Registration Dossier

Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

Currently viewing:

Administrative data

biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
GLP guideline study (OECD guideline 314D for simulation test to assess the biodegradability of chemicals discharged in wastewater). No reference substance was tested during the experiment. No analytical analysis were performed in order to identify the transformation products.
Reason / purpose:
reference to same study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
other: OECD guideline 314D
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
Details on test material:
Batch No.: 3597206
Name of test material (as cited in study report): BACDANOL
Radiolabbelled test substance : [14C]Bacdanol, [Hydroxy Methylene-14C]
Molecular formula: C14H24O
Molecular weight: 208.18 g/mol
Radiochemical purity: 98.8 %
Specific activity (if radiolabelling): 54.31 mCi/mmol
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data

Study design

Oxygen conditions:
Inoculum or test system:
mixture of sewage, soil and natural water
Details on source and properties of surface water:
- Details on collection : Choptank River, Caroline County, Maryland.
- Storage length: 24 h
- pH at time of collection: 5.0
- Hardness (CaCO3): 54 mg/L
- Total organic carbon: 3.5 mg C/L
Details on source and properties of sediment:
not applicable
Details on inoculum:
- Source of inoculum/activated sludge : Cambridge Wasterwater Treatment Facility, Cambridge, Maryland
- Storage length: 20 min
- Preparation of inoculum for exposure: The sludge was sieved a 2 mm mesh, settled for 20 min and the total suspended solids concentration of the sludge supernatant was measured to supplement the river water.
- Concentration of sludge: 3 mg/L
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 10 µg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Details on study design:
- Volume of test solution/treatment:2L for biotic, and 1L for abiotic conditions.
- Solubilising agent : alkyl ethoxylated sulfate (50 µg/mL)
- Test temperature: 20+-3°C.
- pH: 6.9
- pH adjusted: yes, by adding 1.5 N KOH
- Aeration of dilution water: yes, 5 mL/mn
- Suspended solids concentration: 47 mg/L

- Culturing apparatus: -gallon amber glass jugs.
- Number of culture flasks/concentration: 1/ concentration
- Method used to create aerobic conditions: The biotic treatment container was mixed overnight on a saker table open to ambient air.
- Method used to create anaerobic conditions: the abiotic supplemented river water was amended with sufficient 2.5% mercuric chloride buffer solution to achieve a final concentration of 1 g/L. The abiotic treatment container was stoppered with a foam plug and mixed overnight on a shaker table
- Measuring equipment: Liquid Scintillation Counting, and Radio Thin Layer Chromatography.

- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used:polyurethane plugs (PUP)

- Sampling frequency: Biotic samples were collected at 5 minutes (LSC and chemical analysis only), and at the
following intervals for LSC and chemical analysis, evolved 14CO2 and dissolved 14CO2: 30 minutes, 6, 12,
24 hours, 2, 3, 4, 5, 6, 7, 14, 21 and 28 days. Abiotic samples for LSC and chemical analysis were
collected at 5 minutes, 24 hours, 2, 3, 4, 5, 6, 7, 14, 21 and 28 days. The polyurethane plugs (PUP) were
sampled on days 0, 1, 2, 3, 4, 5, 6, 7, 14, 21 and 28.
- Sampling method: Prior to sampling, the test vessels were placed on a magnetic stirrer and the contents were mixed
thoroughly. Samples were removed using 50 mL plastic syringes.

Sampling: Direct LSC
Samples (10 mL) of sludge were removed from the test chambers and three 1 mL aliquots of each
sludge sample were then transferred to scintillation vials containing 15 mL of Ultima Gold™ XR
scintillation cocktail. The cocktailed samples were assayed by LSC.
Sampling: Analytical Characterization
At each sampling interval, three 10 mL samples were removed and filtered through a glass fiber
filter and a C18 cartridge. The aqueous filtrate was collected and analyzed by LSC. The filter and cartridge
were then extracted with 5 mL of methanol followed by 5 mL of (70/30) acetone:methanol. The solvent
extracts were collected together and adjusted to a known volume prior to analysis by LSC.
A subsample of selected biotic and abiotic treatment extracts were spotted onto 150 Å Silica Gel
TLC plates. An aliquot of the test substance was spotted on at least one of the outer lanes. The plates
were developed in (85:15) Ethyl Acetate : Hexane, allowed to dry, and scanned using a Bioscan Imaging
200 System.
- Sample storage before analysis: refrigerated conditions

- Inoculum blank: abiotic control was identical to the biologically active treatment with the exception that it was amended at a nominal concentration of 1 g/L with mercuric chloride.
- Abiotic sterile control: no data
- Toxicity control: no data
- Other: none


Mineralization Data
The cumulative percent 14CO2 evolved was determined at each sampling point by the following
1. % 14CO2 Evolved Since Last Sampling:
= ((Mean dpm/mL in KOH Trap) *100 mL (trap volume)
Total Initial dpm ) * (Initial Test Volume
Current Test Volume )*100

2. % 14CO2 Dissolved:
= (dpm/mL in KOH trapping solution / Initial DPM) * (Initial Test Volume / Current Test Volume)*100

3. % Total Cumulative 14CO2 Produced:
Primary Biodegradation
The percent recovered as parent and metabolites was determined at each sampling point by the
following equations:
= (% CO Evolved) + % CO2 Dissolved (at this sampling period)

4. % of Initial Radioactivity in Extract =
Total dpm/mL in extract (corrected to initial test volume)
Initial dpm/mL X 100

5. % of Initial Radioactivity Recovered as Component:
From the TLC chromatogram, the percent of total radioactivity each component represented was
calculated by the TLC software based upon its relative abundance in the chromatogram. The percent of
initial radioactivity associated with a specific component was calculated as follows:
= Fraction of total radioactivity associated with component (TLC) x % of radioactivity in extract
Guidelines for Integrating and Reporting TLC Peaks
The percent of a particular peak equals the relative abundance of that peak in the TLC
chromatogram times the percent of radioactivity recovered in the extract. When the baseline is flat and
only distinct peaks are present, the relative abundance is based upon only those well-defined distinct
peaks. In the case of distinct but poorly defined peaks, polar and/or non polar groups are identified by
Rf range(s). The Rf is defined as the ratio of the distances traveled by a spot and by the solvent. When
the baseline is noisy and peaks less distinct (e.g., late samples), the relative abundance is based only upon
the total radioactivity in the chromatogram. The background subtraction feature is not normally
employed. In all cases, trends are followed when interpreting the chromatograms, comparing each peak
with those at previous samplings. Unless otherwise directed for specific metabolites, peaks are only
reported when they constitute more than 1% of the initially dosed radioactivity in at least one sample.
Thus, in later samples there may be what appears to be a major peak in the chromatogram, but because
there is so little radioactivity remaining in the extract, it is not reported since it does not equal a
significant fraction of the starting radioactivity. When a peak meets the above criteria as reportable, its
level is reported in every sample where it is evident in the chromatogram. Levels of the parent compound
are always reported, when present in the chromatogram.
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

Kinetic Analysis
The CO2 production data was fitted to various 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) R2, and 5) Standard error of the
different parameters.
Following are the equations used in the evaluations of CO2 production:

First Order
Y = A exp -k1t
Y = % mineralization
t = time
exp = exponent
A = asymptotic yield of CO2
k1 = first order rate constant (hrs-1)

2 Compartment
Y = (A1-exp -k1t) + (B1-exp -k2t)
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 -k1t) + (B1-exp -k2t) + (C1-exp -k3t)
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

Y = 100 - [A(1 - exp -k1t) + k0t]
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 -k1t )-1
Y = % mineralization
t = time
exp = exponent
A = empirical constants
k1 = 1st order rate constant (hrs-1)
Given the rapid depletion of parent material in the biotic treatment, the rate constant (k1) for loss
of parent was calculated as the slope of the natural log of the first two data points. The percent of
material as parent in the abiotic and biotic treatments at 5 minutes were used as the initial (t0) and
5 minute data points. The k1 and DT50 values for loss of parent were approximately 27.6 hrs–1 and
0.025 hours, respectively.
The half-life (DT50) was calculated from the first order rate constant (k1) using the following
(DT 0.693 50) = k1
Reference substance
Reference substance:
not required

Results and discussion

Test performance:
In abiotic samples, mean mass balance was between 73.8% and 86.7% .Mass balance in biotic samples was between 86.2% and 117.64%
Mean total recovery
other: water, material (mass) balance
% Recovery:
St. dev.:
% Degradationopen allclose all
% Degr.:
ca. 100
test mat. analysis
in biotic conditions
Sampling time:
48 h
Remarks on result:
other: in biotic conditions
% Degr.:
ca. 100
test mat. analysis
in abiotic conditions
Sampling time:
504 h
Remarks on result:
other: in abiotic conditions
Half-life of parent compound / 50% disappearance time (DT50)
(pseudo-)first order (= half-life)
Remarks on result:
not measured/tested
not measured
Other kinetic parameters:
other: other: 50% CO2 prodution : 641hrs
Transformation products:
not specified
Details on transformation products:
no data
Evaporation of parent compound:
Volatile metabolites:
not specified
Details on results:
- Transformation of the parent compound in biotic treatment: 59.96% within the first 5 min of the experiment. After 48h of experiment, no more parent compound was detected.

- Range of maximum concentrations in % of the applied amount and day(s) of incubation when observed: 82.07% after 72h in biotic treatment, 37.69% after 72h in abiotic treatment
- Range of maximum concentrations in % of the applied amount at end of study period: n26.02% in biotic treatment, 9.97% in abiotic treatment

- % of applied radioactivity present as CO2 at end of study: 51.43% in biotic treatment. Not measured in abiotic treatment

- % of the applied radioactivity present as volatile organics at end of study: 0.31%, and 1.78% in the biotic and abiotic conditions respectively.

- Transformation of the parent compound: yes
- Formation of transformation products: yes
- Formation of extractable and non-extractable residues: yes
- Volatilization: 0.31%, and 1.78% in the biotic and abiotic conditions respectively.
Results with reference substance:
not applicable

Any other information on results incl. tables

Table 1: Summary of the distribution of 14C activity of the averaged replicates.

    Solvent extract (%)   Aqueous filtrate (%)    
Treatment Time (h) Average total (%) Parent (%) Metabolites (%)   Average total (%) Average C-18 (%) Average 14CO2 (%) Average solids (%) Volatilized (%) Total average recovery (%)
Biotic 0.083 86.98 59.96 32.14 1.47 NA NA 0.12 NA 88.58
0.5 83.57 57.42 28.6 1.55 NA 0.87 0.16 NA 86.16
6 81.65 44.82 37.8 1.62 NA 1.6 0.36 0.4 85.27
12 80.95 34.22 48.16 2.2 NA 2.16 0.72 NA 86.02
24 83.81 25.83 60.28 3.89 NA 4.89 1.07 0.13 93.78
48 77.46 0 79.13 7.92 NA 8.2 1.23 0.17 94.98
72 85.89 0 82.07 11.39 NA 9.61 1.71 0.2 108.79
96 76.4 0 77.27 14.34 NA 11.1 2.34 0.22 104.4
120 72.9 0 72.64 8.86 NA 16.86 2.48 0.25 101.36
144 65.09 0 64.56 17.61 NA 18.46 2.46 0.26 103.88
168 62.9 0 62.12 21.16 NA 20.15 2.77 0.28 107.25
336 45.82 0 46.22 29.99 NA 33.89 3.39 0.29 113.38
504 34.92 0 36.06 35.8 NA 44.34 4.23 3 119.59
672 25.53 0 26.02 35.88 NA 51.43 4 0.31 117.14
Abiotic 0.083 82.11 48.33 36.85 1.36 NA NA 0.15 NA 83.62
24 57.14 25.49 33.58 0.84 NA NA 1.61 0.28 59.87
48 45.72 21.35 28.96 0.87 NA NA 4.29 0.34 51.23
72 56.07 18.85 37.69 1.13 NA NA 5.16 0.6 62.96
96 37.15 13.73 24.43 1.15 NA NA 8.63 0.81 47.74
120 78.75 55.76 24.93 1.12 NA NA 0.39 0.94 81.2
144 26.19 10.95 18.59 1.18 NA NA 17.04 1.09 45.51
168 26.56 8.61 19.81 1.45 32.38 NA 18.86 1.24 80.48
336 13.51 2.79 15.07 10.29 34.28 NA 23.84 1.63 83.5
504 10.4 0 11.41 1.57 32.25 NA 27.28 1.67 73.17
672 9.39 0 9.97   1.86 26.56 NA 33.69 1.78 73.28

Applicant's summary and conclusion

Validity criteria fulfilled:
The results demonstrated that 50% of CO2 production occurred at approximately 641 hours.
Executive summary:

Biodegradation of the test item was tested in treated effluent river-surface water mixing zone, following OECD guideline 314D, in GLP conditions. A known quantity of radiolabeled substance (14C) was incubated in biotic and abiotic conditions. The test substance was dosed at a nominal concentration of 10 µg/L in both conditions (biotic and abiotic). Samples were analyzed after 0.083, 0.5, 6, 12, 24, 48, 72, 96, 120, 144, 168, 336, 504, and 672 hours (i.e. a 28 days period), using radio thin layer chromatography.

The results demonstrated that, with biotic treatment, 50% of CO2 production occurred at approximately 641 hours. The kinetic parameters of parent depletion have not been calculated. No more parent has been measured in solvent extract 48 hrs after treatment and 26.02% of metabolite was still measured at the end of the study.

With abiotic treatment, mineralisation has not been measured. No more parent has been measured in solvent extract 504 hrs (21 days) after treatment and 9.97% of metabolite was still measured at the end of the study.

No ultimate biodegradability of the tested compound and its transformation products has been demonstrated in this study. However, complete degradation of the parent compound has been shown in both biotic and abiotic treatment.