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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-28 to 2017-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(4-amino-1-hydroxy-1-phosphonobutyl)phosphonic acid hydrate
EC Number:
680-378-7
Cas Number:
138624-11-0
Molecular formula:
C4H15NO8P2
IUPAC Name:
(4-amino-1-hydroxy-1-phosphonobutyl)phosphonic acid hydrate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 610-GTH
- Expiration date of the lot/batch: 2019-03-31
- Purity test date: 2017-03-30

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: 8.1 g/L in water

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Since Alendronic acid monohydrate, CAS # 138624-11-0 was easily soluble in water, the test
media were prepared using a stock solution of 1 g/L in Milli-RO water (tap water purified by
reverse osmosis; Millipore Corp., Bedford, Mass., USA). A weighed amount of 499.61 mg of
Alendronic acid monohydrate, CAS # 138624-11-0 was dissolved in Milli-RO water and
made up to 500 mL. After stirring for 3.5 hours, the final stock solution was clear and
colourless. The pH of the stock solution was 2.6. Aliquots of 133 mL of the stock solution
were added to the test item bottles A and B and to the toxicity control. These test bottles
contained medium with microbial organisms (final volume: 2 litres). The test solutions were
continuously stirred during the test, to ensure optimal contact between the test item and the

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage length: The inoclum was used immediately.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (31 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: 3.6 g/L of suspended solids
- Initial cell/biomass concentration: 10 ml/L of mineral medium
- Water filtered: yes
- Filter: Acitvated carbon
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 1 g/L
Based on:
formulation
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium:
A)8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B)22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C)36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D)0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
- Test temperature: 22.2 to 23.7 oC
- pH: 7.4 ± 0.2
- pH adjusted: no
- CEC (meq/100 g):
- Suspended solids concentration: 3.6 g/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: A flask containing the inoculum and test item coupled to 3 CO2 absorbers on the exit air line of the flask. No light was allowed.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Aeration at 30-100 mL/mion with sythetic air (80% N2 20% O2)
- Measuring equipment: See detail of trap for CO2
- Test performed in open system: Yes
- Details of trap for CO2: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers
was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series.
- Sample storage before analysis: Stored overnight and aerated

CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Toxicity control:

STATISTICAL METHODS:
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
No preliminary study was conducted.
% Degradationopen allclose all
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Positive Control
Value:
ca. 0
Sampling time:
1 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Positive Control
Value:
ca. 22
Sampling time:
4 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Positive Control
Value:
ca. 42
Sampling time:
6 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Positive Control
Value:
ca. 58
Sampling time:
8 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Positive Control
Value:
ca. 71
Sampling time:
11 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Positive Control
Value:
ca. 85
Sampling time:
15 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 0
Sampling time:
1 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 0
Sampling time:
4 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
6 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 2
Sampling time:
8 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 2
Sampling time:
11 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 3
Sampling time:
15 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 3
Sampling time:
19 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 3
Sampling time:
22 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 6
Sampling time:
25 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 7
Sampling time:
29 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
1 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
4 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
8 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
11 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
15 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 1
Sampling time:
19 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 2
Sampling time:
22 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 3
Sampling time:
25 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 6
Sampling time:
29 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Toxicity Control
Value:
ca. 0
Sampling time:
1 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Toxicity Control
Value:
ca. 16
Sampling time:
4 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Toxicity Control
Value:
ca. 22
Sampling time:
6 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Toxicity Control
Value:
ca. 26
Sampling time:
8 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Toxicity Control
Value:
ca. 30
Sampling time:
11 d
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
Toxicity Control
Value:
ca. 37
Sampling time:
15 d
Details on results:
On day 29 of the groups that reached that far, all 3 CO2 collecting bottles were collected and analyzed to determine the amount of CO2 produced. For those bottles which only were tested until day 15, the same procedure was done on that day.

BOD5 / COD results

Results with reference substance:
The reference subststance showed a normal biodegredation curve.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Alendronic acid monohydrate, CAS # 138624-11-0 was not readily biodegradable under the conditions of the modified Sturm test presently performed.