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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:
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

 

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.

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

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:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Qualifier:
according to
Guideline:
EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)
GLP compliance:
yes (incl. certificate)
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.
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
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
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

 

Description of key information

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

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:
1 000 mg/kg soil dw

Additional information

CAS 25151-96-6

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.