ARTICLE IN PRESS

POLYMER
TESTING
Polymer Testing 25 (2006) 597–604
www.elsevier.com/locate/polytest

Material Behavior

Comparison of the biodegradation of poly(e-caprolactone),

cellulose acetate and their blends by the Sturm test and
selected cultured fungi
M.R. Calil, F. Gaboardi, C.G.F. Guedes, D.S. Rosa
Programa de Pós-Graduac- ão Stricto Sensu em Engenharia e Ciência dos Materiais (PPG-ECM), Laboratório de Polı´meros Biodegradáveis e
Soluc- ões Ambientais, Universidade São Francisco, Rua Alexandre Rodrigues Barbosa, no 45, Centro, CEP 13251-900, Itatiba, SP, Brazil
Received 20 December 2005; accepted 31 January 2006

Abstract

In this work, we compared the biodegradation of poly(e-caprolactone) (PCL), cellulose acetate (CA) and their blends
using an aerobic biodegradation technique known as the Sturm test, and by their carbon dioxide (CO2) production and
susceptibility to attack by a mixture of fungal strains in solid and liquid media in which samples of each blend were the
only source of carbon. The extent of biodegradation was assessed by the loss of mass. The polymers were also
characterized by thermal analysis using differential scanning calorimetry (DSC). The 40PCL/60CA blend showed faster
biodegradation than the other blends because of its higher CO2 production in aerobic medium (Sturm test). PCL was more
susceptible to attack by a mixture of fungi on solid medium than was CA but showed a lower loss of mass (8.4%) than the
latter polymer; the 60PCL/40CA blend showed the greatest loss of mass during the period of evaluation. In contrast, in
liquid medium, PCL showed a greater loss of mass. Blends of PCL with CA reduced the melting temperature (Tm) of PCL
and increased the Tm of CA, indicating immiscibilty of the polymers. The blends were also less crystalline, which favoured
their biodegradation.
r 2006 Published by Elsevier Ltd.

Keywords: Biodegradation; Cellulose acetate; Fungal cultures; Poly(e-caprolactone); Sturm test

1. Introduction tyrate are examples of biodegradable polymers that

A biodegradable polymer is defined as a plastic
that is degraded primarily by the action of naturally
occurring microorganisms, such as bacteria, fungi
and algae [1]. Materials composed of starch;
polyvinyl alcohol, polylactates and polyhydroxybu-
Corresponding author. Tel.: +55 11 4534 8046;
fax: +55 11 4524 1933.
E-mail address: mrcalil@terra.com.br (M.R. Calil).
offer an attractive solution to the environmental
problem of plastic waste disposal.
Poly(e-caprolactone) (PCL) is an aliphatic polye-
ster that has been widely studied for use in drug
release systems. Extracellular enzymes present in
soil can cleave the extensive chains of PCL before
assimilation of the polymer by microorganisms [2].
However, the high cost of PCL has prevented
its widespread industrial use. Natural polymers,
such as starch and cellulose, are good base materials

0142-9418/$ - see front matter r 2006 Published by Elsevier Ltd.

doi:10.1016/j.polymertesting.2006.01.019
 ARTICLE IN PRESS
598 M.R. Calil et al. / Polymer Testing 25 (2006) 597–604
for the production of inexpensive, rapidly degrad-
able plastics that are easily digested by micro-
organisms.
Cellulose acetate (CA) is one of the most
important plant-derived polymers because of its
wide variety of applications [3]. Although chemi-
cally simple, cellulose is a structurally complex
polymer, and several enzymes are required for the
complete degradation of this molecule [4]. The
chemical modification of cellulose affects its biode-
gradability, and CA has been the most studied
derivative. The high degree of substitution (DS) of
cellulose esters decreases their biodegradability.
Thus, for example, reverse-osmosis membranes
prepared from CA with a DS of 2.5 quickly lose
their semi-permeability because of susceptibility to
microbial attack [5].
Blends of two or more polymeric materials or
copolymers that are not covalently bound [6]
represent a good alternative for reducing the final
cost of industrial products. Various combinations of
such blends have been used to produce conventional
and biodegradable plastics [7,8].
Biodegradation can be defined as the microbial-
catalyzed conversion of a polymeric substrate into
biogas under aerobic ( carbon dioxide [CO2]) and
anaerobic (CH4) conditions in a biologically active
environment. Mineralization should be distin-
guished from biodestructability, which is reflected
in a loss of mass, film or fiber disintegration, or a
loss of physical properties [5]. Hydrolysis is the
principal mechanism by which enzymes degrade
cellulose-based polymers. The first step in depoly-
merization occurs outside of microbial cells through
the action of extracellular enzymes. After cleavage,
the resulting small oligomers can be transported
into cells for final mineralization [4].
Biodegradability tests have been developed to
quantify the ability of microorganisms to degrade
these polymers [9,10]. Several approaches can be
used, including direct incubation in an environment
containing an undefined biocinesis and testing in
highly defined synthetic media with selected cultures
of microorganisms [11].
Standard methods for investigating the biodegra-
dation of plastics under laboratory conditions have
been published by ASTM International [12–14], the
International Organization for Standardization
(ISO) [15–17], and the Deutsche Norm/ European
Standard [18]; all of these tests simulate natural
conditions. The Sturm test is an aerobic biodegra-
dation technique based on CO2 production. The test
substances are exposed to an inoculum of compost
from municipal solid waste, after which aerobic
composting occurs in an environment where tem-
perature, aeration and humidity are closely mon-
itored and controlled [14]. Another method for
evaluating the biodegradation of polymers is by
measuring the loss of mass when exposed to a
mixture of selected fungal strains in culture media
with the plastic sample as sole source of carbon. The
initial phase of biodegradation involves modifica-
tion of the substrate to yield a product that is itself
an intermediate and is subject to further metabo-
lism.
In general, the number of microbial cells or the
biomass of the species acting on the chemical of
interest increases as degradation proceeds [19]. Pure
polymers are usually not degraded because of their
hydrophobic character, which inhibits the enzy-
matic activity of the microorganisms.
In this work, the biodegradation of PCL, CA and
their blends was assessed by CO2 production in the
Sturm test and by the loss of mass of samples
incubated with a mixture of biodegrading fungal
strains in solid and liquid media with the samples of
plastic as sole source of carbon. The polymers were
also characterized by thermal analysis using differ-
ential scanning calorimetry (DSC) to determine the
extent to which crystallinity influenced the biode-
gradation.
2. Experimental
2.1. Materials
2.1.1. Polymers
PCL was supplied in pellet form by Union
Chemical Carbide Ltd. (P-767) (Cubatão, SP,
Brazil) and had a melting index of 1.970.3 [20], a
density of 1145 g/cm3 and a weight-average mole-
cular weight (Mw) of 50,000 g/mol.
CA type 15-4051 was supplied by Rhodia Acetow
Brazil Ltda. (Santo André, SP, Brazil). The average
DS was 2.4, the degree of polymerization was
500–600 and the specific viscosity was 40.295.
Low-density polyethylene (LDPE) was supplied by
Union Carbide Quı́mica Ltd. (DUCB 6000 NT) and
had been used in extrusion processes. This LDPE
was classified as Type I—Class A—5 (ASTM D
1248), with a melting index of 0.370.1 g/10 min [20],
a density of 0.92070.003 g/cm3 (ASTM D 1505)
and an Mw of 36,415 g/mol.
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M.R. Calil et al. / Polymer Testing 25 (2006) 597–604
Grade cellulose type 1.02331.0500 for thin layer
chromatography was supplied by Merck S.A. (São
Paulo, SP, Brazil) and had a particle size of
o20 mm.
2.1.2. Microorganisms
The mixture of fungi consisted of the following
species: Aspergillus niger (ATCC 9642), Penicillium
pinophillum (ATCC 9644), Chaetomium globosum
(ATCC 6205), Aerobasidium pullulans (ATCC
15233) and Gliocadium virens (ATCC 9645). All of
these microorganisms were obtained from the
culture collection of the Fundac- ão Tropical André
Tosello (Campinas, SP, Brazil), except for G. virens,
which was obtained from the Adolfo Lutz Institute
(São Paulo, SP, Brazil).
2.2. Blend preparation
Blends of PCL with CA (60/40 and 40/60 wt%)
were prepared by casting. Pure PCL and CA were
represented by 100/0 and 0/100 wt%, respectively.
The solutions were prepared by dissolving 15% (w/
w) of each polymer in acetone with stirring at
6573 1C for 6 h. The mixtures were then poured
into culture dishes and the solvent was allowed to
evaporate in an atmosphere saturated with acetone.
For the Sturm test, PCL, CA and their blends
were ground using a model TE-625 mill (Tecnal
Equipamentos para Laboratório Ltda., Piracicaba,
SP, Brazil) to provide particles with a size o10
mesh.
2.3. Thermal analysis
Thermal analysis was done using a DSC 204
TASC 414/3A differential scanning calorimeter
(Netzsch-Gerätebau GmbH, Bavaria, Germany) in
a nitrogen atmosphere, at a heating rate of
Ar O2
Ba(OH)2
100 mL
Compressor
Fig. 1. Scheme for monitoring CO2 production.
599
10 1C min
1. Two heating cycles were used for each
film. The PCL was initially heated to 90 1C to
eliminate the thermal history of the sample and then
cooled to room temperature before being immedi-
ately reheated to 250 1C. For CA film, isothermal
treatment (150 1C for 15 min) was followed by
reheating to 250 1C. The second scan was done at
the same heating rate. All of the DSC experiments
were done in triplicate and the thermograms shown
refer to the second heating. The crystallinity of PCL
was determined using a heat of fusion value (DHo)
of ¼ 139.5 J/kg [21] for 100% crystalline PCL.
2.4. Compost inoculum
The compost inoculum consisted of three-month-
old, well-aerated compost derived from the organic
fraction of municipal solid waste and sieved (screen
mesh, o10 mm) to remove large, inert material. The
compost produced 75 mg of CO2 per gram of
volatile solids during the first 10 days of the test,
had a pH of 7.4, a C:N ratio of 43:1, and a total dry
solids content of 52%.
2.5. Biodegradation tests
2.5.1. Biodegradation by the Sturm test
In this test, PCL, CA and PCL/CA blends (60/40
and 40/60 wt%), as well as analytical grade cellulose
for thin layer chromatography (particle size
o20 mm; positive control) and LDPE (negative
control), were used. To assess the CO2 production
by the composted organic matter, four identical
systems were assembled for each polymer analyzed,
with each system consisting of the polymer, a
negative control, a positive control and a blank
containing 600 g of dry solids (inoculum only).
Triplicate samples of each polymer were placed in
recipient B (reactor) (Fig. 1) and immersed in the
CO2
Suction
Reactor
Ba(OH)2
100 mL
Sample
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600 M.R. Calil et al. / Polymer Testing 25 (2006) 597–604

composted organic matter in the follow proportion:
600 g of humus, 100 g of polymer and 50% of
distilled water. The reactor was maintained at
5872 1C [14], and the system was monitored every
24 h for 46 days. The CO2 produced by polymer
degradation was collected in receptacle C and the
extent of aerobic biodegradation during the test was
determined by titration to measure the amount of
CO2.
2.5.2. Biodegradation in culture medium
2.5.2.1. Preparation of culture medium. The liquid
culture medium consisted of the following salts (in
g/l): KH2PO4 0.7, MgSO4  7H2O 0.7, NH4NO3 1.0,
NaCl 0.005, FeSO4  7H2O 0.002, ZnSO4  7H2O
0.002 and MnSO4  H2O 0.001. The pH was adjusted
to 6.5 by adding 0.001 N NaOH. The solid
medium contained the salts indicated above and
15 g of agar/l.
2.5.2.2. Inoculum preparation. The fungi were
grown separately on an appropriate medium at
28 1C for two weeks. Spore suspensions were
collected by flooding the culture plates with 10 ml
of a 0.5% (w/v) solution of Tween 80 followed by
careful removal of the spores with a triangular
Drigalsky rod. The spores were transferred to a
sterile, 125 ml glass-stoppered Erlenmeyer flask
containing 45 ml of sterile water and 10–15 glass
beads 5 mm in diameter, followed by vigorous
shaking. The suspensions were filtered through glass
wool and centrifuged. The supernatant liquid was
discarded and the residue resuspended in 50 ml of
sterile water. This washing procedure was repeated
three times for the spores from each fungal species.
The spore concentration in the final suspension was
counted in a Newbauer chamber (erythrocytomer)
and adjusted to obtain 1072.1  106 spores/ml.
2.5.2.3. Mixed fungal spore suspension. A final
spore suspension for the biodegradation assay was
obtained by mixing equal volumes of spores from
each species prepared as described above.
interval and the samples were then incubated at
28 1C for 63 days. At the end of this period, the
samples were weighed and the loss of mass was
calculated as the difference between the first and
second weighings.
2.5.3. Liquid medium
The loss of mass by pre-weighed samples was
measured after aerobic incubation in liquid medium
inoculated with the mixed spore suspension. Plastic
samples (10 mm  10 mm  1 mm) were disinfected
for 15 min in 70% ethanol, rinsed twice (15 min
each) in sterile distilled water, and incubated at
28 1C for 140 days. Periodically, the samples (in
triplicate) were retrieved aseptically, washed with
sterile distilled water, dried, weighed and then
returned to the flasks containing the culture
medium.
3. Results and discussion
3.1. Thermal analysis
Table 1 shows the melting temperature and
crystallinity values for PCL, CA and PCL/CA
blends, and Fig. 2 shows the DSC thermograms
for the second heating.
The equilibrium melting temperature, i.e., the
melting temperature (Tm) corresponding to the
melting point of an infinitely large lamella, was
68.3 1C for PCL. PCL is a highly flexible polyester
that can crystallize during rapid cooling, and its
blending with CA reduced this Tm by 9%–62.3 1C in
the 60PCL/40CA blend, and by 8.5%–62.5 1C in the
40PCL/60CA blend. In contrast, the presence of
PCL slightly increased (by 3%) the Tm of CA in the
60PCL/40CA and 40PCL/60CA blends, indicating
that PCL and CA were immiscible, as previously
reported [22], probably because there were too few
Table 1
Melting temperatures of PCL, CA, PCL/CA blends, and
crystallinity of PCL and PCL/CA blends

2.5.2.4. Solid medium. The polymer films PCL/CA Melting temperature Crystallinity (%)
(10 mm  10 mm  1 mm) were weighed, disinfected formulation (1C)
(wt%)
for 15 min in 70% ethanol, rinsed twice (15 min
PCL CA PCL Blend
each) in sterile distilled water, and placed on a
3–6 mm deep layer of solidified medium in sterile 100/0 68.3 — 54.2 54.2
dishes. The entire surface of the medium was 60/40 62.3 228.8 60.0 36.0
inoculated with the suspension of mixed spores. 40/60 62.5 230.3 55.0 22.0
0/100 — 224.1 — —
Three replicates were prepared for each time
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M.R. Calil et al. / Polymer Testing 25 (2006) 597–604
0PCL/100CA
40PCL/60CA
60PCL/40CA
Heat flux endo
100PCL/0CA
0 50 100 150 200 250
Temperature (°C)
Fig. 2. DSC curves for PCL, CA, and PCL/CA blends.
hydroxyl groups in CA to interact with PCL. Blends
of poly(L-lactic acid) (PLLA), another biodegrad-
able polyester polymer, with PCL are also immis-
cible [23] but show good properties. PCL is miscible
with poly(vinyl chloride) (PVC) and poly(vinyl
alcohol) (PVA) because the carbonyl group of
PCL can form intermolecular bonds with the polar
functional groups of other polymers [24]. The
thermograms of the 40PCL/60CA and 60PCL/
40CA blends showed a small shoulder after the
main Tm peak, and the addition of CA reduced the
Tm of PCL, probably because of the bulky group
structure of CA and the insufficient content of free
hydroxyls to overcome the structural disorder
produced in the polymers during blending [22].
The intensity of the Tm peak of the blends decreased
as the amount of CA increased, indicating that the
blends were not miscible, although this effect varied
with the blend composition.
CA has a hydroxyl group attached to the
aromatic ring and, although this group could act
as a proton donor to form hydrogen bonds with
proton acceptor polymers, such an interaction is
insufficient to ensure miscibility of the polymers
[24]. El-Shafee et al. [25] reported miscible blends
mediated by interaction of the amorphous regions
of poly(3-hydroxybutyrate) (PHB) with CA buty-
rate (CAB) based on an analysis of the melting
point depression of the crystalline polymer in the
blend. Buchanan et al. [26] observed that when the
Tm of CA was plotted versus the DS, a minimum
was observed close to a DS of 2.3, and the resulting
curve allowed extrapolation to a DS of 2.4,
601
equivalent to approximately 232 1C, which is higher
than the Tm of 224 1C seen here (Table 1).
The crystallinity value for PCL agreed with that
described elsewhere [27], whereas that for CA has
not been reported before. The influence of CA on
the crystallization of PCL was studied by DSC, a
technique that also provides information on the
miscibility between PCL and CA. The addition of
CA increased the crystallinity of PCL in the 60PCL/
40CA and 40PCL/60CA blends, and favored the
organization of the PCL chains; this organization
could reduce the biodegradation of this polymer by
fungi in liquid medium. However, the crystallinity
of the blends decreased as the amount of CA
increased since CA is essentially an amorphous
polymer (Table 1). This decrease in crystallinity
probably facilitated attack by microorganisms
during aerobic biodegradation in the Sturm test,
thereby ensuring early hydrolysis of the blends,
particularly in the amorphous regions of the
polymers.
3.2. Biodegradation
3.2.1. Biodegradation in the Sturm test
Fig. 3 shows the biodegradation of PCL, CA,
PCL/CA blends, LDPE and microcrystalline cellu-
lose in the Sturm test.
PCL showed more biodegradation than CA.
However, the 40PCL/60CA blend produced more
CO2, indicating greater biodegradation of this
formulation compared to the other polymers. This
greater degradation probably reflected the structural
70
100PCL/0CA
60PCL/40CA
60 40PCL/60CA
0PCL/100CA
Cellulose
CO2 production (mg)
50 LDPE
40
30
20
10
0
0 10 20 30 40 50
Time (days)
Fig. 3. Biodegradation of PCL, CA, and PCL/CA blends based
on CO2 production.
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602 M.R. Calil et al. / Polymer Testing 25 (2006) 597–604

disorder between the polymers in this blend, as so did the mass retention, indicating that CA was
shown by DSC. The resulting decrease in crystal- less biodegradable than CA in liquid medium.
linity and the increase in amorphous regions would
in turn facilitate hydrolysis of the polymers and 3.2.3. Solid medium
attack by microorganisms present in the compost Table 2 shows the loss of mass of PCL, CA and
inoculum. After the necessary induction time, which the 60PCL/40CA and 40PCL/60CA blends in solid
corresponded to aging for about 13 days, the
microorganisms acted more efficiently and pro- Table 2
duced more CO2. Loss of mass (%) of LDPE, PCL, CA and PCL/CA blends
incubated with fungi in petri dishes
3.2.2. Liquid medium Samples Loss of mass (%)a
Fig. 4 shows the changes in mass retention for
PCL, CA and their blends in liquid medium LDPE 0.02
inoculated with a mixture of fungi. PCL showed Pure PCL 8.4
60PCL/40CA 25.8
the highest loss of mass (86.5%) and CA, the 40PCL/60CA 20.6
smallest (2.8%), with the 60PCL/40CA and 40/ Pure CA 14.3
PCL/60CA blends having intermediate values
a
(37.5% and 61.2%, respectively). The values are the average of three determinations.
Although CA showed only limited degradation
compared to PCL and the PCL/CA blends, this low
level of degradation was nevetheless evident when
compared with the corresponding control (LDPE)
values. The CA used here had a DS of 2.4, which
indicated a reduced number of free hydroxyls and
an enhanced interaction with water that would PCL CA
inhibit hydrolysis and, consequently, decrease bio-
degradation. Many microorganisms degrade macro-
molecules through the action of hydrolytic enzymes
secreted into the culture medium [28]. The degrada-
tion of PCL and its blends by fungal enzymes seen
here may have been facilitated by the liquid medium
(presence of water). As the amount of CA increased,
20PCL/80CA 40PCL/60CA

100

80
Mass retetion (%)

60PCL/40CA
60
60PCL/40CA

40
100PCL/0CA
60PCL/40CA
20 40PCL/60CA
0PCL/100CA
LDPE

0
0 20 40 60 80 100 120 140 80PCL/20CA 80PCL/20CA
Time (days)

Fig. 4. Biodegradation of PCL, CA, and PCL/CA blends by Fig. 5. Appearance of polymer samples after exposure to fungi in
fungi (liquid medium). petri dishes.
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M.R. Calil et al. / Polymer Testing 25 (2006) 597–604
medium, whereas Fig. 5 shows the fungal growth
under these same conditions.
PCL was attacked by fungi less than CA (Fig. 5),
with the loss of mass being 8.4% and 14.3%,
respectively. The low loss of mass in PCL agreed
with a previous report [29]. Table 2 shows that the
blends were much more biodegradable than pure
PCL and CA. The best substrate was the 60PCL/
40CA blend, which showed a loss of mass of 25.8%,
i.e., 80% and 207% greater than for CA and PCL,
respectively (Table 2). This finding differs from
those of the other two tests (Sturm test and liquid
medium), probably because in this case the solid
medium favored adhesion of the samples to the
substrate surface [19]. In the complex interaction of
microorganisms with polymers on the substrate
surface, the polymers may be degraded into low
molecular mass products and the resulting poly-
meric radicals may combine to form new functional
groups. According to Klemchuk [30], pure polymers
are generally not easily degraded because their
hydrophobic nature tends to inhibit the enzyme
activity of microorganisms. As shown here, the
greater the proportion of CA in the blends, the
greater the dispersion of fungi over the sample
surface.
The mechanical grinding of the polymers in the
Sturm test probably favoured adhesion of the
microorganisms involved in polymer degradation
[19]. In addition, the small size of the samples
provided a high surface area, making attack by
microorganisms easier. These observations could
explain the faster biodegadation of the polymers in
the Sturm test compared to the other methods used
here.
4. Conclusions
The results described here show that increasing
the CA concentration in blends with PCL increased
the crystallinity of PCL and favored the biodegra-
dation of this polymer. However, the Tm of PCL
decreased and that of CA increased in blends of
PCL with CA, suggesting that these polymers were
incompatible. The CA used here was not susceptible
to biodegradation, probably because the reduced
number of free hydroxyls (DS of 2.4) enhanced the
interaction with water and inhibited enzymatic
hydrolysis, thereby reducing the extent of biode-
gradation. In solid culture medium, which con-
tained less water, CA showed some biodegradation
that was still considerably less than for PCL.
603
Acknowledgments
The authors thank Union Chemical Carbide Ltd.
and Rhodia Acetow Brazil Ltda. for supplying the
PCL and cellulose acetate, respectively, and the
Laboratório de Caracterizac- ão de Materiais
(LCAM-USF) for use of their thermal analysis
equipment. This work was supported by FAPESP
(Grant no. 02/13202-6), CNPq (Grant nos. 304577/
2004-9 and 477942/2003-2), and by the Universi-
dade São Francisco.
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