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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-31 to 2010-06-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according Guideline and under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
DIN ISO 11268-2: 1998 (E), BBA Guideline (1994)
Deviations:
yes
Remarks:
The room temperature decreased once and the soil moisture partly deviated by more than 10 % of the initial value at the end of the test.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hostapur SAS 93
- Physical state: Solid, pellets
- Analytical purity: 93.9 % (w/w)
- Purity test date: 2010-03-18
- Lot/batch No.: DEA4MN7946
- Expiration date of the lot/batch: 2011-07-11
- Stability under test conditions: Stable (ca. 0.1 % aqueous solution)
- Storage condition of test material: Room temperature, protected from moisture and light.

Sampling and analysis

Analytical monitoring:
no

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The respective test item concentration was weighed out for each test item concentration and suspended in demineralised water. The suspended test item and additional demineralised water was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 56 % of the maximum water holding capacity.
Subsequently, the test medium was thoroughly mixed to
ensure a homogenous distribution and about 600 g soil dry weight were filled into each test vessel.

- Controls: Artificial soil moistened with demineralised water without test item was used as control medium.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Eisenia fetida SAVIGNY 1826, subspecies Eisenia fetida fetida
- Source: Own breeding
- Age at test initiation (mean and range, SD): synchronized earthworm culture, not deviating more than 1 month
- Weight at test initiation (mean and range, SD): 0.46


ACCLIMATION
- Acclimation period: 2 days prior to test start
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): healthy

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Remarks:
Removement of adults after 28 days

Test conditions

Test temperature:
17 - 22 °C
pH:
Test item concentration [mg test item/kg soil dry weight] pH-value
day 0 Day 57
Control 5.91 6.47
62.5 5.80 6.06
125 5.82 6.15
250 5.81 6.09
500 5.79 6.15
1000 5.80 6.00
Moisture:
Test item concentration [mg test item/kg soil dry weight] Moisture [%] of dry weight

day 0 day 56
Control 36.6 43.5*
62.5 37.5 42.9*
125 39.5 42.5
250 38.6 42.0
500 37.3 44.3*
1000 36.4 42.9*
Details on test conditions:
TEST SYSTEM
- Test container (material, size): round plastic boxes, ID: 15 cm, bottom surface area: 177 cm3, height: 14 cm, transparent and perforated lids enabled sufficient gas exchange and light penetration and prevented the test substrate from drying
- Amount of soil or substrate: 600 g DW
- No. of organisms per container (treatment): 10 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 4



SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.35 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 66
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 8 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 532 +/- 95 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult mortality, biomass, behaviour and pathological symptoms after 28 days
Reproduction (Number of juveniles) after 56 days

VEHICLE CONTROL PERFORMED: not necessary


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Nominal and measured concentrations:
Nominal application rates: 62.5 - 125 - 250 - 500 - 1000 mg test substance/kg soil dry weight (DW)
Reference substance (positive control):
yes
Remarks:
1.0 - 2.5 - 5.0 mg Carbendazim/ kg soil DW

Results and discussion

Effect concentrationsopen allclose all
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
470 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: 93.9% a.i. in test mat.
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
LOEC
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:
EC50
Effect conc.:
813 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: (573- >1000)
Details on results:
- Mortality at end of exposure period: One death adult earthworm was found in the control and at the test item concentration of 125 mg/kg soil dry weight after 28 days of exposure to the test item resulting in a mean mortality of 2.5 %. This level of earthworm mortality is not considered to be of biological significance, and is within the range of experimental variation considered acceptable for the control group.

- Changes in body weigth of live adults (% of initial weight) at end of exposure period: No symptoms or changes in the behaviour of the earthworms were observed in the control and at all test item concentrations

- No. of offspring produced: The mean number of juveniles in the control was 251. The average number of juveniles in the treated groups ranged between 88 and 239. Compared to the control the reproduction of the earthworms was not statistically significantly reduced from 62.5 to 500 mg test item/kg soil dry weight. However, at 1000 mg test item/kg soil dry weight the earthworm reproduction rate was statistically significantly reduced compared to the control. The coefficient of variation calculated for the reproduction of control was lower than 30 %. The EC50 was determined to be at 813 mg test item/kg soil dry weight

- Behavioural abnormalities: No symptoms or changes in the behaviour of the earthworms were observed in the control and at all test item concentrations
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 5 mg Carbendazim/kg soil DW
Reported statistics and error estimates:
One Way Analysis of Variance (ANOVA) was carried out for the determination of statistically significant differences compared to the control. A Normality Test and an Equal Variance Test were conducted prior to running the ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The -value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05. Significant differences as occurred with reproduction data (p ≤ 0.05) were determined by using a Dunnett’s test.

Any other information on results incl. tables

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Test item concentration

[mg test item/kg soil dry weight]

Mortality [%]

Replicate

MV

1

2

3

4

Control

0

0

0

10

2.5

62.5

0

0

0

0

0

125

0

10

0

0

2.5

250

0

0

0

0

0

500

0

0

0

0

0

1000

0

0

0

0

0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Test item concentration    [mg/kg soil dry weight]

Effect

Replicate

1

2

3

4

Control

A

10/10

10/10

10/10

9/10

H

1/10

62.5

A

10/10

10/10

10/10

10/10

125

A

10/10

9/10

10/10

10/10

H

1/10

250

A

10/10

10/10

10/10

10/10

500

A

10/10

10/10

10/10

10/10

1000

A

10/10

10/10

10/10

10/10

Body Weight Changes of the Adult Earthworms      

Test item concentration
[mg test item/kg soil dry weight]

Rep.

Mean body weights per replicate

Mean body weight change of earthworms

Test start

28 days

Per replicate

Mean ± SD

Sign.*

[g]

[g]

[g]

[%]

[g]

[%]

Control

1

0.498

0.581

0.08

16.7

0.15 ± 0.05

33.3 ± 12.0

2

0.436

0.591

0.16

35.6

3

0.456

0.618

0.16

35.5

4

0.432

0.628

0.20

45.3

62.5

1

0.457

0.583

0.13

27.6

0.12 ± 0.04

26.2 ± 9.51

No

2

0.499

0.583

0.08

16.8

3

0.438

0.532

0.09

21.5

4

0.425

0.590

0.17

38.8

125

1

0.427

0.582

0.16

36.3

0.13 ± 0.04

28.6 ± 9.27

No

2

0.461

0.602

0.14

30.6

3

0.501

0.577

0.08

15.2

4

0.439

0.581

0.14

32.4

250

1

0.436

0.571

0.14

31.0

0.14 ± 0.03

31.4 + 9.25

No

2

0.425

0.597

0.17

40.5

3

0.457

0.619

0.16

35.4

4

0.499

0.593

0.09

18.8

500

1

0.499

0.600

0.10

20.2

0.15 ± 0.04

34.2 ± 9.80

No

2

0.435

0.602

0.17

38.4

3

0.425

0.607

0.18

42.8

4

0.463

0.627

0.16

35.4

1000

1

0.433

0.536

0.10

23.8

0.13 ± 0.03

27.7 ± 6.76

No

2

0.496

0.599

0.10

20.8

3

0.435

0.591

0.16

35.9

4

0.455

0.593

0.14

30.3

Reproduction Rate (Number of Juveniles after 8 Weeks)

Test item concentration
[mg test item/kg soil dry weight]

Rep.

Number of juveniles

Mean ± SD

CV               [%]

[%]

of control

Significance compared to control

Control

1

265

251 ± 30.6

12.2

2

232

3

219

4

286

62.5

1

240

239 ± 71.5

29.9

95.3

No

2

315

3

143

4

257

125

1

207

232 ± 27.9

12.0

92.6

No

2

232

3

271

4

218

250

1

217

205 ± 45.3

22.1

81.8

No

2

204

3

145

4

254

500

1

200

189 ± 14.1

7.44

75.6

No

2

202

3

182

4

173

1000

1

118

88.0 ± 28.7

32.6

35.1

Yes

2

106

3

71

4

57

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, Hostapur SAS 93 did not induce evident mortality (2.5 % in the control and at 125 mg/kg soil dry weight) or pathological symptoms or changes of the behaviour of adult earthworms after 28 days of exposure. The earthworm biomass increased in the control and at all test item concentrations without any statistically significant differences. After eight weeks of exposure the earthworm reproduction did not statistically significantly differ to the control between 62.5 and 500 mg test item/kg soil dry weight. However, at 1000 mg test item/kg soil dry the reproduction rate of adult earthworms was statistically significantly reduced compared to the control.

Thus the No Observed Effect Concentration (NOEC) for Hostapur SAS 93 with respect to mortality, body weight and reproduction of Eisenia fetida fetida after 8 weeks of exposure in artificial soil was determined to be at 500 mg test item/kg soil dry weight. The EC50 for reproduction was determined to be at 813 mg/kg soil dry weight.
Executive summary:

Effects of Hostapur SAS 93 (Batch number: DEA4MN7946) on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) from March 31stto June 3rd, 2010 with the definitive exposure phase from March 31st to May 27th, 2010 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany. The study was conducted under static conditions over 8 weeks with the test item concentrations of 62.5, 125, 250, 500 and 1000 mg test item/kg soil dry weight, which were mixed into artificial soil containing 10 % peat and a control using untreated artificial soil. 40 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 4 replicate groups with 10 test organisms per replicate.

After 28 days no evident mortality (2.5 % in the control and at 125 mg/kg soil dry weight) as well as no pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and at all test item concentrations. The earthworm biomass increased in the control and at all test item concentrations without any statistically significant differences compared to the control. After further four weeks the reproduction rate (average number of juveniles) was 251 in the control and ranged between 88 and 239 at the test item concentrations. Compared to the control, the reproduction rate was statistically significantly reduced at 1000 mg test item/kg soil dry weight. The EC50 for reproduction was determined to be at 813 mg/kg soil dry weight. 

Overall, the NOEC of the test item concerning mortality, biomass and reproduction of earthworms was determined to be at 500 mg test item/kg soil dry weight.

All validity criteria recommended by the test guidelines were fulfilled.

Table 1:                       Summary of All Observed Effects in the Tested Doses

Effects

Hostapur SAS 93

[mg test item/kg soil dry weight]

62.5

125

250

500

1000

Mortality of adult earthworms

No

No

No

No

No

Reduction of body weight

No

No

No

No

No

Reduced reproduction rate

  (Number of juveniles)

No

No

No

No

Yes

NOEC and LOEC for Reproduction, Mortality and Body Weight

Hostapur SAS 93

Test item concentration

[mg test item/kg soil dry weight]

NOEC reproduction

500

LOEC reproduction

1000

NOEC mortality, body weight

1000

LOEC mortality, body weight

> 1000

EC50  of reproduction

(95 % Confidence Interval)

813

(573 - >1000)