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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:
The experimental phase of the definitive test was conducted from 27 March to 23 May 2019 (including the last day of data collection)
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
Analytical monitoring:
yes
Details on sampling:
Soil samples were removed on days 0, 2, 28 (before and after adults were removed), and 56 from the control and 1000 mg/kg treatment using a soil core method. Extra test vessels were prepared at initiation for every analytical sample interval. After collecting three soil core samples, the extra test vessel was sacrificed at each interval and the soil samples analysed for L4 concentration. 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): not applicable
- Method of mixing into soil (if used): Direct application by injection below the surface of the natural sandy loam.
- Method of application to soil surface (if used): not applicable
- 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): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evaporation of vehicle before use: not applicable
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): All earthworms used to initiate this exposure were mature with clitellum and were 4 to 7 months old.
- Weight at test initiation (mean and range, SD): control: 0.3780g (0.0227g), 10mg/kg: 0.3797g (no SD), 100mg/kg: 0.3649g (no SD), 1000mg/kg: 0.3719g (0.0266)

ACCLIMATION
- Acclimation period: Not reported
- Acclimation conditions (same as test or not): Same as test
- Health during acclimation (any mortality observed): None reported
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 1000 mg/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.
Post exposure observation period:
n/a
Test temperature:
19-23°C
pH:
6.8-7.0
Moisture:
29-32.6%
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1-L 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: 83%
- % silt: 16%
- % clay: 1%
- Soil taxonomic classification: natural sandy loam
- Soil classification system: N/a
- Composition (if artificial substrate): N/a
- Organic carbon (%): organic content = 6.6%
- Maximum water holding capacity (in % dry weigth): 31.1% at 1/3 bar
- 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 Environment al, Lancaster, Pennsylvania. Results of these analyses established that the laboratory well water, manure, and natural sandy loam did not contain any contaminants that ould 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: 8-hour dark
- Light intensity: 400 to 800 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.

VEHICLE CONTROL PERFORMED: yes/no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study
- Test concentrations: 0, 1000 mg/kg. Two additional concentrations 10 and 100 mg/kg were added to the study, depending on the study results, these would have been part of the range finding studies.
- 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 dw
Measured:
Reference substance (positive control):
yes
Remarks:
benomyl
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:
mortality
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
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:
mortality
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
> 440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
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
Remarks on result:
other: EC50 value was empirically estimated; therefore, corresponding 95% confidence intervals could not be calculated.
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
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:
growth
Remarks:
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:
growth
Remarks:
weight
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:
weight
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
> 440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
weight
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
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:
LOEC
Effect conc.:
> 440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
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:
NOEC
Effect conc.:
440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
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:
EC10
Effect conc.:
> 440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
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
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
> 440 mg/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
as number of juveniles
Details on results:
Details on results
- Mortality at end of exposure period: 0% at 28-d
- Total mass of adults at beginning of test: not reported
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Mean % weight change in control = -16 (SD=3.8), for 10mg/kg = -9.6%, for 100mg/kg = -12%, 1000mg/kg = -0.81 (6.
0)
- No. of offspring produced: Control = 79 mean offspring per replicate (SD=21), 1000 mg/kg = 105 (SD=23)
- 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? yes
- Relevant effect levels: 100% survival in the controls. Significant effects were observed at the 2.5 and 5.0 mg/kg treatment groups. This result is within the range of expected significant values (1 to 5 mg/kg) for reproduction for benomyl as reported in the testing guideline.
- Other: Nominal concentrations of benomyl maintained during the reference test were a solvent (acetone) control and 0.60, 1.3, 2.5, and 5.0 mg/kg. Two replicates, each containing 10 earthworms, were maintained for each treatment level and the solvent control.
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) 28-d  Mean % weight change (SD)  Mean total number of offspring per replicate (SD) 56-d
 0  100%  -16 (3.8)  79 (21)*
 10  100%  -9.6 (N/A)  124 (N/A)
 100  100%  -12 (N/A)  99 (N/A)
 1000  100%  -8.1 (6.0)  105 (23)

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

* One control replicate was considered to be an outlier producing 18 juveniles. According to the study report: "This was likely due to inherent biological variability in reproductive output of invertebrates and the data from this replicate was not included in statistical analysis for this endpoint. The remaining seven replicates from the control group met all relevant guideline criteria."

Validity criteria fulfilled:
yes
Remarks:
Control mortality = 0% at d28, control mean number of juveniles ≥45, CV of reproduction in control = 27%
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 ≥1000 mg/kg which is equivalent to ≥440 mg/kg when expressed as mean 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 ≥1000 mg/kg which is equivalent to ≥440 mg/kg when expressed as the mean measured concentration.

Description of key information

NOEC (28-day): ≥1000 mg/kg dry weight, nominal concentration, equivalent to ≥400 mg/kg dry weight arithmetic mean concentrations, based on growth and survival of Eisenia fetida, highest concentration tested.

EC10 and NOEC (56-day): >1000 and ≥1000 mg/kg dry weight nominal, respectively, equivalent to >440 and ≥440 mg/kg dry weight arithmetic mean concentrations, for the reproduction of Eisenia fetida, highest concentration tested.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
1 000 mg/kg soil dw

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, reproduction or weight 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. The nominal concentration of 1000 mg/kg dry weight is equivalent to 440 mg/kg dry weight expressed as the arithmetic mean measured concentration over the 56 day period.

 

Test substance concentrations were measured at the beginning of the test and on days 2, 28 and 56.Concentrations ranged from 43 to 51% of nominal concentrations at test initiation through day 28 and slightly declined over the last 4 weeks of the 56-day exposure period to a range of 32 to 33%. An overall mean measured concentration was calculated to be 440 mg/kg (44% of nominal concentration).

A homogeneity and stability study was conducted in advance of the earthworm study. The homogeneity results indicate a uniform distribution of the substance in the soil column at the start of the experiment. The stability results indicate that the substance was not stable and sampling did not go beyond day 35. The chromatograms did not indicate that the presence of degradation products, which, together with the steady loss of 14C activity, show that the primary mechanism of test article loss was volatilization of 14C-L4 from the simulated OECD 222 set-up.