Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

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

Remarks:

3 substances available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

see the attached justification in section 13 for the full 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

Analytical monitoring:

not required

Key result

Dose descriptor:

other: The weight of evidence approach is based on a 1997 publication by Banchio and Gramajo. details of the study design and results are found in the 'Any other information and methods incl. tables' and 'Any other information on results incl. tables sections.

Conc. based on:

other: The weight of evidence approach is based on a 1997 publication by Banchio and Gramajo. details of the study design and results are found in the 'Any other information and methods incl. tables' and 'Any other information on results incl. tables sections.

Basis for effect:

other: The weight of evidence approach is based on a 1997 publication by Banchio and Gramajo. details of the study design and results are found in the 'Any other information and methods incl. tables' and 'Any other information on results incl. tables sections.

Remarks on result:

other: Fatty acids of different chain length (C4-C18)

Key result

Dose descriptor:

other: The weight of evidence approach is based on a 1996 publication by Hita et al. Information on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections.

Conc. based on:

other: The weight of evidence approach is based on a 1996 publication by Hita et al. Information on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections.

Basis for effect:

other: The weight of evidence approach is based on a 1996 publication by Hita et al. Information on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections.

Remarks on result:

other: Tristearin; propane-1,2,3-triyl trioctadecanoate

Key result

Dose descriptor:

other: The weight of evidence approach is based on a 2002 publication by Cecutti et al. Details on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections

Conc. based on:

other: The weight of evidence approach is based on a 2002 publication by Cecutti et al. Details on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections

Basis for effect:

other: The weight of evidence approach is based on a 2002 publication by Cecutti et al. Details on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections

Remarks on result:

other: Methyl (Z)-octadec-9-enoate

Triglyceride degradation in soil:

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 monocarboxylic 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.      Bioesterfication 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

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 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

 

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.

Validity criteria fulfilled:

not applicable

Remarks:

read across

Conclusions:

No experimental data evaluating the toxicity of substance subject to registration to soil microorganisms are available.
However, there is convincing evidence that toxicity to soil microorganisms is unlikely to occur. In an experimental study according to OECD 209, no effects on the respiration rate of a microbial sewage treatment community were recorded resulting in a NOEC (3 h) ≥ 1000 mg/L (nominal) (Mead, 1998). Moreover, experimental data taken from a similar source substance indicate that the substance is readily biodegradable. The initial concentration of 10 mg C/L was not toxic to sewage treatment plant microorganisms (Mead, 1998).
The Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance (ECHA, 2012; page 125) states that a test on soil microbial activity will only be additionally necessary for a valid PNEC derivation if inhibition of sewage sludge microbial activity has occurred and this is clearly not the case.
Additionally, this assumption 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.
Taking all information into account in a Weight of Evidence (WoE) approach which is in accordance to the REACh Regulation (EC) No 1907/2006, Annex XI General rules for adaptation of the standard testing regime no further testing with soil microorganisms is deemed necessary.

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Key result

Dose descriptor:

other: The weight of evidence approach is based on a 1997 publication by Banchio and Gramajo. details of the study design and results are found in the 'Any other information and methods incl. tables' and 'Any other information on results incl. tables sections.

Conc. based on:

other: The weight of evidence approach is based on a 1997 publication by Banchio and Gramajo. details of the study design and results are found in the 'Any other information and methods incl. tables' and 'Any other information on results incl. tables sections.

Basis for effect:

other: The weight of evidence approach is based on a 1997 publication by Banchio and Gramajo. details of the study design and results are found in the 'Any other information and methods incl. tables' and 'Any other information on results incl. tables sections.

Remarks on result:

other: The weight of evidence approach is based on a 1997 publication by Banchio and Gramajo. details of the study design and results are found in the 'Any other information and methods incl. tables' and 'Any other information on results incl. tables sections.

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 3

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

weight of evidence

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.

Key result

Dose descriptor:

other: The weight of evidence approach is based on a 2002 publication by Cecutti et al. Details on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections.

Conc. based on:

other: The weight of evidence approach is based on a 2002 publication by Cecutti et al. Details on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections

Basis for effect:

other: The weight of evidence approach is based on a 2002 publication by Cecutti et al. Details on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections

Remarks on result:

other: The weight of evidence approach is based on a 2002 publication by Cecutti et al. Details on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections

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 4

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key 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 sup-plemented 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)

Key result

Dose descriptor:

other: The weight of evidence approach is based on a 1996 publication by Hita et al. Information on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections.

Conc. based on:

other: The weight of evidence approach is based on a 1996 publication by Hita et al. Information on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections.

Basis for effect:

other: The weight of evidence approach is based on a 1996 publication by Hita et al. Information on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections.

Remarks on result:

other: The weight of evidence approach is based on a 1996 publication by Hita et al. Information on the study design and results can be found in the 'Any other information on methods incl. tables' and 'Any other information on results incl. tables' sections.

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 monocarboxylic 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.      Bioesterfication 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

Description of key information

 The chemical safety assessment according to Annex I of Regulation (EC) No. 1907/2006 does not indicate the need to investigate further the toxicity to soil microorganisms. 

Additional information

No experimental data evaluating the toxicity of the substance subject to registration to soil microorganisms are available.

However, there is convincing evidence that toxicity to soil microorganisms is unlikely to occur. In an experimental study according to OECD 209, no effects on the respiration rate of a microbial sewage treatment community were recorded resulting in a NOEC (3 h) ≥ 1000 mg/L (nominal) (Mead, 1998). Moreover, experimental data taken from a similar source substance indicate that the substance is readily biodegradable. The initial concentration of 10 mg C/L was not toxic to sewage treatment plant microorganisms (Mead, 1998).

The Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance (ECHA, 2012; page 125) states that a test on soil microbial activity will only be additionally necessary for a valid PNEC derivation if inhibition of sewage sludge microbial activity has occurred and this is clearly not the case.

Additionally, this assumption 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.

Taking all information into account in a Weight of Evidence (WoE) approach which is in accordance to the REACh Regulation (EC) No 1907/2006, Annex XI General rules for adaptation of the standard testing regime no further testing with soil microorganisms is deemed necessary.