Stevens Ecology
Project Report
PREPARED FOR:
PROJECT NUMBER:
Biodegradability Testing of Ten Oils by
PREPARED BY:
09 September, 2013
Biosynthetic Technologies
4521
OFFICE
1710 State Road
Mosier, OR 97040
USA
VOICE
(541) 478-0594
(866) 942-7601
OECD 301B / ASTM D5864
INTERNET
www.stevensecology.com
EMAIL
info@stevensecology.com
Todd O. Stevens, Ph.D.
Table of Contents
List of Figures
........................................................................................................................................3
List of Tables
..........................................................................................................................................4
Project Summary and Certification
.................................................................................................5
Project Description
.............................................................................................................................6
Task 1. Aerobic Biodegradation by ASTM D 5864 / OECD 301B
...........................................8
Experimental Protocol
........................................................................................................................8
Results
..................................................................................................................................................12
Discussion of Results
........................................................................................................................15
Conclusions
........................................................................................................................................17
References Cited
...............................................................................................................................18
Appendix: Data Tables
.....................................................................................................................19
Stevens Ecology
Project Report 4521 rev.1
Page 2
List of Figures
Figure 1.
Appearance of Samples as Received
Figure 2.
Schematic Drawing of Experimental Apparatus
Figure 3.
Method 301B Microcosms in the Incubator
Figure 3.
Inorganic Carbon Production
Figure 4.
Biodegradation in Project Microcosms
Stevens Ecology
Project Report 4521 rev.1
Page 3
List of Tables
Table 1.
Sample Properties
Table 2.
Experimental Design
Table 3.
Biodegradation Summary
Stevens Ecology
Project Report 4521 rev.1
Page 4
Stevens Ecology
Biogeochemistry Research and Analysis
Project Summary and Certification
Ten samples of oil, submitted by Biosynthetic Technologies, were
tested for aerobic biodegradability by OECD 301B (ASTM method
D 5864-05).
Sample 4521.9 (Diester Hatcol 2901) was mineralized by more than
60% during a 10-day window and can be considered “Readily
Biodegradable.” Sample 4521.7 (POE Hatcol 2926) and the reference
material were mineralized to inorganic carbon by 60% or more
during a 28-day incubation period under stringent standardized
conditions and can be considered “Ultimately Biodegradable.”
Samples 4521.2 (POE Hatcol 5150), 4521.4 (Group II 220R), 4521.5
(Group III Yubase 6), 4521.6 (PAO Synfluid 7), 4521.8 (PAG OSP-32),
and 4521.10 (Bright Stock) were mineralized by 20% or more and
can be considered “Inherently Biodegradable.” Samples 4521.1 (PAO
SpectraSyn 40) and 4521.3 (PAG OSP - 150) were not mineralized
or mineralized less than 20%. However, this assay was more stringent
than “Ultimate” biodegradability assays, and it is possible that the last
two groups might perform better in a OECD 302-series test or
equivalent. Particularly since some samples required long lag periods
before mineralization began.
OFFICE
1710 State Road
Mosier, OR 97040
USA
VOICE
(541) 478-0594
(866) 942-7601
These conclusions are based on the following report of research that
was conducted under my supervision.
INTERNET
www.stevensecology.com
EMAIL
info@stevensecology.com
Sample
Reference
4521.1
4521.2
4521.3
4521.4
4521.5
4521.6
4521.7
4521.8
4521.9
4521.10
Label
Canola Oil
PAO (SpectraSyn 40)
POE (Hatcol 5150)
PAG (OSP-150)
Group II (220R)
Group III (Yubase 6)
PAO (Synfluid 7)
POE (Hatcol 2926)
PAG (OSP-32)
Diester (Hatcol 2901)
Bright Stock
Percent
Degraded
70.1
4.3
25.3
0.0
33.6
37.8
29.1
61.6
26.6
76.2
37.0
Biodegradati Estimated Halfon Rate
Life
0.0250
0.0026
0.0090
0.0000
0.0120
0.0135
0.0104
0.0219
0.0095
0.0272
0.0135
16 days
442 days
67 days
47 days
41 days
56 days
20 days
63 days
14 days
42 days
Estimated Persistence
49 -106 days
1 - 8 years
111 - 445 days
indefinite
71 - 312 days
68 - 272 days
96 - 372 days
45 - 132 days
105 - 419 days
37 - 93 days
74 - 279 days
Todd O. Stevens, Ph.D.
Stevens Ecology
Project Report 4521 rev.1
Page 5
Designation
Ultimately Biodegradable
not biodegradable
Inherently Biodegradable
not biodegradable
Inherently Biodegradable
Inherently Biodegradable
Inherently Biodegradable
Ultimately Biodegradable
Inherently Biodegradable
Readily Biodegradable
Inherently Biodegradable
Project Description
Ten samples of non-aqueous liquid materials, submitted by Biosynthetic Technologies were
subjected to aerobic biodegradability testing by the OECD 301B open-bottle aerobic
biodegradation assay. This protocol simulates an aerobic aquatic environment and is
considered indicative of biodegradability in all aerobic environments. Biodegradation is
measured in relationship to a reference material known to be biodegradable. The
corresponding OECD guidelines provide several benchmark “pass” levels. Substances that
pass the “readily biodegradable” criteria are considered likely to be completely mineralized
during standard waste-water treatment. Ready biodegradability tests may also yield
sufficient data for the “inherently biodegradable” and “ultimately biodegradable”
designations, but are not designed specifically for that purpose.
Sample Description
Nine samples of clear oils were received in the laboratory on 10 June, 2013 and one on 5
August, 2013. The appearance of the samples as received is shown in Figure 1. The
samples were colorless oils of varying viscosity, except that samples 4521.2 and 4521.7 had a
slight yellow color, while sample 4521.10 was amber-colored. Sample descriptions are
shown in Table 1.
Figure 1. Samples As Received
A standard reference material, known to be ultimately biodegradable, was used as a
positive control. For this assay, the reference was Low-Erucic Acid Rapeseed Oil, or Canola
Oil (Aldrich W530228).
Stevens Ecology
Project Report 4521 rev.1
Page 6
Sample Preparation
Aliquots of all samples were submitted to Midwest Microlabs, LLC (Indianapolis, IN)
for elemental analysis. C, H, and N were determined by combustion at 990°C with an
elemental analyzer. Oxygen was determined by Pyrolysis via the Unterzaucher method.
Results are shown in Table 1, and were used to calculate biodegradation from inorganic
carbon production. Density was determined gravimetrically.
sample
Label
Referen Canola Oil
ce
4521.1 PAO
(SpectraSyn 40)
4521.2 POE (Hatcol
5150)
4521.3 PAG (OSP-150)
4521.4 Group II (220R)
4521.5 Group III
(Yubase 6)
4521.6 PAO (Synfluid 7)
4521.7 POE (Hatcol
2926)
4521.8 PAG (OSP-32)
4521.9 Diester (Hatcol
2901)
4521.10 Bright Stock
water
Elemental Composition, Percent
Density C
H
O
N
S
P Cl Na
0.87 78.07 11.68 10.22 0.20 -
Ash total
0 101.04
0.86 85.20
12.98 -
0.00 -
-
-
-
0
98.18
0.97 78.62
12.43 -
0.00 -
-
-
-
0
91.05
0.96 68.22
0.86 85.20
0.85 85.50
10.67 13.60 13.50 -
0.00 0.00 0.00 -
-
-
-
0
0
0
78.89
98.80
99.00
0.88 85.60
1.05 77.20
13.56 11.99 -
0.00 0.00 -
-
-
-
0
0
99.16
89.19
0.94 66.24
0.91 74.90
9.98 11.61 -
0.00 0.00 -
-
-
-
0
0
76.22
86.51
0.80 85.92
1.00 0.00
14.04 0.00
11.11 88.89
0.00 0.00 0.00 0.00 0.00 0.00
99.96
0 100.00
Table 1. Properties of Project Samples
Since the substrates could not be diluted in water to make stock solutions, stock
solutions were prepared by dilution in hexane. Aliquots were then added to microcosms
and the solvent allowed to evaporate. Solvent alone was added to control microcosms in a
similar way.
Seed material, the source of microorganisms for this assay, was collected from the municipal
sewage treatment plant in The Dalles, Oregon. Aerobic inoculum was “mixed liquor”
aerated effluent. Inoculum was transported to the laboratory, diluted to achieve 3 g l-1
suspended solids, and aerated for five days at 25°C, to reduce background metabolizable
carbon.
Stevens Ecology
Project Report 4521 rev.1
Page 7
Task 1. Aerobic Biodegradation by ASTM D 5864 / OECD
301B
This experiment measured the mineralization of the test sample to CO2 in “open” aerated
microcosms that simulated an aerobic aquatic environment, with microorganisms seeded
from a waste-water treatment plant. This is considered representative of most aerobic
environments that are likely to receive waste materials.
Samples were incubated at 25°C in a mineral salts medium containing mature activated
sewage solids in an apparatus that provided continuous aeration, agitation, and trapping of
emitted CO2 (Figure 2). Treatments included media with test material, media with reference
material, or media alone.
Compressed air, free of CO2, was continuously sparged through each test vessel, then
bubbled through a chain of three CO2 traps, containing .05M NaOH. As microorganisms
from the inoculum gradually degraded the test substrates, the resulting CO2 was swept into
the trap bottles and converted to carbonates. The carbonate accumulated in the traps was
measured periodically to determine Biodegradation.
Experimental Protocol
An inoculum of activated “mixed liquor” sewage effluent was obtained from a municipal
sewage treatment plant in The Dalles, Oregon. This material was conditioned in the
laboratory as described above. The inoculum was diluted 1:100 in a mineral salts solution
containing, per liter:
KH2PO4 K2HPO4 .
Na2HPO4 2H2O
NH4Cl
.
CaCl2 7H2O
MgSO4.7H2O
FeCl3.6H2O
0.00850g
0.02175g
0.03340g
0.0050g
0.0364g
0.0225g
0.0025g
Substrates were added to microcosms in amounts that yielded approximately 25 mg of
carbon per liter, as shown in Table 2. Slight variations are due to corrections for density of
the samples.
Stevens Ecology
Project Report 4521 rev.1
Page 8
House Air input
Air purification section
Bubbler flasks containing 1N NaOH
Distribution
Manifold &
Flow meters
Incubator section
vent
Test Vessel
Trap Bottles containing 1N NaOH
vent
Trap Bottles containing 1N NaOH
Test Vessel
vent
Trap Bottles containing 1N NaOH
Test Vessel
Figure 2. Schematic drawing of experimental apparatus.
Stevens Ecology
Project Report 4521 rev.1
Page 9
TREATMENT
AMENDMENT
AMOUNT
NUMBER
CHAMBER
4521C
none
23.1 mg
3
1
4521R
25 mg Canola oil
22.9 mg
3
1
4521.1T
PAO (SpectraSyn 40)
22.8 mg
3
1
4521.2T
POE (Hatcol 5150)
25.8 mg
3
1
4521.3T
PAG (OSP-150)
25.5 mg
3
1
4521.10T
Bright Stock
21.3 mg
3
1
4521.4T
Group II (220R)
22.9 mg
3
2
4521.5T
Group III (Yubase 6)
22.6 mg
3
2
4521.6T
PAO (Synfluid 7)
23.4 mg
3
2
4521.7T
POE (Hatcol 2926)
27.9 mg
3
2
4521.8T
PAG (OSP-32)
25.0 mg
3
2
4521.9T
Diester (Hatcol 2901)
24.2 mg
3
2
Table 2. Design of Aerobic Biodegradation Experiment
Three flasks were prepared for each treatment, each containing 1 liter of inoculated medium
and a test substrate as shown in Table 2. To each flask, a train of three CO2-trap bottle was
connected. Because this experiment required more apparatus than would fit into a single
environmental chamber, they were divided into two groups, as shown in the table.
At each time point, the lead trap bottle was removed from the train, and the remaining two
flasks moved up one position. A fresh trap bottle was added to the end of the chain. The
sacrificed bottle was sealed with a butyl rubber stopper and and acidified to pH <2 by the
injection of 0.5 ml 10% H2SO4 with a hypodermic needle and syringe. After agitation and
equilibration for one hour, headspace samples were removed with a gas-tight syringe and
hypodermic needle, and analyzed by gas chromatography. The instrument used was a
Hewlett-Packard 5880A equipped with dual packed columns (carboseive II, Supelco, Inc.) and
a two-channel thermal conductivity detector, or a Carle GC 8700 equipped with a packed
column (carboseive II, Supelco, Inc.) and a single thermal conductivity detector. Carrier and
reference gases were helium. Instruments were calibrated using mixed-gas standards. The
amount of CO2 produced in each microcosm was used to calculate the percentage of the
test substrate that was mineralized by microorganisms.
%D = (TICt – TICb)
X 100
TOC
Where
TICt = mg inorganic carbon in test bottle at time t
TICb = mg inorganic carbon in blank bottles at time t
TOC = mg organic carbon added initially to the test vessel
Stevens Ecology
Project Report 4521 rev.1
Page 10
At the end of the incubation, the test vessels were acidified with 2 ml of acid solution, to
volatilize any carbonates sequestered in solution. After four hours, the carbonate present in
all remaining trap bottles was determined as described above, and the results added
together for the final time point.
Figure 3. Method 301B Microcosms in the Incubator
Stevens Ecology
Project Report 4521 rev.1
Page 11
Results
Mineralization of reference substances began immediately after the start of incubation, as
determined by accumulation of inorganic carbon in the trap bottles (Figure 4). After
approximately 10 days, the emissions from the test samples diverged into three groups.
Samples 1, 3, 6, and 8 produced approximately the same amount of inorganic carbon (IC) as
the negative control flasks, until the final time point, when samples 6 and 8 began producing
IC. Samples 7 and 9 produced IC at about the same rate as the canola oil reference
material. Samples 2, 4, 5, and 10 produced IC at intermediate rates.
The amount of CO2 produced by controls was subtracted from that in test and reference
microcosms and compared with the theoretical CO2 yield to calculate percent
biodegradation as shown in FIgure 5. The high-rate samples corresponded to the “ultimately
biodegradable” designation. The intermediate rate samples corresponded to the “inherently
biodegradable” designation. The two lowest-rate samples did not appear to be significantly
biodegradable under the conditions of this assay, however between the last two time points,
samples 6 and 8 began to mineralize at a great enough rate that they achieved the
“inherently biodegradable” bench mark. See discussion of lag periods below.
Although the samples were divided into two incubation chambers, this does not appear to
have affected which rate group that a sample fell into. That is, both incubators contained
samples in all three groups and the rates of the samples within each of these groups were
very similar.
Stevens Ecology
Project Report 4521 rev.1
Page 12
Group 1 CO2
3000
2250
1500
750
0
0
7.5
4521C
4521.10
4521R
15.0
4521.1
22.5
30.0
4521.2
4521.3
Group 2 CO2
Inorganic Carbon µmol
3000
2250
1500
750
0
0
7.5
15.0
22.5
30.0
Days Incubation
4521.4
4521.9
4521.5
4521.6
4521.7
4521.8
Figure 4. Inorganic carbon production.
Stevens Ecology
Project Report 4521 rev.1
Page 13
Group 1 Biodegradation
100.00
75.00
50.00
25.00
0
0
7.5
4521R
4521.1
15.0
4521.2
22.5
30.0
4521.3
4521.10
Group 2 Biodegradation
Percent Mineralization
100.00
75.00
50.00
25.00
0
0
7.5
15.0
22.5
30.0
Days Incubation
4521.4
4521.9
4521.5
4521.6
4521.7
4521.8
Figure 5. Calculated Biodegradation
Stevens Ecology
Project Report 4521 rev.1
Page 14
Discussion of Results
The reference material was mineralized normally, and the inorganic carbon accumulation in
control microcosms was less than 20% of that in reference microcosms. The elemental
analysis did not account for all of the mass of samples 4521.3 and 4521.8. There may be one
or more elements present that were not analyzed. However, since little or no inorganic
carbon production was observed from 4521.3, the discrepancy does not affect the calculated
results. If the carbon analysis is in error, the value for the final time point of 4521.8 could be
over-estimated.
The OECD designation of “ready biodegradability” is the most stringent defined by OECD
protocols. It requires at least 60% mineralization of a substance within a 10-day window
during a 28-day incubation. This is depicted by the box superimposed on Figure 5. To meet
the “ready biodegradability” criteria, the degradation curve must enter the box through the
lower left corner and exit through the top of the box. This criteria was satisfied for only
one of the samples, 2521.9 which could be considered “readily biodegradable.”
The OECD guidelines also provide less stringent designations. “Inherently Biodegradable”
materials are those for which unequivocal evidence for mineralization is available. Typically,
20% mineralization, with no time limit is required. “Ultimately Biodegradable” materials are
those for which there is a reasonable expectation that complete mineralization will
eventually be achieved under optimized conditions. Typically 60% mineralization, with no
time limit, and with pre-adapted cultures is required. Although this stringent experiment
was not designed to test “inherent” and “ultimate” biodegradability, it can be seen that some
of the materials passed these criteria.
A wide range of biodegradation rates was obtained from these samples. The reference
material, and Sample 4521.7 passed the “Ultimately Biodegradable” criteria. The observed
rates were typical for vegetable oils. Samples 4521.2, 4521.4 and 4521.5, 4521.6, 4521.8, and
4521.10 passed the “Inherently Biodegradable” criteria. This is the typical range for mineral
oils. It is possible that with pre-acclimated cultures and extended incubation periods, as
allowed for OECD 302 series tests, these three samples would meet the criteria for
“ultimately biodegradable,” but that cannot be seen from this present experiment. The
remaining samples, 4521.1 and 4521.3, did not produce significantly more inorganic carbon
than the negative controls, and do not appear to be biodegradable under the conditions of
the ready biodegradability assay.
Several of the sample curves in this experiment exhibited a “lag period,” or an initial period
of low or no mineralization, before biodegradation began. This also occurred in a prior run
of the experiment, except that the lag periods were approximately one week longer. Lag
periods can be attributed to several possible causes. In some cases, time is required for cells
to biochemically acclimate to the substrate - genes must be turned on and proteins
synthesized. Sometimes only a very few cells capable of degrading the substrate are initially
present, and degradation rates are low until those cells proliferate. These sorts of lag
Stevens Ecology
Project Report 4521 rev.1
Page 15
periods are characteristic of the inoculum. Lag periods might also be due to catabolite
repression. In this case, the enzymes necessary for substrate utilization are suppressed until
some other more energetically-favorable substrate is consumed. Catabolite repression
would seem to be an unlikely cause of lag in this experiment, since no other carbon
substrate was present. However it is at least possible that the substrate itself might repress
some step in mineralization until some initial transformation step (e.g. removal of a sidechain or functional group) has been completed. The exact cause of the lag period is outside
the scope of the current experiment. Note that in dedicated “inherent biodegradability”
tests (OECD 302 series) lag periods are presumed to be reduced or eliminated by preincubating the substrate with aliquots of the inoculum, however that is not allowed for
OECD 301-series assays.
A summary of the results and estimated biodegradation rates are shown in Table 3.
Estimated persistence was calculated for a “best case” scenario as a straight-line
extrapolation and for a worst-case scenario with a half-life kinetic model. The actual rates
in the environment would depend on the starting concentration, potential toxic effects at
higher concentrations, and the environmental conditions. However, the data suggest that in
this concentration range, samples 4521.7, and 4521.9 could require 1 to 4 months for
complete biodegradation, while samples 4521.4, 4521.5, and 4521.10 could require 2 to 14
months, samples 4521.2, 4521.6, and 4521.8 could require 3 to 15 months, and samples
4521.1, and 4521.3 might persist for many years.
Note that these assays assume that the material being tested is a pure substance. The assay
may not provide information about minor components present at a few percent or less. The
OECD guidelines permit the treatment of mixtures of closely related substances - such as
oils - to be considered as pure substances.
Stevens Ecology
Project Report 4521 rev.1
Page 16
Sample
Label
Reference Canola Oil
4521.1
PAO
(SpectraSyn
40)
4521.2
POE (Hatcol
5150)
4521.3
PAG
(OSP-150)
4521.4
Group II
(220R)
4521.5
Group III
(Yubase 6)
4521.6
PAO (Synfluid
7)
4521.7
POE (Hatcol
2926)
4521.8
PAG (OSP-32)
4521.9
Diester
(Hatcol 2901)
4521.10
Bright Stock
Percent Biodegr Estimated
Degraded adation
Half-Life
Rate
70.1
0.0250 16 days
Estimated
Persistence
Designation
49 -106 days
Ultimately Biodegradable
4.3
0.0026 442 days
1 - 8 years
not biodegradable
25.3
0.0090 67 days
111 - 445 days
Inherently Biodegradable
0.0000 -
indefinite
not biodegradable
33.6
0.0120 47 days
71 - 312 days
Inherently Biodegradable
37.8
0.0135 41 days
68 - 272 days
Inherently Biodegradable
29.1
0.0104 56 days
96 - 372 days
Inherently Biodegradable
61.6
0.0219 20 days
45 - 132 days
Ultimately Biodegradable
26.6
0.0095 63 days
105 - 419 days
Inherently Biodegradable
76.2
0.0272 14 days
37 - 93 days
Readily Biodegradable
37.0
0.0135 42 days
74 - 279 days
Inherently Biodegradable
0.0
Table 3. Biodegradation Summary
Conclusions
Sample 4521.9 (Diester Hatcol 2901) was mineralized by more than 60% during a
10-day window and can be considered “Readily Biodegradable.” Sample 4521.7 (POE Hatcol
2926) and the reference material were mineralized to inorganic carbon by 60% or more
during a 28-day incubation period under stringent standardized conditions and can be
considered “Ultimately Biodegradable.” Samples 4521.2 (POE Hatcol 5150), 4521.4 (Group
II 220R), 4521.5 (Group III Yubase 6), 4521.6 (PAO Synfluid 7), 4521.8 (PAG OSP-32), and
4521.10 (Bright Stock) were mineralized by 20% or more and can be considered “Inherently
Biodegradable.” Samples 4521.1 (PAO SpectraSyn 40) and 4521.3 (PAG OSP - 150) were
not mineralized or mineralized less than 20%. However, this assay was more stringent than
“Ultimate” biodegradability assays, and it is possible that the last two groups might perform
better in a OECD 302-series test or equivalent. Particularly since some samples required
long lag periods before mineralization began.
Stevens Ecology
Project Report 4521 rev.1
Page 17
References Cited
ASTM. 2005. Standard test method for determining aerobic aquatic biodegradation of
lubricants or their components. ASTM International. West Conshohocken, PA.
Bitton, G. 1999. Wastewater Microbiology. 2nd ed. Wiley-Liss. New York.
Clesceri, L.S., Greenberg, A.E., and Trussell, R.R. 1989. Standard Methods for the Examination
of Water and Wastewater. 17th ed. American Public Health Association, Washington, D.C.
OECD. 2003. OECD guidelines for the testing of chemicals: Principles and strategies related
to the testing of degradation of organic chemicals. Organization for Economic Cooperation
and Development, Paris.
OECD. 1992. OECD guidelines for the testing of chemicals: Biodegradability in Seawater.
Organization for Economic Cooperation and Development, Paris.
Wackett, L.P. and Hershberger, C.D. 2001. Biocatalysis and biodegradation. Microbial
transformation of organic compounds. American Society for Microbiology, Washington, D.C.
Stevens Ecology
Project Report 4521 rev.1
Page 18
Appendix: Data Tables
Date
Days
Incubation
C
C
C
R
R
R
Aug 7, 2013
0
139.23
142.8
135.66
149.94
146.37
Aug 12, 2013
5
114.24
107.1
117.81
467.67
756.84
628.32
Aug 16, 2013
9
199.92
178.5
260.61
1074.57
1278.06
1088.85
Aug 21, 2013
14
364.14
285.6
310.59
1602.93
1710.03
1406.58
Aug 28, 2013
21
253.47
253.47
410.55
1888.53
1895.67
1995.63
Sep 4, 2013
28
403.41
374.85
392.7
2074.17
2188.41
2227.68
T1
T1
T1
T2
T2
135.66
T2
132.09
146.37
142.8
153.51
135.66
142.8
339.15
207.06
271.32
385.56
303.45
424.83
253.47
267.75
314.16
610.47
517.65
560.49
310.59
349.86
381.99
896.07
842.52
724.71
360.57
439.11
442.68
1024.59
971.04
985.32
514.08
478.38
521.22
1095.99
1088.85
1071
T3
T3
T3
T4
T4
T4
149.94
142.8
146.37
142.8
139.23
135.66
232.05
164.22
149.94
381.99
260.61
349.86
342.72
157.08
157.08
456.96
528.36
406.98
435.54
282.03
249.9
667.59
717.57
689.01
446.25
267.75
392.7
892.5
963.9
1003.17
514.08
310.59
274.89
1253.07
1370.88
1313.76
T5
T5
T5
T6
T6
T6
146.37
149.94
139.23
135.66
142.8
146.37
581.91
449.82
424.83
264.18
242.76
196.35
881.79
639.03
603.33
324.87
317.73
317.73
1342.32
860.37
399.84
442.68
403.41
674.73
1595.79
967.47
656.88
467.67
474.81
803.25
1806.42
1145.97
1278.06
1024.59
1228.08
1367.31
T7
T7
T7
T8
T8
T8
146.37
149.94
142.8
139.23
142.8
146.37
867.51
428.4
778.26
253.47
146.37
149.94
1663.62
885.36
1345.89
346.29
324.87
324.87
2363.34
1888.53
1920.66
399.84
381.99
399.84
2199.12
2145.57
2045.61
674.73
364.14
499.8
2288.37
2166.99
2113.44
960.33
910.35
949.62
T9
T9
T9
T10
T10
T10
210.63
135.66
146.37
135.66
142.8
146.37
274.89
499.8
456.96
321.3
285.6
257.04
1278.06
1295.91
978.18
546.21
474.81
414.12
2102.73
2356.2
1974.21
949.62
738.99
446.25
2034.9
2320.5
2345.49
1281.63
996.03
813.96
2156.28
2259.81
2238.39
1592.22
1338.75
1113.84
Daily CO2 Evolved
Stevens Ecology
Project Report 4521 rev.1
Page 19
Days
Incubation
4521C
4521R
4521.1
4521.2
4521.3
4521.4
4521.5
4521.6
4521.7
4521.8
4521.9
4521.10
0
1
5
1
5
7
0
6
2
7
4
25
2
5
113
618
273
371
182
331
486
234
691
183
411
288
478
9
213
1147
278
563
219
464
708
320
1298
332
1184
14
320
1573
347
821
322
691
868
507
2058
394
2144
712
21
306
1927
414
994
369
953
1073
582
2130
513
2234
1031
28
390
2163
505
1085
367
1313
1410
1207
2190
940
2218
1348
Mean CO2 Evolved
Days
Incubation
4521R
4521.1
4521.2
4521.3
4521.4
4521.5
4521.6
4521.7
4521.8
4521.9
4521.10
0
0.19
0.04
0.17
0.32
0.00
0.22
0.08
0.24
0.17
1.03
5
20.09
5.94
9.51
3.38
7.94
13.95
4.40
19.96
3.55
13.25
6.84
9
37.03
2.46
12.82
0.58
9.15
18.47
3.89
37.25
5.89
40.94
10.32
14
49.61
1.06
18.29
0.42
13.53
20.40
6.72
59.48
3.72
76.00
15.19
21
64.12
4.05
25.04
3.12
23.59
28.52
9.90
62.44
10.13
80.26
28.03
28
70.12
4.27
25.30
-0.74
33.61
37.82
29.09
61.59
26.60
76.15
37.02
Mean Percent Biodegradation
Stevens Ecology
Project Report 4521 rev.1
Page 20
0.09