[No public or meaningful name is available]

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Data waiving:

other justification

Justification for data waiving:

other:

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

other: read-across from supporting substance

Remarks:

read-across from supporting substance

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: Total degradation had occurred after 60 days.

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:

other: read-across from supporting substance

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: 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 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 dehrdrogenase (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 4

Endpoint:

toxicity to soil microorganisms

Type of information:

other: read-across from supporting substance

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: See 'Any other information on results'

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.      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

In accordance with Regulation (EC) No 1907/2006, Annex X, column 2, 9.4 further studies on the effects on terrestrial organisms do not have to be conducted since data for short-term toxicity on soil macroorganism is available for a structurally and chemically closely related source substance showing no effects on survival or biomass during the exposure period.In addition, information gathered from several independent sources is combined in a Weight of Evidence approach, which is in accordance to the REACh Regulation (EC) No. 1907/2006, Annex XI General rules for adaptation of the standard testing regime set out in Annexes VII to X, 1.2, to cover the data requirements of Regulation (EC) No. 1907/2006, Annex IX). This approach provides enough evidence to state that this substance is unlikely to exert toxicity to soil microorganisms.

Fatty acids, C16-18 (even numbered), esters with glycerol oligomers is characterised by a high log Koc (1.31 – 12.67, KOCWIN v2.00; MCI method) indicating a considerable potential for adsorption to soil particles. Therefore, tests with soil-dwelling organisms like earthworm which allows potential uptake via surface contact, soil particle ingestion and pore water (Guidance on information requirements and chemical safety assessment, Chapter R.7c, (ECHA, 2017)) are most relevant for the evaluation of soil toxicity of Fatty acids, C16-18 (even numbered), esters with glycerol oligomers.

According to Chapter R7.b of the Guidance on information requirements and chemical safety assessment (ECHA, 2017), a test on soil microbial activity will be additionally necessary only if inhibition of sewage sludge microbial activity has occurred. In a toxicity control of a biodegradation study no inhibition of sewage sludge microbial activity has been observed (76% after 14 d).

Additionally, literature data evaluating the effects of fatty acid esters, including one Glyceride, to soil microorganisms are available. Hita et al. (1996) investigated the degradation of the model molecule tristearin (Glycerol tristearate) 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. Furthermore, the investigation of ester fractions 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. (2002) and Banchio and Gramajo (1997) for other fatty acid esters. In the first test, one soil sample was chosen and incubated with methyl oleate (plant oil) for 120 days. Methyl oleate and its metabolites were completely degraded after 60 days. 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.

Furthermore, all reliable aquatic acute (fish, invertebrates, algae) and aquatic chronic data (invertebrates, algae) as well as short-term toxicity data for sediment organisms for the suitable source substances indicate no effects up to the water solubility limit. Thus, no selective toxicity was observed for the aquatic compartment. Therefore, no lower toxicity to soil microorganisms than to soil macroorganisms such as the earthworm is expected. According to ECHA guidance (Chapter R.7c: Endpoint specific guidance, 2017, page 149) an invertebrate test (earthworm or collembolan) is preferred in the absence of a clear indication of selective toxicity. Therefore, no further studies on the effects on terrestrial organisms are proposed to be conducted.

According to ECHA guidance (Chapter R.7c: Endpoint specific guidance, 2017) new long-term testing only needs to be conducted where the data on aquatic effects are insufficient to complete the Chemical Safety Assessment. Since acute aquatic toxicity data are available for structurally and chemically closely related source substances for all three trophic levels and chronic toxicity data are available for aquatic invertebrates and algae the Chemical Safety Assessment can be evaluated completely. All reliable aquatic acute and chronic data show no effects up to the water solubility limit.As all acute toxicity effects range above 10 mg/L and no chronic or long-term effects in aquatic organisms up to the water solubility limit were observed, this can be used to waive the data requirements of Annex IX (Chapter R.7c: Endpoint specific guidance, 2017, page 148).

As the substance is considered readily biodegradable (94% biodegradation after 28 days; read-across) confirmed with QSAR calculations for representative constituents (VEGA (1.1.4); Ready Biodegradability Model (v1.0.9)) and no inhibition of sewage sludge microbial activity has been observed in a biodegradation testing (toxicity control: 76% after 14 d), it is expected that an extensive elimination of the substance in sewage treatment plants will occur. According to the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b (ECHA, 2017), the ready biodegradability of a substance can be considered indicative of rapid and ultimate degradation in most environments, including biological sewage treatment plants (STP) where the substance will be extensively removed in the primary settling tank and fat trap. As a result of the high adsorption potential of the substance (log Koc = 1.31 – 12.67, KOCWIN v2.00; MCI method), a removal from the water column to a significant degree by adsorption to sewage sludge can be expected (Guidance on information requirements and chemical safety assessment, Chapter R.7b, (ECHA, 2017)). Thus, only limited amounts will get in contact with activated sludge organisms in STPs and the concentration of the substance in effluents of conventional STPs is presumably marginal. Therefore, discharged concentrations of these substances into the soil compartment are likely to be negligible.Considering this, one can assume that the availability of Fatty acids, C16-18 (even numbered), esters with glycerol oligomers in the soil environment is very low, which reduces the probability of exposure, in particular long-term exposure, of soil organisms in general.

Moreover, the bioaccumulation potential is low. Based on the physico/chemical properties such as poor water solubility and high potential for adsorption a reduced availability in water is expected. In addition, the available literature data showed 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. It can be concluded that the bioaccumulation potential of Fatty acids, C16-18 (even numbered), esters with glycerol oligomers is negligible. This is supported by a low calculated BCF value ranged from 0.94 - 18.58L/kg ww (BCFBAF v3.01, Arnot-Gobas, including biotransformation, upper trophic).With VEGA 1.1.3 BCF values of 0.74 - 2 L/kg were determined (Caesar v2.1.14) for the main components of the substance.

In conclusion, for substances indicating a considerable potential for adsorption to soil particles, tests with soil-dwelling organisms like earthworm are most relevant for the evaluation of soil toxicity. In addition, no inhibition of sewage sludge microbial activity was observed. Generally, long-chain aliphatic esters can be expected to be metabolized by microorganisms. Literature provides evidence that long-chain aliphatic esters undergo common metabolic pathways and are excreted or used as energy source for catabolism by both aquatic and terrestrial microorganisms. No selective toxicity for the aquatic compartment was observed. Furthermore, no chronic or long-term effects in aquatic organisms up to the water solubility limit and no acute effects for aquatic organisms above 10 mg/L were determined. Besides, an extensive elimination of the substance in sewage treatment plants, a low bioavailability and a low bioaccumulation potential is expected for the target substance. Thus, no further study on the effects on terrestrial organisms need to be conducted for Fatty acids, C16-18 (even numbered), esters with glycerol oligomers.