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Toxicity to soil microorganisms

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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
Remarks on result:
other: Precise results cannot be given, see explanation in any other information on results incl. tables.

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-1 mL-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

 

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.
Remarks on result:
other: Precise results cannot be given, see explanation in any other information on results incl. tables.

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.

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 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)
Remarks on result:
other: Precise results cannot be given, see explanation in any other information on results incl. tables.

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 further investigate the toxicity to soil microorganisms.

Key value for chemical safety assessment

Additional information

There are no experimental studies available, in which the toxicity of the target substance Fatty acids, C16, C18 and C18-unsaturated, C12-15 alcohol (linear and branched), esters to soil microorganisms was investigated. Therefore, all available related data is combined in a weight-of-evidence approach, in accordance with REACH regulation (EC) No 1907/2006, Annex XI, 1.2, in order to fulfill the data requirements laid down by Regulation (EC) No 1907/2006 Annex VII – X (ECHA Guidance Chapter R.7c, 2017).

The substance is a long-chain aliphatic ester characterized by a high log Koc (> 5.0), indicating a potential for adsorption to the soil particles. Thus, tests with soil-dwelling organisms that feed on soil particles are therefore most relevant for the evaluation of the soil toxicity of the substance. In the absence of a clear indication of selective toxicity, an invertebrate (earthworm or collembolan) test is preferred, as outlined in ECHA Guidance (Chapter R.7c, 2017). 

In the available earthworm study with the source substance 2-octyldodecyl isooctadecanoate (CAS 93803-87-3), no significant effects on mortality/growth and reproduction of E. fetida were found, resulting in a NOEC (56 d) ≥ 1000 mg/kg soil dw (OECD 222).

Moreover, the ECHA Guidance (Chapter R.7c, 2017) 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.

One reliable study is available, in which the toxicity of the structurally and chemically closely related source substance Dodecyl octadec-9-enoate (CAS 36078-10-1) to aquatic microorganisms was investigated according to OECD 209. At the end of the test no inhibition of the respiration rate of aquatic microorganisms was observed, indicating that the target substance does not cause toxic effects to aquatic and soil microorganisms. Furthermore, the substance is readily biodegradable and will therefore be rapidly degraded in the environment.

Further evidence in support of this conclusion is available from literature. It has been shown that soil microorganism communities can easily degrade fatty acid esters (Hita et al., 1996 and Cecutti et al., 2002) and even use them as energy source (Banchio & Gramajo, 1997). In a study investigating the degradation of tristearin (a triglyceride composed of glycerin, tri-esterified with stearic acid) in three different soils for four weeks it was shown that the amount of stearic acid increased considerably throughout the test, showing the hydrolytic activity of lipases breaking the ester bonds (Hita et al. 1996). The investigation of the ester fractions showed the generation of new alkanoic acids (methyl stearate, ethyl stearate and propyl stearate), which were not present in the controls. After four weeks the molecules were no longer detectable, indicating that degradation by soil microorganisms had occurred. Another study with an incubation period of 120 d showed the complete degradation of methyl oleate (plant oil) and its metabolites by soil microorganisms within 60 d (Cecutti et al. 2002).

The gram-positive soil bacterium Streptomyces coelicolor 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).

Thus, the available literature shows that soil microorganisms are capable of breaking up ester bonds and degrading fatty acids in significant amounts and that fatty acids are used as energy source and are non-toxic.

Taken together in a weight-of-evidence approach in accordance with Annex XI, 1.2, the target substance is not expected to cause toxic effects to soil microorganisms and is therefore not expected to be of concern. Consequently, it is concluded that no further testing is required.