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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:
2015
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))
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
None.
Analytical monitoring:
not required
Details on sampling:
No analytical analysis.
Vehicle:
no
Details on preparation and application of test substrate:
The artificial soil substrate was prepared in-house, by mixing a batch using the constituents and proportions detailed overleaf. The substrate was mixed and stored prior to use in the test. Composition was:

- 10% Sphagnum peat (2 mm sieved)
- 20% Kaolin clay (kaolinite content above 30 per cent)
- 70% Quartz (silica) sand (predominately fine)
- Calcium carbonate was added to adjust the substrate to pH 6.45.

The test substance was dosed using sand, and the dose expressed as mg/kg soil dry weight (dw). The control vessels were mixed with sand only. The weight of the moisture control vessel was checked for moisture loss on a weekly basis, with the exception of day 14, and any losses due to evaporation were corrected by the addition of Reverse Osmosis (RO) water.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
Earthworms were selected arbitrarily from stock cultures with a relatively homogenous age structure (they did not differ in age by more than 4 weeks), from suitable culture stocks received from Bias Labs Ltd, Fife, and maintained at the laboratory's facilities. The earthworms were sexually mature (i.e. between 2 and 12 months old with clitellum), weighing between 250 and 600 mg (wet mass).

ACCLIMATION
Prior to the start of the test, the earthworms were acclimatised for approximately 5 days in basic artificial soil substrate. Earthworms were held in a suitable container, and maintained in test conditions. The earthworms were fed during the acclimatisation period. The earthworms were fed cow dung, collected from organic cows which had not been treated for a minimum of three months prior to collection. Upon receipt, the feed was first frozen, and then thawed as necessary, dried in an oven at 60°C, ground to a powder and sterilised. Feeding occurred ad libitum (approximately 10g) prior to use in the test.
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
no
Total exposure duration:
56 d
Remarks:
None.
Post exposure observation period:
None.
Test temperature:
19.3°C - 21.6°C (min - max)
pH:
6.1 - 6.5 (min - max)
Moisture:
33% - 37% (min - max)
Details on test conditions:
TEST SYSTEM
- Test container (material, size): glass vessel
- Amount of soil or substrate: 500 g dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOIL COMPOSITION

- 10% Sphagnum peat (2 mm sieved)
- 20% Kaolin clay (kaolinite content above 30 per cent)
- 70% Quartz (silica) sand (predominately fine)
- Calcium carbonate was added to adjust the substrate to pH 6.45.

OTHER TEST CONDITIONS
- Photoperiod: 16L:8D
- Light intensity: 400 - 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Adult survival at D28 and juvenile number at D56.

VEHICLE CONTROL PERFORMED: no vehicle

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 5 ; 16 ; 51 ; 164 ; 524 mg/kg soil dry weight
Nominal and measured concentrations:
Nominal concentrations: 5 ; 16 ; 51 ; 164 ; 524 mg/kg soil dw
Measured concentrations: not measured
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 524 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 524 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Details on results:
All key results are presented in tables 2 and 3. Mortality of adults at D28 was almost null, with only one death at the highest concentration (2.5% mortality). This was not significantly different from the control group. After 56 days, the production of juvenile was evaluated. No significant adverse effect was observed on reproduction.
Results with reference substance (positive control):
None.
Reported statistics and error estimates:
Statistical analysis of the adult mortality data, adult weight change data, and reproduction data, was undertaken using CETIS version 1.8.6.8, based on the nominal test concentrations. The following methods were used:
- The NOEC for adult mortality was determined using a Wilcoxon/Bonferroni Adj Test, and the LC50 was determined using Linear Interpolation (ICPIN)
- The NOEC for number of juveniles was determined using a Bonferroni Adj t Test, and the EC50 was determined using Linear Interpolation (ICPIN)

Table 1: Moisture content and pH of test substrate at D0 and D56

Nominal concentrations (mg/kg soil dw)

Moisture content (%)

pH

Day 0

Day 56

Day 0

Day 56

0

33.8

35.3

6.45

6.25

5

33.8

37.2

6.55

6.27

16

33.3

36.4

6.48

6.19

51

33.0

36.5

6.49

6.17

164

33.9

36.0

6.50

6.15

524

34.0

36.6

6.51

6.13

Table 2: Mortality of adult earthworm per treatment after 28 days exposure

Nominal concentrations (mg/kg soil dw)

Number of adult earthworm exposed at D0

Number of mortalities at D28

Total (%)

0

80

0

0

5

40

0

0

16

40

0

0

51

40

0

0

164

40

0

0

524

40

1

2.5

Table 3: Mean number of juvenile worms per replicate at day 56

Nominal concentrations (mg/kg soil dw)

Mean number of juvenile at D56

0

57

5

54

16

69

51

70

164

55

524

47

Validity criteria fulfilled:
yes
Conclusions:
This study investigated the effect of CDBC on E. fetida survival and reproduction according to OECD 222 guideline. The test item had no effect on parent survival or reproductive success up to the highest tested concentration (524 mg/kg soil dw).
Executive summary:

This study investigated the effect of CDBC on E. fetida survival and reproduction, according to OECD 222 guideline. Synchronized adult earthworms (250 - 600 mg initial weight) were used. One control group (8 replicates) and five treatments (4 replicates each) were tested. Nominal concentrations were 5, 16, 51, 164 and 524 mg/kg soil dry weight, mixed in the soil before adding the worms. Adults were let to reproduce in the soil for 28 days. After this period, all adults were removed and counted. Dead adults were counted. Any missing adult was considered dead. After removal of the adults, the soils were further incubated for 28 days after addition of a nutritional source in a single dose at D28. After 28 days, the living juveniles were counted, referring to the reproductive success of the adults. The test item had no effect on parent survival or reproductive success up to the highest tested concentration (524 mg/kg soil dw).

Description of key information

This study investigated the effect of CDBC on E. fetida survival and reproduction, according to OECD 222 guideline. Synchronized adult earthworms (250 - 600 mg initial weight) were used. One control group (8 replicates) and five treatments (4 replicates each) were tested. Nominal concentrations were 5, 16, 51, 164 and 524 mg/kg soil dry weight, mixed in the soil before adding the worms. Adults were let to reproduce in the soil for 28 days. After this period, all adults were removed and counted. Dead adults were counted. Any missing adult was considered dead. After removal of the adults, the soils were further incubated for 28 days after addition of a nutritional source in a single dose at D28. After 28 days, the living juveniles were counted, referring to the reproductive success of the adults. The test item had no effect on parent survival or reproductive success up to the highest tested concentration (524 mg/kg soil dw).

Key value for chemical safety assessment

Additional information