Fatty acids, C5-10, esters with pentaerythritol

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:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted specific principles, acceptable for assessment.

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study investigated the ability of Streptomyces to utilize different chain length fatty acids as sole carbon and energy sources, and to characterize their uptake system biochemically.

GLP compliance:

not specified

Analytical monitoring:

not required

Test organisms (inoculum):

other: Streptomyces coelicolor

Remarks:

Not applicable. In-vitro assay.

Details on test conditions:

EFFECT PARAMETERS MEASURED: In-vivo fatty acid degradation

Reference substance (positive control):

no

The study indicated that S.coelicolor strain M145 can effectively utilize fatty acids of different chain length, from C4 to C18, as sole carbon energy source. The in vivo ß-oxidation studies in cells grown in the presence or absence of fatty acids (Table 1), and in vitro assay of two enzymes of the pathway, acyl-CoA synthetase and acyl-CoA dehydrogenase (Table 2), clearly indicate that S. coelicolor constitutively expressed the enzymes of the ß-oxidation cycle, without the need for the induction by a fatty acid of any chain length. The ß-oxidation pathway in this microorganism, instead of being repressed by glucose was, at least for long-chain fatty acids, stimulated by this metabolite.

Table 1: Rate of ß-oxidation of 300 µM of labeled fatty acids by S.coelicolor M145 grown in SMM-oleate of SMM-glucose

Carbon source	Rate of ß-oxidation (nmol min^-1mL^-1(mg protein)^-1)
	(^14C) palmitate	(^14C) octanoate
Oleate	2.825	2.050
Glucose	4.500	1.950

Table 2: Acyl-CoA synthetase and acyl-CoA dehydrogenase in crude protein extracts prepared from cells of S.coelicolor M145 grown in SMM-glucose or SMM-oleate

Carbon source	Acyl-CoA synthetase (pmol min^-1mL^-1)	Acyl-CoA dehydrogenase (U (mg protein)^-1)
Oleate	8.3 +/- 0.5	35.0 +/- 0.5
Glucose	95.0 +/- 1.0	40.0 +/- 0.5

 

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted specific principles, acceptable for assessment.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The degradation of an oil additive in soil was investigated. Lysimeter was used to follow the migration and progressive biodegradation of the oils by soil microorganisms over time. Also metabolites were identified.

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The lysimeters were cur at various depths into slices of 2.5, 5 or 10 cm thickness. The soil was sieved (mesh size=2 mm) and a 100 g sample was taken according to standard NF X31-100.
- Sample storage conditions before analysis: at -18 °C in a polyethylene bag

Vehicle:

yes

Details on preparation and application of test substrate:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Methyl oleate was applied as an emulsion in water containing 50 g/L R508 (sorbitan ester) as emulsifier.

Test organisms (inoculum):

soil

Total exposure duration:

120 d

Test temperature:

19 - 22 °C (depends on depth of soil)

Details on test conditions:

SOURCE AND PROPERTIES OF SUBSTRATE
- Depth of sampling: 15 to 60 cm
- % clay: 19 %

VEHICLE CONTROL PERFORMED: no

Nominal and measured concentrations:

Methyl oleate was applied to the soil at its recommended rate of 2 L/ha, equivalent to 5.5 mg/10 mL of water for the surface of the lysimeter.

Degradation of methyl oleate in the soil

Total degradation had occurred after 60 days. The half-life was determined as 7 days, during this time, it migrated by only 15 cm. The distributions of these metabolites in space and time changed in a more diffuse manner than that of the parent compound. All those found were shorter carbon-chain fatty acids (Table 1).

Table 1: Characterization of the degradation products of methyl oleate

Fatty acid	Log Kow	Maximum depth (cm)
Oleic acid	-	15
Heptadecanoic acid	-	15
Palmitic acid	7.17	25
Pentadecanoic acid	-	25
Myristic acid	6.11	30
Tridecanoic acid	-	30
Lauric acid	4.6	40
Undecanoic acid	-	50
Capic acid	4.09	50
Pelargonic acid	-	60
Caprylic acid	3.05	50
Heptylic acid	1.92	60
Caproic acid	1.87	60
Valeric acid	1.39	60
Butyric acid	0.79	60
Propionic acid	0.33	60

 

None of the metabolites were detected after 60 days, suggesting that methyl oleate was completely degraded at this point. The plant ester did not migrate very deeply in the soil because it was rapidly broken down by microorganisms in the soil and did not have time to migrate. β-oxidation and ω-oxidation led to the appearance of metabolites that migrated to depths of up to 60 cm and were completely degraded within 60 days.

Reference 3

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted specific principles, acceptable for assessment.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The degradation of the model molecule (pure tristearin) was investigated in three different soil types, to determine the behavior of fatty wastes.

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and application of test substrate:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The three soil samples were first sieved (<2mm), adjusted to 2/3 of the water-holding ca¬pacity of each respective soil and then weighed into 750 cm3 flasks in portions calculated to correspond to 100 g o.d. soil. The soils were subsequently supplemented with a pure triglyceride.

Test organisms (inoculum):

soil

Total exposure duration:

8 wk

Test temperature:

20 °C

Details on test conditions:

TEST SYSTEM
- Test container: flask
- Amount of soil: 100 g
- No. of replicates per concentration: yes, 3 replicates
- No. of replicates per control: yes, 3 replicates

VEHICLE CONTROL PERFORMED: no

Nominal and measured concentrations:

0.2% (wt/wt)

Free lipids were extracted from representative samples and of the combined replicates of each control and supplemented soil. The concentration of free lipids extracted after 1 and 4 weeks from each series. After the first week a low diminution of total free lipids was observed (GOV: 2.7%; CHA: 2.6%; SOR: 3.5%). After 4 weeks, the amount of free lipids decreased (CHA: 11%; SOR: 8%; GOV: no variations). Fluctuations of lipid concentrations observed with time in the control were attributed to an increased activity of soil microorganisms due to the incubation. The main result was the great increase during the first week of the acid + polar fractions. This probably indicates the oxidation and hydrolysis process of the added compound. The amounts decreased when the incubation prolonged to 4 weeks. These compounds did not accumulate as they are certainly intermediate compounds in the biodegradation process. The evolution of the concentrations of monoacid and di-, keto- and hydroxy- acid fractions significantly increased during the first week. After 4 weeks a decrease of quantities was followed. The increase obtained during the first week. Monocarboxylic acids were then predominant over di-, keto- and hydroxylacids in the three soils. The results show that, due to the soil supplementation with tristearin, free fatty acids were produced. After soil microflora adaption, these compounds are utilized as they are freed by enzymatic hydrolysis. A part of the of the monocarboxyclic acids is probably oxidized to form di-, keto- and hydroxyl-acids. Contrary the acid fractions evolution, the amounts of the neutral fractions increased between 1 and 4 weeks in the supplemental soils. This is due to the increase of the quantity of alcohols and polar neutral compounds. Bio-oxidation processes seem to be more efficient after 4 weeks. After 1 week also a low decrease, compared to the controls, in the amounts of hydrocarbons consecutive to a low increase of the ester fractions.

Main result of the monoacid fractions analysis was the rapid formation of stearic acid in considerable amounts. This result showed that an intense hydrolysis reaction with specific lipase of tristearin had occurred after the soil supplementation. The investigations of ester fractions showed that new alkanoic acids (methyl stearate, ethyl stearate, and propyl stearate), not determined in the controls, were generated in the supplemented soils. Among other processes the following hypothesis to explain the formation of these compounds were proposed:

1.      Bioesterification of a part of the free stearic acid, released by an enzymatic hydrolysis reaction

2.      Alcoholysis of the triglyceride to form esters, directly

3.      And/or direct formation of these compounds from tristearin with C-C and C-O bond cleavages

Reference 4

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Sep 2012 - 15 Jan 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study tested with the source substance CAS 25151-96-6. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

GLP compliance:

yes (incl. QA statement)

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: powdered plant material (lupine)

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: a stock solution was prepared by solving the test material in acetone. Subsequently the required amount of stock solution was mixed with quartz sand and powdered plant material. After evaporation of the solvent the quartz sand was mixed with test soil.

VEHICLE:
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: 3 mL acetone /700 g dry mass soil
- Evaporation of vehicle before use: yes

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

19.4 – 20.9 °C

Moisture:

24% WHC

Details on test conditions:

TEST SYSTEM
- Test container: 1 L test vessels
- Amount of soil: 500 g
- No. of replicates per concentration: 3
- No. of replicates per control: 4

SOIL INCUBATION
- Method: bulk / series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Treatments with pesticides or fertilizers: appplication of pesticide >1 year, application of fertiliseres > 3 months before sampling
- Soil texture: loamy sand soil
- % sand: 70
- % silt: 27
- % clay: 3
- pH (in water): 6.0
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 24.6
- Maximum water holding capacity: 233 g H2O/kg soil dry weight
- Cation exchange capacity (mmol/kg): 8.6
- Pretreatment of soil: sieving (2 mm mesh size)
- Storage: at 4 - 8 °C for 9 days
- Initial microbial biomass as % of total organic C: 1.2

EFFECT PARAMETERS MEASURED:
Nitrate content and basal and glucose-induced respiration rate at were determined at test start and termination

VEHICLE CONTROL PERFORMED: no

Nominal and measured concentrations:

Nominal test substance concentration: 62.5, 125, 250, 500 and 1000 mg test item/kg dry mass
soil.

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

176 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

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:

nitrate formation rate

Details on results:

Starting at a test substance concentration of 250 mg/kg a decrease of nitrogen transformation was detected. The effect is presumably due to a physiological adaption and growth of the microbial community due to the availability of the test substance as an easily degradable carbon source.

Mean nitrate content [mg/kg] and deviation from control [%]:

 

	Control	62.5 mg/kg	125 mg/kg	250 mg/kg	500 mg/kg	1000 mg/kg
Test start
Nitrate [mg/kg]	24.6	26.2	25.3	32.3	32.5	31.7
Day 28
Nitrate [mg/kg]	360.5	362.5	351.2	301.5	255.2	239.2
Deviation [%]		- 0.6	2.6	16.4	29.2	33.7

 

Reference 5

Endpoint:

toxicity to soil microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

4 substances are available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

see the justification attached to section 13 for details.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

176 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 2,2-bis(hydroxymethyl)propane-1,3-diyl bisoctadec-9-enoate (CAS 25151-96-6)

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:

nitrate formation rate

Remarks on result:

other: 2,2-bis(hydroxymethyl)propane-1,3-diyl bisoctadec-9-enoate (CAS 25151-96-6)

Fatty acids of different chain length (C4-C18)

The study indicated that S.coelicolor strain M145 can effectively utilize fatty acids of different chain length, from C4 to C18, as sole carbon energy source. The in vivoß-oxidation studies in cells grown in the presence or absence of fatty acids (Table 1), and in vitroassay of two enzymes of the pathway, acyl-CoA synthetase and acyl-CoA dehydrogenase (Table 2), clearly indicate that S. coelicolor constitutively expressed the enzymes of the ß-oxidation cycle, without the need for the induction by a fatty acid of any chain length. The ß-oxidation pathway in this microorganism, instead of being repressed by glucose was, at least for long-chain fatty acids, stimulated by this metabolite.

Table 1: Rate of ß-oxidation of 300 µM of labeled fatty acids by S.coelicolor M145 grown in SMM-oleate of SMM-glucose

Carbon source	Rate of ß-oxidation (nmol min^-1mL^-1(mg protein)^-1)
	(^14C) palmitate	(^14C) octanoate
Oleate	2.825	2.050
Glucose	4.500	1.950

Table 2: Acyl-CoA synthetase and acyl-CoA dehydrogenase in crude protein extracts prepared from cells of S.coelicolor M145 grown in SMM-glucose or SMM-oleate

Carbon source	Acyl-CoA synthetase (pmol min^-1mL^-1)	Acyl-CoA dehydrogenase (U (mg protein)^-1)
Oleate	8.3 +/- 0.5	35.0 +/- 0.5
Glucose	95.0 +/- 1.0	40.0 +/- 0.5

 

Tristearin; propane-1,2,3-triyl trioctadecanoate CAS 555 -43 -1

Three types of soil (Table 1) were chosen from the Poitou-Charentes region, in the western part of France.

Table 1: Characterization of the soil samples used in the experiment (% o.d. soil)

Soil	pH	Clay %	CaCO3 %	TOC %	C/N ratio
Rendzina (CHA)	8.1	29	31	2.7	7.3
Glossic luvisol (SOR)	4.1	13.5	7	7	19
Rendoilic eutrochrept	7.3	25	4	2.2	7.5

 

Mineralization

Mineralization of the substrate was measured, in terms of the CO2 production compared to the controls, weekly over a period of two months.

 

Lipid isolation

After the first and the fourth weeks, samples of the combined replicates were air dried. Neutral and acid fractions, corresponding to simple lipids, were obtained first. Complex lipids were obtained by liquid-liquid extraction. Hydrocarbons and esters were separated from the neutral fractions using column chromatography followed by TLC tests.

Analysis

Naturally occurring esters and ester derivatives from monoacids were analyzed by capillary gas chromatography (GC) and by capillary gas chromatography coupled with a mass spectrometer (GC-MS) after the first and fourth week of the combined replicates.

methyl octadec-9-enoate CAS 112-62-9

Degradation of methyl oleate in the soil

Total degradation had occurred after 60 days. The half-life was determined as 7 days, during this time, it migrated by only 15 cm. The distributions of these metabolites in space and time changed in a more diffuse manner than that of the parent compound. All those found were shorter carbon-chain fatty acids (Table 1).

Table 1: Characterization of the degradation products of methyl oleate

Fatty acid	Log Kow	Maximum depth (cm)
Oleic acid	-	15
Heptadecanoic acid	-	15
Palmitic acid	7.17	25
Pentadecanoic acid	-	25
Myristic acid	6.11	30
Tridecanoic acid	-	30
Lauric acid	4.6	40
Undecanoic acid	-	50
Capic acid	4.09	50
Pelargonic acid	-	60
Caprylic acid	3.05	50
Heptylic acid	1.92	60
Caproic acid	1.87	60
Valeric acid	1.39	60
Butyric acid	0.79	60
Propionic acid	0.33	60

 

None of the metabolites were detected after 60 days, suggesting that methyl oleate was completely degraded at this point. The plant ester did not migrate very deeply in the soil because it was rapidly broken down by microorganisms in the soil and did not have time to migrate.β-oxidationand ω-oxidation led to the appearance of metabolites that migrated to depths of up to 60 cm and were completely degraded within 60 days.

Description of key information

EC50 > 1000 mg/kg dw soil (OECD 216)

Additional information

Since no studies investigating the toxicity to soil microorganisms ofpentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS 68424-31-7)are available for this endpoint, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read across to structurally related category members was conducted.The read-across substance is 2,2-bis(hydroxymethyl)-1,3-propanediyl dioleate (CAS 25152-96-6) a pentaerythritol (mono – tri) ester with predominantly C18 unsatd. fatty acids. Supporting data investigating the toxicity to microorganisms are presented for tristearin (CAS 555-43-1), methyl oleate and for fatty acids of different chain length (C4-C18).

The study with 2,2-bis(hydroxymethyl)-1,3-propanediyl dioleate (CAS 25151-96-6) was conducted investigating the effects on nitrogen transformation according to OECD 216 is available (Simon, 2013). In this GLP study nominal test concentrations of 62.5, 125, 250, 500 and 1000 mg/kg dw were tested. The EC50 for the nitrogen transformation was calculated to be > 1000 mg/kg dw soil (the highest concentration tested). Therefore it can be concluded that the test substance will not exhibit effects to soil microflora.

This is supported by further evidence from literature data. This data showed that soil microorganism communities are well capable of degrading fatty acid esters (Hita et al., 1996 and Cecutti et al., 2002) and use them as energy source (Banchio & Gramajo, 1997). Hita et al. investigated the degradation of the model molecule tristearin which is a triglyceride containing of glycerin tri-esterified with stearic acid in three different soils for 4 weeks. The amount of stearic acid increased in considerable amounts during the experiment showing the hydrolytic activity of lipases breaking the ester bonds. The investigation of ester fractions moreover showed the generation of new alkanoic acids (methyl stearate, ethyl stearate and propyl stearate) which were not determined in the controls. Nevertheless the amounts were no longer present after 4 weeks, which leads to the assumption that degradation by soil microorganisms had occurred. The same was shown by Cecutti et al. (2003). One soil sample was chosen and incubated with methyl oleate (plant oil) for 120 d. Methyl oleate and its metabolites were completely degraded after 60 d. Streptomyces coelicolor, a common gram-positive soil bacterium uses fatty acids (C4-C18) as sole carbon end energy source indicating that fatty acids are not-toxic and can be used for catabolism (Banchio and Gramajo, 1997). The available literature data shows that soil microorganisms are capable to break-up ester bonds and degrade fatty acids in significant amounts. Moreover, the data indicated the non-toxic properties of fatty acids since they can be used as energy source.

Therefore, one can conclude from all available data that toxicity to soil microorganisms is not of concern for PE esters.

 

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.