2-phosphonobutane-1,2,4-tricarboxylic acid

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not indicated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- The purity of the test item is not indicated. - No water-sediment system as described in OECD guideline 308 was used but the inocula gained from river water and river sediment were separately tested for their ability to degrade PBTC. - The strains gained from three ecosystems (river water and sediment and activated sludge) that were identified to degrade PBTC are not clearly assignable (by the reader) to river water and river sediment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 308 (Aerobic and Anaerobic Transformation in Aquatic Sediment Systems)

Deviations:

yes

Remarks:

inocula gained from river water and river sediment were separately tested

Principles of method if other than guideline:

No water-sediment system as described in OECD guideline 308 was used but the inocula gained from river water and river sediment were separately tested for their ability to degrade PBTC.

Background of this study: As PBTC is not easily biodegradable in standard test and not inherently biodegradable according to Zahn-Wellens test, PCBT was investigated for biodegradability if it is the sole source of carbon and of phosphorus of if an alternative carbon source is offered (utilisation of the phosphate only as source of phosphorus) or in the presence of only a low concentration of inorganic carbon. The cleavage of the C-P-bond of PBTC is required since the microorganisms are only able to use inorganic phosphate. The inoculum used was enriched from river sediments and from river waters. In the whole four river sediments, two for aerobic enrichment (Rhein and Wupper) and two for anaerobic enrichment (Rhein and Wupper), and two river waters (Rhein and Wupper) were taken. The river waters and the sediments were used for enrichment of inocula. No water-sediment system as described in OECD guideline 308 was used but the inocula gained from these samples were separately tested for their ability to degrade PBTC.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate / analogue material are not available.

Radiolabelling:

no

Oxygen conditions:

aerobic/anaerobic

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

The river waters were taken from Rhein and Wupper, two rivers in Germany.

Details on source and properties of sediment:

The river sediments were taken from Rhein and Wupper, two rivers in Germany.

Details on inoculum:

Enrichment of inocula from river water (aerobic):
The river waters were added at a rate of 10% per volume (30 mL/300 mL).

Enrichment of inocula from sediment (aerobic and anaerobic):
The inocula were enriched by a certain procedure from two river sediments (Rhein and Wupper).
The sediment samples were suspended in mineral solution (about 10g sediment/ 100 mL solution = inoculum).
The inocula solution were diluted with mineral solution until an OD578 (optical density at 578 nm) of about 0.1 was reached.

For further testing the cultures were highly enriched.
Subsequently, pure cultures (isolates) were gained from these enrichment cultures.
From the isolates certain, effective strains were characterized and used for testing (single or in combination).

The following duration refers to test with the highly enriched cultures.

Duration of test (contact time):

36 - ca. 60 h

Initial conc.:

1 mmol/L

Based on:

test mat.

Initial conc.:

10 mmol/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

other: decrease of Pn (= Phosphonate-phosphorus); Pn = P(total)-Pi; Pi = Orthophosphate

Details on study design:

- The test material PBTC was used as recrystallised disodium salt but the concentrations were calculated on the basis of the free acid.
- The aerobic enrichment was carried out with 300 mL in 1L erlenmeyer flask.
- The anaerobic enrichments were carried out with 100 mL in 120 mL headspace bottles.
- All enrichments were carried out in duplicate.
- After all initial enrichments were tested, two successive transfers of the initial inoculum were made to get highly enriched cultures with a good degradation activity. All enrichments of the first inoculation series were transferred as far as they reached degradation levels of about 15% (after approximately 10 days = 1st transfer). The transfers were done with 1% volume (3 mL/ 300 mL). All enrichments were transferred again after a period of 7 days (2nd transfer).
- The PBTC degradation due to the second transfer ("highly enriched cultures") was measured at 36h and 60h.
- Further test were performed with pure cultures that were isolated from the all enrichment cultures on agar plates using standard techniques.
- 2 pure cultures were isolated of the culture enrichments from river water and 2 pure cultures were isolated of the culture enrichments from river sediments that all were able to use PBTC as a source of phosphorus in the presence of EtOH as an alternative source of carbon. In the whole three isolates were further investigated and their strains characterized.
- Degradation tests were performed with isolation of pure cultures and single strains or a combination of strains.

Compartment:

natural water / sediment: freshwater

Remarks on result:

not measured/tested

Transformation products:

not measured

Remarks:

see 'Details'

Details on transformation products:

If PBTC degrades, orthophosphate and one ore more carbon containing metabolites from C4-backbone are formed. While orthophosphate could analytically be verified, the identification of metabolites as BTCA (butane-1,2,4-tricarboxylic acid) and OH-BTCA (2-hydroxybutane-1,2,4-tricarboxylic acid) was not successful.

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

not specified

Details on results:

Aerobic degradation tests with enrichment cultures:
- PBTC as a sole source of carbon and either with 0.1 mmol orthophospathe or PBTC (1 or 10 mmol) as the source of phosphorus gave negative results. No significant decrease of PBTC concentrations or increase of biomass was observed. - see Table 2
- PBTC (1 mmol) as a sole source of phosphorus but in presence of an alternative carbon source [Ethanol (2mL) or Acetate or Pyruvate (2g/L)] resulted in degradation of PBTC. This result was optimized by more enriched cultures (see below). - see Table 2

Aerobic degradation tests with highly enriched cultures:
- PBTC (1 mmol) as a sole source of phosphorus but in presence of an alternative carbon source [Ethanol (2g/L)] with highly enriched cultures of river water and river sediment resulted in nearly complete degradation after 60 h. - see Table 3

Degradation tests with isolates of pure cultures and single strains or a combination of strains:
- None of the pure cultures was able to degrade PBTC as a sole source of carbon, both with PBTC and orthophosphate as sources of phosphorus.
- Opposed to that, all strains tested, degrade more than 90% of 0.2 mmol of PBTC after 200 h.
- PBTC was rapidly degraded, even (!) in the presence of 0.01 mmol inorganic phosphate (0.95 mg/L) with a certain strain. As this concentration is almost the same as in environmental surface water (river Rhein: 0.8 mg/L), the result indicate that the bacteria isolated from environmental samples may be able to degrade PBTC under environmental conditions.
- The degradation was improved (faster degradation of higher initial concentrations) by combining certain strains.
- In addition to strains identified to have a fast degradation potential, two stains also exhibit low degradation under anaerobic conditions. The publication does not state if the latter strains could be assigned also to river water and river sediment.
- Abiotic degradation was not observed.

Results with reference substance:

NA

Table 2: Results of different combinations of carbon and phosphorus sources added to the mineral media of the enrichment cultures

Carbon Source	No (additional) Phosphorus Source / no Phosphorus	Phosphorus Source: PBTC (1 millimol)	Phosphorus Source: PBTC (10 millimol)	Phosphorus Source: Orthophosphate (0.1 millimol)	Result
Test substance. PBTC (1 millimol) = sole carbon source	-	+	-	-	aerobic: negative (no significant decrease of PBTC concentration, no increase of biomass)
Test substance: PBTC (10 millimol) = sole carbon source	-	-	+	+	aerobic: negative (no significant decrease of PBTC concentration, no increase of biomass)
Test substance PBTC (10 millimol) and alternative source of carbon	+	+	-	-	aerobic: positive (degradation was observed and optimized by highly enriched cultures)
Positive Control: Ethanol (2mL) or Acetate or Pyruvate (2g/L) as alternative source of carbon	-	-	-	+	positive (good growth of inocula on Sodium acetate trihydrate)
Negative Control: Ethanol (2mL) or Acetate or Pyruvate (2g/L) as alternative source of carbon	+	-	-	-	negative (no significant growth of inocula)

Table 3: Level of PBTC-degradation after the 2nd transfer of enrichment cultures with 1 mmol PBTC as the sole source of phosphorus in the presence of EtOH (2 g/L) as an alternative source of carbon

Inocula	Carbon Source	No (additional) Phosphorus Source / no Phosphorus	Phosphorus Source: PBTC (1 millimol)	Result, degradation after 36 h	Result, degradation after 60 h
River water	Test substance PBTC (1 millimol) and Ethanol (2g/ L)	+	+	83	99
River sediment	Test substance PBTC (1 millimol) and Ethanol (2g/ L)	+	+	91	99

Validity criteria fulfilled:

not specified

Conclusions:

2-phosphonobutane-1,2,4-tricarboxylic acid as sole source of carbon, both with 2-phosphonobutane-1,2,4-tricarboxylic acid and orthophosphate as sources of phosphorus, is not biodegradable by enrichment cultures from river water and river sediment but by certain strains gained from these cultures.
2-phosphonobutane-1,2,4-tricarboxylic acid is also biodegradable by enrichment cultures from river water and river sediment if an alternative source of carbon is available, even if inorganic phosphate is present (in sub-ppm range). Both (alternative carbon source, inorganic phosphate) are present in many environmental surface water. The biodegradation is more effective and faster if a certain strain or strain combination gained from this both ecosystems was used.
Slow degradation under anaerobic conditions for cultures from rivers sediment and river water is not clearly stated by the publication but can be strongly be assumed based on the presented information.
In conclusion, 2-phosphonobutane-1,2,4-tricarboxylic acid is biodegradable under environmental conditions.
Abiotic degradation was not observed.

Executive summary:

Opposed to OECD guideline 308, not a water-sediment system but the inocula gained from river water and river sediment were separately tested for their ability to degrade 2-phosphonobutane-1,2,4-tricarboxylic acid.

2-phosphonobutane-1,2,4-tricarboxylic acid as sole source of carbon, both with 2-phosphonobutane-1,2,4-tricarboxylic acid and orthophosphate as sources of phosphorus, was found to be not biodegradable by enrichment cultures from river water and river sediment. Biodegradability was found either by certain strains gained from these cultures or if an alternative source of carbon is available. In the latter case degradation was observed, even if in the presence if inorganic phosphate. Both (alternative carbon source, inorganic phosphate) are present in many environmental surface water. Thus,

2-phosphonobutane-1,2,4-tricarboxylic acid is biodegradable under environmental conditions equivalent / similar to the test conditions. The biodegradation was shown to be more effective and faster if a certain strain or strain combination gained from this both ecosystems was used. Slow degradation under anaerobic conditions for cultures from rivers sediment and river water is not clearly stated by the publication but can be strongly assumed based on the presented information. Abiotic degradation was not observed.

 

The study has shown that biodegradation of 2-phosphonobutane-1,2,4-tricarboxylic acid in river water and river sediment under environmental conditions primarily depends on the presence of an alternative carbon source and could be optimized certain strains that can easily be enriched and isolated from these both compartments.

Description of key information

PCBT as sole source of carbon, both with PBTC and orthophosphate as sources of phosphorus, is not biodegradable by enrichment cultures from river water and river sediment but by certain strains gained from these cultures.
PBTC is also biodegradable by enrichment cultures from river water and river sediment if an alternative source of carbon is available, even if inorganic phosphate is present (in sub-ppm range). Both (alternative carbon source, inorganic phosphate) are present in many environmental surface water. The biodegradation is more effective and faster if a certain strain or strain combination gained from this both ecosystems was used.
Slow degradation under anaerobic conditions for cultures from rivers sediment and river water is not clearly stated by the publication but can be strongly be assumed based on the presented information.
In conclusion, PBTC is biodegradable under environmental conditions.
Abiotic degradation was not observed.