed on day 7, 14 and 21 (onion additionally day 28) by visual observations (number of emerged seedlings and phytotoxic effects) and at test end by shoot height and shoot fresh weight determination.

No statistically significant test item related phytotoxic effects and mortality occurred during the test period for all tested plant species.
For the test species onion, slight phytotoxic effects (stunted growth) in the highest test concentration after 21 and 28 days were visually assessed and documented. Seedling emergence and shoot height in the highest test concentration were lower compared to the control, but inside the range of variability and according to statistical analysis not significant.
Therefore, the NOEC for all tested species and all measured parameters is ≥ 1000 mg/kg DW.

The results of the control for all plant species met the required validity criteria. Therefore, the test is considered to be valid.

Reported statistics and error estimates:
NOEC / LOEC: of biomass One Way Analysis of Variance (ANOVA) was carried out for the growth / emergence determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance, a Normality test (Shapiro-Wilk) and an Equal Variance test (Brown-Forsythe) were done first. P-values for both, Normality and Equal Variance test, are 0.05. The alpha-value for ANOVA (acceptable probability of incorrectly concluding that there is a difference) is = 0.05.
Failure of the normality test and / or the equal variance test can be caused by extremely homogeneous emergence and growthpatterns as opposed to higher variances in other treatments. Due to the high and even number of replicates in the control andtreatment groups, the failure had no influence on the robustness of the calculations. For shoot height, if normality or equal variance failed using the above method, an automated process using the softwareToxRat was used. If the automatic setting confirmed failure of normality or equal variance using Shapiro-Wilk’s Test and the Levene-Test, the statistical test suggested by the program was
used. A multiple sequentially-rejective U-test after Bonferroni- Holm was suggested and used in this case.
EC-values and confidence: No effects ≥ 25 occurred at the end of the study. Therefore, no intervals of biomass growth EC-values were calculable.

The percentage of inhibition of shoot height, shoot fresh weight and rate of emergence is given in Table 1 to Table 5. Test item-related statistically significant inhibitions are given in bold. The tabulated results represent rounded mean values calculated on the exact raw data.

 

negative values = promoted growth

1) Normality test failed

2) Equal variance test failed

Table 1: Soybean: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test

concentration

[mg/kg DW]

Shoot

Height1)

[cm]

Inhibition

 

[%]

Shoot fresh

weight

[mg]

Inhibition

 

[%]

Rate of

Emergence1)

[%]

Inhibition

 

[%]

Control

23.8

-

3009

-

96

-

62.5

25.4

-7

3173

-5

100

-4

125

25.2

-6

3124

-4

100

-4

250

24.2

-2

2923

3

96

0

500

23.8

0

2724

9

96

0

1000

24.4

-3

2978

1

100

-4

 

Table 2: Oats: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test

concentration

[mg/kg DW]

Shoot

Height1)

[cm]

Inhibition

 

[%]

Shoot fresh

weight

[mg]

Inhibition

 

[%]

Rate of

Emergence1)

[%]

Inhibition

 

[%]

Control

42.5

-

1442

-

94

-

62.5

41.3

3

1258

13

95

-1

125

43.2

-2

1245

14

95

-1

250

42.6

0

1293

10

95

-1

500

41.2

3

1268

12

93

1

1000

42.1

1

1208

16

98

-4

 

Table 3: Onion: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test

concentration

[mg/kg DW]

Shoot

Height1)

[cm]

Inhibition

 

[%]

Shoot fresh

weight

[mg]

Inhibition

 

[%]

Rate of

Emergence1)

[%]

Inhibition

 

[%]

Control

16.1

-

176

-

83

-

62.5

15.9

1

155

12

78

6

125

15.0

7

156

11

80

4

250

15.4

4

168

5

98

-18

500

14.4

11

155

12

93

-12

1000

13.1

19

136

23

73

12

 

Table 4: Sugar Beet: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test

concentration

[mg/kg DW]

Shoot

Height1)

[cm]

Inhibition

 

[%]

Shoot fresh

weight

[mg]

Inhibition

 

[%]

Rate of

Emergence1)

[%]

Inhibition

 

[%]

Control

10.0

-

719

-

98

-

62.5

9.5

5

671

7

98

0

125

9.6

4

685

5

100

-2

250

9.5

5

686

5

98

0

500

9.7

3

675

6

100

-2

1000

9.4

6

667

7

95

3

 

Table 5: Rape: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test

concentration

[mg/kg DW]

Shoot

Height1)

[cm]

Inhibition

 

[%]

Shoot fresh

weight

[mg]

Inhibition

 

[%]

Rate of

Emergence1)

[%]

Inhibition

 

[%]

Control

16.2

-

1374

-

98

-

62.5

17.2

-6

1319

4

88

10

125

16.7

-3

1367

1

98

0

250

17.3

-7

1314

4

98

0

500

16.9

-4

1254

9

93

5

1000

16.4

-1

1215

12

98

0

 

Table 6: Lettuce: Inhibition [%] of Shoot Height, Shoot Fresh Weight and Rate of Emergence

Test

concentration

[mg/kg DW]

Shoot

Height1)

[cm]

Inhibition

 

[%]

Shoot fresh

weight

[mg]

Inhibition

 

[%]

Rate of

Emergence1)

[%]

Inhibition

 

[%]

Control

7.6

-

535

-

80

-

62.5

7.2

5

524

2

90

-13

125

7.4

3

593

-11

90

-13

250

7.2

5

540

-1

93

-16

500

7.1

7

478

11

95

-19

1000

7.2

5

539

-1

90

-13

 

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant toxic effects have been observed in studies with terrestric plants at the highest test concentration.
Executive summary:

Potential toxic effects of the test item were assessed on day 7, 14 and 21 (onion additionally day 28) by visual observations (number of emerged seedlings and phytotoxic effects) and at test end by shoot height and shoot fresh weight determination.

For the test species onion, slight phytotoxic effects (stunted growth) in the highest test concentration after 21 and 28 days were visually assessed and documented. Seedling emergence and shoot height in the highest test concentration were lower compared to the control, but inside the range of variability and according to statistical analysis not significant.

Therefore, the NOEC for all tested species and all measured parameters is ≥ 1000 mg/kg DW.

The results of the control for all plant species met the required validity criteria. Therefore, the test is considered to be valid.

Description of key information

No toxic effects have been observed in studies with terrestrial plants at the highest test concentration.

Key value for chemical safety assessment

Additional information

The phytotoxicity of the test item to six terrestrial plant species was determined over a period of 14 days after 50% emergence of the seedlings in the control group according to OECD 208 (July 2006).

The test was conducted for the plant species oats, onion, sugar beet, rape, soybean and lettuce with the test concentrations of

1000 - 500 - 250 - 125 - 62.5 mg/kg soil dry weight (DW).

Potential toxic effects of the test item were assessed on day 7, 14 and 21 (onion additionally on day 28) by visual observations (number of emerged seedlings and phytotoxic effects) and at test end by shoot height and shoot fresh weight determination.

No significant toxic effects were observed for all species at the highest test concentration.

A NOEC >= 1000 mg/kg DW was determined for all species.