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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water and sediment: simulation testing, other
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 scientific principles, acceptable for assessment. Not conducted to GLP.
Principles of method if other than guideline:
Biodegradation test using up-flow conditions in bioreactor; 76-day duration. Parallel tests of substance as such and of substance previously subject to abiotic degradation by UV-exposure were conducted for comparison. Both sets of results are described here.
GLP compliance:
no
Remarks:
The study was not conducted for the purpose of compliance with REACH.
Radiolabelling:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial, non-adapted
Duration of test (contact time):
76 d
Details on study design:
Biodegradation in bioreactor test:
For testing the biological degradation of untreated HDTMP, three equal aerobic bioreactors were set-up operating as up-flow system. The reactors had a working volume of 1.5 liter. The influent flow was set to 0.5 L/d, controlled by a pump (Ismatec, Switzerland). The bioreactors operated at room temperature without temperature control. The reactors were operated for 76 days and fed with synthetic feed composed only of tap water and the specific phosphonate by means 80 mg/L for DDTMP, 100 mg/L for HDTMP and 120 mg/L for EDTMP. The difference in the concentration was in order to severe quantities of similar carbon to the biomass. Thus, the daily reactor load achieved 40 mg for DDTMP, 50 mg for HDTMP and 60 mg for EDTMP respectively. 20 g of the carrier material Lewatit MP-62 (Fluka Analytical, Germany) was added to all bioreactors to support both the adsorption of the phosphonates and attachment of microorganisms to build up a biofilm for optimal degradation. The three reactors were inoculated with 100 ml activated sludge from an industrial wastewater plant (Leuna, Saxon-Anhalt, Germany). After 23 days reactor operation additional 100 ml sludge from the same wastewater plant was added to each
bioreactor. For the reactor monitoring samples were frequently collected from the water bath buffer integrated in the system (Grant GD 120, UK).

Biodegradation of photodegradation by-products test:
The Biological Oxygen Demand (BOD) of HDTMP was analyzed to compare the biodegradability of the by-products subsequent to UV treatment. Three different conditions were tested: 1) without chemical treatment, 2) 5h UV exposure and 3) 5h UV exposure with Mn. For the test were used 250 ml of the phosphonate solution and 1 ml of the activated sludge from an industrial wastewater plant (Leuna, Saxon-Anhalt, Germany). The test was carried out for 20 days.
% Degr.:
58.8
Parameter:
TOC removal
Sampling time:
20 d
Remarks on result:
other: Biodegradation following abiotic treatment (5h exposure, UV alone)
% Degr.:
19.6
Parameter:
other: BOD / COD
Sampling time:
20 d
Remarks on result:
other: Biodegradation following abiotic treatment (5h exposure, UV alone)
% Degr.:
36
Parameter:
TOC removal
Sampling time:
20 d
% Degr.:
20.1
Parameter:
other: BOD / COD
Sampling time:
20 d
% Degr.:
22.1
St. dev.:
5.92
Parameter:
TOC removal
Sampling time:
76 d
% Degr.:
14.7
St. dev.:
7.81
Parameter:
other: "COD"
Sampling time:
76 d
Transformation products:
not measured
Validity criteria fulfilled:
not applicable
Conclusions:
A reliable test demonstrates that biodegradation subsequent to abiotic degradation (UV exposure, 5 h) results in significantly higher removals of HMDTMP (based on TOC) than biodegradation alone, over 20-day duration period. A longer-duration bioreactor test showed significant removals based on TOC.
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:
other: 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 HMDTMP in natural river and lake waters with analysis of ¹⁴CO2 to indicate the extent of biodegradation.
GLP compliance:
no
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.:
>= 3.8 - <= 8.6
Parameter:
radiochem. meas.
Sampling time:
60 d
Remarks on result:
other: Dark conditions
Key result
% Degr.:
>= 7.7 - <= 11.9
Parameter:
radiochem. meas.
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 light

Lake Sterile plus light

River microbial

Lake microbial

River Microbial plus light

Lake Microbial plus light

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

0.37

0.13

3.02

5.36

3.82, 5.17

8.56, 8.04

7.69, 8.34

11.87, 9.75

 


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. 4 - 9% after 60 days was determined in a reliable study conducted according to generally accepted scientific principles. In the presence of natural light, ca. 8 - 12% degradation was observed over the same time period.
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:
other: The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP. The study did not determine the identity of degradation products.
Principles of method if other than guideline:
Natural water - sediment microcosms. ¹⁴C labelled. The study used radiolabelled HMDTMP 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): 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): 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 d
Initial conc.:
429 other: ppb (in well water/Missouri 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.

¹⁴C-labeled test substance (Dequest 2051) added to give 1000 ppb.

Samples of water column removed at day 0 and periodically thereafter and analysed for ¹⁴C activity. At end of test sediment dry weight determined and sediment burned in an oxidizer to determine ¹⁴C activity.
Reference substance:
other: Dextrose
Compartment:
other: water, material (mass) balance
% Recovery:
6.5
Remarks on result:
other: 6.0% and 6.9% in two replicates
Compartment:
other: sediment, material (mass) balance
% Recovery:
54
Remarks on result:
other: 50.4% and 57.5% in two replicates
Key result
% Degr.:
>= 38.9 - <= 41
Parameter:
radiochem. meas.
Sampling time:
38 d
Remarks on result:
other: other: Well water - Missouri river 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 38.9 -41.0%.

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 HMDTMP in a water-sediment microcosm of 38.9 - 41.0 % after 38 days (under sunlight conditions) were determined in a reliable study conducted according to generally accepted scientific principles.
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:
HYPOTHESIS FOR THE CATEGORY APPROACH
HMDTMP acid is a member of the HMDTMP category, which is comprised of the acid form and various salts of the same acid. In the context of the RAAF, read-across of environmental, ecotox, human health and physicochemical data within the HMDTMP Category is considered as Scenario 6.

SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
This category covers various sodium and potassium salts of [hexane-1,6-diylbis-[nitrilobis(methylene)]tetrakisphosphonic acid. The different salts are prepared by neutralising the acid to a specific pH and accordingly the constituents proportions and degree of ionisation are comparable between substances under similar conditions (in vivo and in the environment). All category members are based on the HMDTMP structure.
Since the salts are prepared from the acid, the impurity profile for HMDTMP acid is also typical of the salts in this Category, although acidic impurities would also be present as salts. Typically, impurities include residual inorganic acids/salts and organic by-products from manufacturing.

CATEGORY APPROACH JUSTIFICATION
The category hypothesis is that all the members are various ionised forms of the same parent acid. This category covers various sodium and potassium salts of [hexane-1,6-diylbis-[nitrilobis(methylene)]tetrakisphosphonic acid. The different salts are prepared by neutralising the acid to a specific pH and accordingly the constituents proportions and degree of ionisation are comparable between substances under similar conditions (in vivo and in the environment). All category members are based on the HMDTMP structure. Data are available for the acid form and some salts. Only acid aqueous solutions are commercially available. The properties of the members of the category are consistent across all endpoints.
Contributing effect of counter-ions:
The approach assumes in general that alkaline metal (sodium and potassium) counter-ions are not significant in respect of all the properties under consideration.
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. In dilute aqueous conditions of defined pH and mineral composition, a salt or complex introduced into this medium 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.
Where data are available across different salt forms of HMDTMP, this approach is supported.

DATA MATRIX
A data matrix table of all the data available on the HMDTMP Category members is available in Section 13 of the technical dossier.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
% Degr.:
< 9
Parameter:
radiochem. meas.
Sampling time:
60 d
Remarks on result:
other: Dark conditions
% Degr.:
> 8 - < 60
Parameter:
other: radio chem. meas. or TOC removal
Sampling time:
76 d
Remarks on result:
other: active conditions with light (across 3 studies, 20 - 76 d duration)
Transformation products:
not measured

Description of key information

Three reliable simulation studies of HMDTMP-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 HMDTMP-H and its salts, the role of abiotic removal processes is significant. The key data for soil adsorption are from the study by Michael (1979) (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 (Saeger, 1979). 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 HMDTMP-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 degradation study using radiolabelled HMDTMP in natural fresh waters in sunlight and dark conditions is available (Saeger, 1978). Degradation (mineralisation) in river and lake waters of ca. 3 - 9% after 60 days was determined in a reliable study conducted according to generally accepted scientific principles. In the presence of natural light, 7 - 12% 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 degradation study using radiolabelled HMDTMP in a natural fresh water / sediment microcosm system is available from an existing study (Saeger, 1979). This study used exposure over a duration of 38 days in a water-sediment microcosm using samples sourced from a river location. In this study, degradation (mineralisation) of HMDTMP-H of 38.9 - 41.0% after 38 days (under sunlight conditions) were determined. The radiochemical analysis showed a mass balance of 6.0 - 6.9% in water; 50.4 - 57.5% in sediment; 38.9 - 41.0% released as ¹⁴CO2 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.

A further degradation study using HMDTMP (Brandenburg UT, 2010) compared biodegradation based on TOC following 5 h UV exposure compared to biodegradation without pre-treatment. This showed 52.7 - 58.8% degradation after UV exposure pre-treatment, compared to biological TOC degradation of 36.0% without pre-treatment. 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.

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.