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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
adopted 13 April 2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
The test item is dispersible in water. Therefore, test solutions were made by dispersing weighed amounts of the test item in deionised water, immediately prior to application. The test item was dispersed 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. The treated substrate was thoroughly mixed using a laboratory mixer immediately after application.

Order of application and cleaning procedure
- deionised water (control)
- the test item in ascending order
Test organisms (species):
Eisenia andrei
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)

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

Weight of animals used in the test: 267 – 416 mg/worm
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Test temperature:
18.1 – 21.2 °C
pH:
test start: 6.21 – 6.25
test end: 5.99 – 6.06
Moisture:
test start: 35.0 % (equivalent 55.8 % of WHC)
test end: 34.3 – 34.8 % (equivalent to 54.7 – 55.5 % of WHC)

Max. water holding capacity: 62.7 g/100 g soil dry weight
(WHC)
Details on test conditions:
Test conditions

Test vessel: plastic vessel of Bellaplast (inside dimensions: about 16.5 cm x 12 cm x 6 cm) with a lid 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/ treatment group: 40 (control group: 80)

The test vessels were set up at random in the test room.


Artificial soil
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
- 68.5 % 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)
- 1 % food (dried ground horse manure)
- deionised water

Amount of soil/test vessel: 675 g wet weight corresponding to 500 g dry weight of artificial soil with a water content corresponding to 40-60 % of WHC

Climatic conditions
Light conditions: source: artificial light (Lumilux L58W) intensity: 580 lx
duration: light :dark = 16 h : 8 h

Test duration: 8 weeks (4 weeks adult mortality; 4 weeks juvenile development)


Experimental procedure
One day before test start, the dry artificial soil was pre-moistened by adding deionised water to obtain approximately half of the final water content. Earthworms were acclimatised in a separate batch of the artificial soil (mixed with horse manure) for approximately 24 hours before test start.
On the day of the test start, the test item was introduced by dispersing the quantity of test item required to obtain the desired test concentration in the volume of water required to hydrate the soil to 40-60 % of its WHC. Each test vessel was then filled with the treated soil (625 g wet weight corresponding to 500 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 individually weighed worms (10 worms/vessel) were placed on the surface of the soil. After approximately thirty minutes, the test vessels were closed with perforated transparent lids, which allowed gas exchange between substrate and atmosphere and access of light, but prevented worms from escaping. The test vessels were then set up at random in a controlled-environment test room. One day after application, initially 5 g air-dried and finely ground horse manure was scattered on the soil surface of each test vessel, which was sprinkled with 5 mL deionised water. The feeding interval was weekly during the first four weeks of the test. The weekly amount of manure (5 g) depended on the feeding activity.
After four weeks, the adult worms were removed from the test vessels. The number of surviving worms (adult mortality) and their biomass change were determined, behaviour (including feeding activity) and pathological symptoms were recorded. The adult worms were discarded after counting and weighing. Subsequently, the soil of each vessel was mixed carefully with 5 g manure. This was the last feeding occasion during experiment. The test was then continued for another four weeks. The final assessment included counting of surviving juveniles per test vessel, determination of the water content and pH measurements of the artificial soil. Juveniles were counted by manual inspection of the substrate.

Chronological test schedule
In preparation of the test
Preparation of the dry artificial soil substrate mixed with manure (5 g/500 g d.w.)
Determination of physico-chemical parameters of the artificial soil (WHC, pH)

One day before test start
Preparation of the wet artificial soil substrate
Acclimatisation of the earthworms to the artificial soil substrate (separate batch)

At test start
Preparation of the test item solutions
Weighing out the amount of the artificial soil per test vessel
Addition of the test solution to the soil per test vessel (for each replicated vessel separately), thorough mixing and filling of the treated soil into the test vessel
Washing of the acclimatised test animals with deionised water, gently drying them on a paper towel and weighing them individually; impartial assignment of the required number of test animals to the test vessels (= start of exposure)
Setting up of the test vessels at random in a controlled-environment test room
Determination of physico-chemical parameters of the artificial soil (water content, pH; analysed from pooled samples of each treatment group separately)

24 hours and then weekly after start of exposure (during initial four weeks of exposure)
Feeding with initially 5 g manure per test vessel on the soil surface which was sprinkled with 5 mL deionised water (weekly amount of 5 g manure according to feeding activity)
Observation of the feeding and behavioural activity (worms on the soil surface)

4 weeks after start of exposure
Removal, counting and weighing of surviving adult earthworms (end of adult earthworm exposure)
Feeding with 5 g manure per test vessel mixed with the test substrate (last feeding)

8 weeks after start of exposure
Counting of surviving juvenile earthworms (end of juvenile earthworm exposure and test end)
Determination of physico-chemical parameters of the artificial soil (water content, pH; analysed from pooled samples of each treatment group separately)

Assessments during the test
Time and parameters
⋅ At test start: individual fresh weight (mg/worm) behaviour of earthworms determination of physico-chemical parameters (water content, pH) of the artificial soil

⋅ Weekly: observation of behavioural and pathological symptoms (including the feeding activity)

⋅ 4 weeks after start of exposure: number of surviving adult earthworms per replicate observation of behavioural and pathological symptoms
(including morphological alterations) fresh weight of surviving adult earthworms per replicate

⋅ 8 weeks after start of exposure: number of surviving juveniles per replicate observation of behavioural and pathological symptoms (including morphological alterations) determination of physico-chemical parameters (water content, pH)

Calculation and statistics
The endpoints were mortality, change of biomass (difference in fresh weight of surviving worms between test start and four weeks after treatment) and reproduction (the number of juveniles present). The arithmetic mean and the standard deviation per treatment and per control for each endpoint were calculated.
The statistical analysis was performed with the software ToxRat Professional 2.10.05. Shapiro-Wilk´s Test and Levene´s test, respectively, were used to test the data for normality and homogeneity of variance. Fisher`s Exact Binomial Test with Bonferroni Correction and the Williams Multiple Sequential t-test were used to compare the control with the independent test item groups. For statistical evaluation of the biomass change, the changed mean fresh weight of surviving worms per replicate was used.
Nominal and measured concentrations:
100, 178, 316, 562 and 1000 mg/kg soil dry weight nominal concentrations
Reference substance (positive control):
yes
Remarks:
Carbendazim
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:
mortality
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:
reproduction
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:
growth
Remarks:
Biomass change
Details on results:
At test start, the fresh weight of the earthworms used was in a range of 267 – 416 mg/worm. The physico-chemical parameters measured at the start and at the end of the tests met the guideline requirements.
The test item caused no mortality at concentrations of 100, 316 and 562 mg test item/kg soil d.w. 2.5 % mortality was found at 178 and 1000 mg test item/kg soil d.w., respectively. 1.3 % mortality occurred in the control group. 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 group, i.e. a mean weight increase of 32.2 % was recorded in the control group and 27.1, 35.9, 31.3, 29.6 and 26.3 % at the tested concentrations of 100, 178, 316, 562 and 1000 mg test item/kg soil d.w., respectively.
No statistically significant effects (Williams-test, α = 0.05, one-sided smaller) on the number of juveniles compared to the control group were recorded at any concentration tested.
Based on the statistical evaluation of these results, the overall No-Observed-Effect-Concentration (NOEC) was determined to be ≥ 1000 mg test item/kg soil d.w., and and the overall Lowest-Observed-Effect-Concentration (LOEC) was determined to be > 1000 mg test item/kg soil d.w. The EC50 could not be calculated, but it can be concluded that the EC50 is higher than 1000 mg test item/kg soil dry weight, the highest tested concentration.
Results with reference substance (positive control):
To verify the sensitivity of the test system, the reference item Carbendazim, SC 500 was routinely tested at concentrations of 5 and 10 mg product/kg soil dry weight. In the most recent study with the reference item, the number of juveniles was reduced by 72.7 and 98.8 % at concentrations of 5 and 10 mg product/kg soil dry weight (mean number of juveniles = 23 and 1) after 8 weeks of test duration when compared to control (mean number of juveniles = 84).

The detailed results and corresponding tables are stated below.

 

Effects of the test item on growth (biomass change during 4 weeks exposure) of adult earthworms

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

mg test item/kg d.w.

control

100

178

316

562

1000

Replicate

 

 

 

 

 

 

1

306.3

315.9

314.1

317.2

319.7

313.5

2

320.6

321.3

323.4

320

321.4

321.5

3

330.4

339.9

324.6

328.5

327.3

337.3

4

364.2

344.9

335.5

357.4

349

347.5

5

303.3

 

 

 

 

 

6

322.5

 

 

 

 

 

7

328.4

 

 

 

 

 

8

358.3

 

 

 

 

 

mean

329.3

330.5

324.4

330.8

329.4

330

±SD

22

14.1

8.8

18.4

13.5

15.3

 

Fresh weight/worm [mg] after 4 weeks (mean per replicate)

mg test item/kg d.w.

control

100

178

316

562

1000

Replicate

 

 

 

 

 

 

1

413

380.3

438.2

421

428.2

425.8

2

399.4

424.1

416.5

373.9

429

387.5

3

431.9

445.1

410.7

455.3

401.8

390

4

482.8

431.6

498.9

490.3

447.6

463.1

5

382.5

 

 

 

 

 

6

424.2

 

 

 

 

 

7

469.1

 

 

 

 

 

8

480.2

 

 

 

 

 

mean

435.4

420.3

441.1

435.1

426.7

416.6

±SD

38

28

40.3

49.7

18.8

35.6

 

Biomass change (change in fresh weight after 4 weeks relative to initial fresh weight) weight / worm [mg] (mean per replicate)

mg test item/kg d.w.

control

100

178

316

562

1000

Replicate

 

 

 

 

 

 

1

106.7

64.4

124.1

103.8

108.5

112.3

2

78.8

102.8

93.1

53.9

107.6

66

3

101.5

105.2

86.1

126.8

74.5

52.7

4

118.6

86.7

163.4

132.9

98.6

115.6

5

79.2

 

 

 

 

 

6

101.7

 

 

 

 

 

7

140.7

 

 

 

 

 

8

121.9

 

 

 

 

 

mean

106.1

89.8

116.7

104.4

97.3

86.7

±SD

21.1

18.8

35.3

35.9

15.8

32

 

Biomass change (change in fresh weight after 4 weeks relative to initial fresh weight) [%] (mean per replicate)

mg test item/kg d.w.

control

100

178

316

562

1000

Replicate

 

 

 

 

 

 

1

34.8

20.4

39.5

32.7

33.9

35.8

2

24.6

32

28.8

16.8

33.5

20.5

3

30.7

31

26.5

38.6

22.8

15.6

4

32.6

25.1

48.7

37.2

28.3

33.3

5

26.1

 

 

 

 

 

6

31.5

 

 

 

 

 

7

42.8

 

 

 

 

 

8

34

 

 

 

 

 

mean

32.2

27.1

35.9

31.3

29.6

26.3

 

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

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

mg test item/kg d.w.

control

100

178

316

562

1000

Replicate

 

 

 

 

 

 

1

10

10

10

10

10

10

2

9

10

10

10

10

10

3

10

10

9

10

10

9

4

10

10

10

10

10

10

5

10

 

 

 

 

 

6

10

 

 

 

 

 

7

10

 

 

 

 

 

8

10

 

 

 

 

 

mean

9.9

10

9.8

10

10

9.8

±SD

0.4

0

0.5

0

0

0.5

cv %

3.6

0

5.1

0

0

5.1

mean Mortality in %

1.3

0

2.5

0

0

2.5

 

Number of juveniles per surviving adult worm

mg test item/kg d.w.

control

100

178

316

562

1000

Replicate

 

 

 

 

 

 

1

11.6

10.9

11.8

7.4

10.2

9

2

7.3

11.8

9.3

9.8

11.1

6.8

3

8.6

9.8

7.9

12.9

7.7

6.6

4

11.3

14.1

10.4

11.5

8.9

10.8

5

10.3

 

 

 

 

 

6

12.2

 

 

 

 

 

7

9.1

 

 

 

 

 

8

13.6

 

 

 

 

 

mean

10.5

11.7

9.8

10.4

9.5

8.3

 

Number of juveniles per replicate

mg test item/kg d.w.

control

100

178

316

562

1000

Replicate

 

 

 

 

 

 

1

116

109

118

74

102

90

2

66

118

93

98

111

68

3

86

98

71

129

77

59

4

113

141

104

115

89

108

5

103

 

 

 

 

 

6

122

 

 

 

 

 

7

91

 

 

 

 

 

8

136

 

 

 

 

 

mean

104.1

116.5

96.5

104

94.8

81.3

±SD

22.4

18.3

19.8

23.7

14.9

22.1

cv %

21.5

15.7

20.6

22.8

15.7

27.2

Reproduction compared to control [%]

100

111.9

92.7

99.9

91

78

 

 

Results of statistical evaluation

Biomass change of adult earthworms (mg/worm)

Shapiro-Wilk´s Test and Levene´s test

Shapiro-Wilk´s Test and Levene´s test tests were used, respectively, to test the data for normality and homogeneity of variance. The data were normally distributed and variance homogeneous and therefore a multiple test (Williams Multiple Sequential t-test) was performed.

 

Williams Multiple Sequential t-test Procedure

Comparison of treatments with "Control" by the t-test procedure after Williams. Significance level was Alpha = 0.05, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; LhM: max. likelihood mean; %MDD: minimum detectable difference to Control (in percent of Control); t: sample t; t*: critical t for Ho: μ1 = μ2 = ... = μk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

 

 

Mortality of adult earthworms

Fisher`s Exact Binomial Test with Bonferroni Correction

Pair-wise comparisons between treatment and control on the multiple significance level (alpha is 0.05; one-sided greater). Pair-wise comparisons are performed sequentially using the adjusted Alpha* (= alpha/(k-1); k: number of comparisons (after Holm 1979)). Ho (no effect) is accepted, if the probability p > Alpha*.

 

 

Reproduction (mean number of juveniles per replicate)

Shapiro-Wilk´s Test and Levene´s test

Shapiro-Wilk´s Test and Levene´s test tests were used, respectively, to test the data for normality and homogeneity of variance. The data were normally distributed and variance homogeneous and therefore a multiple test (Williams Multiple Sequential t-test) was performed.

Williams Multiple Sequential t-test Procedure

Comparison of treatments with "Control" by the t test procedure after Williams. Significance level was Alpha = 0.05, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; LhM: max. likelihood mean; %MDD: minimum detectable difference to Control (in percent of Control); t: sample t; t*: critical t for Ho: μ1 = μ2 = ... = μk; the differences are significant in case |t| > |t*| (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

 

 

Result

No statistically significantly adverse effects on mortality, growth and reproduction were observed during the statistical evaluation.

Validity criteria fulfilled:
yes
Conclusions:
Glufosinate-ammonium showed no statistically significantly adverse effects on mortality, growth and reproduction of the earthworm Eisenia fetida in artificial soil up to 1000 mg test item/kg soil dry weight, i.e. the highest concentration tested.
Based on the statistical evaluation of these results, the overall No-Observed-Effect-Concentration (NOEC) was determined to be ≥ 1000 mg test item/kg soil d.w., and and the overall Lowest-Observed-Effect-Concentration (LOEC) was determined to be > 1000 mg test item/kg soil d.w. The EC50 could not be calculated, but it can be concluded that the EC50 is higher than 1000 mg test item/kg soil dry weight, the highest tested concentration.

Description of key information

Glufosinate-ammonium showed no statistically significantly adverse effects on mortality, growth and reproduction of the earthworm Eisenia fetida in artificial soil up to 1000 mg test item/kg soil dry weight, i.e. the highest concentration tested.

Based on the statistical evaluation of these results, the overall No-Observed-Effect-Concentration (NOEC) was determined to be ≥ 1000 mg test item/kg soil d.w., and and the overall Lowest-Observed-Effect-Concentration (LOEC) was determined to be > 1000 mg test item/kg soil d.w. The EC50 could not be calculated, but it can be concluded that the EC50 is higher than 1000 mg test item/kg soil dry weight, the highest tested concentration.

Key value for chemical safety assessment

Additional information

NOEC ≥ 1000 mg test item/kg soil d.w.

EC50 ≥ 1000 mg test item/kg soil d.w.