Registration Dossier

Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 14, 2014 - March 19, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD test guidance in compliance with GLP and reported with a valid GLP certificate.
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
no
Details on sampling:
Not applicable
Details on preparation and application of test substrate:
The artificial soil was prepared in bulk by blending 70% sand, 20% kaolin clay, 10% sphagnum peat and 0.01% calcium carbonate. The pH of the bulk soil at the completion of preparation (prior to hydration) was 5.9. The bulk artificial soil was stored in a sealed container under ambient conditions until used to prepare the test soils. Test soil was prepared by mixing the appropriate amount of test substance with bulk soil and reverse osmosis water to hydrate the soil. Test soil components were mixed for a total of 20 minutes in order to achieve a homogeneous mixture. Negative control soil was prepared in a similar manner as the treated soil but without the addition of test substance or solvent. Solvent control soil was prepared with methanol but without test substance. Seven-hundred-fifty grams of prepared soil were added to each of four replicate chambers for each of the test treatment and control groups. The test concentrations are reported as milligrams of test substance per kilogram of test soil on a dry weight basis (mg/kg).
The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the artificial soil substrate used by Wildlife International have indicated there were no constituents present at levels known to interfere with the purpose or conduct of the study.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Earthworms (Eisenia fetida) for the test were from in-house cultures started with worms originally obtained from the University of Maryland Wye Research and Education Center, Queenstown, Maryland.
Earthworms were cultured in a mixture of moist peat, fed saturated alfalfa and/or cow manure, and held at a bedding temperature of 20 ± 5 °C in continuous light.
Approximately 24 hours prior to the test, 320 adult worms (with clitellum) were selected and placed in a container of prepared artificial soil substrate adjusted to a moisture content of approximately 34% by weight for the acclimation period.
On the day of test initiation, the worms were rinsed briefly with water purified by reverse osmosis water and indiscriminately distributed by pairs into groups of ten worms each. Each group of worms was weighed then placed on the soil surface in the appropriate test chamber. The worms were not fed during testing.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Post exposure observation period:
None
Test temperature:
20 ± 2°C.
pH:
6.7 - 7.3
Moisture:
32.1 - 34.0%
Details on test conditions:
Artificial Soil and Test Soils Preparation
The artificial soil was prepared in bulk by blending 70% sand, 20% kaolin clay, 10% sphagnum peat and 0.01% calcium carbonate. The pH of the bulk soil at the completion of preparation (prior to hydration) was 5.9. The bulk artificial soil was stored in a sealed container under ambient conditions until used to prepare the test soils. Test soil was prepared by mixing the appropriate amount of test substance with bulk soil and reverse osmosis water to hydrate the soil. Test soil components were mixed for a total of 20 minutes in order to achieve a homogeneous mixture. Negative control soil was prepared in a similar manner as the treated soil
but without the addition of test substance or solvent. Solvent control soil was prepared with methanol but without test substance. Seven-hundred-fifty grams of prepared soil were added to each of four replicate chambers for each of the test treatment and control groups. The test concentrations are reported as milligrams of test substance per kilogram of test soil on a dry weight basis (mg/kg).
The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the artificial soil substrate used by Wildlife International have indicated there were no constituents present at levels known to interfere with the purpose or conduct of the study.

Test Chambers
The test chambers were one-liter glass beakers covered with plastic wrap that was perforated for air exchange. All test chambers were identified with the project number, test concentration and replicate.

Physical Properties of Test Soils
Soil temperature was measured in one replicate of each test treatment and control group at test initiation and termination using a hand-held digital thermometer. Moisture content and pH measurements were made on soil samples collected from each batch of soil prepared for control and treatment groups at initiation of the experiment. At test termination, samples for moisture content and pH measurements were collected from one replicate of the control and each treatment group. Measurements of pH were made using a Thermo Orion Model 525APlus pH meter. Soil moisture content was determined by measuring the initial weight of the soil sample then weighing the soil sample after it was dried at approximately 105°C. The percent moisture was calculated using the following formula: % Moisture = [(wet weight - dry weight) ÷ wet weight] * 100.

Environmental Conditions
During the test, the worms were maintained in an environmental chamber set to maintain a temperature of approximately 20 ± 2°C. Air temperature was measured daily in the environmental chamber. The photoperiod during the test was 24 hours of continuous light per day provided by overhead fluorescent bulbs. The target light intensity during the test was approximately 400 to 800 lux, and was verified on Day 14 of the test.

Observations

At test initiation, the worms were placed on the surface of the soil in each replicate test and control chamber and were observed for burrowing behavior. On Days 7 and 14 of the study, the contents of each test chamber were removed to determine the number of surviving earthworms. All surviving earthworms were observed for behavioral or pathological abnormalities and response to mechanical stimulus. On Day 7, following observations, test soil was returned to the same chamber that it was removed from and the worms were placed on the soil surface in order to observe burrowing behavior once again. On Day 14, following observations and body weight determinations, surviving earthworms were stored frozen prior to disposal by incineration.

Body Weights
On Day 0, group weights for all replicate earthworms were collected prior to the earthworms being placed in the test chambers. On Day 14, all surviving worms were removed from each replicate chamber, rinsed with reverse osmosis water and blotted dry on paper. Group body weights were measured for each replicate and average individual body weights were calculated.

Reference Toxicity Test
A non-GLP reference toxicity test was conducted in 2012 at Wildlife International to determine the LC50 value for earthworms exposed to the reference toxicant, chloroacetamide, in artificial soil. The test was conducted with earthworms from the same source to monitor the techniques used and sensitivity of the test population. The earthworms were exposed to chloroacetamide in the soil at nominal concentrations of 7.5, 15, 30 and 60 mg active ingredient/kg dry soil.

Statistical Calculations
The no-observed-effect-concentration (NOEC) was determined by visually inspecting the mortality and clinical observation data. Body weights, and change in body weights, were statistically compared with a Dunnett’s test (α=0.05) using SAS Version 8.2 (2). Prior to conducting Dunnett’s test, the data were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (α = 0.01).
Nominal and measured concentrations:
62.5, 125, 250, 500, 1000 mg/kg
Reference substance (positive control):
yes
Remarks:
chloroacetamide
Duration:
14 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:
14 d
Dose descriptor:
NOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: bodyweight
Details on results:
Environmental Conditions
Air temperature in the environmental chamber was within the desired range of 20 ± 2°C with air temperature ranging from 19 to 20°C, during the exposure period. The earthworms were maintained under continuous lighting at an average intensity of 706 ± 64.7 lux, with a range over the surface of the test chambers of 554 to 797 lux.

Physical Properties of Test Soils
The soil pH ranged from 6.7 to 7.3 in the control and test soils at test initiation and ranged from 7.9 to 8.2 at test termination. Soil temperature ranged from 19.7 to 20.1 °C in the test groups at test initiation and ranged from 20.0 to 20.2 °C at test termination, within the desired range of 20 ± 2 °C. Soil moisture content ranged from 33.7 to 35.3% at test initiation and from 32.1 to 33.5% at test termination, indicating little change in soil moisture content during the test.

Observations
There were no mortalities in the negative control, solvent control or any of the treatment groups during the 14-day test. Because mortality was less than 50% in the highest concentration, the LC50 could not be statistically defined and was judged to be greater than the highest concentration tested. Earthworms in the control and treatment groups were normal in appearance and behavior throughout the test period, with the exception of two earthworms with reduced reaction to mechanical stimulus on Day 14 in the 500 mg/kg treatment group. On Day 0 after the earthworms were placed on the soil surface, some earthworm in the 250, 500 and 1000 mg/kg treatment groups were burrowed just under the soil surface, indicating an aversion to the test soil. In the 1000 mg/kg group they were very slow to burrow and visible against the glass or just under the soil surface. On Day 7, earthworms in the 1000 mg/kg group were found clustered in a ball in the soil, indicating an aversion to the soil. All earthworms burrowed back into the control and test soils after observations were completed on Day 7.

Body Weights
Average individual body weights at test initiation and termination, and the change in body weight from test initiation to test termination, were calculated from the Day 0 and Day 14 replicate measurements. Statistical analyses were performed using SAS Version 8.2 statistical software. The negative and solvent control groups were compared statistically (T-Test of Means) and were not significantly different. Therefore the negative and solvent groups were pooled for comparison with the test substance groups for loss in body weight using a Dunnett’s test (α = 0.05). Data were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (α = 0.05). The initial and change in body weight values were normally distributed and all body weight data analyzed had homogeneous variances. The final body weight values were normally distributed but not homogenous, however Dunnett’s test is robust with respect to homogeneity therefore analyses were considered to be acceptable. Final body weight means and change in body weight (initial - final) means in the treatment groups were statistically significant (p<0.05) at the 1000 mg/kg concentration when compared to the pooled control group mean. Therefore the NOEC was determined to be 500 mg/kg, based on reduced body weight in the 1000 mg/kg level.

Results with reference substance (positive control):
Reference Toxicity Test
The 14-day LC50 value for earthworms exposed to the reference substance, chloroacetamide, in an artificial soil substrate was 28.7 mg a.i./kg dry soil with a 95% confidence interval of 15 and 60 mg a.i./kg dry soil. The LC50 value of 28.7 mg a.i./kg dry soil falls within the range of 20 to 80 mg/kg specified in the ISO 11268-1 document. These results are consistent with those observed in previous studies and verify the adequacy and consistency of the methods used in this study.
Reported statistics and error estimates:
Validity Criteria
The validity criterion for the test was met. Adult mortality of negative control earthworms was less than 10% at the end of the test.
Validity criteria fulfilled:
yes
Conclusions:
The 14-day LC50 estimation for earthworms exposed to Reofos 35 in an artificial soil substrate was determined to be greater than 1000 mg/kg dry soil. The no-observed-effect-concentration (NOEC) was 500 mg/kg, based on reduced body weights in the 1000 mg/kg level.
Executive summary:

The 14-day LC50 estimation for earthworms exposed to Reofos 35 in an artificial soil substrate was determined to be greater than 1000 mg/kg dry soil. The no-observed-effect-concentration (NOEC) was 500 mg/kg, based on reduced body weights in the 1000 mg/kg level.

Endpoint:
toxicity to soil macroorganisms except arthropods, other
Remarks:
Earthworm Reproduction Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 22, 2016 to March 9, 2017
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))
Version / remarks:
Organization for Economic Cooperation and Development. 2004. OECD Guideline for the Testing of Chemicals; Guideline 222: “Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)”.
Deviations:
not specified
Qualifier:
according to
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Version / remarks:
International Organization for Standardization. 1997. ISO/FDIS 11268-2. Soil Quality – Effect of Pollutants on Earthworms (Eisenia fetida) – Part 2: Determination of Effects on Reproduction.
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
No further details specified
Analytical monitoring:
not specified
Details on sampling:
Not specified
Vehicle:
yes
Remarks:
methanol
Details on preparation and application of test substrate:
The test substance was administered to the test organism in soil. This route of administration was selected because it represents the most likely route of exposure to sediment dwelling organisms.
Test soils were prepared by mixing the appropriate amount of test substance with methanol and adding the mixture to dry artificial soil containing finely ground cow manure and mixing for approximately 10 minutes to allow the methanol to evaporate. The RO water was added to hydrate the soil. Test soil components were mixed additionally for approximately 20 minutes for a total mixing time of approximately 30 minutes in order to achieve a homogeneous mixture. Negative control soil was similarly prepared but without the addition of test substance and methanol and without the initial approximate 10-minute mix. Solvent control soil was similarly prepared but without the addition of test substance. Seven-hundred-fifty grams of prepared soil were added to each of four test chambers for each of the treatment and solvent control groups and to each of the eight test chambers for the negative control group. The test concentrations are reported as milligrams of test substance per kilogram of test soil on a dry weight basis (mg/kg dry soil).
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Adult earthworms (Eisenia fetida) for the test were from EAG Laboratories – Easton cultures started with earthworms originally obtained from the University of Maryland Wye Research and Education Center, Queenstown, Maryland. The identity of the original culture was verified by the supplier. Adult earthworms were from a synchronous culture (individuals not differing in age by more than four weeks) maintained in a mixture of moist peat moss with an appropriate amount of calcium carbonate, fed saturated alfalfa, and held at a bedding temperature of 20 ± 5°C in continuous light.
Adult earthworms at least 2 months but less than 4 months old were transferred to the study room and held in a glass aquarium of conditioning bedding (peat) with cow manure as the food source for acclimation to test conditions for 7 days prior to conditioning in the artificial soil substrate. One day prior to test initiation, the adult earthworms (360 with clitellum) were removed from the glass aquarium and divided equally into ten one-liter glass beakers each containing ~ 800 grams prepared artificial soil substrate adjusted to a moisture content of approximately 34% by weight, for the soil acclimation period.
Each beaker was covered with perforated plastic wrap secured with a rubber band. Earthworms were fed cow manure throughout the acclimation period and held under the same environmental conditions as the test.
On the day of test initiation, the earthworms were indiscriminately distributed into holding vessels by pairs into groups of ten earthworms each, rinsed briefly with RO water, gently blotted dry, weighed in groups of 10, and then placed randomly on the soil surface of a test chamber. To minimize bias, which might arise from the selection process, the test chambers were placed randomly on the counter in the test room. The earthworms were fed cow manure during testing. On Day 1 of the test, approximately 5 grams of finely ground cow manure for food, and water to moisten the food, were added to the test chambers. Food was provided approximately weekly during the next three weeks by adding food in a small depression in the soil surface and covering it with a thin layer of soil. The amount of food supplied was reduced if uneaten food remained from the previous feeding interval. On Day 28, after adult earthworms were removed, approximately 5 additional grams of manure was gently mixed into the test soil before it was returned to the test chambers.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
56 d
Post exposure observation period:
No post exposure observation period specified in the study report.
Test temperature:
During the test, the earthworms were maintained in an environmental room set to maintain a temperature of approximately 20 ± 2 °C.
pH:
Soil pH ranged from 7.2 to 7.4 at both test initiation and test termination.
Moisture:
Soil moisture content measured during the test ranged from 33.7 to 33.9% at test initiation and from 32.7 to 34.5% at test termination.
Details on test conditions:
EXPERIMENTAL DESIGN
Adult earthworms (Eisenia fetida) were exposed to a geometric series of five concentrations of the test material in soil. Nominal concentrations of 62.5, 125, 250, 500, and 1000 milligrams of Reofos 35 per kilogram of soil on a dry weight basis (mg/kg dry soil) were selected in consultation with the Sponsor.
Two control groups, a negative control and solvent control, were exposed to soil without the addition of test substance and maintained concurrently. Four replicate chambers were maintained for each test treatment and solvent control group. Eight replicate chambers were maintained for the negative control group. Ten earthworms were randomly allocated in each replicate chamber. On the day of test initiation, the earthworms were rinsed briefly with water purified by reverse osmosis (RO water), blotted dry, weighed in groups of 10, and then placed on the soil surface of a test chamber. The adult earthworms were fed cow manure weekly during the first 28 days of the test, removed from the test chambers on Day 28, and observed for mortality and signs of toxicity. To determine the effects on reproduction, test soil and cocoons were returned to the test chambers for an additional 28 days. Juvenile production in each replicate was assessed at the end of the test (Day 56) and the numbers of any juvenile produced in each replicate was determined on Day 57. Cow manure was also added to the soil once at the beginning of the cocoon exposure period to serve as a source of food for juvenile earthworms emerging from cocoons. The number of juveniles present in each replicate at test termination was used to determine the no-observed-effect-concentration (NOEC) for reproduction.

Test Soil Preparation
The artificial soil was prepared in bulk by blending 70% sand, 20% kaolin clay, 10% sphagnum peat, and 1% calcium carbonate. The pH of the bulk lots of artificial soil at the completion of preparation (prior to hydration) was 6.0 to 6.1. The bulk artificial soil was stored in sealed containers under ambient conditions until used to prepare the acclimation and test soils. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the artificial soil substrate containing finely ground cow manure and the municipal water used by EAG Laboratories - Easton indicated there were no contaminants present at levels known to interfere with the purpose or conduct of the study.

Test Chambers
The test chambers were one-liter low form (108 mm outer diameter (O.D.) X 158 mm height) glass beakers covered with plastic wrap that was perforated for air exchange and was secured with a rubber band. All test chambers were identified with the project number, treatment group, and replicate.
Test chambers were arranged in a randomized block design to minimize bias that might result from a placement effect in the study room.

Physical Properties of Test Soil
Soil temperature was measured in one replicate chamber of each treatment, solvent control, and negative control group at test initiation and termination (Day 56) using a hand-held digital thermometer. Moisture content and pH measurements were made on soil samples collected from each batch of soil prepared for treatment, solvent control, and negative control groups at the time of test soil mixing. At test termination, samples for moisture content and pH measurements were collected from one replicate of each of the negative control, solvent control, and treatment groups. Measurements of pH were made using a Thermo Orion Model Dual Star pH/ISE meter and a Thermo Orion Model 4Star plus pH meter. Soil moisture content was determined by measuring the initial weight of the soil sample and then weighing the soil sample after it was dried at approximately 105°C for at least 18 hours. The percent moisture was calculated using the following formula:

%Moisture = ((wet weight-dry weight) / wet weight) x 100%

Test chambers were weighed periodically to monitor soil moisture loss. Lost soil moisture was replaced by adding RO water to the soil surface until weights approximated those at the start of the test (Day 1) or those weights collected on Day 28 of the test (after adult earthworms were removed).

Environmental Conditions
During the test, the earthworms were maintained in an environmental room set to maintain a temperature of approximately 20 ± 2 °C. Air temperature in the environmental room was measured continuously with a minimum/maximum digital thermometer and recorded at least daily, except for two days, during the exposure period. The photoperiod during acclimation and testing was 16 hours of light and 8 hours of dark per day and was provided by overhead fluorescent bulbs. The target light intensity during the test was approximately 400 to 800 lux, and was verified on Day 0 of the test over the surface of the test chambers using a SPER Scientific Inc model 840006C light meter.

Observations
At test initiation, the earthworms were placed on the surface of the soil in each replicate negative control, solvent control, and test treatment chamber and were observed for burrowing behavior. On Day 28 of test, the test soil in each replicate chamber was removed and spread out onto paper to determine the number of surviving adult earthworms. All surviving adult earthworms were removed and observed for behavioral or pathological abnormalities and response to mechanical stimulus. After the adults were removed, approximately 5 grams of food were evenly spread onto the test soil. By folding the paper into a cylinder, the test soil containing any cocoons and juveniles was gently returned to the test chambers. Following observations and body weight determinations, surviving earthworms were euthanized by freezing and held for disposal by incineration.

Body Weights
To ensure that the earthworms used in the test were of similar size, 20 earthworms were collected indiscriminately from the earthworms acclimated for the test, weighed individually, and the mean weight and standard deviation calculated. Group weights for all earthworms in each replicate were collected prior to being placed in the test chambers on Day 0. On Day 28, all surviving earthworms were removed from each replicate test chamber, rinsed with RO water, and blotted dry. Group body weights were measured for earthworms in each test chamber and the mean individual body weights were calculated.

Collection and Enumeration of Juveniles
Juveniles were removed from the test soil on Day 56 and counted on Day 57. The replicates for each treatment group were placed in a hot water bath (temperature of approximately 60 °C) for at least
20 minutes until the juveniles moved into the top layer, or were on top, of the soil. The juveniles, including some surrounding soil, were removed from each replicate and placed in holding containers with RO water and pieces of paper toweling. The soil from each replicate was also searched by hand to ensure that all juveniles were collected with the heating procedure. Any observed mortality and behavioral or clinical signs were documented. All holding containers were covered with perforated lids and kept in the study room prior to counting. The juveniles were removed from each replicate holding container on Day 57, counted, and examined for any physical abnormalities.
Nominal and measured concentrations:
Nominal concentrations of 62.5, 125, 250, 500, and 1000 milligrams of Reofos 35 per kilogram of soil on a dry weight basis (mg/kg dry soil) were selected.
Reference substance (positive control):
yes
Remarks:
carbendazim
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
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
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Environmental Conditions and Physical Properties of Test Soil
Mean light intensity during the 16-hour light period was 549 ± 48.2 lux, with a range of 499 to 619 lux. The study was housed in an environmental room at a target temperature of 20 ± 2°C. A digital minimum/maximum thermometer was placed in the environmental room to monitor air temperature continuously throughout the study and recorded at least daily, except for two days, during the exposure period. Air temperature recorded on the digital minimum/maximum thermometer in the environmental room ranged from 20 to 22°C during the acclimation and the exposure periods of the study. Since the digital minimum/maximum thermometer recorded temperatures within the target range for the days temperature was not recorded, there was no adverse impact on the study. Soil temperature ranged from 20.7 to 20.8°C in each of the groups at test initiation and from 20.4 to 20.5°C at test termination, and was therefore within the desired range of 20 ± 2°C. Soil pH ranged from 7.2 to 7.4 at both test initiation and test termination. Soil moisture content measured during the test ranged from 33.7 to 33.9% at test initiation and from 32.7 to 34.5% at test termination.

Mortality and Clinical Signs
There were no mortalities in the negative and solvent control groups during the 28-day adult exposure period. There were no mortalities in the treatment groups during the 28-day adult exposure period. All surviving earthworms in the negative control, solvent control, and the treatment groups were normal in appearance and behavior. Earthworms showed no aversion to test soils. Since there was no mortality of adult earthworms in the study, an LC50 value for mortality was not calculated, and was determined to be greater than 1000 mg/kg dry soil, the highest concentration tested.

Body Weights
The earthworms used in the study had a mean wet mass that was within the 300 to 600 mg range specified in OECD 222 on Day 0. The mean weights per replicate ranged from 390 to 480 mg on Day 0. The body weights of the adult earthworms available for use in the test ranged from 251.3 to 474.5 mg with a mean of 327.6 ± 68.39 (standard deviation) milligrams based on 20 representative earthworms that were individually weighed. Mean adult body weights at initiation and termination of the adult exposure period, and the change in body weight from initiation to termination, were calculated from the Day 0 and Day 28 measurements.
Since the negative and solvent control group weight means were not statistically significantly different (p>0.05) when compared with a t-test, treatment group means were compared to the pooled control group mean.
The initial body weight, final body weight, and change in body weight (initial to final) values were normally distributed and the body weight data analyzed had homogeneous variances.
When compared to the pooled control group mean using Dunnett’s two-tailed test of means (p=0.05), there were no apparent effects upon earthworm weight in the treatment groups during the 28-day adult exposure period. There were no statistically significant differences between mean final body weight, and change in body weight, for the treatment groups when compared to pooled control group means. Therefore, based on body weight data, the NOEC was determined to be 1000 mg/kg dry soil, the highest concentration tested.

Reproductive Output
The mean number of juveniles in the negative control group on Day 56 was 162 ± 21.1 (mean ± standard deviation), with a coefficient of variation of 13.1%. In the solvent control group, the mean number of juveniles was 166 ± 17.6, with a coefficient of variation of 10.6%.
Since the mean number of juveniles in the negative and solvent control groups were not statistically significantly different (p>0.05) when compared with a t-test, treatment group means were compared to the pooled control group mean.
In the 62.5, 125, 250, 500, and 1000 mg/kg dry soil treatment groups, the mean number of juveniles was 169, 157, 134, 122, and 118, respectively. The juveniles collected from the negative control, solvent control, and treatment groups were normal in appearance and behavior.
The numbers of juveniles were normally distributed and the data analyzed had homogeneous variances. The mean number of juveniles in the 500 and 1000 mg/kg dry soil treatment groups was statistically significantly reduced from the mean number of juveniles in the pooled control group using Dunnett’s one-tailed test of means (p<0.05). Therefore, the NOEC for reproduction was 250 mg/kg dry soil and the LOEC was nominal 500 mg/kg dry soil. Due to the lack of a 50% reproductive effect at any test level, the EC50 for the juvenile production was determined to be greater than 1000 mg/kg dry soil, the highest concentration tested. The EC10 for juvenile production was determined to be 172 mg/kg dry soil.
Results with reference substance (positive control):
EAG Laboratories - Easton conducted a reference toxicity test with carbendazim in 2006 to document that the earthworms being cultured were sensitive to a known toxicant. The LC50 value for the mortality of the adult earthworms exposed to carbendazim for 28 days was 7.149 mg a.i./kg dry soil, with a 95% confidence interval of 6.338 and 8.273 mg a.i./kg dry soil. There were statistically significant (p <0.01) treatment-related losses in body weight among surviving adult earthworms at test concentrations of 2, 4, and 8 mg a.i./kg dry soil on Day 28. The EC50 value for reproduction was 0.8914 mg a.i./kg dry soil, with a 95% confidence interval of 0.8416 and 0.9718 mg a.i./kg dry soil. The NOEC was 0.5 mg a.i./kg dry soil and the LOEC was 1 mg a.i./kg dry soil, based on the numbers of juveniles produced.
Reported statistics and error estimates:
The test was considered to be acceptable based on the validity criteria. Adult mortality of negative control earthworms was less than 10%, with no mortalities occurring in the negative control group. There were 130 or more juveniles produced in each of the eight replicates for the negative control group, thereby meeting the criterion of 30 or more juveniles per container. There were 147 or more juveniles produced in each of the four replicates for the solvent control group. The coefficient of variation of reproduction in the negative control group was 13.1%, thus satisfying the validity criterion of not exceeding 30%. The coefficient of variation of reproduction in the solvent control group was 10.6%.

Moisture, pH and Temperature of the Test Soil

Nominal Concentration

(mg/kg dry soil)

Test Initiation

Test Termination

Moisture Content1

(%)

pH1

Temperature2

(°C)

Moisture Content2

(%)

pH2

Temperature2

(°C)

Negative Control

Solvent Control

62.5

125

250

500

1000

33.8

33.8

33.7

33.7

33.9

33.8

33.8

7.3

7.3

7.3

7.4

7.3

7.3

7.2

20.7

20.7

20.8

20.7

20.8

20.8

20.8

32.8

34.5

32.7

33.2

33.6

32.9

33.5

7.2

7.2

7.2

7.3

7.4

7.2

7.3

20.5

20.5

20.5

20.4

20.5

20.5

20.5

1Measurements taken from prepared batch soil on Day 0.

2Measurements taken from one replicate test chamber at each concentration.

 

Mortality and Observations of Adult Earthworms Exposed to Reofos 35 in an Artificial Soil Substrate for 28 Days

Nominal Concentration

(mg/kg dry soil)

Rep.

Number Dead/Exposed

Effects1

Replicate Percent Mortality

Group Percent Mortality

Negative Control

(0)

A

B

C

D

E

F

G

H

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

10 AN

10 AN

10 AN

10 AN

10 AN

10 AN

10 AN

10 AN

0

0

0

0

0

0

0

0

0

Solvent Control

(0)

A

B

C

D

0/10

0/10

0/10

0/10

10 AN

10 AN

10 AN

10 AN

0

0

0

0

0

62.5

A

B

C

D

0/10

0/10

0/10

0/10

10 AN

10 AN

10 AN

10 AN

0

0

0

0

0

125

A

B

C

D

0/10

0/10

0/10

0/10

10 AN

10 AN

10 AN

10 AN

0

0

0

0

0

250

A

B

C

D

0/10

0/10

0/10

0/10

10 AN

10 AN

10 AN

10 AN

0

0

0

0

0

500

A

B

C

D

0/10

0/10

0/10

0/10

10 AN

10 AN

10 AN

10 AN

0

0

0

0

0

1000

A

B

C

D

0/10

0/10

0/10

0/10

10 AN

10 AN

10 AN

10 AN

0

0

0

0

0

1Observed Effects: AN = normal in appearance and behaviour.

Note: Cocoons were observed for each replicate of the control and test treatment groups.

 

Mean Body Weights of Adult Earthworms Exposed to Reofos 35 in an Artificial Soil Substrate for 28 Days

Concentration

(mg/kg dry soil)

Rep.

Mean Earthworm Body Weights (g)1

Day 02

Day 282

Change2

(initial to final weight)

Negative Control

(0)

A

B

C

D

E

F

G

H

Mean±Std. Dev.

0.42

0.42

0.42

0.48

0.41

0.40

0.40

0.46

0.43± 0.029

0.57

0.51

0.57

0.62

0.60

0.57

0.59

0.61

0.58± 0.034

+0.15

+0.09

+0.15

+0.14

+0.19

+0.17

+0.19

+0.15

+0.15± 0.032

Solvent Control

(0)

A

B

C

D

Mean± Std. Dev.

0.41

0.43

0.41

0.43

0.42± 0.012

0.63

0.52

0.55

0.54

0.56± 0.048

+0.22

+0.09

+0.14

+0.11

+0.14± 0.057

62.5

A

B

C

D

Mean± Std. Dev.

0.44

0.45

0.39

0.47

0.44± 0.034

0.55

0.56

0.54

0.58

0.56± 0.017

+0.11

+0.11

+0.15

+0.11

+0.12± 0.020

125

A

B

C

D

Mean± Std. Dev.

0.39

0.42

0.41

0.41

0.41± 0.013

0.56

0.56

0.55

0.51

0.55± 0.024

+0.17

+0.14

+0.14

+0.10

+0.14± 0.029

250

A

B

C

D

Mean± Std. Dev.

0.39

0.39

0.42

0.42

0.41± 0.017

0.51

0.55

0.57

0.69

0.56± 0.038

+0.12

+0.16

+0.15

+0.18

+0.15± 0.025

500

A

B

C

D

Mean± Std. Dev.

0.42

0.44

0.40

0.41

0.42± 0.017

0.59

0.60

0.61

0.58

0.60± 0.013

+0.17

+0.16

+0.21

+0.17

+0.18± 0.022

1000

A

B

C

D

Mean± Std. Dev.

0.41

0.41

0.41

0.42

0.41± 0.005

0.54

0.59

0.56

0.55

0.56± 0.022

+0.13

+0.18

+0.15

+0.13

+0.15± 0.024

1Means and standard deviations (mean± standard deviation) were generated by SAS. Manual calculations may differ slightly.

2No treatment group mean was statistically significantly different when compared to the respective pooled control group means with Dunnett’s two-tailed test of means (p >0.05).

 

Number of Juveniles Produced by earthworms Exposed to Reofos 35 in an Artificial Soil Substrate

Nominal Concentration

(mg/kg dry soil)

Replicate

Number of Adults in Replicate

(Day 28)

Number of Juveniles in Replicate1

(Day 56)

Mean Number of Juveniles per Replicate2

(Mean± Standard Deviation)

Negative Control

(0)

A

B

C

D

E

F

G

H

10

10

10

10

10

10

10

10

170

193

159

130

153

151

148

188

162 ± 21.13

Solvent Control

(0)

A

B

C

D

10

10

10

10

177

184

155

147

166 ± 17.64

62.5

A

B

C

D

10

10

10

10

183

181

148

164

169 ± 16.4

125

A

B

C

D

10

10

10

10

127

152

176

171

157 ± 22.2

250

A

B

C

D

10

10

10

10

164

140

127

105

134 ± 24.7

500

A

B

C

D

10

10

10

10

161

140

97

91

122 ± 33.8*

1000

A

B

C

D

10

10

10

10

151

88

110

123

118 ± 26.3*

1All juveniles were normal in appearance and behaviour.

2Means and standard deviations (mean± standard deviation (S.D.)) were generated by SAS. Manual calculations may differ slightly.

3Coefficient of Variation (COV) = S.D./mean*100% = 13.1%. COV was calculated from the mean and standard deviation using Excel 2010 in the full precision mode. Manual calculations may differ slightly.

4COV = 10.6%. COV was calculated using Excel 2010 in the full precision mode. Manual calculations may differ slightly.

* Treatment group means was statistically different (p ≤0.05) from pooled control group mean with Dunnett’s one-tailed test of means. 

Validity criteria fulfilled:
yes
Conclusions:
There was no mortality of adult earthworms exposed to nominal concentrations of Reofos 35 at 62.5, 125, 250, 500, and 1000 mg/kg dry soil for 28 days. Based on body weight and survival data of adult earthworms, the no observed effect concentration (NOEC) was determined to be 1000 mg/kg dry soil, the highest concentration tested. There were no reductions of 50% or greater for the numbers of juveniles produced in the treatment groups in comparison to the pooled control group, therefore the EC50 for reproduction was greater than 1000 mg/kg dry soil, the highest concentration tested. The EC10 for reproduction was determined to be 172 mg/kg dry soil. The NOEC was 250 mg/kg dry soil and the LOEC was 500 mg/kg dry soil, based on the reduced number of juveniles produced.
Executive summary:

The objective of the study was to determine the effects of Reofos 35 on the earthworm, Eisenia fetida, during an 8-week exposure period in an artificial soil substrate. Adults were exposed for 28 days and then removed to evaluate mortality and growth. The cocoons and the soil were returned to test chambers for an additional 28 days to evaluate effects upon reproductive output (number of juveniles at test termination).

 

The study was conducted according to the procedures outlined in the protocol, “Reofos 35: A Reproduction Study with the Earthworm in an Artificial Soil Substrate”. The protocol was based upon procedures outlined in the Organization for Economic Cooperation and Development (OECD) Guideline No. 222: “Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)” and ISO/FDIS 11268-2.

 

Adult earthworms (Eisenia fetida) were exposed to a geometric series of five concentrations of the test material in soil. Nominal concentrations of 62.5, 125, 250, 500, and 1000 milligrams of Reofos 35 per kilogram of soil on a dry weight basis (mg/kg dry soil) were selected in consultation with the Sponsor.

Two control groups, a negative control and solvent control, were exposed to soil without the addition of test substance and maintained concurrently. Four replicate chambers were maintained for each test treatment and solvent control group. Eight replicate chambers were maintained for the negative control group. Ten earthworms were randomly allocated in each replicate chamber. On the day of test initiation, the earthworms were rinsed briefly with water purified by reverse osmosis (RO water), blotted dry, weighed in groups of 10, and then placed on the soil surface of a test chamber. The adult earthworms were fed cow manure weekly during the first 28 days of the test, removed from the test chambers on Day 28, and observed for mortality and signs of toxicity. To determine the effects on reproduction, test soil and cocoons were returned to the test chambers for an additional 28 days. Juvenile production in each replicate was assessed at the end of the test (Day 56) and the numbers of any juvenile produced in each replicate was determined on Day 57. Cow manure was also added to the soil once at the beginning of the cocoon exposure period to serve as a source of food for juvenile earthworms emerging from cocoons. The number of juveniles present in each replicate at test termination was used to determine the no-observed-effect-concentration (NOEC) for reproduction.

 

There was no mortality of adult earthworms exposed to nominal concentrations of Reofos 35 at 62.5, 125, 250, 500, and 1000 mg/kg dry soil for 28 days. Based on body weight and survival data of adult earthworms, the no observed effect concentration (NOEC) was determined to be 1000 mg/kg dry soil, the highest concentration tested. There were no reductions of 50% or greater for the numbers of juveniles produced in the treatment groups in comparison to the pooled control group, therefore the EC50 for reproduction was greater than 1000 mg/kg dry soil, the highest concentration tested. The EC10 for reproduction was determined to be 172 mg/kg dry soil. The NOEC was 250 mg/kg dry soil and the LOEC was 500 mg/kg dry soil, based on the reduced number of juveniles produced.

Description of key information

Toxicity to soil macroorganisms -  earthworms

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
1 000 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
250 mg/kg soil dw

Additional information

The 14-day LC50 estimation for earthworms exposed to Reofos 35 in an artificial soil substrate was determined to be greater than 1000 mg/kg dry soil. The no-observed-effect-concentration (NOEC) was 500 mg/kg, based on reduced body weights in the 1000 mg/kg level.

There was no mortality of adult earthworms exposed to nominal concentrations of Reofos 35 at 62.5, 125, 250, 500, and 1000 mg/kg dry soil for 28 days. Based on body weight and survival data of adult earthworms, the no observed effect concentration (NOEC) was determined to be 1000 mg/kg dry soil, the highest concentration tested. There were no reductions of 50% or greater for the numbers of juveniles produced in the treatment groups in comparison to the pooled control group, therefore the EC50 for reproduction was greater than 1000 mg/kg dry soil, the highest concentration tested. The EC10 for reproduction was determined to be 172 mg/kg dry soil. The NOEC was 250 mg/kg dry soil and the LOEC was 500 mg/kg dry soil, based on the reduced number of juveniles produced.