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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:
Definitive exposure: 5th December 2018 - 30th January 2019
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))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 1000 mg/kg treatment
- Sampling method: soil samples taken on days 0, 2, 28 and 56.
- Sample storage conditions before analysis: Directly after sampling, the cores were placed in glass bottles with Teflon-lined caps prior to analytical processing. Cores were used for the determination of soil test concentration, which was based on the mean of the three individual
results.
Vehicle:
no
Details on preparation and application of test substrate:
- Method of application to filter paper (if used): N/a
- Method of mixing into soil (if used): Direct injection below the surface of the natural sandy loam
- Method of application to soil surface (if used): N/a
- Controls: prepared in the same manner as the treatment except the control medium contained no test substance
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/a
- Concentration of vehicle in test medium (stock solution and final test solution): N/a
- Evaporation of vehicle before use: N/a
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: Aquatic Research Organisms, Hampton, New Hampshire
- Age at test initiation (mean and range, SD): 3-4 months old
- Weight at test initiation (mean and range, SD): control: 0.3529g (0.0173g), 10mg/kg: 0.3485g (no SD), 100mg/kg: 0.3386g (no SD), 1000mg/kg: 0.3447g (0.0162)

ACCLIMATION
- Acclimation period: Not reported
- Acclimation conditions (same as test or not): Same as test
- Health during acclimation (any mortality observed): None
Study type:
laboratory study
Substrate type:
natural soil
Limit test:
yes
Total exposure duration:
56 d
Remarks:
Test was conducted as a limit test at 1000mg/kg (n=8), however two other concentrations were also tested: 10 and 100 mg/kg (n=1). This was done so if effects were observed, the test would serve as a range finder.
Test temperature:
20 ± 2 ºC.
pH:
6.9-7.1
Moisture:
32-39%
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1000-mL glass beakers
- Amount of soil or substrate: approximately 520 g (dry weight) of treated or control soil at a depth of approximately 13 cm. The dimensions of the beakers were such that the soil surface area at the top of the soil layer was 87 cm2.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 8 for 1000mg/kg (plus 5 vessels for chemical analysis), 1 for 10 and 100mg/kg
- No. of replicates per control: 8
- No. of replicates per vehicle control: N/a

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Rochester, Massachusetts
- Pesticide use history at the collection site: N/a
- Collection procedures: N/a
- Sampling depth (cm): N/a
- Soil texture (if natural soil)
- % sand: 89%
- % silt: 8%
- % clay: 3%
- Soil taxonomic classification: natural sandy loam
- Soil classification system: N/a
- Composition (if artificial substrate): N/a
- Organic carbon (%): organic content = 2.1%
- Maximum water holding capacity (in % dry weight): N/a
- CEC: N/a
- Pretreatment of soil: Periodic analysis of representative samples of the natural sandy loam components and cattle manure used during the earthworm culture and testing were analyzed for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. Results of these analyses established that the laboratory well water, manure, and natural sandy loam did not contain any contaminants that
would adversely affect the test organisms in agreement with ASTM (2007) standard practice
- Storage (condition, duration): N/a
- Stability and homogeneity of test material in the medium: N/a

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light, 8-hour dark photoperiod
- Light intensity: 400 to 760 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Food consumption (weekly beginning on d7 until d28) Pooled earthworm weight for F0 at d0 and d28. Mortality and health (e.g. colour changes, lethargy, softness) were performed on d28. Number of juveniles on d56 and d57

VEHICLE CONTROL PERFORMED: not applicable, no vehicle used

TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor of 10
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study
- Test concentrations: 0, 10, 100, 1000 mg/kg
- Results used to determine the conditions for the definitive study: Results were the definitive study (see Study Design Remarks)
Nominal and measured concentrations:
Nominal: 0, 10, 100, 1000 mg/kg
Measured: measured control =
Reference substance (positive control):
yes
Remarks:
Benomyl 56-day reference test from 12th Feb - 10th April 2019
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
growth (as eathworm weight)
Duration:
28 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:
growth (as earthworm weight)
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
Remarks:
as number of juveniles
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
Remarks:
as number of juveniles
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
as number of juveniles
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
as number of juveniles
Details on results:
- Mortality at end of exposure period:
- Total mass of adults at beginning of test:
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Mean % weight change in control = -5.3 (SD=12), for 10mg/kg = -12%, for 100mg/kg = -6.6%, 1000mg/kg = -0.91 (7.8)
- No. of offspring produced: Control = 60 mean offspring per replicate (SD=10)
- No. of unhatched cocoons: N/a
- Morphological abnormalities: None reported
- Behavioural abnormalities: None reported
- Other biological observations:N/a
Results with reference substance (positive control):
- Results with reference substance valid? Unknown as number of offspring not reported
- Relevant effect levels: 100% survival in the controls. Statistical significance observed in the 2.5 and 5mg/kg treatment groups which is within the range of expected significant values in the testing guideline.
- Other: N/a
Reported statistics and error estimates:
As no effects were observed, effect values were empirically estimated.

Table 1 - Summary of biological observations

 Nominal Concentration (mg/kg) Mean % survival (SD)  Mean % weight change (SD) Mean total number of surviving offspring per replicate (SD) 
 Control 96 (7) -5.3 (12)   60 (10)
 10  100 (N/a)  -12  89 (N/a)
 100   100 (N/a)  -6.6  62 (N/a)
 1000  98 (5)  -0.91 (7.8)  62 (20)

N/a - as replicate number was 1 for these treatments, SD cannot be calculated

Validity criteria fulfilled:
yes
Remarks:
Control mortality = 4% at d28, control mean number of juveniles ≥39, CV of reproduction in control = 17%
Conclusions:
No effects were observed on weight or survival after a 28-day exposure of the test substance to Eisenia fetida. Therefore, the 28-day nominal NOECs are ≥1000mg/kg which is equivalent to ≥850 mg/kg when expressed as measured concentrations. No effects were observed on reproduction after a 56-day exposure of the test substance to Eisenia fetida. Therefore, the 56-day nominal NOEC is ≥1000mg/kg which is equivalent to ≥850 mg/kg when expressed as the measured concentration.

Description of key information

NOEC (28 day): 1000 mg/kg dry weight, nominal concentration, growth and survival of Eisenia fetida, highest concentration tested.

EC10 and NOEC (56 day): >1000 and ≥1000 mg/kg dry weight, respectively, reproduction of Eisenia fetida, highest concentration tested.

Key value for chemical safety assessment

Additional information

A 56 day earthworm reproduction test, at concentrations up to 1000 mg/kg soil dry weight, has been conducted in accordance with OECD TG 222 (earthworm reproduction test). No effects on survival or reproduction were observed.

A 28 day NOEC value of 1000 mg/kg dry weight has been determined for the effects of the test substance on growth and survival of adult earthworms, based on nominal concentrations. A 56 day NOEC value of 1000 mg/kg dry weight and EC10 value of >1000 mg/kg dry weight have been determined for the effects of the test substance on reproduction of the earthworm, based on nominal concentrations.

Test substance concentrations were measured at the beginning of the test and on days 2, 28 and 56. Concentrations ranged from 85 to 90% of nominal concentrations at test initiation through to day 28 and declined over the last four weeks of the 56 day exposure period to 77%, with a mean measured concentration of 850 mg/kg (85% of nominal concentration). Results of the replicate samples were generally consistent, indicating homogeneous distribution of the test substance in the exposure vessels.

The 56 day EC10 value is equivalent to >850 mg/kg when expressed as measured concentration.