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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline study but it is not certain whether the study was conducted in accordance with GLP.
Qualifier:
according to guideline
Guideline:
other: Environment Canada, EPS 1/RM/43, June 2004
GLP compliance:
not specified
Remarks:
The data is from a publication and this information was not included.
Analytical monitoring:
yes
Details on sampling:
Soil samples were collected throughout the initial treatment, preparation (i.e., at manure amendment, following the 2-h equilibrium period, and following mixing of the amended biosolid with the Gr10 soil) for the range-finding tests to confirm the analytical methodology used, as well as to evaluate the loss of D5 over time. For the definitive tests, samples were taken for analysis only on day 0 of the test (i.e., when the test organisms were added) to confirm dose concentrations in the test soil. All samples were immediately frozen and stored at -20°C prior to analysis. All samples were analysed for cVMS D4, D5 and D6 concurrently by Quebec Laboratory for Environmental Testing of Environment Canada (Montreal, Quebec).

Triplicate 1.0g (+/-0.2) (wet weight) samples were then measured into glass tubes and well mixed to ensure solids are homogenized. To extract the soil samples, 5 mL of acetonitrile was added to each sample and vortexed followed by the addition of 5 mL of pentane (also vortexed). The sample was then sonicated in an ice bath for 10 min followed by centrifugation at 1500 RPM for 10 min at 15°C. The pentane phase was then transferred to a clean GC vial and 75 μL of naphthalene-D8 solution(lfi) ng/μL in pentane) was added as an internal standard.
Vehicle:
no
Details on preparation and application of test substrate:
A sandy loam soil (Gr10) was obtained from Greely Sand and Gravel (Ottawa Ontario (ON)) in June 2010 and was dried, sieved (4-mm mesh) and homogenized prior to use. The negative control treatment consisted of a formulated artificial soil (AS) comprising 10% air-dried pre-sieved Sphagnum sp. peat, 20% kaolin clay, and 70% silica sand, with calcium carbonate added to standardize the soil pH.

For the toxicity tests, the soil was amended with either an uncontaminated or contaminated surrogate biosolid. Although available, the use of an actual biosolid produced from a water treatment facility was not pursued as an option for use in the toxicity tests as the potential for other contaminants or toxicants present in the sample may have confounded toxicity test results. As a result, mushroom compost and organic cow manure were both considered as options for a surrogate biosolid. Both alternative biosolids were analysed in conjunction with a real biosolid (obtained from the Robert O. Picard Water Treatment facility in Ottawa, ON) for comparison of their physical and chemical characteristics. Based on these characteristics. the organic cow manure was selected for its similarity in percent organic matter to the actual biosolid.

To prepare test samples, the surrogate biosolid was amended with D5 (97% pure liquid; Sigma-Aldrich) to create a contaminated test substance; subsequently, the amended biosolid was manually mixed for 2 min, covered to prevent volatilization, and allowed to equilibrate for 2 h. Following the 2 h equilibration period, the amended biosolid was added to the Gr10 soil at a rate of 5 g/kg (on a dry weight basis), equivalent to 8 t/ha, a common application rate for biosolids on agricultural soils in Ontario to create the test soil. Each test concentration was prepared independently. The average moisture content of the test soil was 38 ± 3%, with an average pH of 7.55±0.15 for all test set-ups.
Test organisms (species):
other: Eisenia andrei
Animal group:
annelids
Details on test organisms:
Two sexually mature adults were added to each test vessel at the start of the test (day 0), and the average initial wet weight of the adult earthworm
was 451.7 +/-108.2 mg.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
20 +/-2°C
pH:
7.55 ±0.15
Details on test conditions:
Test vessels were incubated at 20±2°C with an average light intensity of 10.7 ±2.5 μmol/(m2 t)-1. within the ranges specified in the earthworm method; and food (cooked oatmeal) was added on days 0,14,28 and 42 of the test. On day 28, surviving adults were counted and removed from the test soil, and on day 56 the number of juveniles produced was counted, and the juvenile dry biomass measured and recorded.
Nominal and measured concentrations:
Nominal/measured concentrations (mg/kg dry wt.) of D5 for this test were 0/0, 143/50, 244/91, 414/135, 704/248, 1197/358, 2035/507, 3460/1093, 5882/2362 and 10000/4074.
Reference substance (positive control):
not specified
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 4 074 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
56 d
Dose descriptor:
other: IC50
Effect conc.:
> 4 074 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
other: IC50
Effect conc.:
> 4 074 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: individual juvenile dry mass
Details on results:
There was no efiect on adult survival after 28 d of exposure (100% survival in all treatments, data not shown). While there was a significant reduction in juvenile production at test concentrations ≥507 mg/kg (Fisher's LSD; p<0.05), the lC50 was >4074 mg/kg. There was also no effect on the mean individual dry mass of juveniles; however, the mass increased with increasing exposure to D5. This may be due to less competition for resources (i.e., food) within the test vessel as fewer juveniles were observed at higher test concentrations. Test concentrations > 1093 mg/kg had significantly higher mean individual dry mass compared with lower test cocentrations (Fisher's LSD: p < 0.05).
Reported statistics and error estimates:
The lethal concentration (i.e., LC50s) was calculated for invertebrate mortality using the EPA Probit Analysis Program {version 1.5) or the EPA Trimmed Spearman-Karber (TSK) Program (version 1.5). Inhibitory concentrations (i.e., lC50s) were estimated for invertebrate juvenile production using nonlinear regression analysis via SYSTAT (version 13) software, ensuring that model assumptions (normality and homoscedasticity) were met. When regression models were unable to calculate the IC50, or model assumptions were not met, a "Linear Interpolation for Sublethal Toxicity: The Inhibition Concentration (ICp) Approach" (ICPIN) (version 2.0) was used. ln cases where a 50% effect was not observed, an ANOVA was performed to determine if there was a significant difference in the measured endpoint across treatments. lf a significant difference was found, then Fishers least significant difference test was used to determine where the difference was among treatments.
Validity criteria fulfilled:
yes
Conclusions:
A 28-day LC50 value of >4074 mg/kg dry weight and a 56-day NOEC of ≥4074 mg/kg dry weight have been determined for the effects of the test substance on mortality and reproduction and growth respectively of Eisenia andrei.
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
See endpoint summary for justification of read-across
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 4 074 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
56 d
Dose descriptor:
other: IC50
Effect conc.:
> 4 074 mg/kg soil dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 76 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 41 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 76 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 41 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Data waiving:
other justification
Justification for data waiving:
other:
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 April to 18 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted study according to OECD 222 and difficult substances guidance, with GLP and analysis of media.
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
yes
Remarks:
Natural sandy loam used, as opposed to artificial soil
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal: 0.0076, 0.076, 0.76, 7.6 and 76 mg/kg
- Sampling method: During the definitive exposure, soil samples were removed on days 0, 2, 28 (before and after adults were removed) and 56. An additional test vessel was sacrificed at each interval and the soil sample analysed for D5 concentration as to leave the test organisms and offspring undamaged. Cores were used for the determination of test concentration and the concentration of each sample was based on the mean of the three individual results
- Sample storage conditions before analysis: Directly after sampling, the cores were placed in glass bottles with teflon-lined caps and were presented for analytical processing.
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): D5 was applied directly to natural sandy loam (see Protocol Deviations) with a percent moisture of 2.9%. The soil aliquots were contained in glass jars . The soil was allowed to mix thoroughly at a speed of 15 rpm overnight on a rolling table. Table 1 describes the application of D5 to the natural sandy loam. Due to the limitations of the amount of D5 that could be directly dispensed to the soil, and the volume of the mixing jars, the lower concentrations (0.076 and 0.0076 mg/kg) were prepared by one dilution of treated soil. Table 2 describes the soil dilutions from a 7.6 mg/kg soil concentration prepared the previous day. The treated sandy loam was allowed to mix thoroughly at a speed of 15 rpm overnight on a rolling table. After treated sandy loam was mixed overnight and added to replicate vessels, 200 mL of additional deionized water was added to each replicate to bring the percent moisture to approximately 24%.
- Controls: Sandy loam without test material
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Eisenia fetida
- Source: Original Source of Culture: Happy D Ranch, Visalia, California
- Age at test initiation (mean and range, SD): F0 Generation age: 12 months old
- Weight at test initiation (mean and range, SD): mean individual weight ranged from 0.3855 to 0.4492 g

ACCLIMATION
- Acclimation period: One day
- Acclimation conditions (same as test or not): yes
- Health during acclimation (any mortality observed): No mortality reported
Study type:
laboratory study
Substrate type:
other: natural sandy loam
Limit test:
no
Total exposure duration:
56 d
Remarks:
28-d F0 generation phase teminated
Test temperature:
19 to 23 °C
pH:
Day 0 pH: 6.92-7.55
Day 56 pH: 6.24-6.70
Moisture:
Day 0: 11-22% (Due to the volatility of the test material, soils were not mixed after adding water to the required % moisture and do not represent the actual % moisture of the entire vessel until the water disperses evenly.)
Day 56: 19-28%
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Glass, 1000 ml
- Amount of soil or substrate: 1000 g sandy loam
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: One replicate for the 0.0076, 0.076, 0.76 and 7.6 mg/kg concentrations, eight replicates for the76 mg/kg concentration.
- No. of replicates per control: 8
Each test vessel was covered with perforated transparent plastic wrap and secured with a rubber band.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil) : Periodic analysis of representative samples of the deionized water source, natural sandy loam and cattle manure used during the earthworm culture and testing were analysed for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. Results of these analyses established that the deionized water source, manure and natural sandy loam did not contain any contaminants which would adversely affect the test organisms.
- Geographic location: Not reported
- Pesticide use history at the collection site: Not reported
- Collection procedures: Not reported
- Sampling depth (cm): Not reported
- Soil texture (if natural soil) Sandy loam
- % sand: 75
- % silt: 19
- % clay: 6
- Soil taxonomic classification: Not reported
- Soil classification system: Not reported
- Composition (if artificial substrate): n/a
- Organic carbon (%): The organic matter content of the soil was 5.2%. Awaiting data on OC
- Maximum water holding capacity (in % dry weigth): maximum sorption concentration: 76 mg D5/kg dry soil
- CEC:
- Pretreatment of soil: not reported
- Storage (condition, duration): not reported
- Stability and homogeneity of test material in the medium:Results of the replicate samples were generally consistent indicating homogeneous distribution of the test substance in the exposure vessels.
Losses of the test substance over time were not expected in this exposure system based on previous data collected from samples of treated natural sandy loam. Earthworm metabolism, other biological components associated with the earthworms or microorganisms associated with the manure included as an earthworm food source could have contributed to the concentration decline. Due to these factors, the biological results are reported in terms of nominal concentrations and as geometric mean measured concentrations.


OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8-hour dark photoperiod
- Light intensity: 340 to 750 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations on food consumption were recorded weekly, beginning on day 7, until test day 28. The group weight was determined for earthworms (F0) added to each replicate vessel at test initiation (day 0), and the group weight was determined at the termination of the F0 generation phase (day 28). Earthworm mortality and health (e.g., color changes, lethargy and softness) assessments were also performed on day 28. Any earthworms that were unaccounted for were presumed dead and decomposed.
On day 28, all cocoons were left in the test soil to continue the F1 generation exposure. At test termination (day 56 and again on day 57), the number of juveniles was determined.
pH of each soil batch was determined at test initiation and on day 56 in each replicate test vessel.
Light intensity was measured at the surface of the test vessels on days 0, 7, 14, 28, 42 and 56.
Air temperature inside the environmental chamber which housed the test vessels was monitored continuously.

VEHICLE CONTROL PERFORMED: yes/no: n/a

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 0.0076, 0.076, 0.76, 7.6 were ussed as range finding concentrations
- Results used to determine the conditions for the definitive study: No effects at these concentrations
Nominal and measured concentrations:
Nominal: 0.0076, 0.076, 0.76, 7.6 (as range finding concentrations) and 76 mg/kg as maximum carbon saturation level.
Geometric mean concentration: (only tested for the nominal 76 mg/kg concentration) 41 mg/kg
Reference substance (positive control):
yes
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 76 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 41 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
76 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: survival
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
41 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 76 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight change
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 41 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: weight change
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
76 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight change
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
41 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: weight change
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
> 76 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
> 41 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
76 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
41 mg/kg soil dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 value for reproduction was 1.4 mg/kg
Reported statistics and error estimates:
At the termination of each phase of the study (Day 28 for F0 and Day 56 for F1) data obtained on survival and weight change of F0 adults and the number of F1 offspring were statistically analysed to identify significant treatment-related effects. CETIS-Comprehensive Environmental Toxicity Information SystemTM (Ives, 2013) Version 1.8 software was used to perform the computations.

The No-Observed-Effect Concentration (NOEC), the highest test concentration which demonstrated no statistically adverse effect (p 0.05) when compared to the control data, was determined. The data were first checked for normality using Shapiro-Wilks’ Test (U.S. EPA, 2002) and for homogeneity of variance using Variance Ratio F t Two-Sample Test (U.S. EPA, 2002). If the data sets passed the test for homogeneity and normality, then Equal Variance t Two-Sample Test (U.S. EPA, 2002), was used to determine the NOEC. If the data did not pass the tests for homogeneity and normality, then Unequal Variance t Two Sample Test (U.S. EPA, 2002) was used to determine the NOEC. All statistical conclusions were made at the 95% level of certainty except in the case of the basic assumption tests, e.g., Shapiro-Wilks’ Test and Variance Ratio F Test, in which the 99% level of certainty was applied.
Validity criteria fulfilled:
yes
Conclusions:
28-d F0 LC50 values of >76 mg/kg nominal concentration, >41 mg/kg mean measured concentration, and NOECs of 76 mg/kg nominal concentration, and 41 mg/kg mean measured concentration, have been determined for effects of D5 on survival and weight change of adult earthworms.
56-d F1 LC50 values of >76 mg/kg nominal concentration, >41 mg/kg mean measured concentration, and NOECs of 76 mg/kg nominal concentration, and 41 mg/kg mean measured concentration, have been determined for effects of D5 on reproduction of the earthworm Eisenia fetida.

Description of key information

28-day LC50: >4074 mg/kg dry weight and 56-day NOEC ≥4074 mg/kg dry weight, for the effects of D5 on mortality and reproduction and growth, respectively, of Eisenia andrei.

28-day LC50: >41 mg/kg dry weight and 56-day NOEC ≥41 mg/kg dry weight (measured concentrations), for the effects of D5 on survival and reproduction, respectively, of Eisenia fetida.

Key value for chemical safety assessment

Additional information

There are currently no reliable data describing the long-term toxicity of the registered substance to soil macroorganisms.

 

An OECD TG 222 earthworm reproduction test is being conducted with the registration substance according to ECHA final decision TPE-D-2114497761-36-01/F. The substance dataset and risk assessment will be updated once results of the study are available and the study is finalised. An update is planned to be completed within three months from when the last final report is received. The final test report is expected in March 2021. Please see a letter from the testing laboratory, attached in Section 6.3.1 IUCLID Placeholder EPSR, for further details.

In the interim period, before the data with the registered substance become available, data are read across from the siloxane decamethylcyclopentasiloxane (D5, CAS 541-02-6).

A 28-day LC50 value of >4074 mg/kg dry weight and a 56-day NOEC of ≥4074 mg/kg dry weight (measured initial concentration) have been determined for the effects of the test substance on mortality and reproduction and growth respectively of Eisenia andrei (Velicogna et al., 2011).

No effects at the highest concentration tested were also determined in a second test with D5: 28-d F0 LC50 values  of >76 mg/kg nominal concentration, >41 mg/kg mean measured concentration, and NOECs of 76 mg/kg nominal concentration, and 41 mg/kg mean measured concentration, have been determined for effects of D5 on survival and weight change of adult earthworms.

56-d F1 LC50 values  of >76 mg/kg nominal concentration, >41 mg/kg mean measured concentration, and NOECs of 76 mg/kg nominal concentration, and 41 mg/kg mean measured concentration, have been determined for effects of D5 on reproduction of the earthworm Eisenia fetida (Smithers Viscient, 2015).

Read-across of the terrestrial toxicity data for D5 to Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (CAS 352230-22-9) is considered to be suitable to derive an interim hazard and risk assessment under REACH until the studies with the registration substance are available.

Refer to IUCLID Sections 6.0 and 6.3 (CSR Sections 7.0 and 7.2) for further discussion.