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Ecotoxicological information

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:
2011-2013
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:
2004
Qualifier:
according to guideline
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss federal office of public health
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used):No aqueous application solution could be prepared for the dosing of the test item due to its limited water solubility. Neither was the test item sufficiently soluble in acetone or chloroform (organic solvent) at the required concentrations (according to the results of a solubility pre-experiment). Thus the test item was mixed directly with a portion of sand prior to addition to the artificial soil.

The sand/test item mixtures were prepared separately for each of the four replicates per test concentration. For the preparation of the different test concentrations, the amounts of nominal 31.25, 62.5, 125, 250, and 500 mg were weighed by an analytical balance and mixed with 10 g of sand for each replicate. The weighed portions were mixed into 10 g of sand by means of a roller mixer. The sand/test item mixtures were prepared one day before application and were stored at room temperature, at 20 ± 5 °C overnight.

- Controls: Yes
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: The worms were obtained from a synchronized culture maintained at Harlan Laboratories
- Age at test initiation: 2-3 months old adults with a clitellum
- Weight at test initiation: 308 to 600 mg

ACCLIMATION
- Acclimation period: One day
- Acclimation conditions: Same as test conditions
- Health during acclimation: Yes
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Post exposure observation period:
The study consisted of two parts. Adult earthworms were exposed to the test item for a period of four weeks (first part). After this period, the adults were removed from the test vessels and the cocoons and juvenile earthworms remained in the treated soils for additional four weeks (second part).
Test temperature:
20 ± 2 °C
pH:
6.4 - 6.5
Moisture:
33-34% at test start and 38-40% at test termination after 8 weeks
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Plastic boxes (approximate dimensions of length 16.5 cm, width 12 cm, area 200 cm2, height 9 cm)
- Amount of soil or substrate: 500 g dry weight and 670 g fresh weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)

The artificial soil was prepared according to the guidelines.

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour darkness photoperiod
- Light intensity: 610-711 Lux

EFFECT PARAMETERS MEASURED: Mortality, adverse effects, body weight, food consumption and reproduction.
Nominal and measured concentrations:
Nominal concentrations of 62.5, 125, 250, 500, and 1000 mg test item/kg dry soil.
Reference substance (positive control):
yes
Remarks:
Derosal (Carbendazim)
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
survival and growth
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Other: At the single concentration of the reference item Derosal (8.33 mg formulation /kg dry soil, i.e. 5 mg a.i./kg dry soil), the mortality rate of the adult worms was 0%. The mean decrease of 8% in body wet weight of the earthworms in the positive control during the exposure period was significantly different to the control. The reproduction rate of Eisenia fetida in the positive control was completely inhibited.
Therefore, the results of the positive control demonstrated satisfactory test conditions.

The toxic effects of the test item Amides, C16-C18 (even), N,N’-ethylenebis on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a test period of eight weeks based on the OECD Guidelines for Testing Chemicals, Test No. 222 (April 2004) and the International Standards ISO 11268-2: 1998(E).

Amides, C16-C18 (even), N,N’-ethylenebis was homogeneously mixed into artificial soil at the following test concentrations:

62.5, 125, 250, 500, and 1000 mg test item/kg dry soil.

Additionally, a control treatment (substrate without test item) and a reference item treatment (Derosal®) were tested in parallel.

Adult earthworms were exposed in treated soils for a period of four weeks. After this period, the adults were removed from the test vessels and the survival and growth rate were determined. The cocoons and juvenile earthworms remained in the vessels for additional four weeks. The reproduction rate was determined by counting the number of offspring hatched from the cocoons after this additional test period of four weeks.

Table: Summary of effects on earthworms exposed to Amides, C16-C18 (even), N,N’-ethylenebis

Parameters

Control

Amides, C16-C18 (even), N,N’-ethylenebis [mg/kg dry soil]

Derosal®[mg/kg dry soil]

 

 

62.5

125

250

500

1000

8.33

Mortality [%] after a 4-week exposure

0

0

0

0

0

0

0

Mean change in body fresh weight [%] after a 4-week exposure

20

25

24

20

19

18

-8

Mean reproduction rate (juveniles per replicate)

248

244

263

262

286

223

0.0

Reproduction rate (% of control)

100

98

106

106

115

90

0.0

In conclusion, taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of Amides, C16-C18 (even), N,N’-ethylenebis without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be at least 1000 mg/kg dry soil. The NOEC might even be higher, but concentrations in excess of 1000 mg/kg dry soil were not tested. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.

The reproduction rate of Eisenia fetida in the positive control was completely inhibited, confirming adequate test conditions and the sensitivity of the test animals.

Table 2: Validity criteria for OECD 222.

Criterion from the guideline to be satisfied in the controls

Outcome

Validity criterion fulfilled

Each replicate (containing 10 adults) to have produced ≥ 30 juveniles by the end of the test.

248

yes

The coefficient of variation of reproduction is ≤ 30%.

12%

yes

Adult mortality over the initial 4 weeks of the test is ≤ 10%.

0%

yes


 

Validity criteria fulfilled:
yes
Remarks:
Please refer to table 2 in section "Any other information on results incl. tables"

Description of key information

 The substance is not toxic to earthworms (NOEC (56 d)  1000 mg/kg).

Key value for chemical safety assessment

Additional information

The long-term toxic effects of the substance on survival, growth, and reproduction of the earthworm Eisenia fetida was tested according to OECD guideline 222.

Earthworms were exposed for 8 weeks to test substance concentrations of 62.5, 125, 250, 500, and 1000 mg test item/kg dry soil. The test substance was homogeneously mixed into artificial soil. After an exposure period of 4 weeks, the adults were removed from the test vessels and the survival and growth rate were determined. The cocoons and juvenile earthworms remained in the vessels for additional four weeks. The reproduction rate was determined by counting the number of offspring hatched from the cocoons after this additional test period of four weeks.

Effects on survival, growth and reproduction rates of the exposed adult test organisms were not observed. The NOEC (56 d) was ≥ 1000 mg/kg dry soil.

Based on the available study results, the substance is not considered toxic to terrestrial organisms.

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