Phenol, isopropylated, phosphate (3:1)

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 February 2014 to 4 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with OECD Guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

The test chambers in treatment numbers 5 and 6 were dosed with test substance at nominal concentrations of 318 and 1010 mg/kg dry soil. According to the protocol, treatment numbers 5 and 6 were supposed to be dosed with test substance at nominalconcentrations of 316 and 1000 mg/kg dry soil. In the best judgment of the Study Director, this deviation did not impact the integrity of study.

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

Test substance was not monitored in the test solutions.

Vehicle:

yes

Details on preparation and application of test substrate:

Alfalfa
The alfalfa used to amend the soil in this study was purchased from Petsmart (Easton, Maryland). It was ground and sieved to <710 um, and dried in an oven. A sample was collected and sent to Agvise Labs for carbon/nitrogen analysis. The total carbon was 42.6%, and the total nitrogen was 3.0%. The carbon:nitrogen ratio was 14.2. The dried, ground alfalfa was stored under ambient conditions.

Quartz Sand Carrier
The quartz sand used as a carrier in the study was Q-ROK Graded Quartzite supplied by U.S. Silica (Berkelley Springs, WV, USA).

Preparation of Test Substance/Carrier Mixes
Five dose mixtures were prepared on February 7, 2014. The first mixture was prepared by weighing a total of 1.4998 g of test substance into a tared scintillation vial and adding 10 mL of acetone to dissolve the test substance. The solution was added to 13.5003 g of quartz sand inside a 125 mL round-bottom flask. The scintillation vial was rinsed with 10 mL of acetone two additional times and transferred to the round-bottom flask. The mixture was rotary-evaporated until the acetone was completely evaporated. The remaining test substance coated quartz sand was identified as 11420-020714 and had a nominal test substance concentration of 100 mg/g. The second mixture was prepared by removing an aliquot of 3.1600 g of 11420-020714 and adding quartz sand to bring the total weight to 9.9999 g. The second
Reofos 35 dose mixture was identified as 11420-020714-1. The third mixture was prepared by removing an aliquot of 1.0002 g of 11420-020714 and adding quartz sand to bring the total weight to 10.0000 g.
The third Reofos 35 dose mixture was identified as 11420-020714-2. The fourth mixture was prepared by removing an aliquot of 0.3200 g of 11420-020714 and adding quartz sand to bring the total weight to 10.0000 g. The fourth Reofos 35 dose mixture was identified as 11420-020714-3. The fifth mixture was prepared by removing an aliquot of 0.1001 g of 11420-020714 and adding quartz sand to bring the total weight to 9.9999 g. The fifth Reofos 35 dose mixture was identified as 11420-020714-4. The dose mixtures were stored under refrigerated conditions, and were used to dose test chambers on February 11, 2014.

Application of the Test Substance
Just prior to dosing on February 11, 2014, the moisture content of the soils was adjusted to approximately 50% WHC. Each of the test chambers was amended with approximately 0.75 grams of dried, ground alfalfa. Three test chambers served as controls, and were treated with an average of 1.507 grams of quartz sand. Three test chambers were treated with an average of 1.505 grams of the 1.0 mg/g Reofos 35 carrier mix, resulting in nominal concentrations of 10 mg/kg of dry soil. Three test chambers were treated with 1.504 grams of the 3.2 mg/g Reofos 35 carrier mix, resulting in nominal concentrations of 32 mg/kg of dry soil. Three test chambers were treated with 1.506 grams of the 10.0 mg/g Reofos 35 carrier mix, resulting in nominal concentrations of 100 mg/kg of dry soil. Three test chambers were treated with 1.510 grams of the 31.6 mg/g Reofos 35 carrier mix, resulting in nominal concentrations of 318 mg/kg of dry soil. Three test chambers were treated with 1.515 grams of the 100 mg/g Reofos 35 carrier mix, resulting in nominal concentrations of 1010 mg/kg of dry soil. After dosing, soils were thoroughly homogenized using stainless steel spatulas.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

During acclimation, temperatures were maintained at 19.1 to 22.0 degrees C. During the 28-day test period, temperatures were maintained at 18.9 to 21.8 degrees C.

Moisture:

During the test, the measured moisture contents of the soil samples ranged from 10.5% to 12.8%, and were equivalent to 46.1% to 56.4% of the maximum water holding capacity (WHC).

Details on test conditions:

TEST SYSTEM
Test chambers were 16 ounce glass French square bottles with perforated aluminum foil lids. On February 4, 2014, eighteen test chambers were each filled with approximately 164 grams of moist soil or the equivalent of 150 grams of dry soil. The moisture content of the soils was adjusted to 50% of the water-holding capacity (WHC) or approximately 11.33%.
All test chambers were identified with the Wildlife International project number, a unique test chamber ID number, treatment and test concentration. The test chamber ID numbers were used to identify each treatment and replicate.

SOIL INCUBATION
All test chambers were incubated under aerobic conditions in the dark at approximately 20 degrees C for seven days prior to test start.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The soil used in this study was supplied by Wildlife International. It was collected on April 19, 2012, in Greensboro, Maryland at coordinates N 38 degrees 56.3697 - W 75 degrees 46.3741. The site was an open field between the road and a lightly wooded area, and no pesticides or fertilizers were applied in the previous 5 years. Soil was collected from the top 0-10 cm and sieved to 2 mm. The soil was identified as Greensboro Soil. The soil was received at Wildlife International on April 19, 2012, and stored in an outdoor field plot. The soil was characterized by Wildlife International and Agvise Labs, (Northwood, North Dakota). The soil was classified as a sandy loam with 73% sand, 22% silt, and 5% clay. The soil pH was 5.6, the organic carbon content was 0.97%, and the microbial biomass was 129.8 g/g. The mean water-holding capacity of the soil was determined to be 22.67%.
Sieved : April 25, 2012, May 10, 21, 22, & August 14, 2013
Soil Parameter/Analysis Date/Result:
Sand Content April 27, 2012 73%
Silt Content April 27, 2012 22%
Clay Content April 27, 2012 5%
Textural Class April 27, 2012 Sandy Loam (USDA)
Cation Exchange Capacity April 27, 2012 5.5 meq/100 g
Soil pH April 27, 2012 5.6
Water Holding Capacity April 25-27, 2012 22.67%
Moisture Content May 10, 2013 11.72%
Respiration Rate May 13, 2013 11.6 mg CO2/kg/h
Moisture Content May 21, 2013 7.53%
Nitrate Concentration May 21, 2013 12.3 mg/kg
Moisture Content May 22, 2013 6.14%
Microbial Biomass May 22, 2013 129.8 μg/g


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Analysis of Ammonia Concentrations
Soil samples were removed for analyses of ammonia concentrations immediately after dosing, after 7 days, after 14 days and after 28 days. A single sample of approximately 5.57 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 and placed in an ultrasonic bath for at least 5 minutes. Tubes were placed on a shaker table at 250 rpm for 30 minutes, and then centrifuged at 3000 rpm for 10 minutes. Supernatants were filtered and diluted, as needed, and 2.0 mL aliquots were transferred to Hach TEST ‘N TUBE reactor tubes. One ammonia salicylate reagent packet and one ammonia cyanurate reagent packet were added to each tube. The tubes were shaken to dissolve reagents, vortexed to mix, and allowed to stand for at least 20 minutes. The tubes were inserted in a Hach DR/890 colorimeter, and the ammonia concentrations were recorded.

Analysis of Nitrate/Nitrite Concentrations
Soil samples were removed for analyses of nitrate and nitrite concentrations immediately after dosing, after 7 days, after 14 days and after 28 days. A single sample of approximately 5.57 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 5 minutes. Tubes were placed on a shaker table at 250 rpm for 30 minutes, and then centrifuged at 2000 rpm for 10 minutes on day 0. On days 7, 14, and 28, the tubes were centrifuged at 3000 rpm for 10 minutes. Portions of the supernatants were transferred to micro-centrifuge tubes, and further centrifuged at ~12000 rpm for at least 5 minutes. Aliquots of the supernatants were diluted, as needed, and transferred to auto-sampler vials for 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 0.45 M Na2CO3 plus 0.1 M NaHCO3 in NANOpure water (1:99 v/v).
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 on May 8, 2013, and was identified as KNO3-050813. 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, 25.0, and 50 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 assess method recoveries. The third QC sample was a sample of the 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 days 7, 14 and 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 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 Content Analyses
The moisture content of the soils in the test chambers was adjusted just prior to sampling on days 0, 7, 14 and 28. At each interval, the current weights of the test chambers were measured. Water was added, as needed, to bring the moisture content up to approximately 50% of the water holding capacity. The weights of the test chambers were recorded again, after adjustments.
Soil samples were removed for analyses of moisture content, after moisture adjustments, on days 0, 7, 14, and 28. Samples were weighed into aluminum pans. The pans were placed in an oven set at >100 degrees 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 test chamber was calculated. The measured moisture content was used to determine dry weights of samples removed for analyses of ammonia, nitrite, and nitrate; and total dry weight of soil remaining in the test chambers.
Moisture content was adjusted again after sampling on days 0, 7, 14, and 28, based on the measured moisture content from earlier each day. Moisture content was also adjusted between sampling intervals on day 21.

Nominal and measured concentrations:

Nominal: 10, 32, 100, 318, and 1010 mg/kg dry soil test
Concentrations were not measured.

Reference substance (positive control):

yes

Remarks:

(2-chloro-6(trichloromethyl)pyridine)

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 010 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

> 1 010 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

582.7 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

See tables attached below under background material.
Ammonia Analysis Results
On day 0 there were statistically significant differences (Dunnett’s two-tailed test p<0.05) between the controls and soils treated with the test substance at 318 mg/kg dry soil. On day 7 there were statistically significant differences (Dunnett’s two-tailed test p<0.05) between the controls and soils treated with the test substance at 318 and 1010 mg/kg dry soil. On day 14 there were statistically significant differences (Dunnett’s two-tailed test p<0.05) between the controls and soils treated with the test substance at 1010 mg/kg dry soil. During the study, the ammonia concentrations decreased in the controls and soils treated with the test substance.

Nitrite Analysis Results
Measured nitrite concentrations were less than 5 mg/kg (LOQ) in all samples throughout the study. Comparisons could not be made on days 0, 7, 14, and 28, because none of the soil samples contained measurable amounts of nitrite.

Nitrate Analysis Results
Nitrate concentrations in soils treated with the test substance at 10 mg/kg dry soil showed 0%, -4%, -5%, and -1% inhibition compared with the controls on days 0, 7, 14 and 28, respectively. Nitrate concentrations in soils treated with the test substance at 32 mg/kg dry soil showed -2%, -3%, -1%, and 1% inhibition compared with the controls on days 0, 7, 14 and 28, respectively. Nitrate concentrations in soils treated with the test substance at 100 mg/kg dry soil showed 1%, -3%, -3%, and 0% inhibition compared with the controls on days 0, 7, 14 and 28, respectively. Nitrate concentrations in soils treated with the test substance at 318 mg/kg dry soil showed 2%, -8%, -6%, and 2% inhibition compared with the controls on days 0, 7, 14 and 28, respectively. Nitrate concentrations in soils treated with the test substance at 1010 mg/kg dry soil showed 1%, -23%, -13%, and 23% inhibition compared with the controls on days 0, 7, 14 and 28, respectively. On days 7, 14, and 28 there were statistically significant differences (Dunnett’s two-tailed test p<0.05) between the controls and soils treated with the test substance at 1010 mg/kg dry soil.
There were no interferences observed in the extraction solvent. Recoveries from nitrate fortified samples ranged from 95.7% to 100.6%; therefore, the extraction method was acceptable.

Moisture Content Results
During the test, the measured moisture contents of the soil samples ranged from 10.5% to 12.8%, and were equivalent to 46.1% to 56.4% of the maximum water holding capacity (WHC). 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 (approximately 50% WHC).

Acclimation and Test Conditions
During acclimation, temperatures were maintained at 19.1 to 22.0 degrees C. During the 28-day test period, temperatures were maintained at 18.9 to 21.8 degrees C. The moisture contents of the soils ranged from 10.5% to 12.8% (46.1% to 56.4% of WHC) throughout the test period.

Results with reference substance (positive control):

In a separate study (Wildlife International project number 100E-103)[2], a known inhibitor of nitrogen transformation (2-chloro-6(trichloromethyl)pyridine) was applied to the same soil, and showed 84% inhibition of the nitrate formation rate after 28 days.

Reported statistics and error estimates:

The mean concentrations of ammonia, nitrate and nitrite were statistically analyzed using ANOVA and Dunnett’s “two-tailed” Test to determine statistically significant differences at 95% probability (P<0.05). All calculations were performed using full precision. Manual calculations using these rounded values may produce slightly different results.

Validity criteria fulfilled:

yes

Conclusions:

The long-term effects of Reofos 35 on nitrogen transformation activity of soil microorganisms were minimal at concentrations 10, 32, 100, and 318 mg/kg dry soil. There was an observed effect of Reofos 35 on nitrogen transformation activity of soil microorganisms at concentration level 1010 mg/kg dry soil. After 28 days of exposure, the mean nitrate concentration in soil treated at 10, 32, 100, 318, and 1010 mg/kg dry soil exhibited a -1%, 1%, 0%, 2%, and 23% reduction in nitrate formation, respectively, compared to the controls. The EC10 was calculated to be 582.7 mg/L using linear interpolation between concentrations 318 and 1010 mg/L. The EC25 and EC50 were estimated to be >1010 mg/L, the highest concentration tested.

Executive summary:

The objective of the study was to assess the effects of a Reofos 35 on the nitrogen transformation activity of soil microorganisms. The study was conducted according to the procedures outlined in the protocol “Reofos 35: Soil Microorganisms: Nitrogen Transformation,” Wildlife International Protocol Number 616/011714/216/SUB616. The test substance was Reofos 35. One type of soil, a sandy loam, was used to prepare eighteen individual test chambers, each filled with an equivalent of 150 grams of dry soil. Soil moisture content was adjusted to 11.33% water, or 50% of the maximum water holding capacity. Soil was acclimated in the dark at approximately 20 degrees C. All of the test chambers were amended with dried, ground alfalfa at a rate of 5 g/kg. Three test chambers were treated with quartz sand, and served as controls. Triplicate test chambers were treated with Reofos 35 to prepare the 10, 32, 100, 318, and 1010 mg/kg dry soil test concentrations. All test chambers were maintained at approximately 20 degrees C in the dark under aerobic conditions throughout the 28-day test period. Soil moisture content was maintained between 40% and 60% of the water holding capacity. Samples were collected from each test chamber immediately after dosing and on days 7, 14 and 28 for analyses of ammonium, nitrite, and nitrate concentrations. The mean measured concentrations are given in mg/kg dry soil in the following table:

	Day 0			Day 7			Day 14			Day 28
Treatment	NH4+	NO2-	NO3-	NH4+	NO2-	NO3-	NH4+	NO2-	NO3-	NH4+	NO2-	NO3-
Control	2.4	<LOQ	29.9	2.2	<LOQ	32.6	0.4	<LOQ	119	0.1	<LOQ	184
10 mg/kg	2.3	<LOQ	29.8	2.4	<LOQ	33.8	0.4	<LOQ	125	0.1	<LOQ	187
32 mg/kg	2.3	<LOQ	30.4	2.4	<LOQ	33.7	0.4	<LOQ	119	0.1	<LOQ	182
100 mg/kg	2.4	<LOQ	29.8	2.4	<LOQ	33.6	0.4	<LOQ	122	0.1	<LOQ	184
318 mg/kg	1.9	<LOQ	29.3	2.9	<LOQ	35.3	0.4	<LOQ	126	0.1	<LOQ	181
1010 mg/kg	2.0	<LOQ	29.6	3.3	<LOQ	40.1	0.6	<LOQ	134	0.1	<LOQ	141

LOQ = limit of quantitation was approximately 5 mg NO2-/kg dry soil

After 28 days of exposure, the mean nitrate concentrations in soils treated at 10, 32, 100, 318 and 1010 mg/kg dry soil were 184, 187, 182, 184, 181, and 141 mg NO3/kg dry soil, respectively. The EC10 was calculated to be 582.7 mg/L using linear interpolation between concentrations 318 and 1010 mg/L. The EC25 and EC50 were estimated to be >1010 mg/L, the highest concentration tested.

Description of key information

Toxicity to soil microorganisms

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

1 010 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

582.7 mg/kg soil dw

Additional information

The long-term effects of Reofos 35 on nitrogen transformation activity of soil microorganisms were minimal at concentrations 10, 32, 100, and 318 mg/kg dry soil. There was an observed effect of Reofos 35 on nitrogen transformation activity of soil microorganisms at concentration level 1010 mg/kg dry soil. After 28 days of exposure, the mean nitrate concentration in soil treated at 10, 32, 100, 318, and 1010 mg/kg dry soil exhibited a -1%, 1%, 0%, 2%, and 23% reduction in nitrate formation, respectively, compared to the controls. The EC10 was calculated to be 582.7 mg/L using linear interpolation between concentrations 318 and 1010 mg/L. The EC25 and EC50 were estimated to be >1010 mg/L, the highest concentration tested.