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

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

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

Adequacy of study:

key study

Study period:

September 29, 2015 to March 2, 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 216 (Soil Microorganisms: Nitrogen Transformation Test)

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:

yes

Details on sampling:

Soil samples were removed for analyses of moisture content on days 0 and 28, after moisture adjustments had been made. A single sample was removed from each test chamber at each sampling interval.Three quality control (QC) samples were prepared and analyzed with each set of samples to evaluate the performance of the analytical method. The first QC sample in each set was the extraction solvent (NANOpure water). The extraction solvent samples were designated “REB” for reagent blank, and were used to measure potential interferences in the water. The second QC sample was the extraction solvent fortified with nitrate at an approximate concentration of 80 mg NO3/L. The fortified extraction solvent samples were designated “RES” for reagent spike, and were used to determine method recoveries. The third QC sample was a sample of the untreated (control) soil fortified with nitrate at an approximate concentration of 400 mg NO3/kg. The fortified soil samples were designated “MAS” for matrix spike. The mean nitrate concentration in the control soils was subtracted from the fortified soil result to determine extraction and method recoveries.

Vehicle:

no

Details on preparation and application of test substrate:

Just prior to test substance application, the moisture contents of the soils were adjusted to approximately 50% WHC. Each test chamber was amended with 0.50 grams of dried, ground alfalfa. Three test chambers served as controls, and were not treated with the test substance. Three test chambers were treated with 0.2498, 0.2501, and 0.2498 g of Naugard 445, resulting in nominal concentrations of 2498, 2501, and 2498 mg/kg dry soil, respectively. After dosing, soils were thoroughly homogenized by hand using stainless steel spatulas. After dosing and sampling, the test chambers were returned to the temperature controlled room and incubated under aerobic conditions in the dark for the remainder of the test. Room temperatures were monitored continuously with a min/max thermometer, and temperatures were recorded each working day.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

During the 2-day acclimation period, temperatures ranged from 17.9 to 21.7 °C. During the 28-day test period, temperatures ranged from 18.1 to 21.3 °C.

Moisture:

The measured moisture contents of the soil samples ranged from 11.0% to 12.1%, and were equivalent to 48.7% to 53.5% of the maximum water holding capacity (WHC) during the test. These values were measured after water had been added to the soils at each interval to adjust the moisture contents. The adjusted soil moisture contents were within the range specified in the protocol (40 to 60% WHC).The calculated soil moisture contents during the 2-day acclimation period, the moisture contents ranged from 49.4% to 49.9% WHC. During the 28-day test period, the calculated soil moisture contents before and after adjustments ranged from 40.7% to 54.5% WHC. Throughout the study, the soil moisture contents were maintained within the range specified in the protocol (40%-60% WHC).

Details on test conditions:

Test Soil
The soil used in this study was a Greensboro soil that was collected on April 19, 2012, in Caroline County, Maryland, USA. The site was an open field between the road and lightly wooded area. The vegetation was grasses. Soil collection was not conducted during a drought or immediately following heavy rainfall. No known treatments with pesticides or fertilizers occurred in the past five years. Soil was collected from the top 0-10 cm. The soil was maintained in an outdoor raised plot upon receipt at Wildlife International. The soil was sieved through a 2-mm screen prior to characterization and use in the study. The soil was characterized by Agvise Laboratories (Northwood, ND), and included measurements of the following properties: sand/silt/clay content, soil texture, bulk density, moisture content as 1/3 bar, organic carbon/matter content, soil pH, and cation exchange capacity. In addition, the following soil properties were measured at Wildlife International prior to the start of the study: maximum water holding capacity, respiration rate, nitrate content, moisture content, and microbial biomass. The soil contained 73% sand, the pH was 5.6, the organic carbon content was 1.4%, and the microbial biomass was 225.6 μg/g. The microbial biomass was greater than 1% of the organic carbon content. The maximum water holding capacity (WHC) was 22.67%.

Test Chamber Preparation and Acclimation
Test chambers were 8-ounce French-square glass bottles with aluminum foil used as lids. Lids were perforated to allow circulation of air. The moisture content of the soil was determined to be 10.24%. Six test chambers were each filled with approximately 110 grams of moist soil (equivalent to 100 grams of dry soil). NANOpure water was added to each sample to adjust the moisture content to approximately 11.33%, or approximately 50% of the maximum water holding capacity. All test chambers were identified with the Wildlife International project number, a unique test chamber ID number, test substance ID, replicate, and test concentration. The test chamber ID numbers were used to identify each treatment and replicate. All test chambers were incubated under aerobic conditions in the dark in a temperature-controlled room set at approximately 20 °C for two days prior to application of the test substance. Room temperatures were monitored continuously with a min/max thermometer.

Alfalfa
The alfalfa used in this study was supplied by Tractor Supply Company (Dumor Alfalfa Classic Hay). It was ground and screened through a 710 μm sieve, and dried in an oven. A sample was collected and sent to Agvise Laboratories (Northwood, ND) for carbon/nitrogen analysis. The total carbon was 41.8%, and the total nitrogen was 3.61%. The carbon to nitrogen ratio was 12:1. The dried, ground alfalfa was stored under ambient conditions.

Soil Nitrate Analyses
Soil samples were removed for analyses of nitrate concentrations immediately after dosing, and on day 28. A single sample of approximately 5.6 grams (5.0 grams dry soil) was removed from each test chamber at each sampling interval. Samples were weighed into plastic centrifuge tubes, and 25 mL of NANOpure water were added. Tubes were capped, shaken by hand, and placed in an ultrasonic bath for at least 5 minutes. Tubes were placed on a gyratory shaker table set at 250 rpm for at least 30 minutes, and then centrifuged at 2000 rpm for 10 minutes. Portions of the supernatants were transferred to micro-centrifuge tubes, and further centrifuged at 12000 rpm for 5 minutes. Aliquots of the supernatants were transferred to auto-sampler vials and diluted with NANOpure water, if needed. The vials were submitted for HPLC analysis.

Analyses of nitrate concentrations were performed using an Agilent Series 1260 HPLC system. The HPLC system included an Agilent Series 1260 Binary Pump, 1260 Automated Liquid Sampler (ALS), and 1260 Variable Wavelength Detector (VWD). Separation of ions was achieved using a Dionex IonPac AG22 guard column (4 x 50 mm) and a Dionex IonPac AS22 analytical column (4 x 250 mm). The mobile phase solution contained 4.5 mM Na2CO3 plus 1.0 mM NaHCO3 in NANOpure water. A stock solution of potassium nitrate (KNO3) was prepared in NANOpure water at a concentration of 16.3 mg/mL or 10.0 mg NO3/mL. The stock solution was prepared, and was identified as KNO3-042815. A dilution of the stock solution was prepared in NANOpure water at a concentration of 1.00 mg NO3/mL. The dilution of the stock solution was used to prepare calibration standards in NANOpure water at concentrations of 1.00, 5.00, 10.0, 20.0, 50.0 and 100 mg NO3/L. The stock solution was also used to prepare quality control samples. The stock solution and calibration standards were stored in a refrigerator.

Three quality control (QC) samples were prepared and analyzed with each set of samples to evaluate the performance of the analytical method. The first QC sample in each set was the extraction solvent (NANOpure water). The extraction solvent samples were designated “REB” for reagent blank, and were used to measure potential interferences in the water. The second QC sample was the extraction solvent fortified with nitrate at an approximate concentration of 80 mg NO3/L. The fortified extraction solvent samples were designated “RES” for reagent spike, and were used to determine method recoveries. The third QC sample was a sample of the untreated (control) soil fortified with nitrate at an approximate concentration of 400 mg NO3/kg. The fortified soil samples were designated “MAS” for matrix spike. The mean nitrate concentration in the control soils was subtracted from the fortified soil result to determine extraction and method recoveries.

Soil pH Measurements
Soil samples were removed for pH measurements immediately after dosing and on day 28. A single sample of approximately 5.6 grams (5.0 grams dry soil) was removed from one replicate test chamber in each control or treatment group at each sampling interval. Samples were weighed into scintillation vials, and 5.0 mL of NANOpure water were added. Vials were capped, and shaken by hand. The pH of each soil slurry was measured using an Orion model 520A pH/ISE meter.

Soil Moisture Adjustments and Moisture Content Analyses
The moisture content of the soils in the test chambers was adjusted just prior to dosing on day 0; and at approximately weekly intervals after that. At each interval, the current weights of the test chambers were measured. NANOpure water was added, as needed, to bring the moisture content up to approximately 50% WHC. The weights of the test chambers were recorded again, after adjustments.Soil samples were removed for analyses of moisture content on days 0 and 28, after moisture adjustments had been made. A single sample was removed from each test chamber at each sampling interval. Samples were weighed into aluminum pans. The pans were placed in an oven set at approximately 105 °C for at least one hour, and then transferred to a desiccator to cool. The weights of the dried samples were recorded.
The moisture content of the soil in each sample was calculated. The measured moisture content was used to determine dry weights of samples removed for nitrate analyses, and total dry weights of soils remaining in the test chambers. Moisture contents were adjusted again after sampling on days 0 and 28, based on the moisture content measurements made earlier each day.

Nominal and measured concentrations:

Test systems were dosed with Naugard 445 at nominal concentrations of 0 and 2500 mg/kg dry soil.Three test chambers were treated with 0.2498, 0.2501, and 0.2498 g of Naugard 445, resulting in nominal concentrations of 2498, 2501, and 2498 mg/kg dry soil, respectively.The measured concentrations for all nitrate standards were within 15% of the nominal concentrations throughout the study.

Reference substance (positive control):

yes

Remarks:

2-chloro-6(trichloromethyl)pyridine

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 2 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Range-Finding Test
The range-finding test contained an untreated control soil and soils treated at 10, 100, 1000, and 10,000 mg Naugard 445/kg dry soil. Based on the results of the preliminary range-finding test concentrations of 0 and 2500 mg Naugard 445/kg dry soil were deemed appropriate for the definitive test.

Test Conditions
During the 2-day acclimation period, temperatures ranged from 17.9 to 21.7 °C. During the 28-day test period, temperatures ranged from 18.1 to 21.3 °C.The measured moisture contents of the soil samples ranged from 11.0% to 12.1%, and were equivalent to 48.7% to 53.5% of the maximum water holding capacity (WHC) during the test. These values were measured after water had been added to the soils at each interval to adjust the moisture contents. The adjusted soil moisture contents were within the range specified in the protocol (40 to 60% WHC). During the 2-day acclimation period, the moisture contents ranged from 49.4% to 49.9% WHC. During the 28-day test period, the calculated soil moisture contents before and after adjustments ranged from 40.7% to 54.5% WHC. Throughout the study, the soil moisture contents were maintained within the range specified in the protocol (40%-60% WHC).

Soil pH Measurements

Immediately after application of the test substance and on day 28, there were no pH differences attributed to Naugard 445.

Soil Nitrate Analyses
The mean nitrate concentrations in the control soil were 28.8 and 301.2 mg NO3/kg on days 0 and 28, respectively. The nitrate concentrations increased in the controls throughout the test period. The relative standard deviation (RSD) among control replicates was <15% at each sampling interval; therefore, the test was considered valid. The measured concentrations for all nitrate standards were within 15% of the nominal concentrations throughout the study. All linear calibration curves had r2 values >0.999 for each set of analyses. There were no interferences observed in the extraction solvent. Recoveries from fortified samples ranged from 96.7% to 105.6%; therefore, the extraction method was acceptable. On day 28, there were no statistically significant differences (ANOVA p>0.05) in nitrate concentrations of the soils treated at 2500 mg/kg dry soil when compared to the control (untreated) soil. The mean nitrate formation rate in the control soil was 9.73 mg NO3/kg/day after 28 days. On day 28, there were no statistically significant differences (ANOVA p>0.05) in nitrate formation rates of the soils treated at 2500 mg/kg dry soil when compared to the control soil. On day 28, all soils treated with Naugard 445 had a higher mean nitrate concentration than the control soil. The EC50 value for inhibition of nitrate formation was greater than 2500 mg Naugard 445/kg dry soil (the highest concentration tested).

Results with reference substance (positive control):

In a separate study (Wildlife International project number 100E-104)[2], a known inhibitor of nitrogen transformation (2-chloro-6(trichloromethyl)pyridine) was applied to the same soil, and showed 48% inhibition of the nitrate formation rate after 28 days.

Reported statistics and error estimates:

Statistical comparisons of measured nitrate concentrations were made at each sample interval using the ANOVA function in Microsoft Excel.

Measured Nitrate Concentrations in Soil

Treatment	Replicate	Day 0	Day 28
Controls (0 mg/kg)	A B C Mean: RSD:	31.1 30.2 25.2 28.8 11.0%	280.6 302.1 320.9 301.2 6.7%
Naugard 445 (2500 mg/kg)	A B C Mean: RSD:	25.7 29.3 24.0 26.3 10.2%	312.4 364.5 312.4 329.8 9.1%

Results given in mg NO₃/kg dry soil

RSD = relative standard deviation or coefficient of variation

 

Nitrate Formation Rates in Soil

Treatment	Replicate	Days 0-28
Controls (0 mg/kg)	A B C Mean: RSD:	8.91 9.71 10.56 9.73 8.5%
Naugard 445 (2500 mg/kg)	A B C Mean: RSD:	10.24 11.97 10.30 10.84 9.1%

Results given in mg NO₃/kg dry soil per day

RSD = relative standard deviation or coefficient of variation

 

Mean Nitrate Formation Rates and Inhibition

Treatment		Days 0-28
		Rate	Inhibition
Controls Naugard 445	0 mg/kg 255 mg/kg	9.73 10.84	-- -11.4%

Results given in mg NO₃/kg dry soil per day

Validity criteria fulfilled:

yes

Conclusions:

The calculated EC50 value for inhibition of nitrate formation after 28 days was greater than 2500 mg Naugard 445/kg dry soil (the highest concentration tested). Naugard 445 did not have an adverse effect on nitrogen transformation in soil.

Executive summary:

The study was conducted to assess the effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1- phenylethyl)phenyl] aniline (from this point on referred to as Naugard 445) on the nitrogen transformation activity of soil microorganisms in aerobic surface soils.

 

The study was conducted to comply with the OECD Guideline for Testing of Chemicals, Guideline 216, Soil Microorganisms: Nitrogen Transformation Test.

 

The study was conducted using a single soil type (sandy loam). Test systems were dosed with Naugard 445 at nominal concentrations of 0 and 2500 mg/kg dry soil, and incubated at approximately 20 °C for 28 days. Soil samples were collected on days 0 and 28; and analyzed to determine nitrate concentrations.

 

The nitrate concentrations increased in the controls throughout the test period. The relative standard deviation (RSD) among control replicates was <15% at each sampling interval; therefore, the test was considered valid. Mean nitrate formation rates were calculated, and are presented in units of mg NO3/kg dry soil per day. The percent inhibition of nitrate formation, compared to untreated controls, was calculated for each treatment at each interval.

 

The EC50 value for inhibition of nitrate formation after 28 days was greater than 2500 mg Naugard 445/kg dry soil, the highest concentration tested. Naugard 445 did not have an adverse effect on nitrogen transformation in soil.

Description of key information

A study was conducted in accordance with OECD Guideline 216 to assess the effects of read-across substance, 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1- phenylethyl)phenyl] aniline, (from this point on referred to as Naugard 445) on the nitrogen transformation activity of soil microorganisms in aerobic surface soils.

The study was conducted using a single soil type (sandy loam). Test systems were dosed with Naugard 445 at nominal concentrations of 0 and 2500 mg/kg dry soil, and incubated at approximately 20 °C for 28 days. Soil samples were collected on days 0 and 28; and analyzed to determine nitrate concentrations.

 

The nitrate concentrations increased in the controls throughout the test period. The relative standard deviation (RSD) among control replicates was <15% at each sampling interval; therefore, the test was considered valid. Mean nitrate formation rates were calculated, and are presented in units of mg NO3/kg dry soil per day. The percent inhibition of nitrate formation, compared to untreated controls, was calculated for each treatment at each interval.

 

The EC50 value for inhibition of nitrate formation after 28 days was greater than 2500 mg Naugard 445/kg dry soil, the highest concentration tested. Naugard 445 did not have an adverse effect on nitrogen transformation in soil.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

2 500 mg/kg soil dw