Santicizer 278®

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:

2018-03-14 to 2018-04-20

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)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Soil Micro-organisms: Nitrogen
transformation test, Guideline 216 (January 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: Benzyl 3-isobutyryloxy-1-isopropyl-2, 2-dimethylpropyl phthalate
Alternative name: ®Sancticizer 278
CAS Number: 16883-83-3
Physical State: Pale yellow viscous liquid
Lot Number: 3057
Purity: 98.652%
Expiry: 16 January 2020
Storage: Ambient in darkness (10 - 30ºC)
Date received: 12 February 2018

Details on sampling:

Samples
Triplicate samples of 20 g soil were withdrawn from each of the non-treated control soil and treated soil group replicates within 6 hours of treatment on Day 0, 7, 14 and 28 days after preparation. In addition, on Day 0 a 20 g soil sample was removed from each of the nontreated control replicates (Group 1) and used for spike recoveries. Bulk soils were thoroughly mixed prior to sub-sampling at each time point. The soil samples were extracted on the same day of sampling and sample extracts were transferred to a deep-freezer at approximately -20°C to await analysis. Analysis of the Day 7 and Day 14 soil extracts were not required to meet the test guideline requirements and were therefore not performed.

The soil samples were extracted with 100 mL 0.1M potassium chloride solution by shaking on a mechanical shaker at approximately 150 rpm for 60 minutes. The supernatant solutions were filtered through filter paper and then analysed for their concentrations of nitrate spectrophotometrically. The soil sample extract filtrates were processed as follows: 35 mL of acid mixture (250 ml of ≥95% sulphuric acid was mixed with 250 mL >85% orthophosphoric acid and 20 mg amidosulfonic acid added and mixed to dissolve) was measured into a suitable glass container and then 5 mL of soil extract filtrate added. 5 mL of 2,6-dimethylphenol solution (300 mg of 2,6-dimethylphenol was dissolved in >99% glacial acetic acid and adjusted to 250 mL final volume) was then added, mixed thoroughly and then allowed to stand for 10 – 60 minutes. The absorbance at 324nm was then measured using a Shimadzu UV-1800 spectrophotometer.

Analysis of a blank control used 5 mL 0.1M potassium chloride in place of soil extract filtrate and the absorbance measured as described above.

Analysis of potassium nitrate calibration solutions used 5 mL reference standard in place of soil extract filtrate and the absorbance measured as described above. Reference standard solutions constituting the calibration curve were prepared at concentrations of 1, 5, 10, 15, 20 and 25 µg nitrate N/mL. The absorbance value obtained for the blank control was subtracted from the absorbance values obtained for the calibration solutions. The calibration solution absorbance values were plotted on the y-axis against concentration (µg nitrate N/mL) on the x-axis.

The measured absorbance values for soil extract filtrates were plotted against the calibration curve to derive the sample nitrate nitrogen (N) concentration (µg N/mL). The concentration of nitrate in the test sample (µg/mL) was obtained by multiplying by the conversion constant 4.427.

Vehicle:

no

Details on preparation and application of test substrate:

Soil
The test soil was a sandy loam soil collected from a fallow agricultural site with grass cover near Rugby, Warwickshire, England. The site had received no pesticides or fertilisers for 5 years prior to sampling. Soil was collected from within the top 5 - 20 cm of top soil. Soil collection was conducted by Landlook (Midlands), Leamington Spa, Warwickshire in accordance with the principles of ISO 10381-6. The soil was stored at ambient temperature prior to delivery to Envigo. Soil analyses were conducted by CEMAS Ltd. and performed on representative samples of test soil. Reports giving the full details of the soil collections and analyses are retained with the raw data and the results obtained for the key parameters were recorded. The test soil (Bromsgrove 317/30.01.18) was received at Envigo on 06 February 2018 and stored at 2 – 8 °C in the dark. The test soil was acclimatised at 20 ± 2°C for 15 days prior to testing.

Soil reference number: Landlook (Midlands) soil series/number: Bromsgrove 317, CEMAS Ltd. study number CEMS-8467
Source: Leamington Spa, Warwickshire, England
Texture class: Sandy loam
Particle size distribution (USDA pipette method):
Sand (2.00 – 0.05 mm): 71 % w/w
Silt (0.05 mm – 0.002 mm): 17 % w/w
Clay (<0.002 mm): 12 % w/w
pH (ISO 10390 [1994]): 6.5
Organic carbon (Walkley Black method): 1.43 % w/w
Maximum WHC (ISO 11274:1998[E]) 41.3 % w/w
Cation exchange capacity (EPA method 9081A): 10.4 meq/100 g

Soil characterisation was performed by CEMAS Ltd. as a separate study.


Soil microbial biomass
Soil reference number: Bromsgrove 317/30.01.18
Total biomass (BC) mg C/kg soil: 606
Microbial biomass: 4.24 %

Soil nitrogen concentration*
Soil 317: <0.003 g N/g dry wright soil (<0.3%)

Lucerne meal analysis*
Lucerne meal batch 112: Carbon 42.53%, Hydrogen 6.23%, Nitrogen 3.28%
Carbon: Nitrogen ratio - 13:1
*Soil nitrogen and lucerne meal analysis were performed by Butterworths Ltd. as separate non-GLP compliant studies.

Nitrogen source
For nitrogen transformation, it is usually necessary to amend the test soil with a suitable nitrogen source. Lucerne meal (sieved to 2 mm maximum) having a carbon:nitrogen ratio of 13.0 : 1 was used and 4.07 g was added per kg test soil, having a dry weight of 814 g, to give a final concentration of lucerne meal of 0.5% on a soil dry weight basis. Analysis of the carbon:nitrogen content of the lucerne meal was performed in a separate non-GLP study by Butterworth Laboratories.

Treatment Concentrations
Sancticizer 278, a non-agrochemical, was tested at five treatment concentrations (10, 32, 100, 320 and 1000 mg/kg dry weight soil).

Experimental Design
The following mixtures of soil, nitrogen source and test item were set up:
Group N1: Soil + nitrogen source (non-treated control)
Group N2: Soil + nitrogen source + 10 mg Sancticizer 278/kg dry weight soil
Group N3: Soil + nitrogen source + 32 mg Sancticizer 278/kg dry weight soil
Group N4: Soil + nitrogen source + 100 mg Sancticizer 278/kg dry weight soil
Group N5: Soil + nitrogen source + 320 mg Sancticizer 278/kg dry weight soil
Group N6: Soil + nitrogen source + 1000 mg Sancticizer 278/kg dry weight soil

Preparation and Incubation of Soil Treatment Groups
For nitrogen transformation, the group mixtures described above were prepared in triplicate replicates of 1 kg for each group (N1A, N1B, N1C). Lucerne meal was distributed evenly in several aliquots over the surface of the test soil (spread as a thin layer of approximately 2.5 cm in diameter) and then thoroughly mixed after each aliquot addition.

To achieve satisfactory incorporation of Sancticizer 278 into the test soil, the test item was dissolved in acetone and 20 mL of an appropriate concentration of the dissolved test item was added to sand, evaporated to dryness and then treated sand (at a rate of 10 g sand per kg dry weight soil + test item) distributed evenly in several aliquots over the surface of each soil replicate (spread as a thin layer of approximately 2.5 cm in diameter). The treated test soil was thoroughly mixed after each aliquot addition to give the final nominal soil concentrations. Sufficient distilled water was sprinkled onto the test soil to increase the soil moisture content to 45% of the soil water holding capacity (WHC). The control soil mixture was similarly treated with the same volume of acetone on the same amount of sand, evaporated, mixed, and water added to bring moisture content to 45% of WHC.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 °C +/- 2 °C

Moisture:

45% of the soil water holding capacity (WHC)

Details on test conditions:

Following preparation, the soil replicates were placed into 5 L polypropylene containers and perforated lids fitted to limit water loss and allow gas exchange. The soil group replicates were incubated in the dark in a temperature controlled room set at 20°C ± 2°C. The moisture content of the soil groups was checked weekly by weight and the moisture adjusted by adding sufficient distilled water to bring the samples back to their original moisture content following removal of samples.

Nominal and measured concentrations:

Nominal: 0, 10, 32, 100, 320 and 1000 mg/kg dry weight soil.

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

The test soils were confirmed to have met the OECD guideline requirements for this study type. The microbial biomass of the test soils were >1%.

Nitrogen transformation in non-treated control soil (Group 1) and in the lower concentration test item treatment Groups 2 - 4 (10 mg, 32 mg and 100 mg Sancticizer 278/kg dry weight soil respectively) showed comparable increases in soil nitrate concentrations over the study period. At the end of the 28 day study period the % difference in soil nitrate concentrations between non-treated control soil (Group 1) and treated Groups 2, 3 and 4 was <25%. However at the higher treatment concentrations (Groups 5 and 6, 320 mg and 1000 mg Sancticizer 278/kg dry weight soil respectively) the treated groups showed statistically significant lower nitrate concentrations (p≤0.01, p≤0.001) and showed >25% differences compared to the non-treated control group at the end of the 28 day study period.

The EC50 (857.27 mg/kg), EC25 (258.10 mg/kg) and EC10 (77.65 mg/kg) values have been estimated. However, the confidence interval for the EC50 is outside the range of the data and should be used with caution. The 320 and 1000 mg/kg dry weight soil Test item treated groups had significantly lower nitrate concentrations than the control group (p≤0.003, Dunnett’s test).

The No Observed Effect Concentration (NOEC) tested in this study was considered to be 100 mg Sancticizer 278/kg dry weight soil. The variation between triplicate control soil samples (Group 1) for nitrate analysis was less than ± 15% at Day 28 with a coefficient of variance of 6.17% and was therefore in compliance with the OECD 216 validity criteria.

Nitrate concentrations in soil - group mean values

 

Day	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6
Dose	0 mg/kg dw	10 mg/kg dw	32 mg/kg dw	100 mg/kg dw	320 mg/kg dw	1000 mg/kg dw
0	26.7	35.26	36.01	34.13	41.08	39.96
28	94.39	97.21	97.78	84.43	67.69**	46.73***

¹Nitrate concentrations in soil expressed as mg NO3/kg dry weight soil; each value is a mean figure derived from 3 replicate analyses

**p<0.01

***p<0.001

 

Day	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6
Dose	Control	% deviation from Group 1	% deviation from Group 1	% deviation from Group 1	% deviation from Group 1	% deviation from Group 1
0	-	32.06	34.87	27.83	53.86	49.66
28	-	2.99	3.59	-10.55	-28.29	-50.49

 

Nitrate concentrations in soil* - individual values

Day	Group 1 Non-treated control soil			Group 2 10 mg/kg Sancticizer®278			Group 3 32 mg/kg Sancticizer®278
	1A	1B	1C	2A	2B	2C	3A	3B	3A
0	24.54	27.64	27.92	41.47	26.23	37.79	40.90	25.38	41.74
28	97.02	98.43	87.72	100.69	96.74	94.20	84.33	104.92	104.07

 

Day	Group 4 100 mg/kg Sancticizer®278			Group 5 320 mg/kg Sancticizer®278			Group 6 1000 mg/kg Sancticizer®278
	4A	4B	4C	5A	5B	5C	6A	6B	6A
0	42.87	20.31	39.20	47.67	38.64	36.95	36.10	36.95	46.82
28	86.31	78.13	88.84	77.84	66.28	58.95	51.61	43.72	44.85

*Nitrate concentrations in soil expressed as mg NO3/kg dry weight soil

Validity criteria fulfilled:

yes

Conclusions:

Sancticizer 278 is not anticipated to cause any inhibition or have any adverse long term influence on nitrogen transformation in soil at concentrations up to 100 mg Sancticizer 278 /kg dry weight soil.

Executive summary:

The effects of Santicizer 278 on soil nitrogen transformation by microorganisms were determined in a 28-day soil nitrogen transformation study completed in accordance with OECD TG 216. An estimated EC50 (nitrogen transformation inhibition) of 857.27 mg/kg dw, and NOEC (nitrogen transformation inhibition) of 100 mg/kg dw were determined for Santicizer 278.

Description of key information

The effects of Santicizer 278 on soil nitrogen transformation by microorganisms were determined in a 28-day soil nitrogen transformation study completed in accordance with OECD TG 216. An estimated EC50 (nitrogen transformation inhibition) of 857.27 mg/kg dw, and NOEC (nitrogen transformation inhibition) of 100 mg/kg dw were determined for Santicizer 278.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

857.27 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

100 mg/kg soil dw