Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

August 25, 2015 to January 21, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, which is rated as reliability 2 because it is a read-across study.

Justification for type of information:

Please refer to IUCLID Section 13 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material :None specified

Analytical monitoring:

no

Details on sampling:

Sampling not performed

Vehicle:

no

Details on preparation and application of test substrate:

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 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.

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 14 days prior to test initiation. One day prior to the test the worms (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 (with the exception of one replicate that inadvertently received 11 earthworms), 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:

yes

Total exposure duration:

8 wk

Post exposure observation period:

No post exposure observation period specified

Test temperature:

Soil temperature ranged from 19.7 to 20.8 °C in each of the groups at test initiation and from 20.8 to 21.6 °C at test termination, and was therefore within the desired range of 20 ± 2 °C.

pH:

Soil pH ranged from 7.20 to 7.38 at test initiation, and from 7.26 to 7.43 at test termination.

Moisture:

Soil moisture content measured during the test ranged from 33.2 to 33.7% at test initiation and from 33.3 to 36.7% 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 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline per kilogram of soil on a dry weight basis (mg/kg dry soil) were selected in consultation with the Sponsor. A negative control group was maintained concurrently in soil prepared without the addition of the test material. This route of administration was selected because it was representative of the natural exposure of earthworms to chemicals. Four and eight replicate chambers were maintained for each test treatment and negative control group, respectively, with ten earthworms (one negative control chamber was inadvertently allocated 11 earthworms) in each replicate chamber. On the day of test initiation, the earthworms were rinsed briefly with water purified by reverse osmosis (RO water), blotted on paper toweling, weighed in groups of 10 (with the exception of the one negative control chamber with 11 earthworms), 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. The numbers of any juveniles produced in each replicate was determined at the end of the test (Day 56). 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 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, except for one day, 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 Days 1 and 13 of the test over the surface of the test chambers at the top of each test chamber 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 on paper toweling. 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 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline per kilogram of soil on a dry weight basis (mg/kg dry soil) were selected in consultation with the Sponsor.

Reference substance (positive control):

yes

Remarks:

carbendazim

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:

other: body weight and survival data of adult earthworms

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

based on the numbers of juveniles produced

Details on results:

Environmental Conditions and Physical Properties of Test Soil

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 one day, during the exposure period. Air temperature recorded on the digital minimum/maximum thermometer in the environmental room ranged from 20.0 to 21.9 °C during the acclimation and the exposure periods of the study. Since the digital minimum/maximum thermometer recorded a temperature within the target range for the day temperature was not recorded, there was no adverse impact on the study. Soil temperature ranged from 19.7 to 20.8 °C in each of the groups at test initiation and from 20.8 to 21.6 °C at test termination, and was therefore within the desired range of 20 ± 2 °C. Soil pH ranged from 7.20 to 7.38 at test initiation, and from 7.26 to 7.43 at test termination. Soil moisture content measured during the test ranged from 33.2 to 33.7% at test initiation and from 33.3 to 36.7% at test termination.

For ~3 days of the study, light intensity over some of the test chambers during the 16-hour light period was ~300 lux due to a burnt out fluorescent tube. The tube was replaced and light intensity returned to acceptable limits. Based on the performance of the negative control group, there was no adverse impact on the study due to the light intensity difference from the target range. Mean light intensity during the 16-hour light period, excluding the ~3-day period, was 536 lux, with a range of 497 to 608 lux, which was within the desired range of 400 – 800 lux.

Mortality and Clinical Signs
There were no mortalities in the negative control and treatment groups (except in the 1000 mg/kg dry soil group) during the 28-day adult exposure period. The plastic cover on the test chamber for replicate C of the 1000 mg/kg dry soil group was not perforated and consequently, all 10 adult earthworms were found dead. 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 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, with the exception of the replicate containing 11 earthworms with a mean of 291 mg on Day 0. The mean weights per replicate ranged from 291 to 360 mg on Day 0. The body weights of the adult earthworms available for use in the test ranged from 232.3 to 409.2 mg with a mean of 292.5 ± 54.83 (standard deviation (SD)) 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. The initial body weight values were not normally distributed and the body weight data analyzed had homogeneous variances. Since all 10 adult earthworms were found dead for replicate C of the 1000 mg/kg dry soil group, there was no final body weight data and this replicate was excluded from further analyses. The final body weight and change in 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. 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 surviving juveniles produced per adult earthworm in the negative control group on Day 56 was 11.0 ± 1.90 (mean ± standard deviation), with a coefficient of variation of 17.36%. One juvenile was found dead in the soil of test chamber replicate C in the negative control group. In the 62.5, 125, 250, 500, and 1000 mg/kg dry soil treatment groups the mean number of juveniles produced per adult earthworm was 11.1, 6.8, 6.9, 8.1, and 6.0, respectively. The surviving 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 had homogeneous variances. The mean number of juveniles produced per adult earthworm in three of the five treatment groups, the 125, 250, and 1000 mg/kg dry soil groups, was statistically significantly different from the mean number of juveniles per adult earthworm in the negative control group with Dunnett’s test (p<0.05). While the mean number of juveniles in the 500 mg/kg dry soil group was not statistically different from the negative control, the pattern across the groups indicated that the NOEC for reproduction was 62.5 mg/kg dry soil, and that 125 mg/kg dry soil was the LOEC. Due to the lack of a 50% reproductive effect at any test level, the EC50 for the number of juveniles was 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 87 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 17.4%, thus satisfying the validity criterion of not exceeding 30%.

Results with reference substance (positive control):

Wildlife International conducted a reference toxicity test with carbendazim in 2006 (5) 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.

Reported statistics and error estimates:

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 = 0.05). Results of the statistical tests were used to determine the no-observed-effectconcentration (NOEC) and lowest-observed-effect-concentration (LOEC) for body weight and production of juveniles.

Moisture, pH and Temperature of the Test Soil

Nominal Concentration (mg/kg dry soil)	Test Initiation			Test Termination
	Moisture Content^1 (%)	pH^1	Temperature^2 (°C)	Moisture Content^2 (%)	pH^2	Temperature^2 (°C)
Negative Control	33.5	7.32	20.8	33.3	7.26	21.6
62.5	33.4	7.26	20.0	35.5	7.34	20.9
125	33.4	7.32	19.8	35.6	7.33	20.8
250	33.2	7.20	19.7	34.7	7.36	20.8
500	33.7	7.32	19.8	34.9	7.43	20.9
1000	33.4	7.38	20.0	36.7	7.41	20.9
^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 4-(1-Methyl-1-Phenylethyl)-N-[4-(1-Methyl-1-Phenylethyl)Phenyl]Aniline in an Artificial Soil Substrate for 2 Days

Nominal Concentration (mg/kg dry soil)	Rep.	Number Dead/Exposed	Effects^1	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 0/11^2 0/10	10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 11 AN 10 AN	0 0 0 0 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 -^3 0/10	10 AN 10 AN -^3 10 AN	0 0 -^3 0	0
^1Observed Effects: AN = normal in appearance and behaviour ^2Replicate G of the Negative Control group contained 11 earthworms on Day 28 indicating that the replicate was inadvertently allocated 11 earthworms on Day 0. ^3Replicate C of the 1000 mg/kg dry sol group was excluded. The plastic cover of the test chamber was not perforated and consequently, 10 earthworms were found dead.

 

Mean Body Weights of Adult Earthworms Exposed to 4-(1-Methyl-1-Phenylethyl)-N-[4-(1-Methyl-1-Phenylethyl)Phenyl]Aniline in an Artificial Soil Substrate for 28 Days

Concentration (mg/kg dry soil)	Rep.	Mean Earthworm Body Weights (g)^1
		Day 0^2	Day 28^2	Change^2 (initial to final weight)
Negative Control (0)	A B C D E F G H Mean ± Std. Dev.	0.33 0.32 0.30 0.32 0.31 0.30 0.29^3 0.32 0.31 ± 0.013	0.56 0.61 0.58 0.65 0.62 0.61 0.54^3 0.57 0.59 ± 0.037	+0.23 +0.29 +0.28 +0.33 +0.31 +0.31 +0.25^3 +0.25 +0.28 ± 0.036
62.5	A B C D Mean ± Std. Dev.	0.30 0.31 0.32 0.30 0.31 ± 0.010	0.60 0.62 0.59 0.57 0.60 ± 0.021	+0.30 +0.31 +0.27 +0.27 +0.29 ± 0.021
125	A B C D Mean ± Std. Dev.	0.31 0.33 0.33 0.33 0.32 ± 0.015	0.60 0.63 0.61 0.62 0.62 ± 0.013	+0.29 +0.30 +0.28 +0.32 +0.30 ± 0.017
250	A B C D Mean ± Std. Dev.	0.30 0.31 0.30 0.31 0.31 ± 0.006	0.57 0.59 0.59 0.63 0.60 ± 0.025	+0.27 +0.28 +0.29 +0.32 +0.29 ± 0.022
500	A B C D Mean ± Std. Dev.	0.30 0.36 0.30 0.30 0.32 ± 0.030	0.59 0.58 0.58 0.55 0.58 ± 0.017	+0.29 +0.22 +0.28 +0.25 +0.26 ± 0.032
1000	A B C D Mean ± Std. Dev.	0.31 0.34 0.31 0.30 0.32 ± 0.017	0.55 0.58 -^4 0.63 0.59 ± 0.040	+0.24 +0.24 -^4 +0.33 +0.27 ± 0.052
^1Means and standard deviations (mean ± standard deviation) were generated by SAS. Manual calculations may differ slightly. ^2No treatment group mean was statistically different when compared to the respective negative control group mean with Dunnett’s 2-tailed test of means (p>0.05). ^3Replicate G of the Negative Control group contained 11 earthworms on Day 28 indicating that the replicate was inadvertently allocated 11 earthworms on Day 0. ^4Replicate C of the 1000 mg/kg dry soil was excluded. The plastic cover of the test chamber was not perforated and consequently, 10 earthworms were found dead.

 

Number of Juveniles Produced by Earthworms Exposed to 4-(1-Methyl-1-Phenylethyl)-N-[4-(1-Methyl-1-Phenylethyl)Phenyl]Aniline in an Artificial Soil Substrate

Nominal Concentration (mg/kg dry soil)	Replicate	Number of Adults in Replicate (Day 28)	Number of Juveniles in Replicate^1 (Day 56)	Mean Number of Juveniles per Adult Earthworm in Replicate (Day 56)	Mean Number of Juveniles per Adult Earthworm^2(Mean ± Standard Deviation)
Negative Control (0)	A B C D E F G H	10 10 10 10 10 10 11 10	138 128 115 95 97 108 87 117	13.8 12.8 11.5 9.5 9.7 10.8 7.9 11.7	11.0 ± 1.90^3
62.5	A B C D	10 10 10 10	92 97 113 141	9.2 9.7 11.3 14.1	11.1 ± 2.21
125	A B C D	10 10 10 10	66 70 86 49	6.6 7.0 8.6 4.9	6.8 ± 1.52*
250	A B C D	10 10 10 10	76 54 88 56	7.6 5.4 8.8 5.6	6.9 ± 1.64*
500	A B C D	10 10 10 10	92 87 78 68	9.2 8.7 7.8 6.8	8.1 ± 1.06
1000	A B C D	10 10 -^4 10	62 64 -^4 54	6.2 6.4 -^4 5.4	6.0 ± 0.53*
^1All juveniles were normal in appearance and behaviour with the exception of one found dead in replicate C of the Negative Control group. ^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% = 17.4%. COV was calculated from the mean and standard deviation using Excel 2010 in the full precision mode. Manual calculation may differ slightly. ^4Replicate C of the 1000 mg/kg dry soil group was excluded. The plastic cover of the test chamber was not perforated and consequently, 10 earthworms were found dead. *Treatment group mean was statistically different (p<0.05) from negative control group mean with Dunnett’s 2-tailed test of means.

 

Validity criteria fulfilled:

yes

Conclusions:

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.The NOEC was 62.5 mg/kg dry soil and the LOEC was 125 mg/kg dry soil, based on the numbers of juveniles produced.

Executive summary:

The objective of the study was to determine the effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline 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 Organization for Economic Cooperation and Development (OECD) Guideline No. 222: “Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)” and ISO/FDIS 11268-2.

 

There was no treatment-related mortality of adult earthworms exposed to of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline 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 62.5 mg/kg dry soil and the LOEC was 125 mg/kg dry soil, based on the numbers of juveniles produced.

Description of key information

A study was conducted in accordance with OECD Guideline 222 to determine the effects of read-across substance, 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline, 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 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline 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 62.5 mg/kg dry soil and the LOEC was 125 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:

62.5 mg/kg soil dw