Santicizer P1400

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:

28/05/2021 - 28/07/2021

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 (incl. QA statement)

Specific details on test material used for the study:

Test Substance Name: Santicizer® Platinum 1400
Chemical Name: 1,2-cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) Ester
Common Name: P-1400
CAS Number: 1200806-67-2
Lot Number: 6660
Purity: 99.886 %
Receipt Date: 08 March 2021
Expiration Date: 10 February 2024
Storage on Receipt: Room Temperature (15 - 25°C)

Analytical monitoring:

yes

Details on sampling:

The objective of the study was to determine the effect of the test substance, Santicizer® Platinum 1400, on soil micro-organisms by determination of inhibition of nitrogen transformation over a 28-day exposure period. The study was undertaken according to OECD Guideline 216, Soil Microorganisms, Nitrogen Transformation Test (adopted January 2000). The effect of Santicizer® Platinum 1400 on soil micro-organisms was determined by investigation of nitrate formation in treated soil amended with an organic substrate (lucerne).

Nitrate Extraction
After treatment application on Day 0, and after moisture adjustment on Day 28 of incubation, soil samples were taken and nitrogen extraction was performed. At Day 0 samples were taken from the remaining bulk soil and at Day 28 a sub-sample of soil was taken from each replicate vessel. For each sample approximately 7 g dry weight was taken. Potassium chloride (0.1M KCl, 35 mL) was added to the sample and shaken (ca150 RPM) for approximately 60 minutes on a shaker table. The extracted samples were then centrifuged at ca 3400 RPM for 10 minutes. The supernatant extract was then filtered using 0.45 μm filters. All extracts were analysed on the day of sampling. All remaining extracts were stored frozen post-analysis; frozen samples will be destroyed after issuing the final report.

Nitrate Analysis
The 0.1M potassium chloride extracts were analysed for NO3-N, using a Hach Lange DR 3900 Spectrophotometer. The Spectrophotometer is serviced and calibrated annually ± 1 month by a Hach Lange engineer, as per local SOPs.
For each extract, 1.0 mL of sample was added to a Hach LCK 339 cuvette with 0.2 mL of Hach LCK 339 Solution A. This was then shaken vigorously and allowed to stand for 15 minutes to allow the reaction to take place. Following this, the cuvette
was placed on the Spectrophotometer and the NO3-N reading presented in mg/L was displayed, which was recorded in the raw data. This procedure was repeated for each sample. The Limit of Detection (LOD) for this technique is 0.23 mg/L NO3-N.

Vehicle:

yes

Remarks:

RO water

Details on preparation and application of test substrate:

Test System
The test soil, Lufa 2.3, was received from LUFA Speyer, Germany on 14 June 2021. The soil was received 2 mm sieved and was stored at 4 ± 2oC for less than 3 months prior to use. The soil was consistent with the requirement of the OECD 216 test guideline recommendations. The test soil was acclimatised for 8 days under test environmental conditions.

Soil was amended with a nitrogen source, powdered lucerne, and treated by incorporation of the test substance at 10, 32, 100, 320 and 1000 mg a.i./kg dry soil (a.i. = active ingredient). An untreated control was also included in the study.
Over a 28-day period, the test substance had no adverse effects on the amount of the nitrate produced in the soil type tested.

Soil Collection Site
Geographical Site Reference: “rechts der Landauer Str”, Nr. 826/7
Specification: 2.3 Field Fresh
Organic Fertilisation in 2021:None
Organic Fertilisation in 2020:None
Organic Fertilisation in 2019:None
Organic Fertilisation in 2018:None
Organic Fertilisation in 2017:None
Pesticides Used: (sampling year and previous 4 years): None
Sampling Depth: Approx. 0 - 20 cm

Soil Characteristics
Batch Soil Number:: Lufa 21/003
Nitrogen in % N: 0.08 ± 0.02
Percent Organic Carbon: 0.66 ± 0.09
Cation Exchange Capacity (meq/100g): 6.0 ± 0.9
Maximum Water Holding Capacity (%): 35.7 ± 2.3
Microbial Biomass as a % of Organic Carbon: 1.2 (determined in house)
pH: 5.56 (determined in house)
Wet: Dry Ratio: 1.07 (determined in house)

Lucerne characteristics
Test System Name: Alfalfa 20/001a
Receipt Date: 17 December 2020
Dates of Analysis: 04 - 07 January 2021
Total Nitrogen: 3.61 %
Total Carbon: 43.42 %
Carbon/Nitrogen Ratio 12 : 1

Test Substrate Treatment
Test soil equivalent to 400 g dry weight was weighed out for each test group. The test substance for each treatment level was weighed out onto glass slides. The appropriate glass slide was held over its respective soil group and, using the RO water due to be added to the soil, the test substance was washed off the slide and directly onto the soil. The volume of RO water added to the soil in this way (29 mL) was to increase the moisture content to the required level (40 % of maximum water holding capacity (MWHC)). A 2 g aliquot of powdered lucerne (alfalfa) was also added.The batch of powdered lucerne used was received on 17 December 2020. It was analysed singularly and had an organic carbon: nitrogen ratio of 12:1 as required by the guideline .Once all components had been added 60 seconds were allowed for the test substance to soak into the soil before mixing using an electric hand-mixer. Mixing of test soil was undertaken until evenly distributed, and then approximately 100 g dry soil was weighed from each bulk group of test soil into each replicate vessel. The control group was prepared in a similar way, only without the addition of test substance.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Remarks:

Lufa 21/003 soil with a volume of RO (29 mL) to increase the moisture content to the required level (40 % of maximum water holding capacity (MWHC)). A 2 g aliquot of powdered lucerne (alfalfa) was also added.

Test temperature:

Test vessels were maintained at 20 ± 2°C.

Moisture:

The moisture content of the test substrate was adjusted gravimetrically by the addition of RO water every 7 days, where required, and prior to sampling.

Organic carbon content (% dry weight):

0.66

Nitrogen content (% dry weight):

0.08

Details on test conditions:

Test Design
The definitive test was conducted at nominal concentrations of 10, 32, 100, 320 and 1000 mg a.i./kg dry soil. An untreated control was also included in the test. There were three replicate vessels per test treatment, including the control treatment.

Test Vessels
Test vessels were 500 mL amber glass jars, each filled with a final weight of approximately 100 g dry weight of soil. After filling, the test vessels were sealed with a punctured plastic lid to allow for air exchange.

Environmental Conditions
Test vessels were incubated in the dark throughout the test. The moisture content of the test substrate was adjusted gravimetrically by the addition of RO water every 7 days, where required, and prior to sampling. Test vessels were maintained at 20 ± 2°C. Temperature was recorded throughout the test using a min/max thermometer.

Nominal and measured concentrations:

The definitive test was conducted at nominal concentrations of 10, 32, 100, 320 and 1000 mg a.i./kg dry soil.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg a.i./kg

Nominal / measured:

nominal

Basis for effect:

other: inhibition of nitrogen transformation

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

> 1 000 other: mg a.i./kg

Nominal / measured:

nominal

Basis for effect:

other: inhibition of nitrogen transformation

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 other: mg a.i./kg

Nominal / measured:

nominal

Basis for effect:

other: inhibition of nitrogen transformation

Details on results:

Nitrogen Transformation
The amounts of nitrate produced over 28 days in mg N/kg dry soil are presented in Attchment X
At Day 28, the 10, 32, 100, 320 and 1000 mg a.i./kg nominal concentrations showed a -4.1, -5.7, 13.2, 26.3 and 51.6 % difference in nitrate produced when compared to the control (Attachment 1).
At the higher concentrations of test substance, the amount of nitrates produced is greater than the control. The test substance is not inhibitory to nitrate production, but it is not clear why there was significantly more nitrates produced compared to the control in the highest concentrations. The test substance does not contain nitrogen so could not have been used as a nitrogen source. The increase in nitrate production at the higher test substance rates was likely due to some indirect interaction between the test substance and the soil micro-organisms e.g. providing a feed source for micro-organisms growth etc. Further work would be required to determine any definitive causes for the results observed.
For adverse effects, the NOEC was 1000 mg a.i./kg, and the EC10, EC25 and EC50 were all >1000 mg a.i./kg. As such, the LOEC was >1000 mg a.i./kg.
The individual analysis results at all time-points are presented in Attachment 2.

Validity Criteria
The following validity criterion was met during the definitive test:
The variation in NO3-N concentrations between replicate control vessel samples was <15 % on Day 0 and 28. Therefore, the definitive data is considered to be valid. The coefficient of variation was calculated to be 13.3 and 1.60 % for the Day 0 and 28 extractions, respectively.

Reported statistics and error estimates:

Statistical Analysis
Statistical analysis was performed using the CETIS program v1.8.6.8. Linear interpolation analysis was performed in order to estimate the EC10, EC25 and EC50 values for test substance. It was not possible to calculate the 95 % confidence limits. A Williams multiple comparison test was performed to estimate the test substance no observed effect concentration (NOEC) and lowest observed effect concentration(LOEC) because the data was monotonic and parametric.

Validity criteria fulfilled:

yes

Remarks:

The variation in NO3-N concentrations between replicate control vessel samples was <15 % on Day 0 and 28. Therefore, the definitive data is considered to be valid.

Conclusions:

The effect of the Santicizer® Platinum 1400 on soil micro-organisms was determined by investigation of inhibition of nitrogen transformation over a 28-day exposure period. For adverse effects, the NOEC was 1000 mg a.i./kg, and the EC10, EC25 and EC50 were all >1000 mg a.i./kg. As such, the LOEC was >1000 mg a.i./kg.

Executive summary:

The effect of the Santicizer® Platinum 1400 on soil micro-organisms was determined by investigation of inhibition of nitrogen transformation over a 28-day exposure period. The EC50, EC25 and EC10, the no observable effect concentration (NOEC) and lowest observable effect concentration (LOEC) were determined. At Day 28, the 10, 32, 100, 320 and 1000 mg a.i./kg nominal concentrations showed a -4.1, -5.7, 13.2, 26.3 and 51.6 % difference in nitrate produced when compared to the control. For adverse effects, the NOEC was 1000 mg a.i./kg, and the EC10, EC25 and EC50 were all >1000 mg a.i./kg. As such, the LOEC was >1000 mg a.i./kg. The validity criterion of achieving <15 % variation in nitrate-nitrogen concentrations between the control replicates, was met, therefore the test is considered valid.

Description of key information

The effect of the Santicizer® Platinum 1400 on soil micro-organisms was determined by investigation of inhibition of nitrogen transformation over a 28-day exposure period. For adverse effects, the NOEC was 1000 mg a.i./kg, and the EC10, EC25 and EC50 were all >1000 mg a.i./kg. As such, the LOEC was >1000 mg a.i./kg.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw