[[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 306 (Biodegradability in Seawater)

Deviations:

yes

Remarks:

The nutrient stock solution contained 50.2g (not 48.77g) of Na2HPO4.7H2O

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural water

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): A sample of natural seawater was collected from the Eastern Scheldt in the Netherlands (Jacobahaven) about 2.5 meters above the sea bed.
- Temperature (°C) at time of collection: 20°C
- pH at time of collection: not reported
- Hardness (CaCO3): not reported; Salinity: 29.3%
- Dissolved organic carbon (%): 1.0 mg C/l
- Biomass (e.g. in mg microbial C/100 mg, CFU or other): 5750 and 9000 CFU/ml after 1 and 3 weeks respectively (incubated at 20 +/-2°C)
- Water filtered: After sedimentation of the coarse particle the seawater was decanted over a sieve and aerated until start of test.
- Type and size of filter used, if any: not specified.

Details on inoculum:

no additional inoculum used (refer to 'Details on source and properties of surface water' above)

Duration of test (contact time):

28 d

Initial conc.:

13.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS

- Test temperature: 20.0-20.6°C

- pH: 7.9-8.0

- pH adjusted: no

- Continuous darkness: yes

TEST SYSTEM

- Culturing apparatus: BOD bottles with a volume of about 293ml

- Number of culture flasks/concentration: 5 inoculum blank, 5 test material, 3 reference substance, 3 toxicity control

SAMPLING

- Sampling frequency: 0, 5, 15, 28 days

- Sampling method: a separate set (3 to 5 bottles) of each treatment was sacrificed for each measurement.

CONTROL AND BLANK SYSTEM

- Inoculum blank: yes

- Toxicity control: yes

Reference substance:

acetic acid, sodium salt

% Degr.:

< 0

Parameter:

O2 consumption

Remarks:

Relative to theoretical oxygen demand

Sampling time:

28 d

Remarks on result:

other: <0% biodegradation reported at 5d, 15d and 28d timepoints in the test.

Transformation products:

not measured

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

not measured

Details on results:

The oxygen depletion in the inoculum blank was 2.63 mg O2/l at the end of the test (28 days). This is more than the maximum value of 2mg O2/l prescribed by the guideline but is characteristic of the natural seawater used and is not expected to influence the ready biodegradability.
The oxygen depletion in the toxicity control was lower than that in the reference substance. This indicated that the test material inhibited the degradation of the reference substance.
Oxygen depletion was inhibited by the test substance after 28 days of incubation in natural seawater medium. The test material is therefore considered to be not ready biodegradable in this test.

Results with reference substance:

Rapid degradation (almost complete within five days). A toxicity control test demonstrated the presence of sodium acetate plus test substance indicated inhibition of the inoculum activity, compared to the sodium acetate only, but this was not quantified.

Table 1: Oxygen concentrations in the individual bottles determined after 0, 5, 15, and 28 days incubation with Dequest 2066.

Addition to flask	O2(mg/l) concentration after n days
	0	5	15	28
Inoculum blank	7.47	6.90	5.64	4.79
Inoculum blank		6.89	5.55	4.78
Inoculum blank		6.87	5.60	4.82
Inoculum blank				4.84
Inoculum blank				4.67
Test substance	7.41	6.93	6.64	6.18
Test substance		6.89	6.55	6.15
Test substance		6.85	6.62	6.17
Test substance				6.15
Test substance				6.27
Reference substance	7.37	4.44	2.97
Reference substance		4.42	3.11
Reference substance		4.41	3.20
Toxicity control	7.40	4.47	3.90
Toxicity control		4.47	3.94
Toxicity control		4.49	3.96

Conclusions:

A degradation rate <0% in 28 days was determined in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

Reference 2

Endpoint:

biodegradation in water and sediment: simulation testing, other

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

In an environmental context the speciation will be controlled by the prevailing conditions and ions present, and will be the same regardless of the starting form. The properties of the members of the category are consistent across all endpoints.
The category hypothesis is that all the members are various ionised forms of the same parent acid. The main assumption is that sodium and potassium are not significant in respect of all the properties under consideration. For ammonium salts the properties and role of ammonia are given due consideration. In dilute aqueous conditions of defined pH a salt will behave no differently to the parent acid, at identical concentration of the particular speciated form present and will be fully dissociated. Hence some properties (measured or expressed in aqueous media, e.g. ecotoxicity) for a salt can be directly read-across (with suitable mass correction) to the parent acid and vice versa, and from one salt to another.

Reason / purpose for cross-reference:

read-across source

% Degr.:

< 0

Parameter:

O2 consumption

Remarks:

Relative to theoretical oxygen demand

Sampling time:

28 d

Remarks on result:

other: <0% biodegradation reported at 5d, 15d and 28d timepoints in the test.

Transformation products:

not measured

Reference 3

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

There are some differences in methodology compared to the current version of OECD TG 309 (specifically, the test was conducted at one test concentration (2 mg/l), with non-standard reference substances; the study did not determine the mass balance in the system or identity of degradation products).

Principles of method if other than guideline:

Natural Water Biodegradation and Photodegradation Monsanto shake flask system for CO2 evolution testing (W.E. Gledhill, App. Microbiol. 30, 922 (1975)). The study used radiolabelled DTPMP in natural river and lake waters with analysis of 14CO2 to indicate the extent of biodegradation.

GLP compliance:

no

Radiolabelling:

yes

Oxygen conditions:

other: both aerated and N2-purged conditions were tested.

Inoculum or test system:

natural water

Details on source and properties of surface water:

TEST DETAILS: Natural waters were obtained from the Meramec River (Kirkwood Park) (pH 7.4, TOC 12 mg/l) and Lake No. 34 - Busch Wildlife Area (pH 8.0, TOC 17 mg/l). These were allowed to settle for 2 days and the supernatant liquid used in 500 ml portions for the test.

Duration of test (contact time):

60 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Six water samples were spiked with a stock solution of the test substance to give a test concentration of 2 mg/l (active acid). Control flasks were similarly spiked with either C-14 labelled linear dodecylbenzene sulfonate (LAS) or glucose (glucose used only for lake water exposure as positive
control). Two of the replicates were then sterilised by the addition of 25 mg HgCl2.
An open reservoir containing 10 ml of 0.5N aqueous KOH was suspended in each flask. After sealing, one set of flasks was placed on a rotary shaker and agitated at 80 rpm at ambient temperature (22 °C) in the dark. A second set was taken to an outdoor platform and exposed to natural sunlight and temperatures.

Reference substance:

other: Linear dodecylbenzene sulfonate

Reference substance:

other: glucose

Compartment:

other: water / sediment, material (mass) balance

Remarks on result:

other: The study report states that residual radiochemical activity was determined in the test solution at the end of the exposure duration, but the results are not reported.

Key result

% Degr.:

>= 2.11 - <= 9.88

Parameter:

CO2 evolution

Remarks:

(14CO2)

Sampling time:

60 d

Remarks on result:

other: Dark conditions

Key result

% Degr.:

>= 9.87 - <= 19.82

Parameter:

CO2 evolution

Remarks:

(14CO2)

Sampling time:

60 d

Remarks on result:

other: In active conditions with sunlight

Transformation products:

not measured

Details on results:

Degradation data during the course of the exposure duration are not reported .

Results with reference substance:

The results with the two reference substances used in the study re shown in Table 1 below.

Table 1: Percent degradation values at 60 days for reference substances and test substance in river water and lake water

Type of suspension	% degradation at 60 days
	River Sterile	Lake Sterile	River Sterile plus sunlight	Lake Sterile plus sunlight	River microbial	Lake microbial	River Microbial plus sunlight	Lake Microbial plus sunlight
Reference Linear dodecylbenzene sulfonate	3.67	1.19	3.60	1.92	32.08	6.34	1.88	15.35
Reference Glucose (Lake only)	-	0.31	-	1.15	-	58.82	-	46.66
Test substance - Dequest 2060	2.80	0.08	1.46 (40 -d)	4.52	2.35 2.11	4.98 9.88	10.14 9.87	17.20 19.82

 

The effect of temperature variation in the sunlight exposures is an unknown factor. Water temperature reached as high as 44 °C during the test. This may have had a significant impact on the microbial population and distribution.

In general, the lake water appeared somewhat more active than the river water with respect to Dequest degradation.

For LAS, the reverse was true. Much more acclimation from previous exposure to LAS in the river than the lake would be
expected. The reason for the higher activity of the lake water to Dequest is unknown.

Validity criteria fulfilled:

not applicable

Conclusions:

Degradation (mineralisation) in river and lake waters of ca. 2 - 10% after 60 days was determined in a reliable study conducted according to generally accepted scientific principles. In the presence of natural light, ca. 10 - 20% degradation was observed over the same time period.

Reference 4

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The study did not determine the identity of degradation products.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Natural water - sediment microcosms. C14 labelled. The study used radiolabelled DTPMP in natural freshwater and sediment microcosm systems with analysis of 14CO2 and 14C radiochemical analysis to indicate the fate of the substance and extent of biodegradation.

GLP compliance:

not specified

Radiolabelling:

yes

Oxygen conditions:

aerobic/anaerobic

Remarks:

Some test systems were purged with N2.

Inoculum or test system:

natural water / sediment

Details on source and properties of surface water:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): In the early phase of the study water was collected from the littoral region of a spring fed freshwater lake (Lake 34, Busch Wildlife Area, St. Charles County, Missouri).
In the latter phase of this study, well water (from St. Charles County farm of Monsanto Agricultural Products Company) was used in the microcosm construction.

- Biomass: mean 1580 bacteria/ml (in the well water/river sediment microcosm)

The bottom of each aquarium was covered with a 1/8" Teflons sheet template fitted with fifteen uniformly spaced No. 7 silicone stoppers. 22 liters of water were added to each aquarium.

Details on source and properties of sediment:

- Details on collection (e.g. location, sampling depth, contamination history, procedure): In the early phase of the study sediment was collected from the littoral region of a spring fed freshwater lake (Lake 34, Busch Wildlife Area, St. Charles County, Missouri).
In the latter phase of this study, Missouri River sediment was used in the microcosm construction.

- Sediment samples sieved: yes. The sediment was screened through a steel screen (0.5 in mesh) to remove large particulates.

The bottom of each aquarium was covered with a 1/8" Teflons sheet template fitted with fifteen uniformly spaced No. 7 silicone stoppers. Eight liters of sediment were added to each aquarium.

Details on inoculum:

n/a: native bacterial populations in natural water and sediment only.

Duration of test (contact time):

38 - 50 d

Initial conc.:

1 000 other: ppb (in Lake 34 microcosm)

Based on:

test mat.

Initial conc.:

331 other: ppb (in well water/river sediment microcosm)

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

Microcosms simulating natural water environment constructed using 10-gallon aquaria and a core-chamber technique. Water and sediment from littoral region of a spring fed freshwater lake (Lake 34 water, Busch Wildlife Area, St Charles County, Missouri). 8 litres sediment; 22 litres water. Microcosms were allowed to stabilise for periods ranging fom one month to four months, with gentle aeration and a 16/8 hr light/dark cycle.

pH 8.4 -8.6; conductivity 420 -460 µmhos; dissolved O2 6.5 - 7.5 before coring.

At the end of stabilization period, core chambers were created by inserting sterile glass cylinders (3.8 x 30 cm) through the water column and sediment onto silicone stoppers. Each chanber contained 150 -175 ml water and 20 - 60 g sediment (dwt). Gas manifolds supplied either CO2 -free air or oxygen-free nitrogen about 5cm above the sediment surface. Exhaust gas was passed through a resin trap to remove volatilised organics and then through a CO2 scubbing system.

Sterile microcosms: core chambers were removed from the aquaria, and water and sediment autoclaved seperately and recombined, and 1 ml sodim azide was added.

C-14 labeled test substance (Dequest 2041) added to give 1000 ppb.

Samples of water column removed at day 0 and periodically thereafter and analysed for C14 activity. At end of test sediment dry weight determined and sediment burned in an oxidizer to determine C14 activity.

Reference substance:

other: Dextrose

Compartment:

other: water, material (mass) balance

% Recovery:

6.3

Compartment:

other: sediment, material (mass) balance

% Recovery:

65.6

Key result

% Degr.:

>= 28.6 - <= 30.7

Parameter:

radiochem. meas.

Sampling time:

38 d

Remarks on result:

other: Well water - Missouri river sediment microcosm (sunlight conditions)

Key result

% Degr.:

>= 14.8 - <= 16.8

Parameter:

radiochem. meas.

Sampling time:

50 d

Remarks on result:

other: Lake 34 fresh water sediment microcosm (sunlight conditions)

Transformation products:

not measured

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Results with reference substance:

The dextrose degradation reached 33.7% by day 3, 57.3% by day 11 and 76.1% by day 38.

>90% decrease in water column C14 activity after 10d. Microcosm variables: aeration vs nitrogen purge; light vs dark; did not seem to significantly affect the rate of removal from the water column. Indicates non-degradative mechanism dominates removal from water column under steriile conditions. However exposure under active conditions with light led to much higher removals than sterile conditions with light, starting within a few days after loading.

Autoclaving sodium azide treatment was not adequate for sterilisation.

In active well water systems, CO₂ evolution ranged from 10.4 -25.2%.

C14 activity was not extracted from sediment to any significant degree with o-xylene (unextracted and extracted had similar % of theory, and extract had low %)

Validity criteria fulfilled:

not applicable

Conclusions:

Degradation (mineralisation) of DTPMP-H in two types of water-sediment microcosms of 28.6-30.7% after 38 days and 14.8-16.8 after 50 days (under sunlight conditions) were determined in a reliable study conducted according to generally accepted scientific principles.

Description of key information

Three reliable simulation studies of DTPMP-H in water and sediment systems are available. Low but recordable levels of removal are seen in such systems, particularly in the presence of natural or simulated light.

Although biodegradation in sediment has not been demonstrated for DTPMP-H and its salts, the role of abiotic removal processes is significant. The key data for soil adsorption are from the study by Michael (undated) (refer to Section 5.4.1 for further information about this test). There is no evidence for desorption occurring. Effectively irreversible binding is entirely consistent with the known behaviour of complexation and binding within crystal lattices. The high levels of adsorption which occur are therefore a form of removal from the environment. After approximately 40-50 days, the phosphonate is >95% bound to sediment with only 5% extractable by ultrasonication and use of 0.25N HCl-xylene solvent (based on radiolabelling) in river and lake water microcosms. (Monsanto internal report, cited by Gledhill and Feijtel, 1992). 66-80% removal (binding) is seen after 11 days in the same test. In the context of the exposure assessment, largely irreversible binding is interpreted as a removal process; 5% remaining after 40 - 50 days is equivalent to a half-life of 10 days which is significant for the environmental exposure assessment in the regional and continental scales. This abiotic removal rate is used in the chemical safety assessment of DTPMP-H and its salts.

Key value for chemical safety assessment

Half-life in freshwater:

10 d

at the temperature of:

12 °C

Half-life in freshwater sediment:

10 d

at the temperature of:

12 °C

Additional information

A reliable study, measured with the sodium salt of the parent acid (DTPMP-7Na, CAS 22042-96-2), indicates a low level of biodegradation in seawater, based on 0% biodegradation in 28d (TNO, 1996). This was selected as the key study (TNO, 1996), with a result of 0% in 28d. This conclusion is based upon a reliable study conducted for the read-across substance DTPMP-xNa according to an appropriate test protocol, and in compliance with GLP. The available data support a lack of significant biodegradation under aquatic simulation conditions in the absence of other factors.

In using a read-across result between the parent acid and sodium salt, the main assumption is that sodium is not significant in respect of all the properties under consideration. In dilute aqueous conditions of defined pH a salt will behave no differently to the parent acid, at identical concentration of the particular speciated form present and will be fully dissociated. Hence some properties (measured or expressed in aqueous media, e.g. ecotoxicity) for a salt can be directly read across (with suitable mass correction) to the parent acid and vice versa. Thus it is acceptable to additionally read across between the parent acid and salts. In the present context the effect of the counter-ion (sodium) will not be significant. In biological systems and the environment, polyvalent metal ions will be present, and the phosphonate ions show very strong affinity to them.

A degradation study using radiolabelled DTPMP-H in natural fresh waters in sunlight and dark conditions is available (Saeger, 1978). Degradation (mineralisation) in river and lake waters of ca. 2 - 10% after 60 days was determined in a reliable study conducted according to generally accepted scientific principles. In the presence of natural light, 10 - 20% degradation was observed over the same time period. There are some differences in methodology compared to the current version of OECD TG 309 (specifically, the test was conducted at one test concentration (2 mg/l), with non-standard reference substances; the study did not determine the mass balance in the system or identity of degradation products), however the deficiencies are not considered detrimental to the chemical safety assessment when this study is considered together with other study results as part of a weight of evidence for the overall environmental fate.

A second degradation study using radiolabelled DTPMP-H in two different natural fresh water / sediment microcosm systems is available from an existing study (Saeger, 1979). This study used exposure over a duration of 38 or 50-days in two water-sediment microcosms using samples sourced from lake and river locations. In this study, degradation (mineralisation) of DTPMP-H in two types of water-sediment microcosms of 28.6-30.7% after 38 days and 14.8-16.8 after 50 days (under sunlight conditions) were determined. The radiochemical analysis showed a mass balance of 6.3 - 9.0% in water; 63-65.6% in sediment; 28.6-30.7% released as¹⁴CO₂ at the end of the test. The study did not determine the identity of degradation products, however this deficiency is not considered detrimental to the chemical safety assessment when this study is considered together with other study results as part of a weight of evidence for the overall environmental fate.