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Study of Aliphatic-Aromatic Copolyester Degradation in SandySoil and Its Ecotoxicological Impact

Piotr Rychter,† Michał Kawalec,‡ Michał Sobota,‡ Piotr Kurcok,†,‡ and Marek Kowalczuk*,†,‡

Polish Academy of Sciences, Centre of Polymer and Carbon Materials, 34, M. Curie-Skłodowska Street,41-819 Zabrze, Poland, and Jan Długosz University, Institute of Chemistry, Environment Protection and

Biotechnology, 13/15 Armii Krajowej Avenue, 42-200 Czestochowa, Poland

Received July 30, 2009; Revised Manuscript Received February 16, 2010

Degradation of poly[(1,4-butylene terephthalate)-co-(1,4-butylene adipate)] (Ecoflex, BTA) monofilaments (rods)in standardized sandy soil was investigated. Changes in the microstructure and chemical composition distributionof the degraded BTA samples were evaluated and changes in the pH and salinity of postdegradation soil, as wellas the soil phytotoxicity impact of the degradation products, are reported. A macroscopic and microscopic evaluationof the surface of BTA rod samples after specified periods of incubation in standardized soil indicated erosion ofthe surface of BTA rods starting from the fourth month of their incubation, with almost total disintegration of theincubated BTA material observed after 22 months. However, the weight loss after this period of time was about50% and only a minor change in the Mw of the investigated BTA samples was observed, along with a slightincrease in the dispersity (from an initial 2.75 up to 4.00 after 22 months of sample incubation). The multidetectorSEC and ESI-MS analysis indicated retention of aromatic chain fragments in the low molar mass fraction of theincubated sample. Phytotoxicity studies revealed no visible damage, such as necrosis and chlorosis, or otherinhibitory effects, in the following plants: radish, cres, and monocotyledonous oat, indicating that the degradationproducts of the investigated BTA copolyester are harmless to the tested plants.

Introduction

Biodegradable polymeric packages are meant to be compostedafter disposal. However, it is impossible to avoid improperpractices such as those at illegal and uncontrolled landfill sites.1

Worldwide production of biodegradable polymers is about toreach more than 1 million tons per year.2 However, becauseonly about 60% of this amount is properly disposed of,approximately 400000 tons of waste is still introduced into thesoil environment annually.3

The biodegradation process of aliphatic polyesters in varioustypes of environments was thoroughly investigated.4-14 Thelimited mechanical properties of aliphatic polyesters may beovercome by introducing aromatic units into the main chain ofthese materials. However, the higher the ratio of aromatic unitsin copolyesters, the more resistant the material is to microbes.15-17

The sustainability challenge is to produce relatively cheappolymeric materials possessing the appropriate mechanicalproperties while still maintaining good biodegradation properties.Ecoflex, which is the aliphatic-aromatic copolyester, poly[(1,4-butylene terephthalate)-co-(1,4-butylene adipate)] (poly[(tet-ramethylene terephthalate)-co-(tetramethylene adipate)], BTA),is a very promising material to meet these criteria. Structuralstudies of BTA using NMR have focused mostly on 13C NMR,while the literature information on degradation of this materialin soil is limited and deals with gardening soil.15,18,19

The aim of the present study was to investigate the (bio)deg-radation of BTA in standardized soil. Changes in polymermicrostructure and composition were investigated using 1HNMR, multidetector SEC, and ESI-MS techniques. The suit-ability to the ESI-MS for the molecular level structure elucida-

tion of polyester degradation products has been demonstratedrecently.20-22 Furthermore, determination of changes in thequality of the soil as well as the effects of potential postdeg-radation products on plant growth monitored during thedegradation experiments are reported.

Materials and Methods

Materials. Poly[(tetramethylene terephthalate)-co-(tetramethyleneadipate)] (BTA) in the form of granules was kindly supplied by BASFLudwigshafen. A single screw laboratory extruder (ZMP-TW Gliwice;12 mm screw diameter, 20 D; four heating zones: I, 100 °C; II, 135°C; III, 140 °C; each one 80 mm long; head, 100 °C) was used for thepreparation of polymeric rods. After cooling, extruded BTA was cutinto segments and conditioned at room temperature for 1 week. Allinvestigated samples were in the form of rods weighing 0.1 g each(average diameter 2 mm and length ca. 20 mm).

Each sample was weighed before and after degradation using aRadwag electronic balance (0.1 mg precision). Weight loss wascalculated using the following relationship:

where Mi is the initial mass and Mf is the final mass.Poly(1,4-butylene adipate) (PBA) and poly(1,4-butylene terephtha-

late) (PBT; both from Aldrich) were used as received.NMR Analyses. 1H and 13C NMR spectra were recorded using a

Bruker-Avance spectrometer, operating at 600 MHz for 1H measure-ments, equipped with a BBO probe using CDCl3 as the solvent andtetramethylsilane (TMS) as the internal standard. 1H NMR spectra wereobtained with 64 scans, a 11 µs pulse width, and a 2.65 s acquisitiontime, and 13C NMR spectra were obtained with 20480 scans, a 9.40 µspulse width, and a 0.9088 s acquisition time.

* To whom correspondence should be addressed. Tel.: +48 32 2716077.Fax: +48 32 2712969. E-mail: [email protected].

† Jan Długosz University.‡ Polish Academy of Sciences.

%mass loss )Mi - Mf

Mi× 100

Biomacromolecules XXXX, xxx, 000 A

10.1021/bm901331t XXXX American Chemical Society

The 1H NMR spectrum of BTA showed signals as follows: CH5 at8.10 ppm, CH6

2 (4.44 ppm), CH6′2 (m at 4.38 ppm), CH1′

2 (m at 4.15ppm), CH1

2 (4.09 ppm), CH3′2 (2.33 ppm), CH7

2 (1.97 ppm), CH7′2 (m

at 1.87 ppm), CH2′2 (m at 1.80 ppm), CH2

2 (1.69 ppm), and CH42 (1.66

ppm; Figure 1).SEC Analyses. Molar masses and molar mass distributions of

polymers were determined by SEC experiments conducted in CHCl3

(HPLC grade stabilized with ethanol, purity min. 99.8%, POCh Gliwice)at 35 °C with an eluent flow rate of 1 mL ·min-1, using a set of twoPLgel 5 µm MIXED-C ultrahigh efficiency columns (Polymer Labo-ratories) with a mixed bed and linear range of Mw ) 200-2000000.An isocratic pump (VE 1122, Viscotek) as the solvent delivery system,differential refractive index detector stabilized to a temperature of 35°C (VE3580, Viscotek), a viscometer detector (270 Dual Detector Array,viscometer only, Viscotek), and a UV-vis variable wavelength detectorat a wavelength of 260 nm (Spectra 100, Spectra-Physics) were used.A volume of 100 µL of about 3% w/v sample solution in CHCl3 wasinjected. Polystyrene standards (Calibration Kit S-M-10, PolymerLaboratories) with narrow molecular weight distributions were usedto generate a universal calibration curve according to which sampleswere calculated using OmniSEC 4.6 (Viscotek) software.

The universal calibration method for homopolymer analysis wascalibrated using the PS standard with a peak molar mass Mp ) 31420(dn/dc ) 0.165). Next, the differential refractive index increments withconcentrations (dn/dc) at 35 °C were determined for PBA and PBT

homopolymers. The refractive index increments were found to be 0.046for PBA and 0.126 for PBT. The PBA refractive index increment wasdetermined by analyzing five solutions of PBA in chloroform in theconcentration range of 2.0-0.7% w/v. In the case of PBT, 10 mg ofthe sample was solubilized in 0.5 mL of 1,1,1,3,3,3-hexafluoroisopro-panol and then diluted with CHCl3 to 10 mL. In addition, the differentialabsorbance increment with concentration (dA/dc) for PBT was deter-mined at a wavelength of 260 nm. The dA/dc was found to be equal to122.34 in relation to PS, where the dA/dc was set to 10. PBA did notshow absorbance at this wavelength (dA/dc ) 0).

The copolymer analysis method23,24 was created from the homopoly-mer analysis method by using the determined values of dn/dc and dA/dc for poly(1,4-butylene adipate) (PBA) and poly(1,4-butylene tereph-thalate) (PBT) units.

The concentration of counits in the analyzed sample was calculatedusing a system of two equations:

where RIheight is the intensity of differential refractive index detectorsignal at the respective retention volume, UVheight is the intensity of

Figure 1. 1H NMR spectrum of BTA and expanded regions: 4.60-3.90 ppm; 2.10-1.50 ppm.

RIheight ) conc(A) · dn/dc(A) + conc(B) · dn/dc(B)

UVheight ) conc(A) · dA/dc(A) + conc(B) · dA/dc(B)

B Biomacromolecules, Vol. xxx, No. xx, XXXX Rychter et al.

UV detector signal at the respective retention volume, and conc(i) isthe concentration of unit i at the respective retention volume.

In the case of BTA,

where dn/dc (PBT, 35 °C) ) 0.126, dn/dc (PBA, 35 °C) ) 0.046, dA/dc (PBT, 260 nm) ) 122.34, and dA/dc (PBA, 260 nm) ) 0, therefore,

The solution of this system of equations “transforms” the detectorsignal to analyte concentration. Molar masses were calculated accordingto real analyte concentration. P and Q factors were used with defaultvalues.

The fractionation experiment was conducted using a SEC systemequipped with a set of two PLgel 5 µm MIXED-C ultrahigh efficiencycolumns (Polymer Laboratories), a differential refractive index detectorstabilized at 35 °C (VE3580, Viscotek), and a Foxy Jr. FractionCollector (Teledyne Isco, Inc.). A volume of 100 µL of about 3% w/vsample solution in CHCl3 was injected and divided into five equal partsthat were collected. This procedure was repeated 15 times to collect asufficient quantity of each fraction. The fractions were then character-ized by NMR spectroscopy. For BTA samples were incubated 4 and22 months, respectively, and fractions of SEC analyte were collectedfor ESI-MS analyses at a retention volume between 15 and 18 mL.

ESI-MS Analyses. The electrospray mass spectrometry (ESI-MS)analysis of low molecular weight fractions collected from SEC(retention volume between 15 and 18 mL) was performed for the BTAsamples remaining after 4 and 22 months of degradation, respectively.The analysis was performed using a Finnigan LCQ ion trap massspectrometer (Finnigan, San Jose, CA). The samples of the polyesterswere dissolved in the chloroform/methanol system (2:1 v/v), and thesolutions were introduced to the ESI source by continuous infusionusing the instrument syringe pump at a rate of 3 µL/min. The LCQESI source was operated at 4.5 kV, and the capillary heater was set to200 °C. Nitrogen was used as the nebulizing gas. The analyses wereperformed in the negative-ion mode.

Biodegradation in Soil. Soil Characteristics. The soil used in alldegradation experiments consisted of 84% sand and 10.7% dust andloam. The soil can be qualified as “sandy soil”. The soil propertieswere also determined: acidity (determined according to ISO standard10390), pH (KCl) ) 7.54; salinity (according to ISO 11265 + AC1standard), 79 mg KCl · L-1; and moisture content (according to ISO11465), 15%.25-27

Degradation Experiments. The degradation of prepared rods of BTAwas carried out in the above-described sandy soil, according to ISOstandard 11269-2, in polypropylene pots (capacity 300 cm3) containing200 g of soil per pot.28 The experiments were carried out in four testingperiods over 22 months. A total of 0.2 g of the tested material (1000mg of polymer per kg of soil dry weight) was introduced into a potfilled with soil (placed 2 cm under the surface) under laboratoryconditions. During the exposure period, burial sites were regularlyirrigated with distilled water to maintain a stable humidity level. Aftera predetermined degradation time, polymer specimens were carefullyremoved from the soil to avoid damage, thoroughly cleaned withdistilled water, and dried at room temperature for 1 week. Dried filmswere weighed to calculate the weight loss. The main difficulties withthis method are either loss of sample fragments as a result of thethorough cleaning process or an increase in sample weight due toleftover soil debris. Thus, an average value of three measurements was

used to produce the final result. Macro- and microscopic images of thesurface morphology of the biodegraded polymer specimens wererecorded.

The soil average humidity (15%) and temperature (22 ( 2 °C) ineach pot during all degradation experiments remained constant. Aftera specified period of time (4, 10, 16, and 22 months), sample weightloss, surface erosion, composition, molecular weight, and molecularweight distribution of the polymeric material, as well as changes inselected soil properties such as active pH (H2O), exchangeable aciditypH (KCl), and salinity were determined. Tests for each period of timewere carried out in triplicate.

Phytotoxicity Test. The plant growth test for BTA monofilamentswas performed under laboratory conditions using a monocotyledonousplant oat (AVena satiVa) as well as two dicotyledonous plants, commonradish (Raphanus satiVus L. subVar. radicula Pers.) and cress (LepidiumsatiVum L.), by adapting the OECD 208 Terrestrial Plants Growth Testand the ISO 11269-2 International Standard.29 The medium for theplant growth test was the sandy soil placed in 90 mm diameter pots.After a specified period of time (4, 10, 16, and 22 months), BTAsamples were thoroughly removed from the pots and oat (50 pieces),radish (50 pcs), and cress (100 pcs) seeds were sown in the soil. Incontrol experiments, soil after a given incubation/watering time wasused. Plants were grown for 2 weeks under controlled conditions(constant parameters such as humidity, light intensity, and temperature,20 ( 2 °C, were maintained throughout the whole period of plantgrowth). After a 14 day growing period, seedlings were counted andthe dry and fresh weight of the plants above the soil was determined.The dry weight of the plants was determined after drying at 75 °Cuntil a constant weight was achieved. According to ISO11269-2standards, a visual evaluation of potential growth inhibition, chlorosis,and necrosis occurring in both the control and the test (containingpostdegradation medium) pots was carried out throughout the testperiod.

Evaluation of the significance of the obtained results was performedusing an analysis of variance test (Fisher-Snedecor’s F test), whereasthe LSD0.95 values were calculated using Tukey’s test.

Results and Discussion

Several papers have dealt with the problem of BTAbiodegradation,16,30,31 however, to the best of our knowledge,there has been no report on its degradation in standardized sandysoil or on the effects on plant growth.

BTA is an aliphatic-aromatic random copolyester, which thepolymer chain consists of A (1,4-butandiol adipate) and T (1,4-butandiol terephthalate) units. The microstructure of BTA wasevaluated based on NMR measurements. Previous worksdetermined the molecular structure of this polyester based on13C NMR spectra.31,32 However, 13C NMR with the NOEtechnique is not as quantitative as 1H NMR measurements.Determination of the aromatic/aliphatic monomer units ratiousing 1H NMR spectroscopy was described recently, however,only groups of proton signals were ascribed to the general BTAstructure and no microstructural evaluations were performed.33,34

In the present work, a detailed assignment of 1H NMR signalscorresponding to BTA structure is shown (Figure 1). Molarfractions of terephthalate and adipate were calculated from theratio of intensities of baseline resolved signals 3 and 5, similarto those reported in refs 31 and 32. The number-averagesequence length of both aromatic and aliphatic units wascalculated based on the intensities of the signals of O-CH2-protons (i.e., signals 1, 1′, 6, and 6′) ascribed to the respectivestructures and then the degree of randomness was calculated(see Figure 1 and Table 1).

The proton spectrum revealed one more set of signals forthe central tetramethylene glycol protons. Dyad sequences can

RIheight ) conc(T) · dn/dc(T) + conc(A) · dn/dc(A)

UVheight ) conc(T) · dA/dc(T) + conc(A) · dA/dc(A)

RIheight ) 0.126 · conc(T) + 0.046 · conc(A)

UVheight ) 122.34 · conc(T)

Aliphatic-Aromatic Copolyester Degradation Biomacromolecules, Vol. xxx, No. xx, XXXX C

be observed in the range of about 1.7 to 2.0 ppm, but thesesignals could not be used for calculation directly due tooverlapping of the signals when a 600 MHz apparatus was used.The obtained results revealed that BTA received from themanufacturer is comprised of 47.1 mol % of terephthalate and52.9 mol % of adipate. Furthermore, the number averagesequence length of aromatic and aliphatic structures was around2 and the copolymer randomness was around 1. These resultsindicate the fully randomized structure of the copolyester. Table1 shows a comparison of the BTA structure determined using13C and 1H NMR spectra.

The slight differences found in the 1H and 13C NMR resultscan be attributed to the application of the NOE technique for13C experiments. Small differences in the chemical environmentof the observed carbons could lead to a different gain in theintensity of the signals when the NOE technique was used.However, detailed analysis of the 13C NMR spectrum revealeda signal for the aromatic sequence triad (see SupportingInformation). The terephthalate carbon signal (C1 and C4 inaromatic ring) at 134.0 ppm splits into four signals that wereascribed to the structures T-T-T, A-T-T, T-T-A, and A-T-A,but it was not possible to determine which signal representswhich triad due to lack of the respective model compounds.

Because NMR results are just an average picture of thespecimen, the BTA sample was divided into five fractions usingSEC (Figure 2) to check the composition distribution of thepolymer at various molecular weights of the sample.

Analysis of the fractions clearly shows that the BTAcopolymer has a homogeneous and random distribution ofcounits (see also Supporting Information).

(Bio)Degradation of BTA Rods in Soil. The activity as wellas the number of microorganisms in soil depend on the type ofsoil, humidity of soil, temperature, and organic matter content.35

Soil degradation experiments conducted according to the standardmethods are usually rather long term mainly due to soil’s limitedmicrobial activity as compared to active sludge, compost, orartificial medium inoculated with isolated microorganisms.

Macroscopic visual evaluation of the surface of the BTAsamples during the degradation process indicates erosion of thesurface of the rods starting from the fourth month of theirincubation in soil. At the beginning of sample incubation insoil, that is, after 4 months, slight splits in the surface appeared.After 10 months, local surface erosion in the form of pitsappeared, and finally, after 22 months of sample degradation, ahigh degree of disintegration of the material was observed.Furthermore, the attached microscopic photographs show changesin the surface invisible to the naked eye (Figure 3).

Monitoring of sample weight changes from the beginning ofthe degradation process up to the 10th month of incubationindicated only a slight decrease (ca. 15%) in the plain sampleweight. A more substantial weight loss of about 28% after the16th month and 50% after the 22nd month of incubation in soilwas noted (Figure 4).

These results generally correlate with results obtained forweight loss during the biodegradation test of BTA in environ-ments such as compost or in a medium inoculated withmicroorganisms.31,36 In such an environment, several factors

Table 1. Comparison of BTA Composition and Dyad Sequences Determined Using 1H and 13C NMR Spectrometry

composition (mol %) dyads (mol %)

Aa Tb A-A A-T T-T SLAc SLT

d Re

1H NMR 52.9 47.1 27.9 49.9 22.2 2.14 1.46 1.1513C NMR 57.3 42.7 29.7 50.3 20.3 2.03 1.80 1.05

a 1,4-Butylene adipate. b 1,4-Butylene terephthalate. c Number average sequence length of adipate: SLA ) (I1 + I1′)/I1′. d Number average sequencelength of terephthalate: SLT ) (I6 + I6′)/I6′. e Degree of randomness: R ) 1/SLA + 1/SLT; Ii ) intensity of a signal i.

Figure 2. 1H NMR spectra of resolved BTA fractions.

Figure 3. Macro- and microscopic (magnification 100×) photographsof surface erosion of BTA samples after 0, 4, 10, 16, and 22 monthsof incubation in soil.

D Biomacromolecules, Vol. xxx, No. xx, XXXX Rychter et al.

such as pH, UV, temperature, and the presence of specificmicroorganisms affect the degradation process. The higher rateof BTA degradation in compost and gardening soil (60%humidity) observed by Muller37 is most probably, despite thedifference in humidity, also due to the different shape ofthe sample studied, that is, the ratio of volume to surface of thespecimen. The effect of sample shape is significant in hydro-lytic38 as well as enzymatic degradation.39 Our investigationswere focused on BTA rods. The rod shape was chosen toinvestigate the degradation of thicker samples, which shouldbetter mimic disposable rigid materials. Therefore, the weightloss trend is the same as mentioned in a previous work,37 onlythe degradation time is longer.

Another monitored parameter was the change in molar mass.SEC analysis of the samples with calculations of molar massaccording to the copolymer analysis method revealed a decreasein Mw from an initial 33000 to about 30000 after 4 months ofdegradation. Moreover, there was almost no change in the Mw

of investigated samples after subsequent periods of incubation.There was, however, a slight increase in dispersity from theinitial 2.75 to 4.00 after 22 months of sample incubation. Theincrease in dispersity is due to an increase in the low massfraction, thus, proving that degradation undoubtedly occurs andindicates scission in the polymer chains. The Mn of the samplesdecreased from an initial 12000 to 7500 after 22 months. Thephenomenon of substantial sample weight loss with almost noloss of molar mass would suggest an enzymatic mechanism ofsample degradation. Furthermore, the obtained results indicatethe minor contribution of hydrolytic degradation of the samples.The observed phenomenon could be explained similarly to Tanet al.40 who suggested that the degradation of Ecoflex in thepresence of selected microorganisms was occurring near thechain ends where there is generally more freedom for enzymeattack on the ester groups.

NMR analysis performed for the samples studied after specificincubation times revealed, that the BTA composition barelychanged during the experiment. The mol % of terephthalateremained almost at the same level of about 46-47%, while thenumber average length of terephthalate and adipate as well asthe degree of randomness were also approximately constant(Table 2). These results indicate no compositional preferencesin the degradation process.

Substantial weight loss with almost no change in weight-average molar masses and practically no change in copolymercomposition were observed. The calculated number-averagesequence length of the aliphatic as well as of the aromatic unitis around 2. Witt et al.16 point out that BT oligomers of DP )2 and less may be leached out of the soil due to watering.

Retention of aromatic chain fragments of DP g 3 in the samplemay cause enrichment of the low molar mass polymer fractionin the nondegrading aromatic structures resulting in a broadeningof molar mass distribution as well as a decrease in sample Mn.Therefore, copolymer analysis was used during the SECcalculation to prove this assumption. Application of this methodallowed for monitoring of the chemical composition distributionin the investigated samples. Additionally, in cases where onlyone of the counits (a homopolymer) is present in the analyzedslice of the elugram, it can be detected and identified.

Figure 5 shows results obtained for a plain sample of BTA.Curve 2 represents the calculated concentration of the analyzedsample and curves 3 and 4 are related to A and T unitconcentrations in the polymer. No spectacular changes werevisible here but after NMR analysis of fractions of the suppliedBTA sample this was to be expected. It is worth noticing thata higher concentration of T units was calculated in the low molarmass range (ca. 15-18 mL in Figure 5). This may be due tothe approximately 3 orders of magnitude higher sensitivity ofthe UV detector compared to the RI one.41

The change in the concentration curves of aromatic andaliphatic units for the degraded samples depicted in Figure 6clearly demonstrates a substantial increase in aromatic units (T)in the oligomeric fraction. However, the increase is so low thatthe average chemical composition of the whole sample fromNMR measurements did not change. Nevertheless, when thenumber-average sequence length of the aromatic units is lowerthan 2, one cannot expect there to be a substantial fraction ofinsoluble aromatic blocks of DP g 3. The performed analysis

Figure 4. Weight loss of BTA sample during the degradation processin soil.

Table 2. Characteristics of BTA Copolymers’ Microstructure andMolar Masses after their Respective Degradation Times

composition(mol %)

dyads(mol %)

molarmassdegradation

time(months) A T A-A A-T T-T Mw Mn Mw/Mn

initial 53.7 46.3 27.7 50.3 22.0 33000 12000 2.754 53.2 46.8 26.8 50.9 22.3 30500 10000 3.0510 52.4 47.6 26.7 50.0 23.3 30000 11500 2.6116 52.2 47.8 26.8 50.0 23.2 29500 9500 3.1122 52.6 47.4 26.0 50.2 23.7 30000 7500 4.00

Figure 5. SEC of BTA sample after extrusion in melt: (1) RI detectorresponse; (2) calculated real concentration of analyte; (3) concentra-tion of A units; (4) concentration of T units.

Aliphatic-Aromatic Copolyester Degradation Biomacromolecules, Vol. xxx, No. xx, XXXX E

also indicates that aliphatic units were almost absent in theoligomeric fraction. This may be because the aliphatic units aresusceptible to biodegradation or soluble in water. The curve ofthe A unit concentration reveals fluctuations because of lowersignal/noise ratios of RI detector.

The ESI-MS experiments were performed for low molecularweight fractions collected from SEC analysis (retention volumebetween 15 up to 18 mL, see Figure 6) for the BTA samplesremaining after 4 and 22 months of degradation. The respectiveESI-MS spectra and the table with the list of assigned structuresof the respective parent ions are given in the SupportingInformation. The structures of the parent ions were assignedbased on the studies performed previously42 and on thefragmentation experiments (data not shown). The negative modeESI-MS spectra of low-mass factions reveal signals of parentions assigned to oligomers terminated with one hydroxyl andone carboxyl group as well as two carboxyl groups. Massdifference of 1,4-butylene terephthalate and 1,4-butylene adipateunits is 20 Da. Spectrum of the sample incubated 22 months

reveals distinct signals assigned to 1,4-butylene terephthalatetrimer, tetramer, and pentamer. The same homo-oligomers arehardly observed at spectrum of the sample incubated 4 monthsin soil. The obtained results clearly demonstrated at themolecular level that after 22 months of degradation in soil thelow molar mass polymer fraction was enriched in the aromaticstructures. Moreover, the SEC analysis with UV detection andESI-MS results are complementary, indicating retention of 1,4-butylene terephthalate units in degraded samples.

The presence of possible oligomeric degradation products ofBTA in the postdegradation soil was investigated by nuclearmagnetic resonance techniques (1H NMR) of soil methanol/chloroform (50/50) extracts. No signals of degradation productsof BTA were found in soil extracts after 4 months of degrada-tion. The extract obtained from postdegradation soil after 10months of the experiment revealed only traces of terephthalatesand adipates. However, the extracts obtained after 16 and 22months revealed a higher content of the low molar mass products(Figure 7).

This result suggests that the small amounts of low molecular,oligomeric, or even monomeric products formed during thedegradation of BTA up to 10 months of incubation were eitherinsoluble or assimilated by microorganisms. After that time theconcentration of BTA biodegradation products increased. Fur-thermore, the postdegradation extract was also enriched interephthalate compounds in comparison to adipates. Thisobservation is in good correlation with previously reportedresults17 concerning the biodegradability of aromatic oligomers.

Evaluation of Potential Toxicity of the PostdegradationSoil Sample. The potential toxicity of the residues of theanalyzed copolyester present in postdegradation soil wasinvestigated by measuring changes in soil acidity and salinityas well as by the plant growth test.

Taking into account exchangeable acidity (which refers toacidic cations associated with soil solids that are rapidly releasedinto the soil solution using a concentrated solution of a neutralsalt such as KCl), the change from pH (KCl) ) 7.20 after 4months to 6.81 was observed after 22 months of polymerincubation in soil. However, a comparison of the values of theacidity of postdegradation soil and control soil showed nosubstantial differences (Table 3).

In the case of active acidity (i.e., activity of H+ ions in thesoil solution), a small decrease in pH (H2O) from 7.50 (4months) to 7.05 (22 months) was observed. The most probablereason for the active acidity decrease after 10 months ofincubation of the soil is that the water used for irrigation wasprobably a bit more acidic and caused a decrease in the activeacidity of the soil samples. Active acidity is very “sensitive” to

Figure 6. Calculated real concentration of T units (top) and A units(bottom) in the low molar mass range (elution volume 14-18 mL)and indication of the elution region where samples for ESI-MSanalysis were collected.

Figure 7. 1H NMR spectra of soil extracts of BTA degradation after10, 16, and 22 months (expanded regions 2.20-2.60, 3.40-4.80,and 7.80-8.60 ppm).

F Biomacromolecules, Vol. xxx, No. xx, XXXX Rychter et al.

water pH and therefore tends to fluctuate. On the other hand,exchangeable acidity (cations associated with the soil solids)remained constant. Therefore, it can be concluded that nosignificant changes were observed during the degradation time.It is worth noticing that the pH values for postdegradation soilsamples were lower than for the control samples for each period(one exception was the pH (KCl) after 4 months; the valueswere almost the same), which was probably due to copolymer

samples leaching their water-soluble degradation productsinvolving low molecular oligomers consisting of adipic orterephthalic acid into the soil. The fact that a decrease in pHwas observed not only for post degradation soil but also forcontrol soil is probably related to the depletion of basic nutritivecations, such as calcium, potassium, magnesium, and so on, fromthe soil over time.

Differences in the salinity values of postdegradation soil, ascompared to control soil in each period, during the experimentwere not significant, however, a slight salinity increase in thesoil sample after 22 months was observed, indicating the appear-ance of degradation products of the copolymer. High values ofthe lowest significant difference for soil salinity indicate a largevariance between replications during measurements.

Similar trends concerning soil acidity and salinity werepreviously observed for poly(L-lactide acid), bacterial poly[(R)-3-hydroxybutyrate], and blends of these materials with atacticpoly[(R,S)-3-hydroxybutyrate], where a slight decrease in soilpH was also noticed.8 Changes in soil salinity and acidity alsocorrelate with the data obtained by Rosa et al.43,44 for n-PHBand by Tuominen et al.45 for lactic acid-based polyurethanedegradation in soil and compost, respectively. However, Rosaet al., observed a substantial decrease in soil pH during thedegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Table 4. Changes in Basic Parameters (Avg Values of Three Replicates) of the Plant Growth Test for Oat, Radish, and Cress (CS, ControlSoil; PDS, Post Degradation Soil)a

Cress

No. ofseeds

plantedperiod

(months)soil

sampleNo. ofplants

germinationcompared tocontrol (%)

weight offresh

crop (g/pot)

cropcompared tocontrol (%)

dry weight(mg/g fresh

weight)

dry weightcompared tocontrol (%)

100 4 CS 59 100 1.756 100 0.0541 100PDS 68 115 1.742 99 0.0623 115

10 CS 65 100 1.667 100 0.0576 100PDS 73 128 1.778 107 0.0573 99

16 CS 57 100 1.638 100 0.0592 100PDS 67 118 1.757 107 0.0654 110

22 CS 66 100 1.483 100 0.0667 100PDS 64 97 1.719 116 0.0710 106

LSD0.95 ) 2.12FS ) 14 FS ) 0.435 FS ) 0.0175FP ) 10 FP ) 0.308 FP ) 0.0124

Radish

50 4 CS 40 100 4.023 100 0.0860 100PDS 39 98 4.175 104 0.0896 104

10 CS 38 100 3.950 100 0.0987 100PDS 39 103 3.982 101 0.0916 93

16 CS 36 100 3.859 100 0.0861 100PDS 38 106 4.100 106 0.0812 94

22 CS 39 100 3.960 100 0.0764 100PDS 40 103 4.146 105 0.0820 107


Oat

50 4 CS 47 100 4.143 100 0.0977 100PDS 48 102 4.326 104 0.1033 106

10 CS 48 100 4.382 100 0.1045 100PDS 48 100 4.419 101 0.0992 95

16 CS 47 100 4.207 100 0.0925 100PDS 49 104 4.372 104 0.0972 105

22 CS 47 100 4.082 100 0.0836 100PDS 48 102 4.434 109 0.0865 103


a Additional abbreviations: LSD (lowest significant difference), FS (LSD For samples), FP (LSD For periods).

Table 3. Changes in the Acidity and Salinity of thePost-Degradation Soil (PDS) and Control Soil (CS)a

period(months)

soilsample

pH(H2O)

pH(KCl)

salinity(mg KCl ·L-1)

4 CS 7.40 7.18 91.7PDS 7.50 7.20 82.0

10 CS 7.22 6.93 82.0PDS 7.05 6.84 54.3

16 CS 7.40 6.93 70.7PDS 7.38 6.76 85.0

22 CS 7.30 7.02 102.3PDS 7.05 6.81 133.0

LSD0.95 ) 2.12FS ) 0.29 FS ) 0.23 FS ) 58.9FP ) 0.2 FP ) 0.16 FP ) 41.6

a Average values of three replicates; additional abbreviations: LSD,lowest significant difference; FS, LSD for samples; FP, LSD for periods.

Aliphatic-Aromatic Copolyester Degradation Biomacromolecules, Vol. xxx, No. xx, XXXX G

(PHBV) copolymer, whereas degradation of this copolymer incompost medium caused only a slight increase in pH.43,44 Wittet al.30 found that the biodegradation of BTA stopped in amedium containing actinomycetes because of the dramaticdecrease in pH within a short period of time. The decrease inpH was probably caused by an increase in the concentration ofacidic products of degradation.

Plant Growth Test. According to OECD guidelines for thetesting of chemicals, it is essential to assess the effect of BTAdegradation products on seedling emergence and the earlygrowth of higher plants because plants are ubiquitous in ourenvironment, even in the areas surrounding landfill sites. Thephytotoxicological aspect of the degradation process of com-mercially used biodegradable plastics is extremely importantbecause plants are at the bottom of the food chain in theecosystem. However, the low acute toxicity of BTA degradationintermediates was previously reported.30,46-48

Results concerning the effect of BTA degradation productson seedling emergence and early growth of the selected plantsare shown in Table 4.

All monitored parameters presented in Table 4 indicate thatthere were no statistically significant differences for bothdicotyledonous plants, that is, radish and cress, and for mono-cotyledonous oat. Regardless of the time period, all values ofdry and fresh weight of seedlings as well as the number ofgerminated plants were greater than 90% of those of the plantsgrown in the control experiments. According to the standard, asubstance is nontoxic if the number of seedlings and the overallfresh or dry weight of the plants are at least 90% of the valuesof the plants growing in pots without tested materials. Therefore,BTA and it’s degradation products are nontoxic.

Additionally, a visual evaluation of the plant was performedafter 14 days of plant growth but before counting the numberof seedlings and harvesting the plants (Figure 8). No visibledamages, such as necrosis and chlorosis or other inhibitoryeffects on the plants, were observed during the test.

Conclusions

Although BTA is known to be compostable under industrialcomposting processes, our investigations demonstrated its only

limited degradation in standardized sandy soil where disintegra-tion and partial mineralization49 of BTA samples were observed.

Macroscopic and microscopic evaluation of the surface ofBTA samples after the specified degradation times indicateserosion of the surface of BTA rods starting from the fourthmonth of their incubation in soil. Substantial disintegration ofthe BTA material incubated in standardized soil was observedafter 22 months. The total weight loss after this period of timewas around 50%. Only minor changes in the Mw of BTAinvestigated samples after subsequent periods of incubation wereobserved and the changes were accompanied by a slight increasein dispersity from an initial 2.75 to 4.00 after 22 months ofsample incubation. Such results suggest an enzymatic mecha-nism of BTA rod degradation with only a minor contributionfrom hydrolytic degradation of the samples. After 22 monthsof degradation, multidetector SEC and ESI-MS analyses indi-cated retention of aromatic chain fragments in the degradedsample leading to the enrichment of the low molar mass polymerfraction in the aromatic structures.

The plant growth test revealed no visible damages, such asnecrosis and chlorosis or other inhibitory effects, on the plants(radish, cress, and monocotyledonous oat). The phytotoxico-logical assessment of postdegradation soil suggests that thedegradation products of the investigated BTA copolyester aretotally harmless to the tested plants.

Acknowledgment. The authors are indebted to Dr. GrazynaAdamus for performing and interpretation of the ESI-MSn

experiments. This research project was partially supported byEuropean Union European Regional Development Fund, Con-tract No. POIG.01.03.01-00-018/08-00.

Supporting Information Available. 13C NMR and HMQCNMR spectra, composition, and microstructure characteristicsof prepared fractions of BTA copolyester, comparison of plainBTA sample molar mass calculated according to differentmethods, and ESI-MS spectra of the collected low molar massfraction of specimens incubated 4 and 22 months, respectively.This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 8. Digital photographs of growing plants, (a) oat, (b) cress, (c) radish, after conditioning the soil (1) 10 and (2) 22 months in the absence(control plants) or in the presence of BTA.

H Biomacromolecules, Vol. xxx, No. xx, XXXX Rychter et al.

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