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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:
26-08-2020 to 24-10-2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Two validity criteria were not met due to data from one control replicate. It was stated that the inclusion or removal of this result did not affect the results of the statistical analyses. Other deviations from the OECD 222 guideline: light was measured on days 7, 14, 28 and 56 instead of days 7, 14, 28, 42 and 56.
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
yes
Remarks:
Light was measured on days 7, 14, 28 and 56 instead of days 7, 14, 28, 42 and 56.
Principles of method if other than guideline:
Exceptions to GLP:
- Routine soil, cattle manure, and water screening analyses were conducted at Eurofins Laboratories Lancaster Environmental, Lancaster, Pennsylvania, using standard U.S. EPA procedures and are considered facility records under Smithers’ Standard Operating Procedures. Since the analyses were conducted following standard validated methods, this exception has no impact on the study results.
- The reference toxicant, benomyl, was purchased from a commercial supplier and was not characterised by GLP.
GLP compliance:
yes
Remarks:
See 'Principles of method if other than guideline' for deviations in GLP
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and each treatment levels
- Sampling method: 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 D6 concentration by GC-MSD/EI.
- Sample storage conditions before analysis: Directly after sampling, the cores were placed in glass bottles with Teflon-lined caps prior to analytical processing.
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): Direct injection of test substance below the surface of the natural sandy loam. To aid in test substance dispersal, half the total soil (approximately 1.8 kg wet weight per jar) was injected with the test substance. Afterwards, the remaining half (approximately 1.8 kg wet weight per jar) was added to the jars and the jars were than capped. The soil was allowed to mix thoroughly at a speed of 15 rpm for approximately 3 hours on a rolling table. After rolling table mixing, the contents of the individual rolling jars were respectively combined and each treatment level was mixed using a Hobart mixer for approximately 10 minutes to ensure homogeneity. Following a 10-minute mixing period, the treatment and control soils were added to 1-L beakers to a depth of approximately 12 cm (1043 g dry weight each replicate).
- Controls: The control soil was prepared in the same manner as described for each treatment medium except that the control medium contained no test substance.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle used
- Concentration of vehicle in test medium (stock solution and final test solution): No vehicle used
- Evaporation of vehicle before use: No vehicle used
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: Aquatic Research Organisms, Hampton, New Hampshire (Smithers Lot No. 20A250)
- Age at test initiation (mean and range, SD): All earthworms used to initiate this exposure were mature with clitellum and were 3 to 5 months old.
- Weight at test initiation (mean (SD)): Controls: 0.3961g (0.0280), 63 mg/kg: 0.3981g (0.0211), 130mg/kg: 0.3919g (0.0286), 250mg/kg: 0.3829g (0.0380), 500mg/kg: 0.3968g (0.0115), 1000mg/kg: 0.3858g (0.0108)

ACCLIMATION
- Acclimation period: Not defined
- Acclimation conditions (same as test or not): Different to test conditions: The earthworms were maintained in a vessel containing approximately 600 g of cattle manure, saturated with 1200 mL of laboratory well water, 600 g of finely sieved peat moss, and 1200 g of coconut fibers, saturated with 6000 mL of laboratory well water.
- Health during acclimation (any mortality observed): Earthworms used for testing were healthy and uniform in size.
Study type:
laboratory study
Substrate type:
natural soil
Remarks:
Natural sandy loam
Limit test:
no
Total exposure duration:
56 d
Test temperature:
18 to 21 °C
pH:
6.5 - 7.0 based on measurements taken on day 0 and day 56
Moisture:
18-22% based on measurements taken on day 0 and day 56
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1000mL glass beakers. Each test vessel was covered with transparent plastic wrap held in place with an elastic band and perforated for gas exchange
- Amount of soil or substrate: 1043g (dry weight) of treated or control soil at a depth of approximately 12cm (which exceeded the guideline recommendation of 500 to 600g of soil per test vessel (i.e. 50 to 60g per earthworm)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Not reported
- Pesticide use history at the collection site: Not reported, however analysis of representative samples of the natural sandy loam and manure used during the earthworm culture and testing were analyzed for the presence of pesticides, PCBs, and toxic metals. Results 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
- Collection procedures: Not reported
- Sampling depth (cm): Not reported
- Soil texture (if natural soil)
- % sand: 87%
- % silt: 8%
- % clay: 5%
- Soil taxonomic classification: Sandy loam
- Soil classification system: Not reported
- Composition (if artificial substrate): Not applicable
- Organic carbon (%): 3.6%
- Maximum water holding capacity (in % dry weight): 9.8%
- CEC:
- Pretreatment of soil: Dry manure and deionized water was added to the natural sandy loam and the components were mixed using a Hobart mixer.
- Storage (condition, duration): Not reported
- Stability and homogeneity of test material in the medium: Not reported

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light, 8-hour dark photoperiod
- Light intensity: 400 - 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of food consumption (recorded on days 7, 14, 21 and 28). Pooled weight of F0 earthworms was determined at day 0 and 28. Earthworm mortality and health (e.g., color changes, lethargy, and softness) assessments were also performed on day 28.
On day 56 and 24 hours after, number of juveniles was determined

VEHICLE CONTROL PERFORMED: not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: Not reported
- Results used to determine the conditions for the definitive study: Not reported
Nominal and measured concentrations:
Nominal: control, 63, 130, 250, 500 and 1000 mg/kg dwt
Measured: control, 63, 110, 220, 430 and 930 mg/kg dwt
Reference substance (positive control):
yes
Remarks:
Conducted with benomyl between 5 December 2019 to 30 January 2020
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
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:
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:
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:
growth
Remarks:
as adult weight
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:
as adult weight
Details on results:
- Mortality at end of exposure period: number of surviving adults at day 28 was 98% for controls, 63 mg/kg, 130 mg/kg and 250 mg/kg treatments. 100% survival was observed in the 500 and 1000 mg/kg treatments.
- No. of unhatched cocoons: Not reported, however 24 hours after the study termination (after 56 days), the remaining soil was checked for any remaining juveniles
- Morphological abnormalities: Not reported
- Behavioural abnormalities: Not reported
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: significant effects were observed in the 5.0 mg/kg treatment which is within the range specified in the OECD 222 test guideline.
Reported statistics and error estimates:
% survival, weight change (F0) and number of F1 offspring per replicate were analysed using: CETIS version 1.9. Tests used were Fisher's exact test with Bonferroni-Holm's adjustment for survival, Jonckheere-Terpstra's step-down test for adult weight and Dunnett's multiple comparison test for adult reproduction.

Table 1. Mean biological observations of E. fetida in an exposure to D6


Nominal treatment (mg/kg)

Measured treatment (mg/kg)

Mean survival (SD)

%

Survival CV

 

%

Mean biomass change (SD)

%

Biomass change CV

%

Mean number of juveniles per replicate (SD)

Number of juveniles CV

%

Control

-          

98 (5)

5

3.72 (8.47)

228

74.1 (29.0)

39

63

63

98 (12.6)

13

1.99 (5.01)

252

73.0 (13.5)

19

130

110

98 (5)

5

9.20 (14.49)

157

77.0 (37.5)

49

250

220

98 (5)

5

9.61 (11.34)

118

61.8 (7.8)

13

500

430

100 (0)

0

9.91 (2.39)

24

65.8 (21.7)

33

1000

930

100 (0)

0

16.49 (9.66

59

57.3 (28.0)

49

Validity criteria fulfilled:
no
Remarks:
One control replicate caused failures of two validity criteria (number of juveniles per replicate in the control group at test termination and the %CV of reprodution in the controls). Discounting this result has no effect on the significance of the data.
Conclusions:
A 28-day NOEC value of >1000 mg/kg dry weight and a 56-day NOEC of ≥1000 mg/kg dry weight have been determined for the effects of the test substance on mortality, reproduction and growth respectively of Eisenia fetida. The study failed two validity criteria based on one replicate from the control treatment, however this did not impact the significance of the data and the study was assigned Reliability 2.

Description of key information

LC50 (28-day): >1000 mg/kg dry weight (nominal concentration, highest concentration tested), survival, Eisenia fetida.

NOEC (28-d): >1000 mg/kg dry weight (nominal concentration, highest concentration tested), survival and weight, Eisenia fetida.

NOEC (56-day): ≥1000 mg/kg dry weight (nominal concentration, highest concentration tested), reproduction, Eisenia fetida.

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) and in compliance with GLP. No effects on survival or reproduction were observed.

A 28-day LC50 value of >1000 mg/kg dry weight and a NOEC value of ≥1000 mg/kg dry weight (highest concentration tested) have been determined for the effects of the test substance on survival of adult earthworms, based on nominal concentrations.

A 28-day NOEC value of ≥1000 mg/kg dry weight (highest concentration tested) has been determined for the effects of the test substance on growth of adult earthworms, based on nominal concentrations.

A 56-day NOEC value of ≥1000 mg/kg dry weight (highest concentration tested) has been determined for the effects of the test substance on reproduction of the earthworm, based on nominal concentrations (Smithers 2021).

 

Test substance concentrationswere maintained for the duration of testing and ranged from 80 to 110% of nominal concentrations throughout the entire exposure period. Overall mean measured concentrations ranged from 87 to 100% of nominal concentrations and as it is within 80 to 120% of the nominal target concentration, the results are reported as nominal. The nominal concentration of 1000 mg/kg is equivalent to a measured concentration of 930 mg/kg. 

Results of the replicate samples were generally consistent, indicating homogeneous distribution of the test substance in the exposure vessels.

 

Two of the OECD 222 validity criteria were not met during the test. The following acceptance criteria were required:

Acceptability Criteria

Study Results

Criterion Met

Mean mortality in the control group should be ≤10% at day 28.

Mortality in the control was 2% at day 28.

Yes

At least 30 juveniles per 10 earthworms (single test vessel) by termination in the control group.

In the control, ≥26 juveniles per replicate were produced by test termination.

No, see justification below

The coefficient of variation (% CV) of reproduction in the control should not exceed 30%.

During this study, the % CV for the control was 39%.

No, see justification below

 

Although the coefficient of variation (CV) of reproduction and minimum number of juveniles per replicate vessel do not meet the criteria, there is one control replicate that causes the acceptability criteria to fail. If this one control replicate is removed, all control validity criterion are met (≥46 juveniles per replicate and a CV of 29%). Whether this replicate is included or removed from the statistical analysis, the results are identical with no significant statistical effect of any treatment group compared to the control. Based on these results, it was determined that this study provided a suitable data set to evaluate the test substance for risk assessment, which establishes no risk to earthworm survival and reproduction at a nominal concentration of 1000 mg/kg dry weight.