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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-06-10 to 2008-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 314 B (Simulation Tests to Assess the Biodegradability of Chemicals in Wastewater. B: Biodegradation in Activated Sludge)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(1R)-3-[bis(propan-2-yl)amino]-1-phenylpropyl]-4-(hydroxymethyl)phenol
EC Number:
929-946-0
Cas Number:
286930-02-7
Molecular formula:
C22 H31 N O2
IUPAC Name:
2-[(1R)-3-[bis(propan-2-yl)amino]-1-phenylpropyl]-4-(hydroxymethyl)phenol
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 122465-71-dlb001

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: >99%
- Specific activity: 26.7 mCi/mmol, 78.3 µCi/mg

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: Refrigerated
- Solubility and stability of the test substance in the solvent/vehicle: Completely soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluted to a concentration of 99.7 ug/mL in 10:90 absolute ethanol:water
- Preliminary purification step (if any): Purity and radioactivity were verified immediately prior to dosing

FORM AS APPLIED IN THE TEST (if different from that of starting material)
diluted to a concentration of 99.7 ug/mL in 10:90 absolute ethanol:water
Radiolabelling:
yes

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on source and properties of surface water:
None was added to the activated sludge.
Details on source and properties of sediment:
None was added to the activated sludge.
Details on inoculum:
- Oxygen concentration (mg/l) initial: 6.3 mg/L
- Source of activated sludge (e.g. location, contamination history): Collected from the Cambridge Wastewater Treatment Facility located in
Cambridge, Maryland one day prior to the start of the study
- Storage conditions: Aerated and mixed overnight on a shaker table with the vessel open to ambient air
- Storage length: ~24 horus
- Preparation for exposure: Aerated at a rate of 5 mlL/min
- Biomass concentration (mg/L) used in test: 2500 mg/L
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 50 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
Details on study design:
TEST CONDITIONS
- Volume of test solution/treatment: 1004 µL-biotic; 501 µL-abiotic; 815 µL-positive control were added to the vessel
- Composition of medium: Sludge
- Solubilising agent (type and concentration if used): 10:90 ethanol/distilled water
- Test temperature: 21-22 oC
- pH: 7.5
- pH adjusted: yes for the abiotic control
- Suspended solids concentration: 2500 mg/L
- Continuous darkness: no
- Any indication of the test material adsorbing to the walls of the test apparatus: None

TEST SYSTEM
- Culturing apparatus: 1 gallon amber glass jugs were used as the test vessels
- Number of culture flasks/concentration: 3/abiotic,biotic, and positive control
- Method used to create aerobic conditions: aeration at 5 mL/min
- Measuring equipment: KOH trap at the exit of the vessels measured evolved CO2. Otherwise prior to sampling, the test vessels were placed on a magnetic stirrer and the contents were mixedthoroughly. Chemical analysis, LSC and dissolved 14 CO2 samples were removed from the biotic treatments through a sampling port constructed of Teflon tubing that extended to the bottom of the test vessel and a stopcock. Prior to the removal of the sample, the stopcock was opened and the test mixture was repeatedly pulled up and pushed back into the flask to clear the line. The abiotic treatment was sampled using a wide mouth pipette.
- Test performed in closed vessels due to significant volatility of test substance: No, though the glass jugs were stoppered.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Evolved CO2 was trapped using 3 consecutive CO2 KOH traps on the exit line from the bottles.

SAMPLING
Biotic samples were collected at 1 minute (LSC and chemical analysis only), and at the following intervals for LSC and chemical analysis, evolved 14CO2 and dissolved 14CO2: 60 minutes, 2, 3, 5, 8, 12, 24 hours, 2, 3, 4, 6, 7, 14, 21 and 28 days. Abiotic samples for LSC and chemical analysis were collected at 1 and 60 minutes, 12, 24 hours, 2, 3, 4, 5, 6, 7, 14, 21 and 28 days. In addition, positive control samples for liquid scintillation counting, evolved 14 CO2 and dissolved 14 CO2 were collected at 60 minutes, 2, 3, 5, 8, 12, 24 hours, 2, 3, 4, 5, 6, 7, 14, 21 and 28 days. LSC counting was performed at the 1 minute interval to measure the test concentration.
- Sample storage before analysis: Overnight incubation or flash freezing where applicable.
- Other:

DESCRIPTION OF CONTROL AND/OR BLANK TREATMENT PREPARATION
CONTROL AND BLANK SYSTEM
- Abiotic sterile control: An abiotic control of sludge was prepared the same as the biotic sludge, except for the addition of the test substance to the vial.
- Positive Reference control:A stock solution of [14C] benzoic acid was prepared by nitrogen evaporating a 2.0 mL aliquot of stock solution (ID = 8429-001) to near dryness then reconstituting to 1.0 mL (10:90) absolute ethanol:water. The radioactive concentration of the solution was determined immediately prior to dosing by LSC analysis. The following is a description of the radiolabeled form of the positive control substance:
Name: Benzoic Acid, [ 14 C(U)]
Lot Number: 070822
Specific activity: 100 mCi/mmol (0.8188 mCi/mg)
Solvent: Ethanol Concentration: 1 mCi/mL
Molecular Formula: C6H5COOH
Molecular Weight: 122.12

STATISTICAL METHODS:
The loss of parent data was fitted to various decay equations using nonlinear regression. Regression analysis was performed using Jandel Table Curve 2D (version 3.00) software. The criteria used to judge the best kinetic model for the observed data are: 1) F-value, 2) Visual observation of the fit on the printed graph, 3) Examination of error residuals for the regression model, 4) R 2 , and 5) Standard error of the different parameters.

Following are the equations used in the evaluations of CO2 production:

FIRST ORDER
Y = A*exp(-k1*t)
Where:
Y = % mineralization
t = time
exp = exponent
A = asymptotic yield of CO2
k1 = first order rate constant (hrs^(-1))

2 COMPARTMENT
Y = (A1 - exp(-k1*t)) + (B1 - exp(-k2*t))
Where:
Y = % mineralization
t = time
exp = exponent
A1 = % mineralized at first order rate k1
B1 = % mineralized at first order rate k2

3 COMPARTMENT

Y = (A1-exp(-k1*t)) + (B1-exp(-k2*t)) + (C1-exp(-k3*t))
Where:
Y = % mineralization
t = time
exp = exponent
A1 = % mineralized at first order rate k1
B1 = % mineralized at first order rate k2
C1 = % mineralized at first order rate k3

THREE-HALF-ORDER
Y = 100 - [A(1 - exp(-k1t) + k0*t]
Where:
Y = % mineralization
t = time
exp = exponent
A = deflection point at which the rate changes from first order to zero order
k0 = zero order rate constant (hrs^(-1))
k1 = 1st order rate constant (hrs^(-1))

FIRST ORDER LOGISTIC (SIGMOIDIAL)
Y = A (1-exp(-k1*t)^-1 )
Where:
Y = % mineralization
t = time
exp = exponent
A = empirical constants
k1 = 1st order rate constant (hrs^(-1))

Following are the equations used in the evaluations of the loss of parent data:

FIRST ORDER
Y = A*exp(-k1*t)
Where:
Y = % of intiial 14C present as parent
t = time
exp = exponent
A = initial parent concentration (%)
k1 = first order rate constant (hrs^(-1))

2 COMPARTMENT
Y = (A exp(-k1*t)) + (B exp(-k2*t))
Where:
Y = % of initial 14C present as parent
t = time
exp = exponent
A = % parent concentration degraded at first order rate k1
B = % parent concentration degraded at first order rate k2

3 COMPARTMENT
Y = (A exp(-k1*t)) + (B exp(-k2*t)) + (C exp(-k3*t))
Where:
Y = % of initial 14C present as parent
t = time
exp = exponent
A = % parent concentration degraded at first order rate k1
B = % parent concentration degraded at first order rate k2
C = % parent concentration degraded at first order rate k3

THREE-HALF-ORDER
Y = 100 - [A(1 - exp(-k1*t)) + k0*t]
Where:
Y = % of initial 14C present as parent
t = time
exp = exponent
A = deflection point at which the rate changes from first order to zero order
k0 = zero order rate constant (hrs^(-1))
k1 = 1st order rate constant (hrs^(-1))

ASYMPTOTIC FIRST
Y = A + [(B-A) exp(-k1t) ]
Where:
Y = % of initial 14C present as parent
t = time
exp = exponent
A,B = empirical constants
k1 = 1st order rate constant (hrs^(-1))
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
The measured activity in the 1 and 60 minute biotic sludge samples were 97.0% and 98.1% of the target activity,
respectively. The measured activity in the 1 minute and 60 minute abiotic sludge samples were 94.7% and 95.7% of the target activity, respectively. The measured activity in the 1 minute and 60 minute positive control sludge samples were 88.9% and 82.5% of the target activity, respectively. The temperature range recorded during the test was 21 to 22°C.
Mean total recoveryopen allclose all
Compartment:
abiotic control measured at end of test
% Recovery:
2.67
Compartment:
biologically active treatment at end of test
% Recovery:
82.14
% Degradationopen allclose all
Key result
% Degr.:
ca. 5.2
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
0.017 h
Key result
% Degr.:
ca. 3.62
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
1 h
Key result
% Degr.:
ca. 2.24
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
2 h
Key result
% Degr.:
ca. 2.71
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
3 h
Key result
% Degr.:
ca. 0.48
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
5 h
Key result
% Degr.:
ca. 1.58
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
8 h
Key result
% Degr.:
ca. 7.61
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
12 h
Key result
% Degr.:
ca. 1.98
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
24 h
Key result
% Degr.:
ca. 2.33
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
48 h
Key result
% Degr.:
ca. 3.16
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
72 h
Key result
% Degr.:
ca. 3.87
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
96 h
Key result
% Degr.:
ca. 4.7
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
120 h
Key result
% Degr.:
ca. 4.54
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
144 h
Key result
% Degr.:
ca. 6
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
168 h
Key result
% Degr.:
ca. 8.76
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
336 h
Key result
% Degr.:
ca. 9.98
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
504 h
Key result
% Degr.:
ca. 10.39
Parameter:
other: Measure of CO2 evolved + percent radioactivity remaining on solids
Sampling time:
672 h
Half-life of parent compound / 50% disappearance time (DT50)
Key result
Compartment:
biologically active treatment at end of test
DT50:
ca. 0.45 h
St. dev.:
1.16
Type:
second order
Temp.:
21.5 °C
Mineralization rate (in CO2):
1.55 h-1
Other kinetic parameters:
first order rate constant
third / second order rate constant
Transformation products:
no
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
yes
Remarks:
Radioactivity Remaining with the Solids The amount of radioactivity remaining with the extracted solids was calculated as follows: ((Total dpm/mL from combusted solids (corrected for test volume)) / (Time zero nominal dpm/mL)) X 100
Details on results:
TEST CONDITIONS
- Aerobicity (or anaerobicity), moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:
on the - the and -th day of incubation, respectively. At the end of the study period, the corresponding concentrations were - and -- % of the applied amount, respectively.

MINOR TRANSFORMATION PRODUCTS
- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed:
- Range of maximum concentrations in % of the applied amount at end of study period:

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT:

EXTRACTABLE RESIDUES
- % of applied amount at day 0:
- % of applied amount at end of study period:

NON-EXTRACTABLE RESIDUES
- % of applied amount at day 0:
- % of applied amount at end of study period:

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 4.28%

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study:

STERILE TREATMENTS (if used)
- Transformation of the parent compound:
- Formation of transformation products:
- Formation of extractable and non-extractable residues:
82.20% Recovered in Extracts in Water as the Parent
- Volatilization:
Results with reference substance:
The [C] benzoic acid dosed to the positive control treatment achieved 30.43% 14CO2 production at the 60 minute interval, 61.05% on day 14 and 65.49% at the termination of the test on day 28.

Applicant's summary and conclusion

Validity criteria
Validity criteria fulfilled:
yes