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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-09-17 - 2008-11-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an internationally accepted guideline. All study parameters are based on the specific guideline.
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
The test item is soluble in water. Therefore, test solutions were made by dissolving weighed amounts of the test item in deionised water, immediately prior to application. Test item was dissolved in sufficient deionised water such that the addition of the test solutions to the test substrate resulted in a final water content of 40-60 % of WHC.
Treated substrate was thoroughly mixed using a laboratory mixer (see appendix 1 for details) immediately after application.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Test organism: earthworm Eisenia fetida (Savigny) 1926, subspecies Eisenia fetida andrei (Bouche)

Origin of the test organism: reared under ambient laboratory conditions in the test facility (original breeding animals were purchased from "W. Neudorff GmbH KG", An der Muhle 3, 31860 Emmertal, Germany)

Breeding under laboratory conditions; Breeding medium: mixture of manure : straw : peat (1:1:1) origin: horse manure and straw were purchased from farmers, peat was purchased from the company "Fioragard"

Breeding conditions: breeding containers (50 cm x 40 cm x 30 cm), constant diffuse light, temperature: approximately 20 °C, moist soil, pH: approximately 7

Age of animals used in the test: adult worms (approximately 4 months old with clitellum)

Time for acclimatisation to test conditions: approximately 24 hours
Weight of animals used in the test: 328 - 493 mg/worm
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Post exposure observation period:
no
Test temperature:
18-22 °C
pH:
test start: 5.79 - 5.80
test end: 5.73 - 5.77
Moisture:
Max. water holding capacity (g/100 g dry soil): 62.2
Water content (%):
Test start: 35.0-35.1 % (equivalent to 56.3 - 56.4 % of WHC)
Test end: 34.8-34.9% (equivalent to 55.9 - 56.1 % of WHC)
Details on test conditions:
Adult earthworms of the species Eisenia fetida were kept in a precisely defined artificial soil to which the test item had been applied. The aim of the test was to determine possible effects of the test item within a period of 14 days, i.e. mortality and changes of fresh weight of the surviving worms, and to determine the LC50, if possible. In addition observations were made on the morphology and behaviour of the worms during the test.
Approximately 24 hours before test start, the artificial soil was prepared and deionised water was added to the dry components to adjust the water content to approximately 40-60 % of WHC. The selected worms were allowed to acclimatise in a separate batch of the untreated artificial substrate for approximately 24 hours before test start.
The test substrate consisted of artificial soil, the test item (except in controls) and deionised water. The test item was introduced by dispersing the quantity of the test item to obtain the desired test concentration in a portion of deionised water necessary to wet the soil substrate to the required water content. The control substrate contained the corresponding amount of deionised water only. Each test vessel was then filled with the treated soil (approximately 751 g wet weight corresponding to 556 g dry weight). After a randomising procedure according to the worm fresh weight, selected groups of 10 worms were then randomly assigned to each treatment group.
The acclimatised test animals were washed, gently dried on a paper towel, weighed and randomly placed onto the test substrate (10 animals per test vessel). For the whole test duration the worms were not fed. Assessments were performed after incubation periods of 7 and 14 days in a controlled- environment test room.
Nominal and measured concentrations:
Nominal: 100 and 1000 mg/kg dw
Reference substance (positive control):
yes
Remarks:
2-chloroacetamide
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
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:
mortality
Results with reference substance (positive control):
To verify the sensitivity of the test system, the reference item 2-chloroacetamide was routinely tested at concentrations of 14.1, 18.3, 23.8, 31.0 and 40.3 mg a.i./kg soil d.w. In the most recent study with the reference item the 14-day LC50 was calculated as 21.2 mg a.i./kg soil d.w. with 95 % confidence limits ranging from 20.2 to 22.2 mg a.i./kg soil d.w. The test results indicate that the laboratory conditions are adequate and have not changed over time.

Treatment group

mg P70/05/S-4/kg soild.w.

 

control

100

1000

Replicate

Initial fresh weight/worm [mg] (mean per replicate)

1

373.0

380.1

385.7

2

395.3

393.7

397.5

3

409.8

404.2

379.9

4

422.8

437.0

414.9

mean

400.2

403.8

394.5

SD

21.3

24.3

15.4

cv%

5.3

6.0

3.9

 

Fresh weight / worm [mg] after 14 days (mean per replicate)

1

356.4

363.7

371.6

2

383.7

371.2

384.3

3

387.0

397.7

354.4

4

390.6

389.3

385.1

mean

379.4

380.5

373.9

SD

15.6

15.7

14.4

cv%

4.1

4.1

3.8

 

Biomass change (change in fresh weight after 14 days relative to initial fresh weight)

 

weight/worm [mg] (mean per replicate)

1

-16.6

-16.4

-14.1

2

-11.6

-22.5

-13.2

3

-22.8

-6.5

-25.5

4

-32.2

-47.7

-29.8

mean

-20.8

-23.3

-20.7

SD

8.9

17.6

8.3

 

Biomass change (change in fresh weight after 14 days relative to initial fresh weight)

 

 

[%](mean per replicate)

 

1

-4.5

-4.3

-3.7

2

-2.9

-5.7

-3.3

3

-5.6

-1.6

-6.7

4

-7.6

-10.9

-7.2

mean

-5.1

-5.6

-5.2

SD: standard deviation cv%:coefficient of variation


Validity criteria fulfilled:
yes
Conclusions:
The NOEC was determined to be 1000 mg P70/05/S-4/kg soil d.w..
The 14-d LC50 could not be calculated, but it can be concluded that the LC50 is higher than 1000 mg P70/05/S-4/kg soil d.w.
Executive summary:

Effects of P70/05/S-4 on survival and growth of the earthworm Eisenia fetida andrei were determined according to OECD guideline 207 at test concentrations of 100 and 1000 mg P70/05/S-4/kg soil d.w. after an exposure period of 14 days. A water-treated control group was included. The study was performed with four replicates per treatment group and 10 adult worms per replicate.

At the beginning of the test, the fresh weight of the earthworms was in a range of 328 - 493 mg/earthworm. The physico-chemical parameters of the test substrate measured at the start and at the end of the test met the guideline requirements. The maximum burrowing time was 5 minutes in the control group and in the P70/05/S-4 treated groups.

The test item caused no mortality at concentrations of 100 and 1000 mg P70/05/S-4/kg soil d.w. No mortality (0 %) occurred in the control group. No abnormal behaviour or toxic symptoms of the worms were observed in the test item treatment groups or control groups during the test.

The test item caused no statistically significant change in biomass (difference in fresh weight of surviving worms between test start and day 14 of exposure) compared to the control (5.1 % biomass decrease) at concentrations of 100 and 1000 mg P70/05/S-4/kg soil d.w. (weight decreases of 5.6 and 5.2 % were recorded).

The NOEC was determined to be 1000 mg P70/05/S-4/kg soil d.w.

The 14-d LC50could not be calculated, but it can be concluded that the LC50is higher than 1000 mg P70/05/S-4/kg soil d.w.


Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-02-01 - 2016-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Chemical name: N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)
Analysed purity: 99.8 %
Water solubility: ca. 8 % = 87 g/L
Appearance: white solid
Analytical monitoring:
no
Vehicle:
yes
Remarks:
deionised water
Details on preparation and application of test substrate:
The required quantity of test item was dissolved in the amount of deionised water by which the water content of the artificial soil was adjusted. The test solution was then mixed by intensive stirring with the artificial soil in a laboratory mixer.
The mixer was cleaned with a brush following application of each test item concentration and with cleaning agents after the last mixing. Cleaning agents used were Mucasol (alkaline cleaning agent for laboratory equipment), water, deionised water and spirit for glass equipment.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Test organism: earthworm Eisenia fetida (SAVIGNY, 1826) subspecies Eisenia andrei (BOUCHÉ, 1972)
Origin of the test organism: reared under ambient laboratory conditions in the test facility (original breeding of animals was purchased from “W. Neudorff GmbH KG”, An der Mühle 3, 31860 Emmerthal, Germany)
Holding and breeding under laboratory conditions
Breeding medium: mixture of horse manure, straw, peat (1:1:1)
origin: horse manure and straw were purchased from farmers, peat was purchased from the company “Torfwerk Moorkultur Ramsloh”
Breeding conditions:
breeding containers (50 cm x 40 cm x 30 cm) diffuse light temperature: about 20 °C moist soil; pH: about 7
Age of animals used in the test:
adult worms (approximately 3 months old with clitellum) Weight of animals used in the test: 299 – 441 mg/worm
Time for acclimatisation to test conditions:
approximately 24 hours in the artificial substrate (with food)
Food used during the test: air-dried and finely ground horse manure
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
18.1 – 20.8 °C
pH:
guideline requirement: 6.0 ± 0.5
test start: 5.94 – 6.06
test end: 5.85 – 5.97
Moisture:
guideline requirement: 40-60 % of WHC
test start: 34.9 – 35.1 % (equivalent to 54.8 – 55.1 % of WHC)
test end: 34.6 – 34.8 % (equivalent to 54.3 – 54.6 % of WHC)
Details on test conditions:
Test vessel: plastic vessel of Bellaplast (inside dimensions: about 16.5 cm x 12 cm x 6 cm) with a lid which was pervious to air and light Number of animals/vessel (= replicate): 10
Number of replicates/ control group: 8
Number of replicates/ treated group: 4
Number of animals/ treated group: 40 (control group: 80)
The test vessels were set up at random in the test room.
Artificial soil substrate
Composition of artificial soil: - 10 % sphagnum peat; origin: Torfwerk Moorkultur Ramsloh, 26683 Saterland, Germany, classified according to DIN 11540 (as close to pH 5.5-6.0 as possible, no visible plant remained, finely ground, dried to measured moisture content) - 20 % kaolin clay (kaolinite content > 30 %); type: Kaolin W, origin: ERBSLÖH Lohrheim GmbH, 65558 Lohrheim, Germany - 0.5 % calcium carbonate; origin: MERCK KGaA, 64271 Darmstadt, Germany - 69.5 % industrial quartz sand; type: Millisil W3, origin: Quarzwerke GmbH, 50207 Frechen, Germany
(fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
- deionised water
Laboratory mixer: mixing machine KitchenAid
mixing time: 2 x 2 minutes for each replicate rotations per minute: 240
Amount of soil/test vessel: 810 g wet weight corresponding to 600 g dry weight of artificial soil with a water content corresponding to 40-60 % of WHC
Max. water holding capacity
WHC (g/100 g soil d.w.): 63.7
Water content (%):* guideline requirement: 40-60 % of WHC
test start: 34.9 – 35.1 % (equivalent to 54.8 – 55.1 % of WHC) test end: 34.6 – 34.8 % (equivalent to 54.3 – 54.6 % of WHC)
pH-value:* guideline requirement: 6.0 ± 0.5
test start: 5.94 – 6.06 test end: 5.85 – 5.97
Climatic conditions (test room) Temperature: 18.1 – 20.8 °C
Recording: continuously by a data logger
Light conditions: source: artificial light (Lumilux L58W) intensity: 570 lx
duration: light : dark = 16 h : 8 h Test duration: 8 weeks (4 weeks adult mortality; 4 weeks juvenile development)
Nominal and measured concentrations:
Nominal:
62.5, 125, 250, 500, 1000 mg test item/kg soil dry weight (not corrected for purity, spacing factor: 2.0)
Reference substance (positive control):
yes
Remarks:
Nutdazim 50 FLOW (Carbendazim, SC 500)
Key result
Duration:
56 d
Dose descriptor:
NOEC
Remarks:
mortality
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
182 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
632 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Results with reference substance (positive control):
In a separate study (BioChem project No. R 12 10 48 004 S, dated October 29, 2012) the reference item Nutdazim 50 FLOW (Carbendazim, SC 500) had a significant effect on biomass increase and reproduction of Eisenia fetida. The reproduction rate was clearly inhibited by 72.7 % and 98.8 % compared to the control at the tested concentrations of 5 and 10 mg product/kg soil dry weight.

Effects of the test item on mortality and reproduction of adult earthworms

Treatment group

mg test item/kg soil dry weight

 

control

62.5

125

250

500

1000

Replicate

Number of surviving adult worms per replicate (4 weeks after test

initiation)

1

10

10

10

10

10

10

2

10

10

10

10

10

10

3

10

9

10

10

10

10

4

10

10

10

10

10

10

5

10

 

 

 

 

 

6

10

 

 

 

 

 

7

9

 

 

 

 

 

8

10

 

 

 

 

 

mean

9.9

9.8

10.0

10.0

10.0

10.0

± SD

0.4

0.5

0.0

0.0

0.0

0.0

cv %

3.6

5.1

0.0

0.0

0.0

0.0

 

Mortality [%]

mean

1.3

2.5

0

0

0

0

 

Number of juvenile worms per replicate

1

103

125

104

68

57

27

2

138

92

87

89

64

51

3

128

82

101

75

53

39

4

92

131

109

92

69

13

5

96

 

 

 

 

 

6

77

 

 

 

 

 

7

67

 

 

 

 

 

8

108

 

 

 

 

 

mean

101.1

107.5

100.3

81.0

60.8*

32.5*

± SD

23.9

24.1

9.4

11.4

7.1

16.3

cv %

23.6

22.5

9.4

14.1

11.7

50.1

Reduction of

 

 

 

 

 

 

reproduction compared to control (%)

-

-6

1

20

40

68

Validity criteria fulfilled:
yes
Conclusions:
In a 56-day earthworm reproduction study with the test item no statistically significantly adverse effects on mortality and biomass of the earthworm Eisenia fetida in artificial soil were determined up to and including 1000 mg test item/kg soil dry weight, i.e. the highest concentration tested. The NOEC for biomass and reproduction was determined to be 1000 and 250 mg test item/kg soil dry weight, respectively. The EC50 (based on reproduction) was calculated to be 632 mg test item/kg soil dry weight.
Executive summary:

In a 56-day earthworm reproduction study with P70/05/S-5 no statistically significantly adverse effects on mortality and biomass of the earthworm Eisenia fetida in artificial soil were determined up to and including 1000 mg test item/kg soil dry weight, i.e. the highest concentration tested. The NOEC for biomass and reproduction was determined to be 1000 and 250 mg test item/kg soil dry weight, respectively. The EC50 (based on reproduction) was calculated to be 632 mg test item/kg soil dry weight.

No statistically significant mortality compared to the control was observed at any test item concentration. No effects on behaviour (including feeding activity) of the worms were observed during the test.

The test item caused no statistically significant change in biomass (change in fresh weight after 4 weeks relative to initial fresh weight) compared to the control treatment at any concentration tested.

Statistically significant effects (Williams-t-test, α = 0.05, one-sided smaller) on number of juveniles compared to the control group were recorded at concentrations of 500 and 1000 mg test item/kg d.w.

All validity criteria for the study were met.

NOEC (mortality)

1000 mg test item/kg soil d.w.

NOEC (biomass)

1000 mg test item/kg soil d.w.

NOEC (reproduction)

250 mg test item/kg soil d.w.

LC50(mortality)

> 1000 mg test item/kg soil d.w.

EC10(reproduction)

182 mg test item/kg soil d.w.

(95 % confidence limits 131 to 252 mg test item/kg soil d.w.)

EC20(reproduction)

279 mg test item/kg soil d.w.

(95 % confidence limits 221 to 352 mg test item/kg soil d.w.)

EC50(reproduction)

632 mg test item/kg soil d.w.

(95 % confidence limits 541 to 738 mg test item/kg soil d.w.)

Description of key information

Effects of P70/05/S-4 on survival and growth of the earthworm Eisenia fetida andrei were determined according to OECD guideline 207 at test concentrations of 100 and 1000 mg P70/05/S-4/kg soil d.w. after an exposure period of 14 days. The NOEC was determined to be 1000 mg P70/05/S-4/kg soil d.w..

The 14-d LC50 could not be calculated, but it can be concluded that the LC50 is higher than 1000 mg P70/05/S-4/kg soil d.w.

In a 56-day earthworm reproduction study with the test item no statistically significantly adverse effects on mortality and biomass of the earthworm Eisenia fetida in artificial soil were determined up to and including 1000 mg test item/kg soil dry weight, i.e. the highest concentration tested. The NOEC for biomass and reproduction was determined to be 1000 and 250 mg test item/kg soil dry weight, respectively. The EC50 (based on reproduction) was calculated to be 632 mg test item/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
250 mg/kg soil dw

Additional information