Stevens Ecology Project Report

09 September, 2013

PREPARED FOR:




Biosynthetic Technologies

PROJECT NUMBER:
4521

OFFICE
1710 State Road
Mosier, OR 97040

Biodegradability Testing of Ten Oils by
USA
VOICE
(541) 478-0594



OECD 301B / ASTM D5864
(866) 942-7601
INTERNET
www.stevensecology.com

EMAIL
[email protected]

PREPARED BY:

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

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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

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List of Tables

Table 1.
Sample Properties

Table 2.
Experimental Design

Table 3.
Biodegradation Summary

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 Stevens Ecology Project Summary and Certification
Biogeochemistry Research and Analysis
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
OFFICE
than “Ultimate” biodegradability assays, and it is possible that the last
1710 State Road
two groups might perform better in a OECD 302-series test or
Mosier, OR 97040
USA equivalent. Particularly since some samples required long lag periods
before mineralization began.
VOICE
(541) 478-0594
These conclusions are based on the following report of research that
(866) 942-7601
was conducted under my supervision.
INTERNET
www.stevensecology.com

EMAIL
[email protected]
Sample Label Percent Biodegradati Estimated Half- Estimated Persistence Designation
Degraded on Rate Life

Reference Canola Oil 70.1 0.0250 16 days 49 -106 days Ultimately Biodegradable
4521.1 PAO (SpectraSyn 40) 4.3 0.0026 442 days 1 - 8 years not biodegradable
4521.2 POE (Hatcol 5150) 25.3 0.0090 67 days 111 - 445 days Inherently Biodegradable
4521.3 PAG (OSP-150) 0.0 0.0000 - indefinite not biodegradable
4521.4 Group II (220R) 33.6 0.0120 47 days 71 - 312 days Inherently Biodegradable
4521.5 Group III (Yubase 6) 37.8 0.0135 41 days 68 - 272 days Inherently Biodegradable
4521.6 PAO (Synfluid 7) 29.1 0.0104 56 days 96 - 372 days Inherently Biodegradable
4521.7 POE (Hatcol 2926) 61.6 0.0219 20 days 45 - 132 days Ultimately Biodegradable
4521.8 PAG (OSP-32) 26.6 0.0095 63 days 105 - 419 days Inherently Biodegradable
4521.9 Diester (Hatcol 2901) 76.2 0.0272 14 days 37 - 93 days Readily Biodegradable
4521.10 Bright Stock 37.0 0.0135 42 days 74 - 279 days Inherently Biodegradable

Todd O. Stevens, Ph.D.

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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).

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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.

Elemental Composition, Percent

sample Label Density C H O N S P Cl Na Ash total
Referen Canola Oil 0.87 78.07 11.68 10.22 0.20 - - - - 0 101.04
ce
4521.1 PAO 0.86 85.20 12.98 - 0.00 - - - - 0 98.18
(SpectraSyn 40)
4521.2 POE (Hatcol 0.97 78.62 12.43 - 0.00 - - - - 0 91.05
5150)
4521.3 PAG (OSP-150) 0.96 68.22 10.67 - 0.00 - - - - 0 78.89
4521.4 Group II (220R) 0.86 85.20 13.60 - 0.00 - - - - 0 98.80
4521.5 Group III 0.85 85.50 13.50 - 0.00 - - - - 0 99.00
(Yubase 6)
4521.6 PAO (Synfluid 7) 0.88 85.60 13.56 - 0.00 - - - - 0 99.16
4521.7 POE (Hatcol 1.05 77.20 11.99 - 0.00 - - - - 0 89.19
2926)
4521.8 PAG (OSP-32) 0.94 66.24 9.98 - 0.00 - - - - 0 76.22
4521.9 Diester (Hatcol 0.91 74.90 11.61 - 0.00 - - - - 0 86.51
2901)
4521.10 Bright Stock 0.80 85.92 14.04 0.00 0.00 - - - - - 99.96
water 1.00 0.00 11.11 88.89 0.00 0.00 0.00 0.00 0.00 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.

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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

0.00850g

K2HPO4

0.02175g

Na2HPO4 2H2O

. 0.03340g

NH4Cl


0.0050g

CaCl2 7H2O


. 0.0364g

MgSO4.7H2O

0.0225g

FeCl3.6H2O

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.

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 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.

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 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

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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

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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.

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 Group 1 CO2
3000

2250

1500

750

0
0 7.5 15.0 22.5 30.0

4521C 4521R 4521.1 4521.2 4521.3

4521.10

Group 2 CO2
3000
Inorganic Carbon µmol

2250

1500

750

0
0 7.5 15.0 22.5 30.0
Days Incubation

4521.4 4521.5 4521.6 4521.7 4521.8

4521.9

Figure 4. Inorganic carbon production.

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 Group 1 Biodegradation
100.00

75.00

50.00

25.00

0
0 7.5 15.0 22.5 30.0

4521R 4521.1 4521.2 4521.3 4521.10

Group 2 Biodegradation
100.00
Percent Mineralization

75.00

50.00

25.00

0
0 7.5 15.0 22.5 30.0
Days Incubation

4521.4 4521.5 4521.6 4521.7 4521.8

4521.9

Figure 5. Calculated Biodegradation

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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

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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 side-
chain 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 pre-
incubating 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.

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 Sample Label Percent Biodegr Estimated Estimated Designation
Degraded adation Half-Life Persistence
Rate
Reference Canola Oil 70.1 0.0250 16 days 49 -106 days Ultimately Biodegradable
4521.1 PAO 4.3 0.0026 442 days 1 - 8 years not biodegradable
(SpectraSyn
40)
4521.2 POE (Hatcol 25.3 0.0090 67 days 111 - 445 days Inherently Biodegradable
5150)
4521.3 PAG 0.0 0.0000 - indefinite not biodegradable
(OSP-150)
4521.4 Group II 33.6 0.0120 47 days 71 - 312 days Inherently Biodegradable
(220R)
4521.5 Group III 37.8 0.0135 41 days 68 - 272 days Inherently Biodegradable
(Yubase 6)
4521.6 PAO (Synfluid 29.1 0.0104 56 days 96 - 372 days Inherently Biodegradable
7)
4521.7 POE (Hatcol 61.6 0.0219 20 days 45 - 132 days Ultimately Biodegradable
2926)
4521.8 PAG (OSP-32) 26.6 0.0095 63 days 105 - 419 days Inherently Biodegradable
4521.9 Diester 76.2 0.0272 14 days 37 - 93 days Readily Biodegradable
(Hatcol 2901)
4521.10 Bright Stock 37.0 0.0135 42 days 74 - 279 days Inherently Biodegradable

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.

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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.

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Appendix: Data Tables
Date Days C C C R R R
Incubation
Aug 7, 2013 0 139.23 142.8 135.66 149.94 146.37 135.66
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 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

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 Days 4521C 4521R 4521.1 4521.2 4521.3 4521.4 4521.5 4521.6 4521.7 4521.8 4521.9 4521.10
Incubation
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
9 213 1147 278 563 219 464 708 320 1298 332 1184 478
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 4521R 4521.1 4521.2 4521.3 4521.4 4521.5 4521.6 4521.7 4521.8 4521.9 4521.10
Incubation
0 0.19 0.04 0.17 0.32 0.00 0.22 0.08 0.24 0.17 1.03 0.09
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