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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:
August 26, 2016 to January 21, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid study conducted according to established protocol
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
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 1% calcium carbonate. The pH of the bulk lot of artificial soil at the completion of preparation (prior to hydration) was 6.1. The bulk artificial soil was stored in a sealed container under ambient conditions until used to prepare the test soils.

Test soils were prepared by mixing the appropriate amount of test substance with dry artificial soil before adding the finely ground cow manure and the RO water to hydrate the soil. Test soil components were mixed for a total of approximately 20 minutes in order to achieve a homogeneous mixture. Negative 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 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).

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 Wildlife International indicated there were no contaminants present at levels known to interfere with the purpose or conduct of the study. Detailed results of the analyses are stored in archives at Wildlife International.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Adult earthworms (Eisenia fetida) for the test were from Wildlife International 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/or cow manure, and held at a bedding temperature of 20 ± 5°C in continuous light.

Adult earthworms between 2 months and 1 year old were transferred to the study room and held in a glass aquarium for acclimation to test conditions for 7 days prior to test initiation. One day prior to the test the earthworms (340 with clitellum) were removed from the glass aquarium and divided into ten one-liter glass beakers containing 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.

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 on paper toweling, weighed in groups of 10 and then placed randomly on the soil surface of a test chamber. The test chambers were placed randomly in order to minimize bias, which might arise from the selection process. 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:
no
Total exposure duration:
28 d
Test temperature:
During the test, the earthworms were maintained in an environmental room set to maintain a temperature of approximately 20 ± 2°C.
pH:
The pH of the bulk lot of artificial soil at the completion of preparation (prior to hydration) was 6.1.
Moisture:
Soil moisture content measured during the test ranged from 32.8 to 33.7% at test initiation and from 32.3 to 34.9% at test termination.
Nominal and measured concentrations:
Nominal soil concentrations included 0 (Negative Control), 62.5, 125, 250, 500, and 1000 mg Alkenes, C11-12, hydroformylation products, distn. residues/kg dry soil.
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Mortality and Clinical Signs
There were no mortalities in the negative control and treatment groups during the 28-day adult exposure period. All surviving earthworms in the negative control and the treatment groups were normal in appearance and behavior (Table 2). In addition, some of the adults in two test chambers (replicate H of the negative control and replicate B of the 500 mg/kg dry soil treatment groups) had very distended clitellum. Earthworms showed no aversion to test soils. Since there was no treatment-related 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 wet mass that was within the 300 to 600 mg range specified in OECD 222. The mean weights per replicate ranged from 300 to 400 mg on Day 0. The body weights of the adult earthworms available for use in the test ranged from 223.1 to 441.6 mg with a mean of 318.8 ± 64.13 (SD) milligrams based on 20 representative earthworms that were individually weighed. The mean weights per replicate ranged from 550 to 700 mg on Day 28 with a mean change in body weight per replicate ranging from +19 to +39 mg. The adult earthworms, some with very distended clitellum, from replicate H of the negative control and replicate B of the 500 mg/kg dry soil treatment groups had the largest mean weights per replicate for both final weight (670 and 700 mg, respectively) and weight gain (36 and 39 mg, respectively).

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 (Table 3). The initial and change in body weight values were not normally distributed and the body weight data analyzed had homogeneous variances. The final body weight values were normally distributed and the body weight data analyzed had homogeneous variances. When compared to the negative control group, 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 negative control group means.
Separate statistical analyses were conducted to determine if the final body weight and change in body weight data for the two replicates with the adults with very distended clitellum had an effect on the results. When replicate H of the negative control group was excluded, there were also no statistically significant differences between mean final body weight, and change in body weight, for the treatment groups when compared to negative control group means. However, both the final and change in body weight values were normally distributed and the body weight data analyzed had homogeneous variances. The results were the same when both replicate H of the negative control and replicate B of the 500 mg/kg dry soil treatment groups were excluded from the analyses. Therefore, body weight data of all replicates for each negative control and treatment group were used in the determination of the NOEC, which was 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 91. In the 62.5, 125, 250, 500, and 1000 mg/kg dry soil treatment groups the mean number of juveniles was 94, 74, 77, 115, and 93, respectively (Table 4). The juveniles collected from the negative control and treatment groups were normal in appearance and behavior. The juveniles produced in replicate H of the negative control and replicate B of the 500 mg/kg dry soil treatment groups had the lowest numbers (43 and 40, respectively). The adults with the distended clitellum may not have deposited their cocoons when observations were performed on Day 28. This may be the cause of the large final weights and weight gains for the adults and the low number of juveniles present on Day 56 for these two replicates.

The numbers of juveniles were normally distributed and the data analyzed had homogeneous variances. There were no statistically significant differences between mean numbers of juveniles produced for the treatment groups when compared to negative control group means. Separate statistical analyses were conducted to determine if the number of juveniles produced for the two replicates with the adults with distended clitellum had an effect on the results. When replicate H of the negative control group was excluded, there were also no statistically significant differences between mean numbers of juveniles produced for the treatment groups when compared to negative control group means. In addition, the numbers of juveniles were normally distributed and the data analyzed had homogeneous variances. The results were the same when both replicate H of the negative control and replicate B of the 500 mg/kg dry soil treatment groups were excluded from the analyses. Therefore, the number of juveniles produced in all replicates for each negative control and treatment group was used in the determination of the NOEC and EC50 for reproduction, which were 1000 mg/kg dry soil and greater than 1000 mg/kg dry soil, the highest concentration tested, respectively.

Reference Toxicity Test
Wildlife International conducted a reference toxicity test with carbendazim in 2006 (4) 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 effects upon adult earthworm weight at concentrations of 0.3 mg a.i./kg dry soil and in the 2, 4, and 8 mg a.i./kg dry soil groups. 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.
Validity criteria fulfilled:
yes
Conclusions:
There was no treatment-related mortality of adult earthworms exposed to Alkenes, C11-12, hydroformylation products, distn. residues 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 negative control group, therefore the EC50 for reproduction was greater than 1000 mg/kg dry soil, the highest concentration tested. The NOEC was determined to be 1000 mg/kg dry soil, based on the numbers of juveniles produced.
Executive summary:

Wildlife International conducted this study for Sasol Italy S.p.A. at the Wildlife International toxicology facility in Easton, Maryland. The in-life portion of this test was conducted from October 13 to December 8, 2015. Soil moisture measurements and juvenile counts were completed on December 9, 2015.

The objective of this study was to determine the effects of Alkenes, C11-12, hydroformylation products, distn. residues 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).

There was no treatment-related mortality of adult earthworms exposed to Alkenes, C11-12, hydroformylation products, distn. residues 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 negative control group, therefore the EC50 for reproduction was greater than 1000 mg/kg dry soil, the highest concentration tested. The NOEC was determined to be 1000 mg/kg dry soil, based on the numbers of juveniles produced.

Description of key information

There was no treatment-related mortality of adult earthworms exposed to Alkenes, C11-12, hydroformylation products, distn. residues 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 negative control group, therefore the EC50 for reproduction was greater than 1000 mg/kg dry soil, the highest concentration tested.  The NOEC was determined to be 1000 mg/kg dry soil, based on the numbers of juveniles produced.  

Key value for chemical safety assessment

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

Additional information

An earthworm reproduction study was conducted at the Wildlife International toxicology facility in Easton, Maryland. The in-life portion of this test was conducted from October 13 to December 8, 2015. Soil moisture measurements and juvenile counts were completed on December 9, 2015.

The objective of this study was to determine the effects of Alkenes, C11-12, hydroformylation products, distn. residues 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).

There was no treatment-related mortality of adult earthworms exposed to Alkenes, C11-12, hydroformylation products, distn. residues 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 negative control group, therefore the EC50 for reproduction was greater than 1000 mg/kg dry soil, the highest concentration tested. The NOEC was determined to be 1000 mg/kg dry soil, based on the numbers of juveniles produced.