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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
Study was conducted in accordance with a recognized scientific procedure for determining biodegradability. Study was conducted in compliance with GLP. The study meets national and international scientific methods and provides sufficient information to support the conclusion.
Qualifier:
according to
Guideline:
OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
 Activated sludge from the HRC sewage treatment plant. The nature of the influent to this treatment plant is not known. The activated sludge was filtered through Whatman No 1 paper (first 200 ml discarded) and the filtrate was kept aerated until used. 
Duration of test (contact time):
28 d
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
The carbon content of the test substance was calculated from the empirical formula and sufficient added to 2 litres of culture medium to give a nominal carbon content of 40 mg C/l. The solution was then split into two 1 litre replicates and inoculated with activated sludge at a rate of 0.5 ml/l. Duplicate negative controls were also prepared. The test was conducted in  darkness. Vessels were stirred using a magnetic stirrer. The test solutions were maintained at a nominal temperature of 22 °C, with cooling by cold-finger condenser. Evaporative losses were made up with distilled water.  Samples were taken for analysis on days 0, 7, 14, 21, 27 and 28.  
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (DOC removal)
Value:
0
Sampling time:
28 d
Details on results:
Briquest 543-33S attained 0% degradation after 28 days and was not considered readily biodegradable under OECD Guideline No. 301-E. The control substance attained 85% degradation after 14 days and 97% after 28 days, confirming suitability of the inoculum and culture conditions. Degradation products: no
Results with reference substance:
Day 7: 79%
Day 14: 85%
Day 28: 97%
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance achieve 0% biodegradation in 28 days using a relevant test method. The result is considered to be reliable.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
Study was conducted in accordance with a recognized scientific procedure for determining biodegradability. Study was conducted in compliance with GLP. The study meets national and international scientific methods and provides sufficient information to support the conclusion.
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Details on results:
RESULTS: Briquest 543-45AS attained 0% degradation after 28
days and was not considered biodegradable under OECD
Guideline No. 302B. The standard Digol attained 98%
degradation after 28 days confirming suitability of the
inoculum and culture conditions.
Interpretation of results:
under test conditions no biodegradation observed
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2006-07-05 to 2006-10-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was conducted according to an appropriate OECD test guideline, with acceptable restrictions. The restrictions were preadapted inoculum was used and inorganic phosphate was left out of the nutrient medium.
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
preadapted inoculum was used and inorganic phosphate was left out of the nutrient medium.
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Principles of method if other than guideline:
NB It was concluded from a previous study that the OECD 306 test (biodegradability in seawater) does not appear to be a suitable screening test for the selection of phosphonates that have a high potential to biodegrade. It is believed that the capability of micro-organisms to break the C-P bonds in the phosphonates may play an important role in the contradictory results found in an OECD 306 study. The composition of the micro-organisms in the seawater may fluctuate and there is a chance that in one batch of seawater the “right” micro-organisms are present, whereas these are absent in another batch. Another factor of influence is the presence of phosphate in the test media. As C-P cleavage activities by microorganisms can be induced when the availability of inorganic phosphate is limited, a more optimal degradation of phosphonates is expected in test media with no or low concentrations of phosphate.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: secondary effluent produced in Husmann unit from sludge from domestic WTP
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Domestic WTP in Driebergen, The Netherlands.

- Pretreatment: Filtered 2mm. Suspended solid concentration brought to 2.5 g dry weight/l by dilution with tap water. Sludge placed in Husmann unit and was stabilized for one week and pre-adapted for two weeks to a mixture of the ten test substances of which the total influent concentration of each test substance gradually increased to 0.1 mg COD/l within one week. 5 ml aliquot of the supernatant of the Husmann unit was used to inoculate one litre of nutrient medium. The supernatant is considered equivalent to the secondary effluent described as an inoculum in the OECD 301D guideline
Duration of test (contact time):
28 d
Initial conc.:
1 mg/L
Based on:
COD
Initial conc.:
2.5 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Dilutions of test substance stock solution were made in natural seawater, enriched with nutrients to give final nominal test substance concentrations. This series was completed with a blank control, containing only inoculated nutrient medium.

- pH: 6.5 to 7.7

- Test temperature: 20 ±2 °C

TEST SYSTEM
- Culturing apparatus: 300 ml volume BOD bottles.

- Number of culture flasks/concentration: Two concentrations (1.0 and 2.5 mg COD/l; 6.72 and 16.79 mg/l) of test substance, a blank control, an inoculum activity control, a toxicity control, a sterile control and a sterile blank control. Four replicates of each.

- Method used to create aerobic conditions: Incubation solutions saturated with oxygen prior to incubation.

SAMPLING
- Sampling frequency: The O2 concentration was determined with a Luminescent Dissolved Oxygen sensor at the start of the test and at regular intervals during the test: 0, 7, 14, 21 and 28d.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
- Other: blank sterile control
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
17
Sampling time:
7 d
Remarks on result:
other: 6.72 mg/l
Parameter:
% degradation (O2 consumption)
Value:
7
Sampling time:
28 d
Remarks on result:
other: 16.79 mg/l
Details on results:
Reference substance: Degraded 72% after 14 d.
Toxicity control: More than 25% degradation within 14d.
The test fulfills the conditions of validity of the guideline.
Conclusions:
Biodegradation rates of 17% in 7 days (6.72mg/l) and 7% in 28 days (16.79mg/l) were determined in a reliable study conducted according to an appropriate test protocol, but not conducted according to GLP. Preadapted inoculum was used and inorganic phosphate was left out of the nutrient medium.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
EU Method C.12 (Biodegradation: Modified SCAS Test)
Deviations:
yes
Remarks:
(extended test duration, lower test substance concentration)
Principles of method if other than guideline:
TEST TYPE:  Semi-continuous Activated Sludge Procedure using C-14 labelled test material.
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge from a domestic sewage treatment plant. Mixed liquor was taken (activated sludge and supernatant) from a domestic STP at  a nominal suspended solid concentration of 2500 mg/l. 
Duration of test (contact time):
220 d
Initial conc.:
3.3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Remarks:
(based on direct liquid scintillation counting in liquor at the beginning and end of the cycle)
Details on study design:
Mixed liquor was taken (activated sludge and supernatant) from a domestic STP at  a nominal suspended solid concentration of 2500 mg/l. At the beginning of each cycle (24 hours per day, 72 hours at the weekend), 5 mg of radiolabelled Dequest 2060 (not: it is not clearly stated in the report whether this is as pure phosphonate or as aqueous phosphonate i.e. equivalent to 2.5 mg active acid) in 2 ml water was added to the mixed liqour along with synthetic sewage sludge. Aeration was maintained until the end of each cycle, at which time the sludge was settled and 1 litre of supernatant liquid removed. The cycle was then re-initiated by  the addition of tap water, sewage and a further aliquot of test material solution. The pH and settled sludge volume were monitored for each cycle and the suspended solids concentration weekly.  Testing was carried out over a 7-month period.
Reference substance:
not required
Parameter:
% degradation (radiochem. meas.)
Value:
3.51
Sampling time:
24 h
Remarks on result:
other: ± 0.94 % (95% confidence limits). Results for 24h cycle
Parameter:
% degradation (radiochem. meas.)
Value:
0.87
Sampling time:
72 h
Remarks on result:
other: ± 2.28 % (95% confidence limits). for 72-hour cycle.
Details on results:
The length of time the SCAS unit was acclimated to the test material had no significant effect on the observed degradation rates.
Results with reference substance:
Not required
Validity criteria fulfilled:
not applicable
Interpretation of results:
not inherently biodegradable
Conclusions:
The results suggest that Dequest 2060 cannot be treated with any great success in a secondary sewage treatment plant.
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
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:
read-across: supporting information
Key result
Parameter:
% degradation (radiochem. meas.)
Value:
3.51
Sampling time:
24 h
Key result
Parameter:
% degradation (radiochem. meas.)
Value:
0.87
Sampling time:
72 h

Description of key information

The substance is not readily or inherently biodegradable, based on several reliable studies (0 - 7% biodegradation in 28 days)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A reliable ready biodegradability study is available (OECD 301E, Huntingdon, 1984), indicating 0% biodegradation in 28 days based on DOC removal.

A second reliable ready biodegradability study using pre-adapted inoculum (OECD 301D, TNO, 2006) indicated 7% biodegradation in 28 days based on O2 consumption. (Note: This is a standard study with some acceptable deviations: pre-adapted inoculum was used and inorganic phosphate was omitted from the nutrient medium. In addition, solutions of test substance stock solution were made in natural seawater).

A reliable inherent biodegradation test study (Safepharm 1999) indicated 0% biodegradation in 28 days.

A reliable SCAS test (Saeger, 1978) indicated 3.5% biodegradation in a 24 h cycle.

A reliable anaerobic biodegradability study (Zeneca, 1995) indicated 6 - 8% biodegradation in 56 days.

Furthermore, the results of a reliable biodegradability in seawater study (TNO, 1996) (refer to Section 5.2.2) supports the conclusion that the substance is not expected to be readily biodegradable.

There are many biodegradation results available. Only the most reliable are listed here. PFA (2010) gives a fuller set of results and an overview. The other studies, of low reliability, are not reported in detail here since they add nothing further to the overall understanding.