Phosphoric acid, mono- and di-C16-18-alkyl esters

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2017 to 1 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Physical State/Appearance: White waxy solid
Purity: 100%
Storage Conditions: Room temperature in the dark

Analytical monitoring:

no

Details on sampling:

The inhibitory effect of the test material on carbon transformation was assessed by the determination of the glucose-induced respiration rate in the soil samples on Days 0 and 28 and compared to data obtained from solvent control soil samples.

On Days 0 and 28 three aliquots (50 g*) of soil were removed from the control, solvent control and test material vessels for nitrate analysis. A further three aliquots (50 g*) were removed from the control, solvent control and test material vessels were removed for measurement of the glucose-induced respiration rate on Days 0 and 28.

To determine the glucose induced respiration rate of each replicate soil sample, glucose (50 mg) was ground with quartz sand of particle size 0.1 to 0.5 mm (250 mg) prior to mixing with the soil sample (50 g*) to give final concentrations of 1000 mg glucose/kg soil and 10 g sand/kg soil. The soil sample was then transferred to a 250 mL ground glass stoppered conical flask and placed in a CES Respirometer to measure the glucose induced respiration rate.
The CES Respirometer system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. As respiration progressed, the micro-organisms converted oxygen to carbon dioxide, which was absorbed into the CO2 trap (ethanolamine solution, 50% v/v), causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data collected by the respirometer’s battery-backed memory was then transferred to the hard disk drive of a non-dedicated computer.

After an initial 5-minute stabilization/initialization period, oxygen consumption values were measured for at least 12 hours.

* Dry weight

Vehicle:

yes

Remarks:

Chloroform

Details on preparation and application of test substrate:

Preliminary solubility work showed that the test material had limited solubility in water, but that a preliminary solvent stock solution could be prepared in chloroform. Therefore, for the purposes of the test, the test concentrations were prepared from a preliminary solvent stock solution prepared in chloroform. In order to aid dispersal of the test material in the soil, an aliquot of the preliminary solvent stock solution was adsorbed onto dry quartz sand and the solvent allowed to evaporate to dryness prior to dispersal in the test soil.

The test material/sand combinations was then added to final bulk soil along with an aliquot (30.3 mL) of water and thoroughly mixed to give the required test concentrations of 10, 32, 100, 320 and 1000 mg/kg, with a nominal moisture content of 45% of the Water Holding Capacity (WHC). The soil for each test concentration was then mixed again to ensure homogeneity prior to removing three aliquots for the Day 0 analysis. The remaining soil bulk was incubated in separate glass Duran bottles.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

18 and 22 deg.C

Moisture:

45% of the Water Holding Capacity (WHC)

Organic carbon content (% dry weight):

0.68

Details on test conditions:

The soil samples were incubated in glass Duran bottles. The test vessels were sealed with polyurethane foam stoppers in order to maintain aseptic conditions and minimize moisture loss by evaporation and maintained in a temperature controlled room at temperatures of between 18 and 22 deg.C in darkness.

The test soil was a sandy loam soil obtained from a site in Rugby, Warwickshire, England that had received no pesticides or fertilisers for 5 years prior to sampling.

On arrival at Envigo Research Limited, UK, the soil was stored at temperatures of between 3 and 4 deg.C prior to use. Prior to the start of the test, the soil was removed from refrigeration and stored at temperatures of between 15 and 21 deg.C for 22 days and shielded from light.

The moisture content of the soil was determined prior to the start of the two consecutive dry weights within 4% were attained. The moisture content of the soil, expressed as a percentage of the dry weight, was determined to be 8%. The Water Holding Capacity (WHC) of the soil supplied by LUFA Speyer was 35.4 ± 1.0 g/100 g and hence 30.3 mL of deionized reverse osmosis water per 0.4 kg* of soil was added. This gave a final water content of 15 g/100 g, i.e. 45% of the WHC as recommended by the Testing Guidelines.

* Dry weight

Reference substance (positive control):

yes

Remarks:

2-Chloro-6-(trichloromethyl)pyridine

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:

respiration rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

ca. 320 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Results with reference substance (positive control):

The results from the positive control with 2-Chloro-6-(trichloromethyl)pyridine (i.e., Nitrapyrin) were within the normal ranges for this test material.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the carbon transformation activity of the soil microorganisms, resulted in an EC50 of greater than 1000 mg/kg.  The NOEC for carbon transformation in soil was determined to be 320 mg/kg.

Executive summary:

A study was performed to assess the long-term effect of the test material after a single application, on the carbon transformation activity of soil microorganisms. 

Soil microorganisms were exposed to the test material at concentrations of 10, 32, 100, 320 and 1000 mg/kg for 28 days at temperatures of between 18 to 22 deg.C in the dark. 

The inhibitory effect of the test material on carbon transformation was assessed by the determination of the glucose-induced respiration rate in the soil samples on Days 0 and 28 and compared to data obtained from solvent control soil samples.

Based on the observed effects of the test material on the carbon transformation activity of the soil microorganisms, the EC₅₀was determined to be greater than 1000 mg/kg. The NOEC for carbon transformation in soil was determined to be 320 mg/kg.

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2017 to 1 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Physical State/Appearance: White waxy solid
Purity: 100%
Storage Conditions: Room temperature in the dark

Analytical monitoring:

no

Details on sampling:

The inhibitory effect of the test material on nitrogen transformation was assessed by the determination of nitrate concentration in the soil samples on Days 0 and 28 and compared to data obtained from solvent control soil samples.

On Days 0 and 28 three aliquots (50 g*) of soil were removed from the control, solvent control and test material vessels for nitrate analysis. A further three aliquots (50 g*) were removed from the control, solvent control and test material vessels were removed for measurement of the glucose-induced respiration rate on Days 0 and 28.

* Dry weight

Vehicle:

yes

Remarks:

Chloroform

Details on preparation and application of test substrate:

Preliminary solubility work showed that the test material had limited solubility in water, but that a preliminary solvent stock solution could be prepared in chloroform. Therefore, for the purposes of the test, the test concentrations were prepared from a preliminary solvent stock solution prepared in chloroform. In order to aid dispersal of the test material in the soil, an aliquot of the preliminary solvent stock solution was adsorbed onto dry quartz sand and the solvent allowed to evaporate to dryness prior to dispersal in the test soil.

The test material sand combinations was then added to final bulk soil along with an aliquot (30.3 mL) of water and thoroughly mixed to give the required test concentrations of 10, 32, 100, 320 and 1000 mg/kg, with a nominal moisture content of 45% of the Water Holding Capacity (WHC). The soil for each test concentration was then mixed again to ensure homogeneity prior to removing three aliquots for the Day 0 analysis. The remaining soil bulk was incubated in separate glass Duran bottles.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

18 and 22°C

Moisture:

45% of the Water Holding Capacity (WHC)

Organic carbon content (% dry weight):

0.68

Details on test conditions:

The soil samples were incubated in glass Duran bottles. The test vessels were sealed with polyurethane foam stoppers in order to maintain aseptic conditions and minimize moisture loss by evaporation and maintained in a temperature controlled room at temperatures of between 18 and 22°C in darkness.

The test soil was a sandy loam soil obtained from a site in Rugby, Warwickshire, England that had received no pesticides or fertilisers for 5 years prior to sampling.

On arrival at Envigo Research Limited, UK, the soil was stored at temperatures of between 3 and 4 deg.C prior to use. Prior to the start of the test, the soil was removed from refrigeration and stored at temperatures of between 15 and 21°C for 22 days and shielded from light.

The moisture content of the soil was determined prior to the start of the two consecutive dry weights within 4% were attained. The moisture content of the soil, expressed as a percentage of the dry weight, was determined to be 8%. The Water Holding Capacity (WHC) of the soil supplied by LUFA Speyer was 35.4 ± 1.0 g/100 g and hence 30.3 mL of deionized reverse osmosis water per 0.4 kg* of soil was added. This gave a final water content of 15 g/100 g, i.e. 45% of the WHC as recommended by the Testing Guidelines.

* Dry weight

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/kg

Reference substance (positive control):

yes

Remarks:

2-Chloro-6-(trichloromethyl)pyridine

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Results with reference substance (positive control):

The results from the positive control with 2-Chloro-6-(trichloromethyl)pyridine (i.e., Nitrapyrin) were within the normal ranges for this test material.

Validity criteria fulfilled:

yes

Conclusions:

The test material showed no significant adverse effect on the nitrogen activity of soil microorganisms at a test concentrations of up to and including 1000 mg/kg over a 28-Day period and therefore is considered to have no long-term effect on nitrogen transformation in soil. The No Observed Effect Concentration (NOEC) for nitrogen transformation in soil was determined to be 1000 mg/kg.

Executive summary:

A study was performed to assess the long-term effect of the test material after a single application, on the nitrogen transformation activity of soil microorganisms.

Soil microorganisms were exposed to the test material at concentrations of 10, 32, 100, 320 and 1000 mg/kg for 28 days at temperatures of between 18 to 22°C in the dark. The test soil was a sandy loam soil obtained from a site in Rugby, Warwickshire, England that had received no pesticides or fertilisers for 5 years prior to sampling. The soil used in the nitrification test was amended with powdered Lucerne-green-grass meal to act as a respiratory substrate.

The inhibitory effect of the test material on nitrogen transformation was assessed by the determination of nitrate concentration in the soil samples on Days 0 and 28 and compared to data obtained from solvent control soil samples.

Variation in nitrification rates and glucose-induced respiration rates among the respective control replicates was less than 15% and therefore satisfied the validation criteria in this study. 

The test material showed no significant adverse effect on the nitrogen activity of soil microorganisms at a test concentrations up to and including 1000 mg/kg over a 28-day period and therefore is considered to have no long-term effect on nitrogen transformation in soil. The No Observed Effect Concentration (NOEC) for nitrogen transformation in soil was determined to be 1000 mg/kg.

Description of key information

The effect of the test material on the carbon transformation activity of the soil microorganisms, resulted in an EC50 of greater than 1000 mg/kg.  The NOEC for carbon transformation in soil was determined to be 320 mg/kg.

The test material showed no significant adverse effect on the nitrogen activity of soil microorganisms at a test concentrations of up to and including 1000 mg/kg  over a 28-Day period and therefore is considered to have no long-term effect on nitrogen transformation in soil.  The No Observed Effect Concentration (NOEC) for nitrogen transformation in soil was determined to be 1000 mg/kg.