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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 18, 2017 to March 30, 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))
Deviations:
not specified
Qualifier:
according to
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
Lot Number 2015210546
Analytical monitoring:
no
Vehicle:
not specified
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/soil dwelling organisms.

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 approximately 20 minutes in order to achieve a homogeneous mixture.

Negative control soil was similarly prepared but without the addition of test substance. Seven-hundredfifty grams of prepared soil were added to each of four test chambers for each of the treatment group 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 6 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 (340 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:
28 d
Post exposure observation period:
28 days to evaluate effects upon reproductive output
Test temperature:
20 ± 5°C
pH:
Soil pH ranged from 7.4 to 7.7 at test initiation and from 7.3 to 7.4 at test termination.
Moisture:
33.2 to 33.7%
Details on test conditions:
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 Trixylyl phosphate per kilogram of soil on a dry weight basis (mg/kg dry soil) were selected in consultation with the Sponsor. One control group, a negative control, was exposed to soil without the addition of test substance and maintained concurrently. Four replicate chambers were maintained for each test treatment 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 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 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 and treatment groups. Measurements of pH were made using a Thermo Orion Model Dual Star pH/ISE 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.

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 during the exposure period. The photoperiod during acclimation and testing periods 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 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.

Data Analyses
Differences between the treatment groups and the control group were evaluated to assess potential effects on reproduction, body weight, and changes in body weight. Initial and final adult body weight and the number of juvenile earthworms were determined for each replicate. Treatment group weight means were calculated and compared to the control group mean using Dunnett’s two-tailed test of means in order to determine which group(s), if any, differed significantly from the control at the 0.05 level of significance. Treatment group means for juvenile production were calculated and compared to the control group mean using Dunnett’s one-tailed test of means in order to determine which group(s), if any, had significantly reduced juvenile production from the control at the 0.05 level of significance. Prior to conducting the 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). Results of the statistical tests were used to determine the no-observed-effect-concentration (NOEC) and lowest-observed-effect-concentration
(LOEC) for body weight and production of juveniles. The NOEC was defined as the maximum test concentration which showed no treatment-related mortality or sub-lethal effects, including clinical signs of toxicity, reduction or increase in adult body weight, or reduction in the number of juveniles. The LOEC was defined as the lowest test concentration that showed effects for the same categories. Statistical calculations were made using a personal computer and commercially available software (3). The EC/LC50 is defined as the concentration of test substance that caused a 50% reduction relative to the control group for a variable of concern. No EC/LC50 calculations were warranted for juvenile production or adult mortality due to the lack of a 50% effect at any test level. The EC10 is defined as the concentration of test substance that caused a 10% reduction relative to the control group for juvenile production. No EC10 calculations were warranted for juvenile production in due to the lack of a 10% effect at any test level.
Nominal and measured concentrations:
Nominal concentrations of Trixylyl phosphate at 62.5, 125, 250, 500, and 1000 mg/kg dry
Reference substance (positive control):
yes
Remarks:
Historical data for a positive control used as a reference
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
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:
reproduction
Key result
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:
reproduction
Key result
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
(adult)
Key result
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
Key result
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
Details on results:
Environmental Conditions and Physical Properties of Test Soil
Mean light intensity during the 16-hour light period was 577 ± 58.2 lux, with a range of 506 to 667 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 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. Soil temperature ranged from 19.8 to 20.6°C in each of the groups at test initiation and ranged from 21.0 to 21.1°C at test termination, and was therefore within the desired range of 20 ± 2°C. Soil pH ranged from 7.4 to 7.7 at test initiation and from 7.3 to 7.4 at test termination. Soil moisture content measured during the test ranged from 33.2 to 33.7% at test initiation and from 32.4 to 36.0% at test termination.

Mortality and Clinical Signs
There were no mortalities in the negative control group 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 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 320 to 440 mg on Day 0. The body weights of the adult earthworms available for use in the test ranged from 250 to 484 mg with a mean of 370.2 ± 72.66 (standard deviation) milligrams based on 20 representative earthworms that were individually weighed.

The initial body weight values were not normally distributed and the body weight data analyzed had homogeneous variances. The final body weight and change in body weight (initial to final) values
were normally distributed and the body weight data analyzed had homogeneous variances. Mean final body weight and change in body weight were compared to the control group mean
using Dunnett’s two-tailed test of means (p=0.05). There was a statistically significant reduction in mean final body weight for the 125 and 250 mg/kg dry soil treatment groups when compared to the control group mean. There was a statistically significant reduction in mean change in body weight for the 125, 250, and 500 mg/kg dry soil treatment groups compared to the control group mean. There appeared to be no discernible dose-response pattern for final body weight and change in body weight for the treatment groups. There were no apparent effects upon reproductive output due to these significant weight differences from the control group for the 125, 250, and 500 mg/kg dry soil treatment groups. 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 143 ± 25.7 (mean ± standard deviation), with a coefficient of variation of 17.9%. In the 62.5, 125, 250, 500, and 1000 mg/kg dry soil treatment groups, the mean number of juveniles was 147, 140, 159, 135, and 120, respectively (Table 4). The juveniles collected from the negative control and treatment groups were normal in appearance and behavior. The numbers of juveniles were normally distributed and the data analyzed did not have homogeneous variances. There were no statistically significant differences between the mean number of juveniles for the treatment groups when compared to the control group mean using Dunnett’s one-tailed test of means (p>0.05). Therefore, the NOEC for reproduction was 1000 mg/kg dry soil and the LOEC was greater than 1000 mg/kg dry soil. Due to the lack of a 10% and 50% reproductive effect at any test level, both the EC10 and EC50 for the juvenile production were determined to be greater than 1000 mg/kg dry soil, the highest concentration tested.

Validity Criteria
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 111 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. The coefficient of variation of reproduction in the negative control group was 17.9%, thus satisfying the validity criterion of not exceeding 30%.
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.
Validity criteria fulfilled:
yes
Conclusions:
There was no mortality of adult earthworms exposed to nominal concentrations of Trixylyl phosphate 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 10% or greater for the numbers of juveniles produced in the treatment groups in comparison to the control group, therefore the EC10 and EC50 for reproduction were each greater than 1000 mg/kg dry soil, the highest concentration tested. The NOEC was 1000 mg/kg dry soil and the LOEC was greater than 1000 mg/kg dry soil, based on body weight and juvenile production.
Executive summary:

The objective of this study was to determine the effects of Trixylyl phosphate 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).

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 Trixylyl phosphate per kilogram of soil on a dry weight basis (mg/kg dry soil).

One control group, a negative control, was exposed to soil without the addition of test substance and maintained concurrently. Four replicate chambers were maintained for each test treatment 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 Trixylyl phosphate 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 10% or greater for the numbers of juveniles produced in the treatment groups in comparison to the control group, therefore the EC10 and EC50 for reproduction were each greater than 1000 mg/kg dry soil, the highest concentration tested. The NOEC was 1000 mg/kg dry soil and the LOEC was greater than 1000 mg/kg dry soil, based on body weight and juvenile production.

RESULTS BASED ON NOMINAL TEST CONCENTRATIONS:

EC50 for juvenile production: >1000 mg/kg dry soil

95% confidence limits: not calculable

EC10 for juvenile production: >1000 mg/kg dry soil

95% confidence limits: not calculable

NOEC for juvenile production: 1000 mg/kg dry soil

LOEC for juvenile production: >1000 mg/kg dry soil

LC50 for adult mortality: >1000 mg/kg dry soil

95% confidence limits: not calculable

NOEC for body weight: 1000 mg/kg dry soil

LOEC for body weight: >1000 mg/kg dry soil

Description of key information

There was no mortality of adult earthworms exposed to nominal concentrations of Trixylyl phosphate 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 10% or greater for the numbers of juveniles produced in the treatment groups in comparison to the control group, therefore the EC10 and EC50 for reproduction were each greater than 1000 mg/kg dry soil, the highest concentration tested. The NOEC was 1000 mg/kg dry soil and the LOEC was greater than 1000 mg/kg dry soil, based on body weight and juvenile production.

Key value for chemical safety assessment

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

Additional information

The objective of this study was to determine the effects of Trixylyl phosphate 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).

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 Trixylyl phosphate per kilogram of soil on a dry weight basis (mg/kg dry soil).

One control group, a negative control, was exposed to soil without the addition of test substance and maintained concurrently. Four replicate chambers were maintained for each test treatment 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 Trixylyl phosphate 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 10% or greater for the numbers of juveniles produced in the treatment groups in comparison to the control group, therefore the EC10 and EC50 for reproduction were each greater than 1000 mg/kg dry soil, the highest concentration tested. The NOEC was 1000 mg/kg dry soil and the LOEC was greater than 1000 mg/kg dry soil, based on body weight and juvenile production.

RESULTS BASED ON NOMINAL TEST CONCENTRATIONS:

EC50 for juvenile production: >1000 mg/kg dry soil

95% confidence limits: not calculable

EC10 for juvenile production: >1000 mg/kg dry soil

95% confidence limits: not calculable

NOEC for juvenile production: 1000 mg/kg dry soil

LOEC for juvenile production: >1000 mg/kg dry soil

LC50 for adult mortality: >1000 mg/kg dry soil

95% confidence limits: not calculable

NOEC for body weight: 1000 mg/kg dry soil

LOEC for body weight: >1000 mg/kg dry soil