ORIGINAL PAPER

PLA and Montmorilonite Nanocomposites: Properties,Biodegradation and Potential Toxicity

Patrıcia Moraes Sinohara Souza •

Ana Rita Morales • Maria Aparecida Marin-Morales •

Lucia Helena Innocentini Mei

� Springer Science+Business Media New York 2013

Abstract The concern related to solid waste increases

efforts to develop products based on biodegradable mate-

rials. At present, PLA has one of the highest potentials

among biopolyesters, particularly for packaging. However,

its application is limited in some fields. In order to optimize

PLA properties, organo-modified montmorilonites have

been extensively used to obtain nanocomposites. Although

PLA nanocomposites studies are widely reported in the

literature, there is still few information about the influence

of organoclays on de biodegradation process, which is a

relevant information, since one of the main purposals

related to the final disposal of biopolymers as PLA is

composting. Besides, in the last years some research has

been conducted in order to evaluate the potential toxicity of

montmorilonite, unmodified or organo-modified. Since the

use of montmorilonite is expanding in different applica-

tions, human exposure and risk assessment are important

issues to be investigated. In this context, this review

intends to compile available information related to com-

mon organoclays used for PLA nanocomposites, its prop-

erties, biodegradation analysis and potential toxicity

evaluation of nanocomposites, focused on montmorilonite

as filler. Two issues of relevance were pointed out. The first

is food safety and quality, and the second consideration is

the environmental effect.

Keywords PLA � Organoclays � Nanocomposites �Biodegradation � Toxicity

PLA

PLA or poly (lactic-acid) was discovered in 1932 by Car-

others (at DuPont). He was only able to produce a low

molecular weight PLA by heating lactic acid under vacuum

while removing condensed water. PLA was first used in a

blend with polyglycolic acid, as suture material and sold

under the name ‘Vicryl’ in the USA in 1974 [1].

Lactic acid has two optical isomers (L-lactic acid and

D-lactic acid). Chemical synthesis of lactic acid is mainly

based on the hydrolysis of lactonitrile by strong acids, which

provides only the racemic mixture of D-lactic acid and

L-lactic acid. The interest in the fermentative production of

lactic acid has increased due to the prospects of environ-

mental friendliness and the use of renewable resources

instead of petrochemicals. Besides, fermentation allows

obtaining desired optically pure L-lactic acid or D-lactic acid.

Low cost of substrates, low production temperature and low

energy consumption are some advantages of biotechnolog-

ical production compared to chemical synthesis [2].

Many microbes can produce lactic acid, but a competi-

tive commercial process requires a fast-growing and high-

yield strain with low-cost nutrient requirements. Typically

Lactobacillus fermentation, which occurs under anaerobic

conditions, fulfills these requirements [3]. The diversity of

carbohydrates that can be utilized in the fermentation

depends on the particular strain of Lactobacillus. In gen-

eral, most of the simple sugars obtained from agricultural

byproducts can be used. These sugars include (1) maltose

and dextrose from corn or potato starch; (2) sucrose from

cane or beet sugar; and (3) lactose from cheese whey [4].

P. M. S. Souza � A. R. Morales (&) � L. H. I. Mei

Department of Materials Engineering and Bioprocess, School

of Chemical Engineering, State University of Campinas,

Campinas, SP, Brazil

e-mail: morales@feq.unicamp.br

M. A. Marin-Morales

Department of Biology, Institute of Biosciences, Sao Paulo State

University, Rio Claro, SP 13083-852, Brazil

123

J Polym Environ

DOI 10.1007/s10924-013-0577-z

The main fermentation pathways in lactic acid bacteria

are well known and can be divided into homofermentative

or heterofermentative. Homofermentative bacteria are

classified as those which produce lactic acid through the

Embden-Meyeorf pathway, converting as much as 1.8 mol

of lactic acid per mol of hexose ([90 % yield lactic acid

from glucose). Heterofermentative bacteria are classified as

those which produce less than 1.8 mol of lactic acid per

mol of hexose, with minor levels of other metabolites being

formed, including acetate, ethanol, glycerol, formate,

mannitol, and carbon dioxide. The homofermentative

pathway is of industrial importance in lactic acid manu-

facture, due to the greater yield of lactic acid and the lower

levels of fermentation byproducts [5].

The stereospecificity of the lactic acid depends on the

enzyme which is involved in its production: L-lactate

dehydrogenase or D-lactate dehydrogenase [2]. The

organisms that predominantly yield the L-lactic acid are

Lactobacilli amylophilus, Lactobacilli bavaricus, Lacto-

bacilli casei, Lactobacilli maltaromicus and Lactobacilli

salivarius. Strains such as Lactobacilli delbrueckii, Lac-

tobacilli jensenii, or Lactobacilli acidophilus yield the

D-isomer or mixtures of both [6].

In general, there are three methods which can be used to

produce high molecular mass PLA of about 100,000 Daltons:

direct condensation polymerization; azeotropic dehydrative

condensation and polymerization through lactide formation

[7]. The condensation polymerization is the least expensive

route but it is difficult to obtain high molecular weights,

which makes necessary the use of coupling agents or ester-

ification-promoting adjuvants, adding cost and complexity

[8–11]. Different coupling agents added in order to increase

the molecular weight can form either hydroxyl-terminated

PLA (condensation in presence of small amount of multi-

functional hydroxyl compounds such as glycerol) or car-

boxyl-terminated PLA (condensation in presence of

multifunctional carboxylic acids such as maleic acid) [4].

Azeotropic dehydrative condensation of lactic acid per-

mits yield high molecular weight poly (lactic acid) without

using chain extenders or adjuvants. The general procedure

consists in the reduction of distillation pressure of lactic

acid for 2–3 h at 130 �C in order to remove condensation

water. Catalyst and diphenyl ester are added. After that, a

tube packed with 3 A molecular sieves is attached to the

reaction vessel, and the reflux solvent is returned to the

vessel via the molecular sieves for an additional 30–40 h at

130 �C. The polymer is then isolated as it is or dissolved

and precipitated for further purification. This technique

permits obtaining high-molecular-weight polymers, but

with considerable catalyst impurities due to high levels

needed for acceptable reaction rates [4, 7, 12, 13].

Polymerization by lactide formation is the current

method used by Cargill Dow LLC to obtain high molecular

weight polymers for commercial applications. From dex-

trose fermentation, either D-lactic acid, L-lactic acid or a

mixture of both are pre-polymerized to obtain an inter-

mediate low molecular weight poly (lactic acid). Under

lower pressure, the pre-polymer is catalytically converted

into a mixture of lactide stereoisomers. Lactide, which is

the cyclic dimer of lactic acid, is formed by the con-

densation of two molecules, combining the isomers as

follows: L-lactide (two L-lactic acid molecules), D-lactide

(two D-lactic acid molecules) and meso-lactide or

D,L-lactide (an L-lactic acid and an D-lactic acid mole-

cule) [7].

The different percentages of the formed lactide isomers

depend on the lactic acid isomer feedstock, temperature

and catalyst. The D-lactide and L-lactide enantiomers can

form 1:1 racemic stereocomplex (D,L-lactide), which melts

at 126–127 �C, significantly higher than the pure isomer.

This complex is commonly referred to as D,L-lactide to

differentiate it from meso-lactide [14].

Before the polymerization process, the lactide flow is

split into a low D-lactide stream and a high D/meso lactide

stream. A ‘family’ of polymers, characterized by the

molecular weight distribution and by the amount and the

sequence of D-lactide in the polymer backbone, can be

produced by ring opening polymerization of optically

active types of lactide [15]. Both meso- and D-lactide

induce twists in other very regular molecular architecture

of poly (L-lactide). The molecular imperfections are

responsible for decrease in both rate and extent of poly

(L-lactide) crystallization [7].

According to the model proposed by Kowalski et al.

[16], the lactide ring can be opened by nucleophilic attack

on the ester bond to initiate polymerization. Water and

alcohols are suitable initiators (nucleophiles), including the

hydroxyl group of lactic acid.

The catalyst tin(II) bis-2-ethylhexanoic acid, usually

referred as tin octanoate-Sn(Oct)2, has been widely used

for PLA synthesis. This is mainly due to its solubility in

many lactones, low toxicity, Food and Drug Administration

(FDA) approval, high catalytic activity and ability to give

high-molecular-weight polymers with low racemization

[6, 17, 18]. Results of kinetics studies of cyclic esters

polymerization revealed that Sn(Oct)2 needs activa-

tion with R-OH and the actual initiator is the tin(II) alk-

oxide formed as described in schemes Scheme 1a and

Scheme 1b [16].

Sn Octð Þ2þROH� OctSnORþ OctH ðScheme1aÞ

OctSnORþ ROH� Sn ORð Þ2þOctH ðScheme1bÞ

The tin alkoxide, Sn(OR)2, is the initiating species in the

ring-opening polymerization of the cyclic ester monomer

through coordination-insertion mechanism [19].

J Polym Environ

123

The stereochemical composition of the lactide monomer

stream can determine the stereochemical composition of

the resulting polymer since bonds to the chiral carbons are

not broken in the polymerization. Polymerization of

L-lactide produces poly(L-lactide) and polymerization of

D-lactide yields poly(D-lactide). Poly(L-lactide) and poly

(D-lactide) have identical properties except their stereo-

chemistry. However, racemic (50 % D- and 50 % L-Lac-

tide) mixture gives poly (DL-lactide), that is an amorphous

polymer. In addition, PLA can be produced with varying

fractions of L and D lactide. It is well established that the

properties of polylactides vary to a large extent, depending

on the ratio and the distribution of the two isomers and

molecular weight of the polymer [20]. Isotactic PLLA

homopolymer, comprising L-lactide only, is a semi-crys-

talline material with the highest melting point, while PLA

copolymers with higher D-isomer content exhibit lower

melting points and dramatically slower crystallization rate

[21–23].

PLA can be processed by injection molding, sheet

extrusion, blow molding, thermoforming, and film blowing

and it is approved by the FDA for its intended use in

fabricating articles in contact with food [6]. Currently, PLA

is being commercialized as a food packaging polymer for

short shelf life products with common applications such as

containers, drinking cups, sundae and salad cups, overwrap

and lamination films, and blister packages [24].

PLA/Montmorilonite Nanocomposites

Montmorilonite (MMT) belongs to a family of clays known

as smectite with crystal structure made up of two silica

tetrahedral sheets sandwiched with an edge-shared octa-

hedral sheet of either aluminum or magnesium hydroxide.

The thickness of a single layer is about 1 nm, while the

lateral dimension of the crystals can range from 30 nm to

several microns or greater. The crystal layers are stacked

regularly to provide van der Waals gaps, known as galleries

[25, 26]. The silicate surface of MMT is relatively more

hydrophilic than PLA, which justifies the use of an

organically modified nanoclay to compatibilize and facili-

tate its dispersion in the polymer matrix. One useful

characteristic of MMT is the presence of cations in the

galleries, typically Na?, Li?, Ca2?, Fe2? and Mg2?, that

can be readily substituted through ion exchange with

organic cations, by treating the clay with surfactants

including primary, secondary, tertiary or quaternary alky-

lammonium or alkylphosphonium cations [25].

Table 1 presents some common modified montmorilo-

nites used for preparing PLA nanocomposites, the modifier

structure and commercial names.

Regarding to the preparation of polymer–clay nano-

composites, the interaction mechanism of the polymer and

clay depends on the polarity, hydrophobicity and reactive

groups of the polymer, and the clay mineral type [27]. The

main techniques for preparing polymer-clay nanocompos-

ites are in situ intercalative polymerization, intercalation of

polymer from solution (or solvent casting) and melt inter-

calation. At in situ polymerization method the monomer is

primarily absorbed into clay interplanar spaces and then

polymerization occurs, allowing formation of polymer

chains between the intercalated sheets. The intercalation of

polymer from solution method requires a solvent capable

of solubilizing the polymer and swelling the silicate layers

in order to promote intercalation of polymer from solution.

When the layered silicate is dispersed within a solution of

the polymer, the polymer chains intercalate and displace

the solvent within the gallery of the silicate. By the solvent

evaporation the nanocomposite can then be obtained

[28, 29].

Recently, melt intercalation has become the method of

choice because it is the most industrially viable approach

that can lead to commercialized processes using the

infrastructure already existing in the plastic industries. In

this method, a dry mixture of the polymer pellets and the

nanoclay powders is blended under shearing action, at a

temperature above the melting point of the polymer [30].

The shearing action is very important because it facilitates

the diffusion of the polymer chains from the bulk polymer

melt into the galleries between the silicate layers of

nanoclay [31, 32].

The absence of solvent makes melt compound technique

an economical and environmentally sound method. As a

result of the organic modification, the nanoclays are

intercalated with alkyl ammonium cations bearing long

alkyl chains, which increase the interlayer spacing and can

improve the compatibility of this nanofiller with the poly-

mer matrix [30]. Depending on the chemical modification

and structure of the modifier used in organoclays, disper-

sion of their layers in the polymer matrix might be pro-

moted. For instance, the interaction of hydrogen-bonding

between hydroxyl groups in the organic modifier of the

organoclay with the carbonyl group of PLA chain segments

contributes to their deagglomeration and dispersion in the

PLA matrix induced by shearing forces [33]. Strong

interactions between the PLA hydroxyl end groups and the

MMT platelet surfaces, or the hydroxyl groups of the

ammonium surfactant in the organically modified MMT

could also occur, as reported by Jiang et al. [34].

The properties of MMT nanocomposites are highly

dependent on how well the clay disperses in the polymer

matrix. In general, clay dispersion can be distinguished in

three modes: (1) the clay particles are not delaminated. In

this case, the resulting materials tend to exhibit similar

J Polym Environ

123

properties as conventional microcomposites. The unsepa-

rated MMT layers surrounded by the polymer are often

referred to as tactoids; (2) the polymer chains are inserted

into the galleries of the swollen silicate layers, the clay is

known as intercalated, leading to decreased polymer chain

mobility and resulting in material reinforcement; and

(3) the clay is completely delaminated and homogeneously

dispersed in the continuous polymer matrix, the layered

silicates are termed exfoliated nanocomposites, giving rise

to the maximal potential for physical properties enhance-

ment [35].

The combination of PLA and montmorilonite-layered

silicate may result in a nanocomposite with good barrier

properties, due to the tortuous path created by clay parti-

cles. A theoretical model of the gas permeability of the

nanocomposite films was formulated by Nielsen in 1967

[36]. For his theoretical expressions, Nielsen assumed that

the sheets are placed perpendicular to the diffusive

pathway.

In this model, the gas permeability of the nanocom-

posites (PPLACN) is related to the permeability of the neat

polymer (PPLA), the volume fraction (/clay), length (Lclay),

and width (Dclay) of the clays, as in Eq. 1:

PPLACN

PPLA¼ 1

1þ ðLclay=2DclayÞ/clay

ð1Þ

The tortuosity factor (s) was defined as:

s ¼ 1þ Lclay

2Dclay

� �/clay ð2Þ

The gas permeability of nanocomposite films depends

primarily on the dimension of the layered silicate particles,

its dispersion in the polymer matrix and the percentage of

silicate particles loaded in the film [37]. Some results

reported in the literature, which indicate an improvement in

barrier properties to CO2, O2 and water vapor permeability

(WVP) in PLA/MMT nanocomposites, are listed in

Table 2.

Table 1 Common modified montmorilonites used for PLA nanocomposites preparation

Modifier Modifier structure Commercial name

Methyl, tallow, bis-2-hydroxyethyl, quaternary

ammonium

N+

TCH3

CH2CH2OH

CH2CH2OH

CLOISITE 30B/Southern Clay Products

Dimethyl, dehydrogenated tallow, 2-ethylhexyl

quaternary ammonium

CH3 N+

CH2CHCH2CH2CH2CH3

CH2CH3

CH3

HT

CLOISITE 25A/Southern Clay Products

Dimethyl, dihydrogenated tallow, quaternary

ammonium

CH3 N+

HT

CH3

HT

CLOISITE 20A/Southern Clay Products

CLOISITE 15A/Southern Clay Products

Methyl, dihydrogenated tallow, quaternary

ammonium

CH3 N+

HT

H

HT

CLOISITE 93A/Southern Clay Products

Octadecyl ammonium

H N+

H

H

C18H37

NANOMER 1.30P/Nanocor

Stearyl dihydroxyethyl ammonium NANOFIL 804/Sud-Chemie

* T is tallow having around 65 % C18, 30 % C16 and 5 % C14

J Polym Environ

123

Although PLA is primarily used in packaging, its

expected market should be rapidly extended to electrical

and electronic equipment sector, which requires flame re-

tardancy. A possible way of decreasing PLA flammability

is the incorporation of montmorilonite in the polymer

matrix [46]. Bourbigot et al. [47] reported a significant

reduction of 40 % in the peak heat release rate (PHRR) in

PLA when dispersing 3wt.% of montmorilonite platelets. A

mechanism commonly used to explain flame retardancy in

nanocomposites is a clay-containing barrier, which is

formed gradually by the residue of the clay particles during

the pyrolysis and combustion of the polymer. However,

another hypothesis has been reported in the literature.

According to this new hypothesis the clay particles can

migrate to the surface before pyrolysis and combustion

take place, so that the major new ingredient of the charred

barrier accumulates on the surface before the beginning of

the sample gasification. Therefore, the migration process of

the clay could happen at temperatures below the pyrolysis

temperature [48].

The migration process of clays in polypropylene/or-

ganoclay nanocomposites at elevated temperatures was

investigated by Lewin et al. [48], which conducted X-Ray

Diffraction (XRD) and Attenuated Total Reflectance Fou-

rier Transform Infrared (ATR-FTIR) measurements on

isothermally heated samples. Upon annealing of samples it

was observed that extent of migration diminishes with

increasing temperature of annealing (in the range of 200

until 300 �C). At 200 �C, almost no decomposition of

surfactant occurs. In this case, the clay particles are col-

loidal nanostructures governed by colloidal forces. A

dominant force in the nanocomposite polymer melt is the

surface free energy (SFE). The clay can migrate to the

surface due to difference in the interfacial tension between

clay and polymer, and the surface free energy of the

polymer itself. Above 200 �C the nanosctructure collapses

along with the decomposition of the surfactant, and mi-

crocomposite particles are formed. These particles, which

are not colloidal, cannot be propelled to the surface by the

SFE difference, and they stay suspended (by the viscosity

and temperature gradients, as well as by the relatively

abundant decomposition gases and bubbles) or form sedi-

ment on the sample. The microcomposite phase coexists

with the nanocomposite. With the increase of the propor-

tion of the microcomposite, the amount of the nanocom-

posite in the system decreases, therefore the reservoir of

migrating clay particles decreases until at 300 �C. This

aspect was evidenced by the concentration of clay on the

surface which at 300 �C was smaller than that on the

bottom.

The study conducted by Tang et al. [49] revealed that

extent of migration is much slower in case of nylon-6

(PA6)/organoclay nanocomposites than those of polypro-

pylene previously reported [48], which was explained by

the lower mobility of the exfoliated particles in the PA6

matrix, probably due to strong linkages formed by end

amine groups of PA6 and negative sites of clay surfaces.

Studies conducted at temperatures below the onset of

surfactant and polymer decomposition, in the range of

180–200 �C, indicated that the extent of migration is

shown to increase with the increase in the percentage of

polymer polarity, mainly as consequence of increasing the

percentage of air added to the nitrogen gas used for purging

of the samples during annealing. The authors pointed out

five steps in the oxidation-migration cycle, resulting in the

migration of exfoliated clay to surface. First step is

Table 2 MMT/PLA nanocomposites: preparation methods, organomodifiers and barrier properties

Modifier Preparation method (organoclay wt %) Gas permeability Reference

Dimethyl, dihydrogenatedtallow,

quaternary ammonium

Solvent casting (5.0) WVP reduced by 36 % Rhim et al. [38]

Solvent casting (0.8) CO2 permeability reduced by 30 % Koh et al. [39]

Methyl, tallow, bis-2- hydroxyethyl,

quaternary ammonium

Solvent casting (5.0) WVP reduced by 5 % Rhim et al. [38]

Melt intercalation (4.5) O2 permeability reduced by 62 %

WVP reduced by 41 %

Katiyar et al. [40]

Solvent casting (0.8) CO2 permeability reduced by 50 % Koh et al. [39]

Octadecyl ammonium Melt intercalation (4.0) O2 permeability reduced by 14 % Ray and Okamoto [41]

Melt intercalation (5.0) O2 permeability reduced by 46 % Chowdhury [42]

Dimethyl, hydrogenated tallow,

2-ethylhexyl quaternary ammonium

Melt intercalation (5.0) O2 permeability reduced by 48 %

WVP reduced by 50 %

Thellen et al. [43]

Dioctadecyl, dimethyl ammonium Melt intercalation (4.0) O2 permeability reduced by 14 % Ray and Okamoto [41]

Octadecyl, trimethyl ammonium Melt intercalation (4.0) O2 permeability reduced by 11 % Ray and Okamoto [41]

n-dodecyl tri-n-butyl phosphonium Melt intercalation (3.8) O2 permeability reduced by 39.5 % Maiti et al. [44]

Hexadecylamine Solvent casting (4.0) O2 permeability reduced by 42 % Chang et al. [45]

Dodecyl, trimethyl ammonium Solvent casting (4.0) O2 permeability reduced by 41 % Chang et al. [45]

J Polym Environ

123

diffusion of oxygen into the melt. The second is oxidation

of polymer matrix molecules producing polar groups. The

third involves intercalation of these molecules into the clay

gallery. The fourth is exfoliation. Finally, the fifth is

migration of the exfoliated layers [50, 51].

PLA Biodegradation

A schematic overview of polymers biodegradation reaction

under aerobic conditions is given in Eq. 3 [52].

Cpol þ O2 ! CO2 þ H2Oþ Cres þ Cbio ð3Þ

Microbes use oxygen to oxidize carbon and form carbon

dioxide as one major metabolic end-product. Therefore, the

carbon in the polymer (Cpol) is primarily converted into the

inorganic form (CO2) [52]. The biodegradation can also

result in the production of microbial cellular constituents

(biomass) [53]. The biomass yield (Cbio) is typically

between 10 and 40 % depending on the substrate which is

converted. The Cres consists of partially undegraded

residues or metabolites. In any case the Cres cannot be

considered as being fully biodegraded [52].

Under anaerobic biodegradation the carbon in the

polymer (Cpol) is converted into biogas, a mixture of

methane and carbon dioxide, as shown in the Eq. 4 [52].

Cpol ! CH4 þ CO2 þ Cres þ Cbio ð4Þ

The degree of aerobic biodegradation can be determined

by measuring the total amount of carbon dioxide gas (CO2)

evolved from the polymer. The degradation of biopolymers

is generally defined as percentage of mineralization, which

is the proportion of cumulative CO2 gas actually generated

by the sample tested to the theoretical CO2 content of the

material [54]. According to ASTM D6400 [55], a

compostable plastic demonstrate a satisfactory rate of

biodegradation when achieving 90 % of the organic carbon

in the whole item or for each organic constituent, which is

present in the material at a concentration of more than 1 %

(by dry mass), is converted to carbon dioxide by the end of

the test period when compared to the positive control or in

the absolute. The ratio of conversion to carbon dioxide can

be found within 180 days using the test methods D5338

[56], ISO 14855–1 [57], or ISO 14855–2 [58].

The standard ASTM D5338 decribes a test method

which determines the degree and rate of aerobic biodeg-

radation of plastic materials on exposure to a controlled-

composting environment under laboratory conditions, at

thermophilic temperatures [56]. Based on this standard,

respirometric systems can be categorized by measuring

technique: cumulative measurement respirometric (CMR)

and direct measurement respirometric (DMR). Acid-base

titration is normally used in the cumulative measurement

technique. The evolved CO2 gas is trapped in a sodium

hydroxide (NaOH) or barium hydroxide (Ba(OH)2) solu-

tion. Then, the trap solution is titrated using a known

concentration of hydrochloric acid. For the direct mea-

surement technique, the concentration of CO2 is measured

directly from the exhaust or outlet of the bioreactor using

gas chromatography (GC), or an in-line infrared gas ana-

lyzer, such as a nondispersive infrared (NDIR) gas analyzer

[54]. Standard test ISO 14855-1 gives similar guidelines to

that of ASTM D5338, and ISO 14855-2 is similar to ISO

14855-1 except for the method of CO2 measurement and

the amount of compost and sample used. In addition, inert

materials such as sea sand or vermiculite can be used with

the compost for providing better aeration and retention of

moisture content [59].

The biodegradation of plastics depends on both envi-

ronmental factors (i.e., temperature, moisture, oxygen, pH,

microorganisms consortium) and the chemical structure of

the polymer [60]. PLA, as one member of the polyester

family, presents ester linkages in its polymer backbone

which are susceptible to hydrolysis [24]. During the initial

step of degradation, the high molecular weight PLA chains

hydrolyze to lower molecular weight oligomers. The rate of

this hydrolysis can be accelerated by acids or bases and is

dependent on both moisture content and temperature [61].

Generally, microorganisms do not assimilate high molec-

ular weight molecules (e.g., for PLA, Mw C 20,000 Da)

because they are too large to penetrate into the microor-

ganisms’ cells [60]. On contrary, low molecular weight

substances can be assimilated, and converted into metab-

olites [62]. The upper limit of molecular weight that

microbes can metabolize differs by polymer. A critical

molecular weight (Mw) for PLA is 10,000–20,000 Da

[7, 63].

The molecular weight of a polymer affects the biodeg-

radation rate in two different ways. As the molecular

weight increases, the polymer’s glass transition tempera-

ture (Tg) also increases, which makes the polymer less

flexible. The flexibility of a polymer chain is related to the

energy required to rotate molecules around bonds and to

the facility on moving atoms closer to or further away from

others. If a polymer chain is more conformationally flexi-

ble, more water can penetrate the interchain spaces and

increase the rate of hydrolytic degradation. Furthermore, a

higher molecular weight polymer also has a longer chain

length, and consequently more bonds should be cleaved in

order to form the water-soluble oligomers or monomers

that are small enough to be consumed by microorganisms

[60].

It is well known that the degradation of PLA occurs

slowly in soil. Soil burial tests have shown that its degra-

dation takes a long time to start. Meanwhile, PLA can be

degraded in a composting environment where it is

J Polym Environ

123

hydrolyzed into smaller molecules (oligomers, dimers, and

monomers) after 45–60 days at 50–60 �C. These smaller

molecules are then degraded into CO2 and H2O by

microorganisms in the compost [64].

Some studies have been conducted at large scale and

interesting results were obtained comparing natural and

composting conditions. Rudnik and Briassoulis [65]

reported visual analysis of PLA films of different thick-

nesses during long-term biodegradation under Mediterra-

nean natural soil environment. The first significant changes

were observed only after 7 months. It was also verified that

the thinner the films, the more pronounced biodegradation

could be observed. After 11 months trials, the films were

disintegrated to a low degree. Kale et al. [24] evaluated

three PLA packages, a bottle, a tray, and a deli container in

a compost pile. A significant degradation of the PLA

packages was confirmed within 30 days under composting

conditions by an exponentially decrease of molecular

weight and an evident disintegration, verified by visual

analysis.

Hydrolitic Degradation

From the molecular viewpoint, ester hydrolysis is a well

known reaction in organic chemistry. The hydrolytic

reaction of esters can be catalyzed by acids and bases. The

reaction product of hydrolysis is able to accelerate ester

hydrolysis by autocatalysis. In the case of aliphatic poly-

esters, as PLA, chain cleavage at the ester bond is auto-

catalyzed by carboxyl end groups initially present or

generated by the degradation reaction [66].

Tsuji [67] studied effects of L-lactide unit content, tac-

ticity, and enantiomeric polymer blending on autocatalytic

hydrolysis of different PLA films. The non-blended

PDLLA, PLLA and PDLA films and PLLA/PDLA(1/1)

blend film were prepared to be at amorphous state. The

samples were investigated in phosphate-buffered solution

(pH 7.4) at 37 �C for up to 24 months. The analysis of

mass loss, Gel Permeation Chromatography and tensile

testing showed that the autocatalytic hydrolyzabilities of

PLAs in the amorphous state decreased in the following

order: nonblended PDLLA [ nonblended PLLA, non-

blended PDLA [ PLLA/PDLA(1/1) blend. The high

hydrozability of the nonblended PDLLA film compared

with those of the nonblended PLLA and PDLA films was

attributed to the lower tacticity of PDLLA chains, which

decreases their intramolecular interaction and therefore

PDLLA chain are susceptible to the water molecules

attack. In contrast, the retarded hydrolysis of PLLA/

PDLA(1/1) blend film compared with those of the non-

blended PLLA and PDLA films was attributed to the

peculiar strong interaction between PLLA and PDLA in the

blend film, resulting in the disturbed interaction of PLLA

or PDLA chains and water molecules.

The structure of PLLA consists on left-handed helical

chains [68]. Since PDLA must have a right-handed helical

crystalline structure, it is likely that the stereocomplex is

formed through van der Waals forces such as dipole–dipole

interaction between the two different helical chains in

solution where molecular motion is sufficiently great [69].

In stereocomplex (racemic) crystallites, formed as a result

of stereocomplexation, equimolar L-lactide and D-lactide

unit sequences, are packed side-by-side or can crystallize

separately in different crystallites (homo-crystallites), even

when PLLA and PDLA are equimolarly present in the

system. Stereocomplexation (racemic crystallization) and

homocrystallization dominates in solutions and from the

melt when the molecular weights of the two enantiomeric

polymers are low and high, respectively [70, 71].

Reducing the interaction between PLLA and PDLA

could be attained by the homo-crystalization before in vitro

hydrolysis. In this case the hydrolysis rate of the enantio-

meric blend specimen would expect to become similar to

those of nonblended specimens. This hypothesis was

evaluated in the study reported by Tsuji [72], in which

hydrolysis of homo-crystallized enantiomeric blend and

nonblended PLLA and PDLA films were compared. The

films were melted at 250 �C for 3 min and then homo-

crystallized at 140 �C for 600 min, followed by quenching

at 0 �C, as suggested by Tsuji and Ikada [71]. The samples

were submitted to in vitro hydrolysis in phosphate-buffered

solution (pH 7.4) at 37 �C for up to 24 months. The

hydrolytic rate constant values were estimated for hydro-

lysis period of 0–12 and 12–24 months. In the period of

0–12 months, the effects of enantiomeric polymer blending

on hydrolysis were very small indicating that in the enan-

tiomeric blend film separate homo-crystallization of PLLA

and PDLA into the respective crystallites have reduced the

peculiar strong interaction between PLLA and PDLA in the

amorphous region between the homo-crystalline regions.

However, in the period of 12–24 months, the polymer

blending significantly retarded the autocatalytic hydrolysis

of the enantiomeric blend film compared to PLLA and

PDLA films. This could be attributed to the increased chain

mobility and reduced entanglement effects due to the chain

cleavage to a great extent, resulting in the enhanced

interaction between PLLA and PDLA chains [72].

Hydrolytic reactions are controlled in part by the rate of

water diffusion, which preferentially occurs into the flexi-

ble amorphous regions in the polymer bulk, since the dif-

fusion through crystalline regions is very low. In general,

amorphous regions are more susceptible to hydrolysis than

crystalline regions [73]. Interestingly, it has been verified

by different authors that during hydrolytic degradation

PLA tend to present whitening [60, 74, 75]. This behavior

J Polym Environ

123

can be a result of increased samples crystallinity [74].

Hydrolytic degradation occurs in two stages: the first stage

occurs in the amorphous regions and the remaining unde-

graded chain segments win more space and mobility, which

lead to reorganizations of the polymer chains and an

increased crystallinity; the second stage of the hydrolysis

takes place in the crystalline regions, leading to an increase

in the rate of mass loss [76, 77].

The remaining crystalline regions have extended-chain

crystallite structures and are called ‘‘crystalline residues’’.

Few studies have focused on the hydrolytic degradation of

these remaining regions. Since medical applications of

PLA include scaffolds for tissue regeneration and matrices

for drug delivery systems, this kind of investigation is

relevant, because crystalline residues could remain for a

long period in the human body [78, 79]. Tsuji and Ikarashi

[80] prepared PLLA crystalline residues rapidly by high-

temperature hydrolysis of crystallized PLLA films in a

phosphate-buffered solution (PBS) at 97 �C and formed

crystalline residues, which were further hydrolyzed in PBS

at 37 �C for the periods of time up to 512 days. The

authors reported that crystalline residues could remain for a

long period such as ca. 2 9 103 days (ca. 5.5 years) in the

human body even after PLLA loses its functions as bio-

materials. The study conducted by Tsuji and Tsuruno [81]

estimated that the activation energy for hydrolytic degra-

dation of stereocomplex crystalline residues of a PLLA/

PDLA blend (97.3 kJ mol-1) was significantly higher than

75.2 kJ mol-1 reported for a-form of PLLA crystalline

residues, indicating that stereocomplex crystalline residues

showed the higher hydrolysis resistance compared to that

of a-form of PLLA crystalline residues.

PLA with different degrees of crystallinity has different

hydrolytic degradation rates, as a result of different isomers

contents [7]. D-lactide, by inducing twists in the very reg-

ular poly (L-lactide) molecular architecture, reduces poly-

mer crystallinity resulting PLA polymers with higher

contents of D-lactide to degrade much faster [60]. Saha and

Tsuji [82] reported that the incorporation of small amounts

of D-lactic acid units (0.2 and 1.2 %) drastically enhanced

the hydrolytic degradation of PLLA due to the enhanced

neutral hydrolytic degradation (NHD), that is, hydrolytic

degradation under controlled neutral pH. The authors

demonstrated by X-ray Diffraction and by Differential

Scanning Calorimetry that even low contents of D-lactic

acid units at PLLA structure are capable to disorder the

chain regularity, increasing the amorphous regions and

leading to a high supply rate of water through the polymer

matrix. Also, high concentration of hydroxide ions in

alkaline media strongly accelerates the hydrolytic degra-

dation of PLA-based materials [83, 84]. According to Jung

et al. [85] PLA exhibit very slow degradability in moderate

acidic and basic conditions, as well as in neutral conditions.

PLA polymer chains are more easily degraded in strongly

acidic and basic solutions (pH \ 1 and pH [ 13, respec-

tively), whereas hydrolytic degradation is more significant

in basic conditions.

Degradation products, such as water-soluble lactic acid

from PLA, can change the pH of the exposure environment

and affect not only the rate of hydrolysis, but also the

growth of microorganisms [60]. Temperature is also a

significant factor in controlling polymer biodegradation

since both hydrolysis reaction rates and microbial activity

increase as temperature increases. A study performed by

Cargill Dow LLC showed that the hydrolysis rate of PLA

increased dramatically above the Tg [63]. The hydrolysis

process is affected by the rate of diffusion of water through

the polymer [60]. The diffusion coefficient of water in an

amorphous or semicrystalline polymer is related to

molecular dynamics or segmental motions of the amor-

phous regions. Above Tg, the motion will be rapid and free

volume increases [86]. Consequently, water molecules

would be able to access the amorphous regions easily,

resulting in a higher degradation rate [87].

Microbial Degradation

Biodegradation of PLA and its copolymers are usually

done by esterases, proteases and lipases secreted from

microorganisms [6]. Two steps occur in the microbial

polymer degradation process: depolymerisation or chain

cleavage step; and mineralization. In the first step extra-

cellular enzymes act either endo (random cleavage on the

internal linkages of the polymer chains) or exo (sequential

cleavage on the terminal monomer units in the main chain).

Due to the size of the polymer chain and the insoluble

nature of many polymers, this step normally occurs outside

the organism [88].

A useful technique for evaluating biological degradation

is the clear zone method with agar plates. In this case, agar

plates containing emulsified polymers are inoculated with

microorganisms. If degrading microorganisms are present,

it is possible to verify formation of clear halo zones around

the colonies, which is related to the excretion of extracel-

lular enzymes that can diffuse through the agar and degrade

the polymer into water soluble materials [89]. Ecological

studies using clear zone method evaluated the abundance

of PLA-degrading microorganisms in different environ-

ments, confirming that PLA is less susceptible to microbial

attack compared to the following synthetic and microbial

polyesters: poly(e-caprolactone)-PCL, poly(hexamethylene

carbonate)-PHC, poly (tetramethylene succinate)-PTMS

and poly(b-hydroxybutyrate)-PHB [90, 91].

An interesting aspect related to PLA degradation by

microorganism studied by Tokiwa et al. [92] is the ability

of a silk degrading actinomycete, Amycolatopsis strain

J Polym Environ

123

KT-s-9, to also degrade PLLA. This hypothesis was eval-

uated by the clear zone method and it was confirmed. The

Amycolatopsis is the same genus as that of PLLA

degrading microorganisms reported by Paramuda et al.

[90]. The main amino acid constituents of silk fibroin are

L-alanine and glycine, and there is a similarity between the

stereochemical position of the chiral carbon of L-lactic acid

unit of PLA and L-alanine unit in the silk fibroin [89]. Most

strains degrading PLA also degrade silk fibroin, suggesting

that the strains may recognize the repeated L-lactic acid

unit of PLA as an analogue of L-alanine unit in silk fibroin

[93].

Jarerat et al. [94] investigated PLLA degraders among

41 genera (105 strains) of actinomycetes from the culture

collections by determining the clear zone forming ability of

these strains on PLLA agar plates. From the 41 genera

screened, only 5 genera (Amycolatopsis, Kibdelosporan-

gium, Saccharothrix, Lentzea, and Streptoalloteichus) of

the family Pseudonocardiaceae and related genera were

able to form clear zones on PLLA agar plates. The authors

provided evidence that PLLA degrading microorganisms

are not restricted only to the genus Amycolatopsis. Of the

total 12 strains of genus Saccharothrix, 9 strains could

make clear zones on silk fibroin and also in PLLA plates

implying that many PLLA degraders are also widely dis-

tributed within this genus.

The stereochemistry of the monomer units in the poly-

mer chains influences the biodegradation rates, since an

inherent property of many enzymes is their stereochemical

selectivity [88]. Reeve et al. [95] compared enzymatic

degradation of PLA with different %L repeat unit contents

of 100 (PLA-100), 99, 96, 94 and 92. PLA films of these

samples were prepared by solution casting and then

annealing at 75 �C for 36 h to crystallize. PLA-0 (100 % D

isomer) film showed no apparent weight loss because

D-lactic acid units containing polymer chains are not rec-

ognized by proteinase K, which is selective for degradation

of L-lactic acid units. Although it would be expected a

decreasing in the rate of K proteinase degradation with

decreased %L repeat unit composition, the opposite

occurred, probably because of the degree of crystalline

order of the samples, since enzyme-catalyzed film degra-

dation process occurs preferentially at amorphous domains.

Optimal rates for both film weight loss and initial surface

degradation were found for PLA-92, which presented a

critical disruption of the crystalline order resulting from the

introduction of 8 % D repeat units.

Li et al. [96] studied three stereo copolymers, namely

PLA40, PLA25, and PLA10, synthesized by ring opening

polymerization of 40/60, 25/75 and 10/90 L-lactide/D-lac-

tide feeds, respectively. PLA10 was semi-crystalline, while

PLA25 and PLA40 were intrinsically amorphous materials.

Enzymatic degradation of the films was investigated in the

presence of proteinase K. It was confirmed that proteinase

K preferentially degraded L-lactic acid units as opposed to

D-lactic acid ones. By analyzing weight loss data, degra-

dation of PLA40 and PLA25 was much more pronounced

than for PLA 10. The ratio of absorbed water was also used

as a parameter of degradation, and it was showed to be

related to Tg values of polymers. The Tg decreased in

order to PLA10 [ PLA25 [ PLA40. The lower the Tg, the

higher the water absorption ratio because the free volume

between chains in the polymer matrix increases above Tg.

Torres et al. [97] tested lactic acid consumption in liquid

cultures media inoculated by 14 filamentous fungal strains

belonging to the genera Aspergillus, Fusarium, Rhizopus,

Penicillium, and Trichoderma, which are usually found in

natural soils. Consumption of lactic acid (one of the ulti-

mate intermediate products of PLA degradation) and lactic

acid oligomers was monitored by high-performance liquid

chromatography (HPLC). After 7 days in culture media,

inoculated with filamentous fungi in the presence of lactic

acid and its oligomers, only three strains were able to

totally use lactic acid as sole carbon and energy sources:

two strains of Fusarium moniliforme and one strain of

Penicillium roqueforti.

Biodegradation of PLA/Montmorilonite

Nanocomposites

In general, molecular weight variation and weight loss are

reported in literature as the main parameters used for

evaluating PLA/montmorilonite nanocomposites biode-

gradability in compost, although some other techniques for

microbial degradation analysis have also been reported [41,

98–104].

Ray et al. [98, 99] reported biodegradability of neat PLA

and the corresponding nanocomposites prepared with 4

wt % of octadecyltrimethylammonium-modified montmo-

rilonite (C18C3–MMT). The samples were degraded in

compost at 58 ± 2 �C. Taking into account visual analysis,

weight-average molecular weight (Mw) measurements and

weight loss monitoring, it was verified that the biode-

gradability of neat PLA was significantly enhanced after

nanocomposite preparation with C18C3–MMT. Interest-

ingly, within 1 month, both extent of Mw and the extent of

weight loss were at the same level for both neat PLA and

the nanocomposite. However, after 1 month, a sharp

change occurred in the nanocomposite weight loss, which

was completely degraded in compost within 2 months. It

was proposed that 1 month was the critical timescale to

start heterogeneous hydrolysis of PLA matrix (promoted by

hydroxyl groups in silicate layers after nanocomposite

absorption of water from the compost). This assumption

was confirmed by conducting the same experimental

J Polym Environ

123

procedure with PLA nanocomposite prepared with dime-

thyldioctadecylammonium salt modified synthetic mica,

which has no terminal hydroxylated edge group. This

material presented the same degradation trend of PLA

[100].

Ray and Okamoto [41] used a repirometric test to study

the biodegradation of PLA matrix in a compost environ-

ment. The authors determined mineralization (CO2 evolu-

tion) of pure PLA and PLA/clay nanocomposites with

montmorilonites (4 wt %) modified with octadecylammo-

nium cation (PLA/C18) and octadecyltrimethylammonium

cation (PLA/qC18) at composting conditions (58 ± 2 �C).

PLA component in PLA/C18 showed a slightly higher

biodegradation rate, while the rate of degradation of pure

PLA and PLA/qC18 was almost the same level. The authors

also analyzed molecular weight, and residual mass for the

samples. The rate of molecular weight change for pure

PLA and PLA in various nanocomposites was almost the

same. After 2 months in compost a PLA-based nanocom-

posite PLA/qC18 was completely degraded, while the

unfilled PLA and PLA/C18 sample were recovered in the

form of very small pieces.

Paul et al. [101] evaluated hydrolytic degradation by

monitoring molecular weight for PLA nanocomposites

with CLOISITE 25A (more hydrophobic) and CLOISITE

30B (more hydrophilic). The samples were put in flasks

containing phosphate buffer at pH 7.4. The flasks were

immersed in a water bath at 37 �C. The hydrolysis time led

to a modification in relative opacity of the materials, an

expected behavior due to the preferential degradation of

amorphous regions and the increasing of crystallinity. After

five and half months of hydrolytic degradation, nanocom-

posites turned white and became extremely brittle. The

number-average molecular weight (Mn) of the unfilled

PLA sample decreased by 41.6 % with respect to its initial

value, and 71.2 and 79.2 %, for intercalated nanocompos-

ites based on CLOISITE 25A and CLOISITE 30B,

respectively. It has been highlighted that the degradation is

due to the relative hydrophilicity of the filler. The more

hydrophilic the filler, the more pronounced is the

degradation.

Fukushima et al. [102] evaluated biodegradation of PLA

and its nanocomposites with two modified montmorilonites

(CLOISITE 30B and NANOFIL 804). Compression mol-

ded test samples were put in contact to compost at 40 �C

and relative humidity between 50 and 70 %. By visual

analysis, it was verified that the samples exhibited con-

siderable deformation and whitening. This study showed a

considerable decrease on number-average molecular

weight and weight-average molecular weight (Mn and Mw,

respectively) values for all samples, indicating the effective

potential of PLA and nanocomposites biodegradation in

compost. The addition of nanoclays was found to increase

PLA degradation rate, which was attributed to hydroxyl

groups belonging to the silicate layers of these clays. PLA/

CLOISITE 30B nanocomposite showed the highest

decrease in Mn and Mw values (79 and 52 %, respec-

tively), which was attributed to a higher dispersion in PLA

as compared to NANOFIL 804.

PLA microbial degradation by Bacillus licheniformis

was also evaluated by Fukushima et al. [102]. According to

Size Exclusion Chromatography, it was not observed

considerable differences in the PLA and PLA/NANOFIL

804 degradation trend, at least within 10 days of microbial

degradation. Whereas, contrarily to the degradation in

compost, the incorporation of CLOISITE 30B slightly

delayed the process of PLA degradation by the bacterium

Bacillus licheniformis, probably due to a certain rejection

effect of this bacterium towards CLOISITE 30B. In order

to investigate this issue organoclays were submitted to

plate test. The principle of plate test involves placing the

test material on the surface of a mineral salts agar in a petri

dish containing no additional carbon source. The test

material and agar surface are sprayed with a standardized

mixed inoculums of known bacteria and/or fungi. The test

material is examined, after a predermined incubation per-

iod at constant temperature, for the amount of growth on its

surface [88].

In the case of NANOFIL 804, microbial growth occur-

red in the surroundings of the organoclay, indicating a

positive response of the bacterium to this clay, probably by

using the NANOFIL 804 modifier as carbon and energy

source for its growth. In the presence of CLOISITE 30B,

the Bacillus licheniformis colonies generated a transparent

halo around the organoclay. It was supposed that the

organic moiety of CLOISITE 30B inhibited the bacteria

growth. According to the authors the phenomenon should

not be regarded as a toxic effect of CLOISITE 30B, since

no mortality in the colonies was observed but only an

absence of bacterial growth [102].

Fukushima et al. [102] also used the technique of Dif-

ferential Scanning Calorimetry to assess degradation of the

polymer and nanocomposites in both cases (degradation in

compost and microbial degradation). After degradation it

was verified a reduction in cold crystallization temperature,

and the presence of an additional melting peak. This

behavior was attributed to the formation of crystals with

different structure or less perfect crystals, due to chain

scission caused by hydrolytic degradation.

Arena et al. [103] investigated PLA and its nanocom-

posites degradation by Bacillus licheniformis. Biodegra-

dation was performed on compression molded films in a

medium for the microbial growth. The samples were

incubated aerobically at 32 �C, in the dark, and the test was

carried out for 5 months. The average percentage of mass

loss for each material was calculated against time of

J Polym Environ

123

incubation. PLA degradation by Bacillus licheniformis was

accelerated by the presence of organoclays. After

5 months, PLA exhibited considerable whitening, while all

its nanocomposites lost completely their physical stability.

Sangwan et al. [104] determined the biodegradability of

the neat PLA and three PLA/montmorilonite nanocom-

posites, modified with octadecylammonium (ODA),

dimethyl dialkyl amine (DDA) and trimethyl stearyl

ammonium (TSA), acccording to ISO 14855, at 58 �C.

After 60 days, the total degradation levels for PLA/TSA,

PLA/ODA, PLA/DDA nanocomposites and the neat PLA

samples were approximately 35, 32, 30 and 31 %,

respectively. The authors also conducted DNA sequencing

for compost samples, collected from the surface of PLA

and nanocomposites after 32, 50 and 60 days, to determine

microorganisms involved in the degradation process of

these materials. The microbial diversity in the blank

compost samples (before degradation process) was con-

sidered as a reference information in order to detect

changes after the materials’ biodegradation. Members of

the phylum Actinobacteria were the most dominant group

in the degraded samples. These microorganisms were not

identified in the blank compost samples, probably due to

their lower population. Related to the diversity of fungi, the

degraded neat PLA and the three PLA nanocomposites

were found mainly populated with fungi belonging to

phylum Ascomycota. Due to their numerical abundance,

members of the microbial groups Actinobacteria and

Ascomycota seem to play an important role during bio-

degradation process.

Montmorilonite: Safety Concerns and Toxicity

Evaluation

Evaluation of potential toxicity of montmorilonite is nec-

essary, since its use is expanding, which implies in human

exposure. The exposure could occur during its develop-

ment, manufacture and use, or disposal [105, 106].

Although the nanoscale components of montmorilonite-

containing composite products are often bound to the

material, and not in a free-form, there is still potential for

release of these materials during their wear [107]. Bio-

polymer nanocomposites are a field of emerging interest in

food-packaging applications. From a food-safety point of

view, it is important to characterize migrants from nano-

composites containing clay as fillers [108]. As a general

rule, nanocomposites must comply with the total migration

limit of 10 mg/dm2 (10 milligrams per square decimeter of

surface area of the material or article) [109].

An analytical platform consisting of asymmetrical flow

field-flow fractionation (AF4) coupled with multi-angle

light-scattering (MALS) and inductively coupled plasma

mass spectrometry (ICP-MS) was used by Schmidt et al.

[110] in a migration study of PLA/CLOISITE 30B (5 %)

nanocomposites, conducted according to European Stan-

dard [109]. Based on AF4-MALS analyses, the authors

found that particles with varying radii of 50–800 nm

indeed migrated into a mixture of 95 % ethanol and 5 %

glass-distilled water used as a food simulant (according to

current European Union legislation). The full AF4-MALS-

ICP-MS system showed, however, that none of the char-

acteristic clay minerals was detectable, and it was con-

cluded that clay nanoparticles were absent in the migrant.

Schmidt et al. [111] tested three films for total and

specific migration of all major constituents in the various

prepared PLA/laurate-modified Mg–Al layered double

hydroxide (PLA-LDH-C12) nanocomposite films. In

addition to silicate clay minerals as fillers in nanocom-

posites, layered double hydroxides (LDHs) have attracted

interest during the last few years. LDH-C12 was introduced

into PLA either by direct compounding with PLA or by

addition of previously prepared masterbatch. All the three

tested films showed migration of nanosized LDH. Migra-

tion of tin was detected in two of the film samples prepared

by dispersion of LDH-C12 using a masterbatch technique.

This result was expected, since these films were made by

in situ intercalative ring opening polymerization, using tin

catalyst. The results indicated migration of tin at about the

limit of European Comission [109]. Migration of the lau-

rate organomodifier took place from all three film types. It

was concluded that the material properties were in com-

pliance with the migration limits for total migration and

specific lauric acid migration as set down by the European

legislation for food contact materials. However, there are

other factors that should be analyzed before considering the

materials as suitable for food packaging, such as the effect

of LDH organomodifiers on organoleptic properties of

packaged food, including possible consequences for film

use, and which sort of food packaging might be appropriate

given the migration properties of any particular PLA-LDH

nanocomposite type.

Simon et al. [112] reported thermodynamics aspects

related to migration of nanoparticles from the package to

food. Migration from nanocomposites would take place

until an equilibrium distribution of nanoparticles between

package and food is established. By considering amor-

phous phase of polymer as a fluid, the authors calculated

the diffusion coefficient of nanoparticles and the amount of

migrated nanoparticles for different polymers matrices

(low-density polyethylene-LDPE, high-density polyethyl-

ene-HDPE, polypropylene-PP, polystyrene-PS and poly-

ethyleneterephtalate-PET). The authors concluded that any

significant migration of nanoparticles from package to food

could be expected just in the case of small nanoparticles

(radius of about 1 nm), from polymer matrices with a

J Polym Environ

123

relatively low dynamic viscosity, which also do not interact

with nanoparticles. One example would be the migration of

nanosilver from polyolefines (LDPE, HDPE, PP), which

meets these conditions. On the other side, for bigger

nanoparticles bound in polymer matrices with a relatively

high dynamic viscosity, such as montmorilonite with sur-

face modification, embedded in various polymer matrices,

migration would not be detectable. According to the

authors, the expectation would be that the nanoparticles

would not impose any significant risk to the consumer.

However, there is a lack of experimental data to confirm

this hypothesis.

According to the current European Regulation on food

contact materials ‘‘substances deliberately engineered to

particle size which exhibit functional physical and chemical

properties that significantly differ from those at a larger

scale’’ should be risk assessed on a case-by-case basis, until

more information is available about such new technology

[113]. The most widely investigated clays for nanocom-

posites are montmorilonites, commonly modified with qua-

ternary ammonium compounds. None of organomodifiers of

this type is yet to be found on the positive list in the current

European Union food-contact plastics legislation [110].

Nigmatullin et al. [114] reported the possibility of

quaternary ammonium compounds migration from CLOI-

SITE 10A, CLOISITE 20A, CLOISITE 93A, CLOISITE

15A and CLOISITE 30B in aqueous media, by monitoring

electrical conductivity for 6 h. The same study was made

for polyamide and organoclays nanocomposites, in a period

of 60 days. As expected, migration of surfactants from the

polymer composite was drastically slowed compared with

corresponding release rate of the surfactants from nanoc-

lays. Similar to the organoclays results, leaching of the

most hydrophilic surfactants used for the preparation of

CLOISITE, 10A and 30B, was quicker for the corre-

sponding polymer/clay composites. Taken into account the

possibility of modifiers migration the authors recommend

the use of nanocomposites in fields where surfactant

migration is acceptable.

There are three primary routes of human exposure to

nanomaterials: inhalation, ingestion and dermal absorption.

Inhalation is the most studied pathway and a critical research

gap is the identification of conditions that will cause

nanomaterials that are in the gas phase, in liquids, or

embedded in solids to become airborne. The ability of

nanomaterials to penetrate skin is influenced by the condition

of the skin and the physicochemical properties of the

nanomaterials. Data suggest that nanoparticles greater than

10 nm in diameter are unlikely to penetrate human skin

[115]. However, uptake may occur if skin is damaged or

diseased [116, 117], although data on penetration of

nanomaterials into damaged skin is limited. Critical research

questions related to ingestion include the propensity of

nanomaterials to survive in the gastrointestinal tract. In this

case, the extent of absorption and assimilation into the

organism is also a pertinent issue to be investigated [115].

Efforts have been done by researchers using different

methods to assess information about nanomaterials and its

possible health effects. Some investigations focused on clays,

unmodified and organo-modified, have been conducted and

the results were recently reported [106, 118–121]. Consid-

ering the current studies, some relevant definitions as cyto-

toxicity, genotoxicity and mutagenicity are important for the

better understanding of potential toxic effects and risks

coming from clays.

The term cytotoxicity means the effect of chemical

agents as evidenced by altered cellular characteristics, by

failure of the cell to attach to surfaces, and by the altera-

tions in the rate of cell growth, cell death, and cell disin-

tegration [122]. The terms genotoxic and genotoxicity

apply to agents or processes that alter the structure, infor-

mation content, or segregation of DNA, including those

that cause DNA damage by interfering in the normal rep-

lication processes; or those that, in a non-physiological

manner, alter temporarily its replication [123].

The term mutation applies both to heritable genetic

changes that may be manifested at the phenotypic level,

and the underlying DNA modifications when known, as

specific base pair changes or chromosomal translocations,

as examples. The terms mutagenic and mutagen will be

used for agents that give rise to an increased occurrence of

mutations in populations of cells and/or organism. Muta-

genesis refers to those changes in the genetic materials in

cells brought about spontaneously either by chemical or by

physical means whereby successive generations differ in a

permanent and heritable way from their predecessors [123].

The main assays, which have been used in recent analysis

of nanomaterials to detect its toxic potential, are briefly

described below.

Ames Test (Bacterial Reversion Mutation Test)

The Ames test is a widely accepted short-term test for

detecting chemicals that induce mutations in the DNA of

organisms [124]. It uses auxotrophic, mutant microorgan-

isms that require a particular kind of nutrient (histidine

amino acid) and are not capable for growing on a medium

missing this nutrient, unless they have a reverse mutation to

the wild type. The reverse mutation occurs when a gene is

restored to the mutation [125]. Various strains of the Sal-

monella typhimurium histidine dependent contain mutations

in the genes that impair synthesis of histidine required for cell

growth. In the Ames test, substances or compounds are added

to different areas on the agar plate, and the bacterium is then

plated onto the minimal histidine media. The test compound

is considered to have mutagenic potential if it is able to cause

J Polym Environ

123

mutations that permit the bacterium to revert back its histi-

dine synthesis ability [126].

The toxicity can also be analyzed by microscopic

examination of the background lawn. The absence of tox-

icity will reveal the presence of densely packed micro-

colonies which form a thin film. By the other side, when a

chemical is toxic there may be ‘‘thinning’’ or complete

absence of the background lawn compared to the negative

control [123].

Comet Assay/Single-Cell Gel Electrophoresis Assay

The comet assay is a relatively simple, but sensitive and

well validated tool for measuring strand breaks in DNA in

single cells. Cells are embedded in a thin layer of agarose

on a microscope slide and lysed with detergent and high

salt solution. This procedure also removes proteins and

histones, leaving a nucleoid from each embedded cell lying

within a cavity in the gel. The presence of breaks in DNA

causes a local relaxation in the supercoiled loops of DNA

in the nucleoid. After passing a small electrical charge

through the gel, the relaxed areas of the DNA loops are

pulled towards the anode, forming a comet ‘tail’, while the

DNA in the nucleoid forms the comet ‘head’. Comets are

visualized by fluorescent microscopy, and the amount of

DNA in the tail, relative to the head, is proportional to the

amount of strand breaks. Cells can be incubated in vitro

with an agent of interest prior to the comet assay, and the

resulting DNA damage can be then measured [127].

Ostling and Johanson [128] introduced by the first time

the concept of microgel electrophoresis. This was a neutral

version of the Comet Assay [129]. Singh et al. [130]

published a modified version, which used alkaline condi-

tions. This method combined DNA gel electrophoresis with

fluorescence microscopy to visualize migration of DNA

strands from invidual agarose-embedded cells. If the neg-

atively charged DNA contains breaks, DNA supercoils are

relaxed and the broken ends are able to migrate toward the

anode during a brief electrophoresis. If the DNA was

undamaged, the lack of free ends and large size of the

fragments prevent migration. Determination of the relative

amount of DNA that migrated provides a simple way to

measure the number of DNA breaks in an individual cell

[131].

MTT Viability

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoli-

um bromide) is a water soluble tetrazolium salt, which is

converted to an insoluble purple formazan by cleavage of

the tetrazolium ring by succinate dehydrogenase within the

mitochondria. The formazan product is impermeable to the

cell membranes and it is accumulated in healthy cells

[132]. First described by Mosmann [133] to detect mam-

malian cell survival and proliferations, it is a rapid color-

imetric method. Since it measures the amount of formazan

generated directly proportional to the number of viable

cells, this method has spread out in the scientific commu-

nity and has been adopted largely for its precision and

rapidity in measuring cell survival and proliferation [134].

LDH

This assay involves the measurement of a cytoplasmic

enzyme that is released following target cell death so that

its activity in the cellular supernatant indicates the pro-

portion of dead cells [135]. The LDH leakage assay is

based on the measurement of lactate dehydrogenase

activity in the extracellular medium. The loss of intracel-

lular LDH, and its release into the culture medium, is an

indicator of irreversible cell death due to cell membrane

damage [132].

ROS

Reactive oxygen species (ROS) is a collective term that

broadly describes O2-� derived free radicals such as

superoxide anion (O2-), hydroxyl (HO�), peroxyl (RO2

�),

and alkoxyl (RO�) radicals, as well as O2-derived nonrad-

ical species such as hydrogen peroxide (H2O2) [136].

Several xenobiotics interact with the mitochondrial elec-

tron transport chain, increasing the rate of O2-� production

by two different mechanisms. Some of these compounds

stimulate oxidative stress because they block the electron

transport, increasing the reduction level of carriers located

upstream of the inhibition site. Other xenobiotics may

accept an electron from a respiratory carrier and transfer it

to molecular oxygen (redox cycling), stimulating O2-�

formation without inhibiting the respiratory chain [137].

Direct measurement of ROS in cell media has been

achieved by two similar fluorescein-compound-based tests

or by electron paramagnetic resonance [138].

Recent results on toxicity related to montmorilonite and

the main conclusions are summarized in Table 3.

Lordan et al. [106] evaluated cytotoxic effects induced

by two different clays, the unmodified (CLOISITE Na?)

and the organically modified (CLOISITE 93A), in human

hepatoma HepG2 cells. The characterization by SEM

demonstrated notable differences in platelet sizes between

the clays. In general, CLOISITE Na? consisted of mainly

large tactoid structures with lengths ranging from

30–100 lm, whereas CLOISITE 93A clay demonstrated a

broader dispersion of smaller particles sizes ranging in the

length of 3–35 lm. Based on SEM analysis, the authors

concluded that air drying procedure used to sterilize

nanoclays resulted in formation of micro-sized

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agglomerates. Henceforth, CLOISITE Na? and CLOISITE

93A could no longer be referred to as ‘nano’clays. The

authors conducted MTT, LDH release and ROS formation

assays. Some findings, briefly described below, indicated

the clays are highly cytotoxic and may be a possible risk to

human health.

MTT Assay

HepG2 cells were treated with increasing clay concentration

(from 1 to 1,000 lg/mL). A dose response effect was evident,

following treatment with each one of clays, with a significant

decrease in viable cells. The highest concentration of CLOI-

SITE Na? and CLOISITE 93A (1,000 lg/mL) reduced cell

viability to approximately 23 and 37 %, respectively.

LDH Release Assay

CLOISITE 93A appeared to have a more pronounced

effect on LDH leakage, with significant levels of LDH

released from cells following exposure to 50, 100, 500 and

1,000 lg/mL. Results from LDH release assay also con-

firmed the detrimental effect of CLOISITE Na? on the

cells; however, no dose-response effect was detected.

Table 3 Studies about montmorilonite toxicity

Clay Modifier Methodology Main conclusions Reference

CLOISITE Na? – MTT assay

LDH release assay

ROS production assay

Citotoxicity in human hepatoma HepG2

cells

Lordan et al. [106]

CLOISITE 93A (methyl,

dihydrogenated

tallow,quaternary

ammonium)

CLOISITE Na? – Salmonella microssome

assay (strains TA 98 and

TA 100)

Non-cellular generation of

ROS

Comet assay

Both clays tested did not indicate

mutagenic activity in TA 98 and TA

100

Genotoxic effect of CLOISITE 30B on

human colon-cancer cell line (Caco-2

cells). The genotoxicity was not mediate

by non-cellular ROS production

The genotoxic effect of CLOISITE 30B

can be due to the modifier

Sharma et al. [118]

CLOISITE 30B (methyl, tallow,

bis-2-

hydroxyethyl,

quaternary

ammonium)

Nanosilicate platelets

(NSP) derived from

natural

montmorilonite clay

– Salmonella microssome

assay (strains TA98,

TA100, TA102, TA1535

and TA1537)

MTT assay

LDH release assay

Comet assay

Micronucleus (MN) assay

in vivo

Acute oral toxicity in rats

With all five strains of Salmonellatyphimurium, none of mutations was

found

Low cytotoxicity on Chinese Hamster

Ovary (CHO) cells

Comet assay test on CHO cells showed no

DNA damage

The MN test indicated no significant

micronucleus induction in the CHO

cells

LD50 acute oral toxicity of NSP was

more than 5700 mg/kg for the SD rats

Li et al. [119]

Montmorilonite-MMT – WST-1 assay

Clonogenic assay

LDH release assay

ROS production assay

Oral toxicity in rats

Pharmacokinetic study in

rats

Cytoxicity tests in human normal

intestinal cells (INT-407) indicated that

MMT could cause some cytotoxic

effects in at high concentration after

long-time exposure

Toxicity was not found in mice receiving

up to the highest dose tested in

rats(1,000 mg/kg)

MMT could be absorbed into the body

within 2 h, but it did not significantly

accumulate in any specific organ in rats

Baek et al. [120]

Uniform particle size

NovaSil-UPSNa– Oral toxicity in rats. Dietary inclusion of UPSN at levels as

high as 2 % (w/w) did not result in overt

toxicity

Marroquın-Cardona

et al. [121]

a Mainly composed of montmorilonite but it also contains mica, feldspars and quartz minerals

J Polym Environ

123

ROS Production Assay

CLOISITE Na? induced intracellular reactive oxygen spe-

cies (ROS). Conversely, CLOISITE 93A had little effect on

ROS, suggesting that ROS production does not appear to be

related to the organoclay’s cytotoxic properties.

Sharma et al. [118] investigated natural clay mineral

montmorilonite (CLOISITE Na?) and an organo-modified

montmorilonite (CLOISITE 30B) for genotoxic potential,

as crude suspensions and as suspensions filtrated through a

0.2 lm pore-size filter to remove particles above the

nanometer range. Some interesting results are reported

bellow.

Salmonella Microssome Assay

The Salmonella/microsome assay was performed for

CLOISITE 30B suspensions (filtered and unfiltered) and

CLOISITE Na? suspensions (only unfiltered samples). For

unfiltered suspensions, eight concentrations were tested

(1.2, 5, 10, 20, 40, 79, 106 and 141 lg/mL). In the filtered

30B solution, the stock solution before filtration was

141 lg/mL and eight concentrations were tested in the

following dilutions: undiluted, 1.33x, 1.78x, 3.5x, 7x, 14x,

28x, 118x. None of suspensions showed indications of

mutagenic activity in this assay. Toxicity, measured as a

decrease in background lawn and in revertant frequency,

was not observed at any concentration tested.

Comet Assay

The unfiltered CLOISITE 30B and CLOISITE Na? sam-

ples were, each one, tested at four concentrations: 56.5, 85,

113 and 170 lg/mL. Filtered 30B and Na? samples were

also tested in four concentrations: the highest concentration

(stock solution) was 226 lg/mL before filtration, and the

stock solution was then diluted 2x, 2.7x and 4x. Filtered

and unfiltered CLOISITE Na? suspensions in culture

medium (Dulbecco’s Modified Eagle Medium-DMEM) did

not induce DNA strand-breaks in Caco-2 cells, as tested in

the alkaline comet assay. However, both the filtered and the

unfiltered samples of CLOISITE 30B induced DNA strand-

breaks, in a concentration-dependent manner; and the two

highest test concentrations produced statistically signifi-

cantly different results, from those seen with negative

(culture medium) control samples.

Since CLOISITE Na? was not genotoxic, and CLOI-

SITE 30B was genotoxic also in filtered suspensions

without clay particles, the genotoxicity may be due to

quaternary ammonium compound (QAC). The authors

investigated this issue by synthesizing the QAC detected at

filtered suspensions and by testing it in comet assay. The

genotoxic potency of the synthesized organo-modifier was

in the same order of magnitude at equimolar concentrations

of organo-modifier in filtrated CLOISITE 30B samples,

which, according to the authors, partly explain its geno-

toxic effect.

Non-Cellular Generation of ROS

CLOISITE particles were tested in six concentrations

(0, 14, 28, 57, 113 and 226 lg/mL) and ROS production

was assessed in two independent unfiltered 30B and Na?

samples experiments, each containing 3–4 replicates. Fil-

tered 30B and Na? samples were assessed in one experi-

ment at the same concentrations used at comet assay, and

carbon black was used as the positive control. Unfiltered

Na? and 30B samples did not induce ROS production.

Filtered Na? and 30B samples were also tested in one

experiment at the same concentrations as that used in the

comet assay, and also did not induce ROS. It was con-

cluded that genotoxicity of 30B samples determined with

the comet assay was not mediated by non-cellular ROS

production.

Li et al. [119] investigated the toxicity of nanosilicate

platelets (NSP) derived from natural montmorilonite clay,

with an average dimension of 80 9 80 9 1 nm3. Overal,

the study has demonstrated the safety of the NSP for

potential uses in biomedical areas, for instance, as drug

carrier. The description of assays and some results are

related below.

MTT Assay

Chinese Hamster Ovary (CHO) cells were treated with

increasing clay concentration (from 62.5 to 1,000 lg/mL).

The results indicated a slight decrease in cell viability for

the NSP-exposed CHO cells at high concentration and

exposure time (3, 12, and 24 h). However, the NSP pre-

sented a low toxicity below concentrations of 1,000 lg/mL

for 70 % cell viability in the period of 12 h. The exposure

up to the concentration of 1 mg/mL for 24 h showed a

40 % loss of viability on CHO cells. The half maximal

inhibitory concentration (IC50) value at 24 h for CHO cells

was more than 1,000 lg/mL.

LDH Release Assay

Chinese Hamster Ovary (CHO) cells were treated with

increasing clay concentration (from 62.5 to 1,000 lg/mL).

The release of enzyme lactate dehydrogenase into cell cul-

ture medium was measured quantitatively. Cell membrane

leakage was reflected in the elevated LDH levels in cell

culture medium after exposure to NSP solution for 24 h. The

LDH levels of CHO cells were increased from 0 to 40 %

when exposing at 62.5 to 1,000 lg/mL concentration.

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123

Comet Assay

The CHO cells were cultured in culture plates at a density

of 5 9 105 cells/plate for 24 h and treated with different

amounts of NSP at 62.5, 125, 250, 500, and 1,000 lg/mL.

A negative control (with double-distilled water) and a

positive control (with H2O2 100 lM addition) were per-

formed for comparisons. The DNA damage index (ratio of

tails to total comet bodies) indicated no significant DNA

damage for the NSP exposure.

Salmonella Microssome Assay

The Salmonella/microsome assay was performed for the

test substance suspensions at 0.625, 1.25, 2.5, 5, and

10 mg/mL. With all five strains of Salmonella typhimurium

tested (TA98, TA100, TA102, TA1535 and TA1537), none

of mutations was found.

Micronucleous Assay In Vivo

The mice were fed with NSP at the concentrations of 20,

200, and 500 mg/kg body weight. The negative control

group received double-distilled water while the positive

control group received intraperitoneal injection of mito-

mycin C at the dose of 1 mg/kg. Peripheral-blood cells were

collected after 24 and 48 h. Micronucleus formation of

polychromatic erythrocytes in the cells was counted under

microscope. The test indicated no significant micronucleus

induction in the CHO cells at the concentrations tested.

Oral Toxicity

A total of 24 rats in each sex were divided into control

(distill-water injection), low dose (1,500 mg/kg), interme-

diate dose (3,000 mg/kg), and high dose (5,700 mg/kg)

groups. For feeding to rats, the acute oral toxicity was shown

a low lethal dose (LD50) or greater than 5,700 mg/kg body

weight for both male and female rats.

Baek et al. [120] evaluated the toxicity of montmorilo-

nite in human normal intestinal cells in short- and long-

terms as well as in mouse model. The authors pointed out

that although many attempts have been made to develop

this clay as delivery carrier for small bioactive molecules,

information about the potential toxicological effects of

montmorilonite in vitro and in vivo is rather lacking. The

main findings reported in this study are described below.

WST-1 Assay

The short-term cytotoxicity was measured by WST-1

assay, a colorimetric method based on the principle that

WST-1 is reduced by viable cells, eventually producing a

soluble red formazan salt. Thus, if cell proliferation was

influenced by toxic materials, small amount of WST-1

converts to red formazan in comparison with control group.

Human normal intestinal cells (INT-407) were exposed to

various clay concentrations (0.5, 5, 50, 125, 250, 500, and

1,000 lg/mL) for 24, 48, and 72 h. The clay significantly

inhibited cell proliferation after 24–72 h incubation in a

concentration- and time-dependent manner at the concen-

tration levels of above 100 lg/mL. The IC50 (inhibition

concentration 50 %) values for 24, 48, and 72 h incubation

were determined to be 565.50, 428.71, and 192.52 lg/mL,

respectively. The long-term cytotoxicity of montmorilonite

was evaluated by applying clonogenic assay, using various

concentrations (5, 25, 50, 75, 100, 125, and 150 lg/mL). A

colony was defined as a cell group consisted of at least 50

cells. The concentration tested of MMT significantly

inhibited normal colony formation after 10 days exposure,

even at the lowest concentration of 5 lg/mL.

LDH Release Assay

INT-407 cells were treated with increasing clay concentra-

tion (from 0.5 to 1,000 lg/mL). The potential cytotoxic

effect of montmorilonite was assessed by measuring released

level of intracellular lactate dehydrogenase (LDH) into the

extracellular medium upon membrane damage. Montmo-

rilonite remarkably induced LDH release from INT-407 cells

only at the highest concentration tested, 1,000 lg/mL after

48-72 h. Significant increase in LDH leakage was not found

up to the concentration of 500 lg/mL after 48-72 h. It is

worth noting that the clay did not caused LDH leakage within

24 h.

ROS Production Assay

The INT-407 cells were exposed to montmorilonite

(0.5–1,000 lg/mL) for 24, 48, and 72 h. It was observed that

ROS was not generated in the cells treated with MMT up to

the dose of 500 lg/mL within 24 h. Montmorilonite signif-

icantly generated ROS in INT-407 cells at the concentration

above 50 lg/mL after 48–72 h incubation. Only high con-

centration of 1,000 lg/mL produced significantly high level

of ROS at all the time points tested (after 24–72 h).

Oral Toxicity

The acute oral toxicity of montmorilonite was evaluated in

mice after single dose administration of four different doses

(5, 50, 300, and 1,000 mg/kg). Any remarkable abnormal

behaviors, symptoms, and body weight loss were not observed

in mice treated with montmorilonite during 14 days post-

administration. The lethal dose (LD50) values were estimated

to be more than 1,000 mg/kg on the basis of the fact that no

J Polym Environ

123

mortality was found in all the mice treated with different doses

of clay.

Pharmacokinetic Study

Three different single doses (50, 300, and 1,000 mg/kg) of

montmorilonite were administered in each group of three

mice. The blood samples were collected via orbital vein at

several time points and centrifuged to obtain the plasma.

For tissue distribution study, the tissue samples such as

kidney, liver, lung and spleen were collected at designated

times after oral administration. Pharmacokinetics of mont-

morilonite was assessed by measuring the plasma concen-

tration of Al, a main clay component, by inductively

coupled plasma atomic emission spectroscopy (ICP-AES)

after single dose oral administration in mice. The plasma

level of montmorilonite rapidly decreased within 2 h post-

oral administration even at high dose of 1,000 mg/kg. The

tissue biodistribution study demonstrated that montmorilo-

nite content increased in the lung at 0.25–1 h, but the clay

did not significantly accumulate in any specific organs such

as kidney, liver, lung, and spleen at 8 h post-administration.

Marroquin-Cordona et al. [121] studied uniform particle

size NovaSil (UPSN). NovaSil (NS) is a common anti-

caking agent in animal feeds and it has been shown to

adsorb aflatoxin B1 (AFB1), which is a group 1 carcinogen

(IARC, 2002) mainly produced by fungi present at con-

taminated maize and peanuts (CAST, 2003). UPSN is a

refined material derived from parent NS; it is mainly

composed of montmorilonite and it is a candidate for

potential use as aflatoxins sorbent. Compared to the parent

NovaSil, UPSN contains lower levels of dioxins/furans,

and has been selected for a more consistent uniform par-

ticle size. The study investigated the efficacy and potential

toxicity of USPN for long-term use. The results showed

that AFB1 sorption characteristics were the same to UPSN

and NS. In order to obtaining information about toxicity, it

was conducted a 3-month rodent study after UPSN inges-

tion. The rats were fed rations free of clay (control) and

containing UPSN low dose (0.25 %) and high dose (2 %)

for 13 weeks. The authors reported that all rats remained

healthy and no apparent adverse effects due to consump-

tion both doses tested were noticed throughout the study.

Environment Effects of PLA and its Nanocomposites

Possible environment benefits of PLA-based materials can

be identified by using the life cycle assessment (LCA),

which is a method to account for the environmental

impacts associated with a product or service. The term ‘life

cycle’ indicates that all stages in a product life are con-

sidered, from resource extraction to ultimate disposal [15].

Among the positive points of PLA production, in com-

parison with the other hydrocarbon-based polymers, is the

decrease of CO2 emission. Vink et al. [139] showed that

the net greenhouse gas emissions of NatureWorks� PLA

polymers decreased from 2 kg CO2 eq./kg polymer in 2003

to 0.3 kg in 2006. The authors also estimated the -0.7 kg

of CO2 for the next PLA generation, using wind energy in

the near future. PLA is also interesting by environmental

standpoint taking into account the amount of water con-

sumption and energy requirements for its production, when

compared to fossil based polymers [15].

Negative effects coming from biodegradable polymers

should also be considered. One of the major difficulties

when designing degradable polymers is obtaining good

properties and stability during the service-life, whereas, at

the same time, the material should be completely degrad-

able in a desired time frame and produce harmless degra-

dation products that do not accumulate in the environment

[140].

New techniques need to be developed to identify the low

molecular weight degradation products. These methods

comprise techniques for isolation of the products from the

polymeric material or the degradation media (water and

soil), pre-concentration of the compounds, methods for

further separation, and finally identification [141]. Inkinen

et al. [140] described the potential of some analytical

techniques for evaluation of degradation products, such as

gas chromatography-mass spectrometry, liquid chroma-

tography, electrospray ionization-mass spectrometry and

capillary zone electrophoresis. However, there is a lack of

information from chemical analyses of PLA metabolites in

compost or soil matrix.

It is also important to consider that each material

modification could change the degradation product pattern

by introducing new water-soluble products. Different

modifiers such as citrate esters, tributyl citrate, oligomeric

malonate esteramides, 4,4-methylene diphenyl diisocya-

nate, polyglycerol esters, polyethylene glycol, bifunctional

cyclic ester and poly (1,3-butylene adipate) have been

studied in order to improve PLA properties such as strength

or flexibility, stiffness, barrier properties and thermal sta-

bility [142]. Residual catalysts in the polymer matrix could

also be liberated during the biodegradation process. Plastic

degradation by-products, such as dyes, plasticizers or cat-

alyst residues in compost can potentially migrate to

groundwater and surface water bodies via run-off and

leachate [143].

One of the interesting discarding purposes for PLA-

based materials is the composting process, in which

organic materials are treated to generate an agriculture

fertilizer. According to ASTMD6400 [55] a compostable

plastic undergoes degradation by biological processes

during composting to yield CO2, water, inorganic

J Polym Environ

123

compounds, and biomass at a rate consistent with other

known compostable materials and leave no visible, dis-

tinguishable or toxic residue. The tested materials shall not

adversely impact on the ability of composts to support

plant growth. Additionally, the polymeric products or

materials must not introduce unacceptable levels of heavy

metals or other toxic substances into the environment, upon

sample decomposition.

Composted biodegradable plastics will expose plants,

soil dwelling organism (such as worms) and aquatic

organisms to polymer degradation products. Indeed, there

is a concern related to toxic compounds into the final

compost in the sense to prevent negative effects on the

environment, organisms and humans via the food-chain.

Due to the complex nature of polymer break down, it is not

possible to indentify all the compounds present in a mix of

degradation products, some of which may be toxic [143].

For evaluating potential toxicity of compost different

standards tests are reported in the literature.

The OECD Guideline 208 [144] assesses effects on

seedling emergence and early growth of higher plants

following exposure to the test substance in the soil (or other

suitable soil matrix). In this test, seeds are placed in contact

with soil treated with the test substance and evaluated for

effects following usually 14–21 days after 50 % emer-

gence of the seedlings in the control group. The endpoints

measured are visual assessment of seedling emergence, dry

shoot weight (alternatively fresh shoot weight) and in

certain cases shoot height, as well as an assessment of

visible detrimental effects on different parts of the plant.

These measurements and observations are compared to

those of untreated control plants. It is required testing two

plant species (i.e., Dicotyledonae and Monocotyledonae).

ASTM D6002-96 [145] also describes a terrestrial eco-

toxicity test suggested for obtaining evidence regarding

compost effects on plant life. Compost from contact test

(for instance ASTM D5338) may be evaluated at the

beginning and end of the test to establish the potential

effect of microbial degradation products. The compost is

extracted with water and filtered. The supernatant is used

for the germination test. Various dilutions of the superna-

tant are prepared, and aliquots are added to Petri dishes

lined with filter paper. Cress seeds are placed on the wet

paper and left to germinate in the dark over 4 days at room

temperature. The percentage of germinated seeds is deter-

mined after 4 days and compared to a water control.

Compost containing test materials should not be signifi-

cantly different from the blank soil at 95 % confidence

interval.

In the USA, a compostability certification and logo

program was started in 2000 by a joint effort of Interna-

tional Biodegradable Products Institute (BPI), an industry

organization of bioplastic producers, and the US

Composting Council (USCC), that represents the interests

of the composting industry [52]. The Biodegradable

Products Institute has attributed the ‘‘compostable’’ label

for bags, food service items, resins and packaging materi-

als, taking into account the requirements described at

ASTM D6400 [55]. However, ecotoxicity aspects related to

biodegradable polymers are scarcely investigated. Some

examples of ecotoxicological results described in the lit-

erature are related bellow.

Cesar et al. [146] evaluated ecotoxicological aspects

related to poly (e-caprolactone) and adipate modified starch

blend after biodegradation tests in soils with different

textures by determining germinated seeds and seed ger-

mination velocity, using rice seeds. The speed of germi-

nation was determined according to a method described by

Maguire [147]. The authors also conducted dry matter mass

determinations, which were obtained for plants harvested

after 14 days of sowing. These analyses were conducted

for soils with different content of the materials and for the

control treatment (without plastic). Rice seedling emer-

gence was not affected by the addition of plastic to the

substrate and no significant differences were observed

between the dry mass of the shoots and roots at 14 days

and the seed germination velocity across the different

treatments in relation to the control.

Mitelut and Popa [148] evaluated toxicity of the com-

post using the germination bioassay for 6 types of biode-

gradable materials, based on poly (lactic acid)/poly

(butylene adipate-co-terephthalate blends (50:50), with

different composition and thickness after 90 composting

days. The characteristics of the compost were compared

with the control (synthetic solid waste without material

after 90 composting days). After the composting process,

the phytotoxicity of the composts was evaluated using the

seed germination bioassay according to Gariglio [149]. The

extraction process was conducted by mixing 100 mL of

distilled water with 50 g media from compost samples.

After that, different dilutions of compost extract were

prepared for the germination assay conducted at Petri

dishes lined with filter paper. Each dish received 5 mL of

the extracts. In these tests it was used radish seeds ‘White

Icicle’ (Raphanus sativus). By evaluating the germination

index, based on germinated seeds and root growth, it was

showed direct correlation of phytotoxicity with the content

of the compost extract, all samples being more toxics than

the control.

Alauzete et al. [150] investigated some compounds that

can be formed ultimately during the hydrolytic degradation

in compost, namely lactic acid, sodium DL-lactate and

calcium DL-lactate. An artificial soil with test substances

was placed in an aerated jar maintained in dark at 20 �C for

14 days. The number of surviving worms initially present

was then counted, and lethal concentration, corresponding

J Polym Environ

123

to 50 % surviving worms, was evaluated approximately

and expressed in grams of substance/100 g of dry artificial

soil. The LC50 of the various compounds for Eisenia andrei

were evaluated. Lactic acid seemed to be toxic and lactate

was almost as toxic as lactic acid, while calcium lactate

was less toxic than sodium lactate.

Related to PLA/organoclays nanocomposites there is a

potential application of these materials for packing.

Therefore, as composting may be a possible route of dis-

posal for PLA-based nanocomposites, its fillers could also

be released at compost. No studies on the ecotoxicological

effects of nanoclays have been identified. In soil science,

the term ‘clay fraction’ (or simply ‘clay’) refers to a class

of materials, whose particle size is \2 lm in equivalent

spherical diameter. The clay fraction also includes ‘nano-

clays’ particles (\100 nm in diameter) [151].

United States Environmental Protection Agency (EPA)

[152] reported the relevance to manage potential risks

across the life cycle of nanomaterials, from production

through the disposition of wastewaters and residuals con-

taining nanomaterials. It is widely recognized that there are

many obstacles to model development and, in general, to

conducting environmental risk assessments. In order to

address effects of nanomaterials on the living components

of ecosystems the new field of ‘‘nanoecotoxicology’’ has

emerged [153]. In a recent paper review of Rico et al. [154]

some information about the absorption, translocation,

accumulation, and biotransformation of nanomaterials

(mainly metal based and carbon based) was compiled.

Since clay is a naturally occurring material for which

environmental organisms have adapted throughout evolu-

tion, the inherent toxicity is expected to be low [155].

Related to organoclays, Material Safety Data Sheets of the

commercial available products, such as CLOISITEs, report

that these products have no known ecotoxicological effects.

The compounds commonly used in organoclays preparing

are quaternary ammonium compounds, which belong to an

important class of industrial chemicals that have been also

applied for other applications such as antistatic agents,

disinfectants, corrosion inhibitors and textile softeners.

After use, these compounds are discarded and may enter in

the waste water treatment systems, waterways and/or soils

[156, 157], which have attracted concerns about environ-

ment pollution and investigations about this kind of pol-

lutant. Therefore, it is important to consider the available

knowledge in order to predict some effects of these com-

pounds released due to nanocomposites degradation.

According to Ginkel et al. [158], the risks to the envi-

ronment of cationic surfactants are very limited. Microor-

ganisms have probably evolved enzymes to readily degrade

these substances because these compounds contain chem-

ical structures in common with those occurring naturally

(alkyl groups and methylamines). On the other hand, since

some cationic surfactants are water insoluble, bioavail-

ability is perhaps the key to determine their biodegradation.

Bioavailability is defined as that fraction that is readily

accessible to microbial degradation and exists in a dynamic

equilibrium between the aqueous and solid phases.

Reduced bioavailability is also responsible for reduced

toxicity of cationic surfactants to other organisms [159,

160].

Taking into account the low concentration in nano-

composites (in general up to 5 %) and low solubility of

organoclays modifiers, low ecotoxicity effects could be

expected. However, by considering the data previously

reported [106, 118], the potential toxicity related to qua-

ternary ammonium compounds should not be discarded. By

the lack of information and considering that an important

discarding purpose for these PLA-based materials is com-

posting, studies might investigate the compost resulting

from their degradation process. The potential genotoxic

effects of compost to plants and animals, and the possible

synergistic or antagonistic effects of the mixtures on bio-

logical systems are scarcely known. In order to assess the

impacts of the compost on human health and the ecosys-

tems, biomonitoring must be carried out using bioassays

which are able to detect harmful effects [161].

Higher plants are recognized as excellent genetic models

to detect environmental mutagens and are frequently used

in monitoring studies. Among the plant species, Allium

cepa has been used to evaluate DNA damages, such as

chromosome freaks and disturbances in mitotic cycle. The

Allium cepa has been applied for environmental monitoring

to detect different classes of pollutants, such as metals,

pesticides, aromatic hydrocarbons, textile industry dyes,

products used to disinfect drinking water and also complex

mixtures, such as water and soil samples from contami-

nated areas [162].

The A. cepa test enables the assessment of different

genetic endpoints, such as mitotic index, chromosome

freaks and micronucleus [161]. The mitotic index (MI) is

characterized by the total number of dividing cells in the

cell cycle and it can be used as parameter to assess the

cytotoxicity of several agents. The cytotoxicity levels of an

agent can be determined by the increase or decrease in the

MI [163]. To evaluate chromosome abnormalities (CA) by

the Allium cepa test, several types of disturbances are

considered in the different phases of cell division (pro-

phase, metaphase, anaphase and telophase). CA are char-

acterized by changes in either chromosomal structure or in

the total number of chromosomes, which can occur both

spontaneously and as a result from exposure to physical or

chemical agents [164]. Micronucleous result from dam-

ages, not or wrongly repaired, in the parental cells, being

easily observed in daughter cells as a similar structure to

the main nucleus, but in a reduced size. Thus,

J Polym Environ

123

micronucleous arises from the development of some CA,

like chromosome breaks and losses. Micronucleous has

been considered by many authors as the most effective and

simplest endpoint to analyze the mutagenic effects [165].

Besides all the advantages already mentioned, the Allium

cepa test has shown high sensitivity and good correlation

when compared with other test systems, e.g. mammals. The

sensitivity and correlation among test systems are funda-

mental for a more accurate evaluation of the environmental

risks, as well as extrapolation of data to other target

organisms, e.g. man. [162]. Therefore, Allium cepa test is a

potential tool for evaluating toxic potential of compost after

degradation process of compostable materials.

Conclusion

PLA-based materials are already produced at large scale,

and the use of organoclays fillers could even promote its

applications in packing by enhancement of gas barrier

properties. The concerns about healthy safety of these

materials have received efforts from the scientific commu-

nity. Some of the studies have demonstrated potential

cytotoxic and genotoxic effects from organoclays. Further

investigations are necessary to clarify toxicity potential of

nanomaterials aiming the safe use in food-packing products.

Estimating environment effects coming from organoc-

lays/polymer nanocomposites is also a challenge issue. As

a discarding purpose for PLA and PLA nanocomposites

packages, composting have been suggested as an important

route. The biodegradation of PLA is well described as

favorable to occur in composting conditions, in which

temperature and humidity accelerate hydrolytic degrada-

tion. Related to nanocomposites, just few studies evaluat-

ing the role of clays on polymer degradation process were

done, which makes necessary more investigation to better

understanding this issue.

Other important, but scarcely studied issue to be asses-

sed is whether there is some negative effect promoted by

composts in which biodegradation of PLA and its nano-

composites proceed. A potential tool that could permit this

kind of evaluation is biomonitoring. Furthermore, the use

of Allium cepa as an test organism is suggested for compost

samples analysis, since it has demonstrated a great poten-

tial for environmental monitoring.

Acknowledgments The authors are grateful to FAPESP (process

number 2011/14250-3 and 2012/00227-2) and CNPq, for the financial

support.

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