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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:
23 April - 21 May 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study (2000) without verification of test concentration. Composition given in appendix, but batch no. does not match batch no of supplier. Temperature maximum very high, no data on moisture and humidity, which is essential since this influenced what happened in highest dose groups (dry top soil and consequent effects on emergence). Conclusion EC50 valid, but no reliable NOEC for germination due to uncertainty direct/indirect effect. EC50 and NOEC for vegetative growth > 1000 mg/kg dry soil.
Qualifier:
according to guideline
Guideline:
OECD Guideline 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)
Deviations:
yes
Remarks:
2000 guideline was used, no moisture content given
Principles of method if other than guideline:
Current guideline (2006) would require confirmation of test concentration and moisture/humidity data. Also EC25, NOEC data should be determined if data allow. Also a test with a reference substance should be available. Trimming 9 shoots to 5 is not justified.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
On 9 January 2001, a 25 kg bag of horticultural grade fine silver sand was washed, sieved and dried.
Only sand passing through a 1 mm sieve was used.
As the test substance was insoluble in water, chloroform was chosen as the most suitable solvent for this study as the test substance was
readily miscible with it at the concentrations to be tested.
Test solutions for the nominal 1.0, 10, 100 and 1000 mg/kg test concentrations were prepared by the addition of the appropriate amount
of test substance to a 250 ml volumetric flask and topping up the flask to the 250 ml mark with chloroform.
The above solutions were transferred to 1 litre labelled amber glass jars. To each of these, in turn, weighed quantities of silver sand were added in two stages (approximately half the total quantity of the required amount of sand each time). The jars were capped and shaken between additions.
After each jar had been thoroughly shaken, the contents of the jars were transferred into aluminium foil trays and the chloroform allowed to
evaporate off in a fume cupboard for 24 hours.
The solvent control was also prepared in a similar way, but with the addition of the sand to 250 ml of chloroform only.
After the 24 hour evaporation period, the dry sand/test substance mixtures were returned to the labelled glass jars which were then
capped and placed in an insulated box in a cold room until being transferred to Jealott's Hill Research Station.
This mixture was incorporated into the WC-B soil on day 0 of the study. Each sand/test substance mixture was added to the appropriate
amount of WC-B soil in the ratio 1:9 (dry weight basis) and mixed thoroughly in a large polyethylene sack.
Species:
Avena sativa
Plant group:
Monocotyledonae (monocots)
Details on test organisms:
- Common name: Oat
Species:
Helianthus annuus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Sunflower
Species:
Phaseolus aureus
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Mung bean
Test type:
seedling emergence toxicity test
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
28 d
Test temperature:
17.4-43 C
pH:
5.3-6.4
Moisture:
No data given
Details on test conditions:
The test containers were 9 cm square plastic plant pots. For each test concentration the
replicate pots were retained in a pre-formed plastic tray (Kari-TraysTM) with a perforated
base. The trays containing the pots were placed in plastic trays previously lined with
capillary matting, with individual trays for each test concentration and controls. This
ensured that any leachate from the pots would be retained and be available for reabsorption
into the pots via the sub-irrigation system. Water was supplied primarily by
sub-irrigation through this capillary matting which was kept wet throughout the study.
Additional water was supplied by top-down watering each morning and afternoon, as required, throughout the test period.
The test was carried out in glasshouse 135 at Syngenta, International Research Station,
Jealott's Hill, Bracknell, Berkshire, RG42 6ET.
Nominal and measured concentrations:
nominal: 1.0, 10, 100, 1000 mg/kg (dry test soil)
Reference substance (positive control):
no
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
germination
Species:
Helianthus annuus
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
germination
Species:
Phaseolus aureus
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
germination
Species:
Avena sativa
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Species:
Phaseolus aureus
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Species:
Helianthus annuus
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Species:
Avena sativa
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
germination
Species:
Helianthus annuus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
germination
Species:
Phaseolus aureus
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
germination
Details on results:
Based on 2 x 2 contingency test procedures, emergence was significantly reduced (P=0.05, one-sided) in the 1000 mg kg-1 test concentration for sunflower and mung bean, compared with the mean of the control and solvent control. This reduction is considered to be due to watering difficulties described below. Emergence of oat was not significantly reduced (P=0.05, one-sided) at any concentration tested.
Visual observation of the soil in all the replicate plots showed that at the test concentration of 1000 mg kg-1 (dry soil) the water-repellent properties of the test material was sufficient to inhibit the percolation of water through the soil. This would possibly cause the moisture sitting on the surface to evaporate quickly and not allow the seed to be in contact with the water long enough to trigger germination. Where water did successfully pass through the pots sporadic germination took place.
The 14 day emergence of seeds in the controls during this test was 100% for oat and mung bean and 94% for sunflower. These values were all above the satisfactory value of 65% recommended in the relevant OECD Guideline. In spite of the physical effect of the test material on germination at the top concentration of 1000 mg/kg (dry soil), the results indicate that the EC50 for emergence (germination) is >1000 mg/kg (dry soil) for all species.

The total fresh weight of shoots as a percentage of the controls indicate that there was no dose related response for any of the species tested, in any of the test concentrations. Furthermore, for oat, the total of the shoot weight in the 1000 mg kg-1 test concentration was greater than the total shoot weight in the solvent control; for mung bean it was greater than both the control and solvent control; and for sunflower, the average weight per plant was greater than both the control and solvent control. Hence, statistical analysis was not considered appropriate. The results show that the 28 day EC50 for vegetative growth is >1000 mg/kg (dry soil) for all species.

On day 7, a preliminary assessment of shoots emerging was made. All appeared healthy, although seed emergence was delayed in some of the replicates in the highest concentration. In spite of the standard watering procedure across all plant pots, the soil in the top concentration was observed to be dry to a depth of approximately 0.5 cm. By day 14, more seeds in the top concentration had emerged and the earlier emerged seeds had developed into healthy plants. On day 28, there were some visible effects to the plants in the 1000 mg kg-1 (dry soil) as all species varied in size, particularly in some of the replicates for oat and sunflower. Some of the mung bean plants in the top concentration also had thinner stems than those in other test concentrations. These effects were considered to be due to the later emergence of seeds, caused by the water repellent nature of the test material, since approximately the top 0.5 cm soil was observed to be dry compared to the other treatments. All plants, however, including those which had emerged in the top concentration, appeared healthy and showed no symptoms of toxicity.
Reported statistics and error estimates:
No data given.

Emergence

Replicate number

Concentration of AJ0019 mg/kg (dry soil)

Control

Solvent control

1

10

100

1000

Oat (Avena sativa)

1

9

9

8

9

8

6

2

9

9

9

9

9

9

3

9

6

9

9

9

8

4

9

9

9

9

8

9

Total

36

33

35

36

34

32

% of seeds sown

100

92

97

100

94

89

Sunflower (Helianthus annuus)

1

9

9

9

9

8

9

2

8

9

8

9

6

2

3

9

9

9

9

7

7

4

8

9

8

9

9

2

Total

34

36

34

36

30

20

% of seeds sown

94

100

94

100

83

56

Mung bean (Phaseolus aureus)

1

9

9

9

9

9

7

2

9

9

9

9

9

3

3

9

9

9

9

9

9

4

9

8

9

9

9

8

Total

36

35

36

36

36

27

% of seeds sown

100

97

100

100

100

75

Biomass fresh weight

Replicate number

Concentration of AJ0019 mg/kg (dry soil)

Control

Solvent control

1

10

100

1000

Oat (Avena sativa)

1

10.86

10.21

12.40

11.60

11.13

6.23*

2

12.15

9.80

10.85

7.93

10.85

8.72*

3

15.50

8.82

10.52

9.60

7.12

11.07

4

12.46

10.36

18.01

6.93

11.26

14.51

Total

50.97

39.18

46.77

36.06

40.36

40.53

% of control

-

77

92

71

79

80

Sunflower (Helianthus annuus)

1

19.62

15.94

13.03

22.47

17.95

21.28

2

19.67

23.55

16.38

20.14

24.15

11.31a

3

16.02

22.60

19.09

11.21

18.31

27.77

4

17.48

17.93

16.71

15.05

26.21

8.37b

Total

72.79

80.02

65.22

68.87

86.62

68.38

% of control

-

109

90

95

119

94

Mung bean (Phaseolus aureus)

1

18.53

21.68

18.74

19.64

20.75

20.52

2

17.56

16.03

18.14

15.89

18.63

12.73b*

3

13.66

16.49

16.80

15.98

18.09

25.70

4

22.33

16.81

20.07

14.76

15.53

29.00

Total

72.08

71.00

73.72

66.27

73.00

82.95

% of control

-

99

102

92

101

115

a = Only 2 seedlings

b = Only 3 seedlings

* = Very variable size plants  

Validity criteria fulfilled:
yes
Conclusions:
Study primarily done according to guideline 2000. However a potential issue with moisture occurred that could influence NOEC determination (direct/indirect) for germination. EC50 values can be used as well as NOEC for vegetative growth. Visual assessment of condition of plants not given individually. Temperature was extreme in first week of study, moisture data and humidity data are lacking. Indirect or direct cause of reduced germination can not be concluded due to dry top soil, which should not have happened.
Executive summary:

The OECD 208 terrestrial toxicity test on plants was performed with AQUAPEL 364 for 28 days. Test concentrations were 1.0, 10, 100, 1000 mg/kg dry soil, nominal. The test species were Oat (Avena sativa), Sunflower (Helianthus annuus) and Mung bean (phaseolus aureus). 4 replicate pots were prepared for controls and test concentrations each containing 9 seeds, later thinned to maximally 5. Soil (50% sterilised loam and 50% gravel) was 9:1 mixed with silver sand previously coated with test substance by means of a vehicle (chloroform). The solvent was evaporated prior to the start of the study. Both germination (up to day 14) and growth (day 28) were studied as well as a visual assessment of plant condition was made on day 14 and 28. In the highest dose groups (1000 mg/kg dry soil) the top soil was dry, likely due to the water repellent properties of the test substance. This likely caused - only at this dose - a reduced germination. The results show that both the EC50 (14days) for germination as well as the EC50 (28 days) for growth for all species is > 1000 mg/kg dry soil based on nominal concentration.

Description of key information

Key study is Brixham Environmental Laboratories (2002). Effects seen in highest dose for germination could have been due to physical conditions (indirect effect) therefore NOEC germination/emergence at 100 mg/kg to be considered as worst-case. EC50's for emergence and vegetative growth > 1000 mg/kg, NOEC veg growth >= 1000 mg/kg.

Key value for chemical safety assessment

Short-term EC50 or LC50 for terrestrial plants:
1 000 mg/kg soil dw
Long-term EC10, LC10 or NOEC for terrestrial plants:
100 mg/kg soil dw

Additional information

Key study is Brixham Environmental Laboratories (2002). Effects seen in highest dose for germination could have been due to physical conditions (indirect effect) therefore NOEC germination/emergence at 100 mg/kg to be considered as worst-case. EC50's for emergence and vegetative growth > 1000 mg/kg, NOEC veg growth >= 1000 mg/kg. According to OECD Guideline 208 from 2000 without verification of test concentration. Composition given in appendix, but batch no. does not match batch no. of supplier. Temperature maximum very high, no data on moisture and humidity, which is essential since this influenced what happened in highest dose groups (dry top soil and consequent effects on emergence). Conclusion EC50 valid, but no reliable NOEC for germination due to uncertainty direct/indirect effect.