Alcohols, C20-22, reaction products with phosphorus oxide (P2O5)

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:

March, 16, 2020 - July, 28, 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

January 21, 2016

Deviations:

yes

Remarks:

Three deviations were observed during the study. These deviations are not critical and have no influence on the results presented in the report.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: 190604011722
- Expiration date of the lot/batch: June 03, 2022
- active ingredients: 99.91% w/w (0.09% water)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20 ± 5°C)

Analytical monitoring:

not required

Vehicle:

yes

Details on preparation and application of test substrate:

The test item was not soluble in water, a special solubilisation protocol was carried out before
the experimentation, according to the OECD n°23 guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals OECD n°23.
This method consisted in dissolving the test item in an appropriate volume of organic solvent such as dichloromethane and switch to ultrasounds during maximum 45 minutes at 30°C. Then, the preparation was added in around 10 to 20 g of sand to evaporate the solvent, in the dark and ambient temperature.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2°C

Moisture:

40 – 60% of the water holding capacity ± 5%

Organic carbon content (% dry weight):

0.65

Nitrogen content (% dry weight):

0.07

Details on test conditions:

TEST SYSTEM

- Amount of soil: 450g
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- tested concentrations: the study was conducted on five concentrations levels: 50 – 80 – 120 – 180 and 270 mg test item / kg dry soil.The highest tested concentration corresponded to the maximum Predicted Environmental Concentration(PEC) and five times that concentration, according to OECD 2016 §21.

SOIL INCUBATION
- Method: bulk / series of individual subsamples: The soil was divided into six portions of equal weight. Five portions were mixed with the test item at the five test concentrations and one portion, moistened with water (Control).
450 g of soil samples were amended with powdered Lucerne alfalfa mea, with a C/N ratio between 12/1 and 16/1 and placed in test containers with sufficient headspace, under aerobic conditions. The applied Lucerne-soil ratio was 5 g of Lucerne per kilogram of dry soil . The ratio C/N was equal to 11.3 / 1.

Three replicates for five treated and untreated soils (= Controls) were transferred in containers.
A special solubilisation protocol was carried out before the experimentation, this method consisted in dissolving the test item in an appropriate volume of organic solvent such as dichloromethane and switch to ultrasounds during 45 minutes at 30°C. Then, the preparation was added in around 15 g of sand to evaporate the solvent, in the dark and ambient temperature.
Three controls were performed in the same conditions (15g of sand and dichloromethane).
After the treatment applications, the untreated (control) and treated (50, 80, 120, 180 and 270 mg/Kg soil dry weight) samples were thoroughly mixed and incubated in the dark at 20+/- 2°C during 28 days.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): LUFA 2.3 supplier of soil is located in Germany.
- Depth of sampling: 20 cm
- Soil texture:
- % sand: particle size between 0.005 to 2.0 mm in %: 59.4 ± 0.7
- % silt: particle size between 0.002 and 0.05 mm in %: 33.3 ± 0.6
- % clay: particle size < 0.002 mm in %: 7.3 ± 0.9
- Soil taxonomic classification: sand loam
- Soil classification system: according to USDA
- pH (in water): 6.4
- Organic Carbon content (% dry weight) 0.65 ± 0.08
- Initial nitrate concentration for nitrogen transformation test (% dry weight): 0.07 ± 0.02
- Maximum water holding capacity (in % dry weigth): 31.4
- Cation exchange capacity (meq/100g): 6.8 ± 1.4
- Pretreatment of soil:
- Initial microbial biomass as % of total organic C: 0.28 g Microbial Carbon/ 100g Organic Carbon

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The formed NO 3 -N in each treatment was compared with NO 3 -N in the control. A dose-response curve versus time was prepared. Statistical evaluation of the test results was performed with an ANOVA analysis at 5% significant level (Minitab 17, statistical software) and NOEC/LOEC were determined.

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

yes

Remarks:

Name* : Multi Anion Standard 3 for IC Composition*: Nitrate (NO 3 - ) Phosphate (PO 4 3- ) Sulfate(SO 4 2- ) Supplier*: SIGMA ALDRICH Deadline to use*: April 2022 Batch number*: BCBX0466 Purity*: See certificate of analysis *data given by the supplier

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

> 270 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

The results are summarized in the following table:

Treatment group (mg test item/kg soil dry weight		0 day after applications	14 days after applications	28 days after applications	Deviation from control at 28 days (%)*
Control	NO 3 -N [mg/Kg soil dw)	13.05	47.74	96.15	/
50	NO 3 -N [mg/Kg soil dw)	13.40	46.70	90.81	-5.6
80	NO 3 -N [mg/Kg soil dw)	13.70	44.74	92.79	-3.5
120	NO 3 -N [mg/Kg soil dw)	13.63	43.62	90.32	-6.1
180	NO 3 -N [mg/Kg soil dw)	10.37	44.22	92.02	-4.3
270	NO 3 -N [mg/Kg soil dw)	6.96	38.84	92.05	-4.3

* Based on NO 3 -nitrogen production ,
dw: Dry weight

On day 0, nitrate contents were similar to the Control group and for the treated groups. The soil LUFA 2.3 contains nitrate and the repartition between the Control group and treated groups was homogeneous.

After 14 days of incubation, the formed nitrate content increased in the control group and in the treated groups. No significant difference in the nitrogen transformation were found for any concentration of the test item after 14 days of incubation period in comparison to the control.

According to the OECD guideline 216, the variation between the three controls replicates should be less than 15%. This criterion was met after 28 days of incubation (variation: 2.3 %) but also, at initial time (9.0 %) and after 14 days of
incubation (2.8 %). Therefore, the testing was considered to be valid.

The micro-organisms responsible of the formation of nitrate were not affected at any tested concentration.

Reported statistics: The formed NO 3 -N in each treatment was compared with NO 3 -N in the control. Statistical evaluation of the test results was performed with an ANOVA analysis at 5% significant level (Minitab 17, statistical software) and a
statistical difference was determined at the levels 50 and 120 NOEC but no statistical difference was determined at the levels 80, 180 and 270.

The results did not permit to calculate a regression in order to determine an estimation of the ECx values because no significant difference in the nitrogen transformation were found for any concentration of the test item at the end of the 28 days incubation period in comparison to the control.

Validity criteria fulfilled:

yes

Remarks:

Validity for OECD 216: Variation between replicate Control sanples. Acceptance criteria: ≤ ± 15% Conformity for the test: Ok 9.0% at initial time 2.8% after 14 days of incubation 2.3% after 28 days of incubation

Conclusions:

All the validity criteria were successful.
No adverse effects of the test item “REACTION PRODUCTS BETWEEN C20/22 (even numbered) ALCOHOL AND PHOSPHORIC ANHYDRIDE” on nitrogen transformation in soil was observed at any tested concentration (50, 80, 120, 180 and 270 mg/Kg soil dry weight) after 28 days of incubation.

Executive summary:

The aim of this study was to investigate long term potential effects of exposure of the test item “REACTION PRODUCTS BETWEEN C20/22 (EVEN NUMBERED) ALCOHOL AND PHOSPHORIC ANHYDRIDE” according to the OECD 216
guideline, on Nitrogen transformation activity of soils microorganisms according to the OECD 216 guideline (January 21, 2000).
A sandy loam soil low in organic matter was used. According to the sponsor, the test item is a non-agrochemical product and five concentrations was used.

Validity criteria conformity:

Validity for OECD 216	Acceptance criteria	Conformity for the test
Variation between replicate Control samples	≤ ± 15%	Ok 9.0% at initial time 2.8% after 14 days of incubation 2.3% after 28 days of incubation

All the validity criteria were successful.

The results of this study are summarized below:

Test conditions:
Test item: REACTION PRODUCTS BETWEEN C20/22 (EVEN NUMBERED) ALCOHOL AND PHOSPHORIC ANHYDRIDE – Non-agrochemical product
Nominal concentrations: 0 (control), 50, 80, 120, 180 and 270 mg/Kg dry soil
Soil: LUFA 2.3 Sandy loam

450 g of soil samples were amended with powdered Lucerne alfalfa meal; 5 g of alfalfa meal per Kilogram of soils and placed in test containers. The untreated (control) and treated (50, 80, 120, 180 and 270 mg/Kg soil dry) samples were
incubated in the dark at 20+/- 2°C. The moisture content of the samples was checked and adjusted two or three times a week. NO 3 -N content was determined in the control and treated soils at 0, 14 and 28 days after treatment by ionic
chromatography.

The results are summarized in the following table:

Treatment group (mg test item/kg soil dry weight		0 day after applications	14 days after applications	28 days after applications	Deviation from control at 28 days (%)*
Control	NO 3 -N [mg/Kg soil dw)	13.05	47.74	96.15	/
50	NO 3 -N [mg/Kg soil dw)	13.40	46.70	90.81	-5.6
80	NO 3 -N [mg/Kg soil dw)	13.70	44.74	92.79	-3.5
120	NO 3 -N [mg/Kg soil dw)	13.63	43.62	90.32	-6.1
180	NO 3 -N [mg/Kg soil dw)	10.37	44.22	92.02	-4.3
270	NO 3 -N [mg/Kg soil dw)	6.96	38.84	92.05	-4.3

According to the OECD guideline 216, the variation between the three controls replicates should be less than 15%. This criterion was met because the variation was only 2.3 % after 28 days of incubation but also, at initial time (9.0%) and
after 14 days of incubation (2.8%). Therefore, the testing was considered to be valid.

Conclusion:
No adverse effects of the test item “REACTION PRODUCTS BETWEEN C20/22 (even numbered) ALCOHOL AND PHOSPHORIC ANHYDRIDE” on nitrogen transformation in soil was observed at any tested concentration (50, 80, 120,
180 and 270 mg/Kg soil dry) after 28 days of incubation.

 

Description of key information

The aim of this study was to investigate long term potential effects of exposure of the test item “REACTION PRODUCTS BETWEEN C20/22 (EVEN NUMBERED) ALCOHOL AND PHOSPHORIC ANHYDRIDE” according to the OECD 216 guideline, on Nitrogen transformation activity of soils microorganisms according to the OECD 216 guideline (January 21, 2000).

No adverse effects of the test item “REACTION PRODUCTS BETWEEN C20/22 (even numbered) ALCOHOL AND PHOSPHORIC ANHYDRIDE” on nitrogen transformation in soil was observed at any tested concentration (50, 80, 120, 180 and 270 mg/Kg soil dry) after 28 days of incubation.