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  • This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

    and sharing with colleagues.

    Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

    websites are prohibited.

    In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

    regarding Elseviers archiving and manuscript policies areencouraged to visit:

    http://www.elsevier.com/copyright

    http://www.elsevier.com/copyright
  • Author's personal copy

    Comparison of biodegradability of various polypropylene filmscontaining pro-oxidant additives based on Mn, Mn/Fe or Co

    Stphane Fontanella a, Sylvie Bonhomme a, Jean-Michel Brusson e, Silvio Pitteri d, Guy Samuel f,Grard Pichon g, Jacques Lacoste a,h, Dominique Fromageot a, Jacques Lemaire a, Anne-Marie Delort b,c,*aCentre National dEvaluation de Photoprotection (CNEP), Universit Blaise Pascal, 63 174 Aubire cedex, FrancebClermont Universit, UBP, ICCF, F-63000 Clermont-Ferrand, FrancecCNRS, UMR 6504, F-63177 Aubire, Francee Total Petrochemicals Research Feluy, Zone industrielle C, B-7181 Feluy, BelgiumdBasell Poliolefine Italia, 44100 Ferrara, ItalyfAssociation NEOSAC, F-43600 Ste Sigolne, FrancegGroupe Extrusion Soufflage de Ste Sigolne (E3S) F-43600 Ste Sigolne, FrancehClermont Universit, ENSCCF, ICCF, F-63000 Clermont-Ferrand, France

    a r t i c l e i n f o

    Article history:Received 26 October 2012Received in revised form28 December 2012Accepted 3 January 2013Available online 11 January 2013

    Keywords:PolypropyleneBiodegradationPro-oxidantsThermooxidationPhotooxidation

    a b s t r a c t

    The biodegradability of two polypropylene films with low content of ethylene (a statistical copolymer(PPs) and a block copolymer (PPb) with balanced additions of phenolic antioxidant and pro-oxidantsbased on Mn, Mn/Fe or Co was studied. Abiotic pre-treatments by accelerated artificial photooxidationand thermooxidation representing about 3e4 years of outdoor weathering, including 3e4 months ofexposure to daylight and 3 years in soil were followed by FTIR and SEC measurements. When a controlledoxidation was reached in the films, they were inoculated, in a second step, with the strain Rhodococcusrhodochrous in mineral medium and incubated up to 180 days. The metabolic activity of bacteria wasevaluated by measuring ATP content, ADP/ATP ratio and cell viability. Complementary 1H NMR experi-ments were conducted on the incubation media, with and without cells, in order to monitor the con-sumption of soluble compounds excreted from the oxidized polymers by R. rhodochrous cells. The mainconclusions are that the Co derivatives (with Co content 150 ppm) must be considered toxic forR. rhodochrous. PP films containing pro-oxidants based on Mn and Mn Fe give positive results for thebiotest (low ADP/ATP ratio, post-development in Petri dishes). However the biodegradability of oxidizedPP films is less efficient in comparison to oxidized PE films (see paper published in this journal). Thisobservation may be correlated with the accumulation in the incubation media of oxidized oligomers thatcannot be metabolized rapidly by the bacterial cells and/or by the residual crystallinity of PP derivatives.

    2013 Published by Elsevier Ltd.

    1. Introduction

    The replacement of inert and non biodegradable materials bybiodegradable alternative is significantly increasing in hydro-biodegradable materials and oxobiodegradable polyolefin mate-rials. However the domain of oxobiodegradable materials ispresently essentially limited to HDPE, MDPE, LDPE formulations.Addition to PE films of pro-oxidant agents, basically transitionmetal salts or complexes inducing photo and thermal oxidation,area widespread technique largely used in industry. The fine

    balance of antioxidant and pro-oxidant contents guarantees thatafter the preset period of service life, relatively fast abiotic oxida-tion begins. As a consequence, the material loses its mechanicalproperties and disintegrates into small fragments, providing ananswer to the problem of visual pollution by plastic litter that isconstantly in the center of public attention.

    More importantly, the oxidized fragments of PE films wereproven to be slowly biodegradable as reported in extensive reviewson the topic [1e3]. The biodegradability of oxidized PE films hasbeen assessed by two different approaches: complex media (soil,compost, sludge) with microbial consortia, or pure microbialstrains in controlled mineral medium.

    In complex media the biodegradability of PE containing pro-oxidant can be evaluated by a conventional approach, which eval-uates the mineralization percent based on CO2 formation. These

    * Corresponding author. CNRS, UMR 6296 F-63171 Aubire, France. Tel.: 33 47340 77 14; fax: 33 473 40 77 17.

    E-mail address: [email protected] (A.-M. Delort).

    Contents lists available at SciVerse ScienceDirect

    Polymer Degradation and Stability

    journal homepage: www.elsevier .com/locate /polydegstab

    0141-3910/$ e see front matter 2013 Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.polymdegradstab.2013.01.002

    Polymer Degradation and Stability 98 (2013) 875e884

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    studies weremainly performed by the groups of Chiellini [4e6] andJakubowicz [7, 8]. Usually the microbial population was not iden-tified, except in the recent study of Jakubowicz in 2011 [8]. Exten-sive information can be found in the review of Ammala [3].This conventional approach is the basis of some internationalstandards [3].

    An alternative approach is to use pure strains: the most fre-quently studied microbial strains belong to the bacterial generaRhodococcus, Pseudomonas, Arthrobacter, Bacillus, Streptomyces or tothe fungi genera Phanerochaete, Penicillium. Aspergillus [1,3]. Forpure cultures some authors assessed the biodegradability of theoxidized PE films by visualizing the microorganisms by microscopy[9e12] or by measuring the change of crystallinity of thematerial [13].

    In the approach developed by our group, the biodegradability ofoxidized particles is evaluated in controlled experimental condi-tions i.e. experiments with identified microbial strains in an aque-ous medium formulation with defined chemical compounds. Tomonitor both growth and development of microbial culturesadenosine triphosphate (ATP) and adenosine diphosphate (ADP)concentrations were assayed. ATP is the key molecule of all livingcell energetic metabolism. Its level reflects metabolic activity ofa culture. This test method is extremely sensitive and quantitative,and it allowed us to show that the cell populations in the presenceof the oxidized PE samples were in a better energetic state than thecontrol cultures that did not contain any polymer. It suggested thatthe cells were able to grow and to maintain their energetic statusover months [14,15]. This approach, using Rhodococcus rhodochrousATCC 29672 has been validated recently as the French AgreementAFNOR AC T 51-808 for the evaluation of oxidized PE materials [16].

    Although the biodegradability of PE films containing pro-oxidants is now well documented, to our knowledge no paperhas been published on the biodegradability of polypropylene filmscontaining pro-oxidants.

    The main goal of this work was to evaluate the biodegradabilityof polypropylene films containing different pro-oxidant additivesusing the same methodology based on ATP, ADP/ATP test (alreadysuccessfully applied to PE films); the idea is to check additive effi-ciency on both oxidation/fragmentation and biodegradation. Twotypes of PP matrices were examined: a statistically random PPcopolymer at low content of ethylene (PPs) and a block PPcopolymer at a low content of ethylene (PPb). Three different pro-oxidant additives PP1, PP2 and PP3 containing various metal salts(based on Mn/Fe, Co and Mn respectively) were tested and com-pared with additive free PPs and PPb used as reference samples.

    The objective of the study was to compare these various filmsto determine which factors are more favorable for theirbiodegradability.

    In addition to ATP content and ADP/ATP ratio measurements,cell viability was assayed.

    Complementary 1H NMR experiments were conducted on theincubation media, with and without cells, in order to monitor themetabolism of soluble compounds excreted from the oxidizedpolymers by R. rhodochrous cells.

    Moreover size exclusion chromatography (SEC) measurementshave been done on all the studied polymer samples initially, afterabiotic oxidation and after biodegradation.

    2. Materials and methods

    2.1. Tested materials

    Statistical PP copolymers at low content of ethylene (PPs) weresupplied by Total Petrochemicals, block PP copolymers at lowcontent of ethylene (PPb) were supplied by Lyondell Basell. Bothsamples were modified with an iron based photo-inducer supply-ing radicals through a photooxidoreduction process, and/or witha cobalt or manganese based thermo-inducer, organometallic type,catalyzing the primary hydroperoxide decomposition, and werelater extruded into transparent films with thickness ranging from51 to 63 mm (Table 1).

    To antagonize the prodegradant activity of both photo- andthermo-inductors during the first year of storage and use underindoor conditions, phenolic antioxidant was also added to theblends, extending the thermal-induction period at 60 C in the darkto over 400 h.

    Metal contents in each film were determined by inductivelycoupled plasma/atomic emission spectroscopy (ICP/AES) with anOriba/Jobin Yvon Ultima C instrument. To perform nebulization inthe plasma chamber, a few grams of PPb and PPs film were firstcalcined during 24 h with temperature programming going up to750 C (2 min). The resulting ash was dissolved into an aqueoussolution of nitric acid (34%). Calibration curves of each elementwere built with certified standard solutions (1000 ppm) and con-tents were measured by interpolation method. According to thevariability of industrial made samples and the experimental pro-tocol, a 20% relative error should be considered on the values givenin Table 1.

    2.2. Abiotic treatment

    The mechanism of photo-thermal oxidation of PP has beenextensively studied and is well understood [17e21]. The criticalphotoproducts reflecting the oxidation extent were identified ascarboxylic acid groups absorbing at 1713 cm1. Fig. 1 presents theFTIR spectra in a domain of specific interest which characterizes thechemical evolution of PPb film exposed in an artificial accelerateddevice AMETEK-SEPAP 12.24 [22] (l 290 nm, temperature of theexposed surface set at 60 1 C) during 0, 5, 10, 20, 40 and 60 h.Fig. 2 presents plots that illustrate the variations of absorbance at

    Table 1Sample description, abiotic degradation conditions and biodegradation results for PPs and PPb containing or not pro-oxidant additives.

    Additives Metal content ppm Samples Filmthickness(mm)

    Time of exposurein SEPAP 12.24 (h)

    Time of treatmentin aerated oven at60 C (h)

    Absorbance increaseat 1715 cm1

    Aspect Viabilitytest

    [ADP]/[ATP]

    Fe Mn Co

    PP1 126 85 PPs 56 30 840 0.95 2.0146 104 0 PPb 57 20 336 0.61 0.7

    PP2 12 17 149 PPs 55 20 600 1.40 e 4.36 4 157 PPb 58 20 360 2.01 e 17.5

    PP3 1 753 1 PPs 62 30 552 1.17 1.62 638 0 PPb 57 20 408 2.02 1.4

    None 0 0 0 PPs 63 45 936 0.34 3.80 0 0 PPb 51 45 2712 0.06 0 1.8

    Aspect: (0) not fragmented, () fragmented, () very fragmented.Viability test: () no growth in TS medium after 180 days, () growth in TS medium after 180 days.

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    1713 cm1 vs the exposure duration in the three additive-containing films PPbePP1, PPbePP2 and PPbePP3 and in theadditive-free PPb. As we can see in Fig. 2 for the additive freesample, 20e45 h of exposure corresponds to the photochemicalinduction period (total photo-transformation of phenolic antioxi-dants into inactive compounds).

    The films with additives were exposed into the SEPAP artificialaccelerated device for photothermal oxidation during 20e45 hfollowed by 336e840 h of treatment in an aerated oven at 60 C.Based on the activation energy determinedwhen thermo-oxidationproceeds (after the total consumption of phenolic antioxidants, i.e.the end of the total induction period), 300 h of thermo-oxidation at60 C were considered equivalent to 2e3 years of thermooxidationat room temperature in the dark (particles buried in the soil orparticles not exposed to sunlight). The thermal treatment of filmswith no additive was extended to 936e2712 h.

    In the phase of true thermo-oxidation beyond the inductionperiod, the apparent activation energy was determined to be100 10 kJ mol1. The control of the oxidation extent was carriedout by transmission FTIR spectrophotometry for the nonefragmented films and by micro FTIR spectrophotometry (FTIRspectrometer equipped with IR microscope) for the oxidized par-ticles obtained after fragmentation.

    After the exposure in the SEPAP 12.24, the samples were con-sidered sterile and treated in sterile way.

    2.3. Strains of bacteria

    R. rhodochrous ATCC 29672 was purchased from American TypeCulture Collection.

    2.4. Medium and conditions of the incubations

    Mineral medium utilized throughout the study had the fol-lowing composition: 3.8 g Na2HPO4.12H2O, 1.8 g KH2PO4, 0.02 gMgSO4.7H2O, 0.03 g Fe(NH4)2(SO4)2.6H2O, 0.01 g CaCl2.2H2O, 0.5 gNaCl, 0.3 g NH4Cl and 1 ml of trace element solution per liter. Thetrace element solution contained 0.20 g MnSO4, 0.029 g H3BO3,0.022 g ZnSO4.7H2O, 1.0 g Na2MoO4, traces of Co(NO3)2, and tracesof CuSO4 dissolved in 500 ml of water.

    Thimerosal at final concentration 0.01% (w/w) was added intoabiotic control cultivations as a growth inhibitor.

    In general incubations for SEC and NMR spectroscopy observa-tions were done in closed 100 ml glass flasks with 20 ml of media.Incubations for ATP and ADP concentration determination weredone in 4 ml closed glass vials with 0.4 ml of media. In both casesthe head-spaces were sufficiently large to provide the cultures withoxygen, and in addition the flasks and vials were opened weekly sothat the head-space air could be refreshed. Gastight sealing of thevessels was necessary to prevent water evaporation during the longincubation. The cultures were kept at 27 C with gentle shaking. PPsubstrate concentrations were about 4 mg/ml for flask cultures andabout 5 mg/ml for cultures in vials.

    2.5. FTIR measurements

    Small fragments with sections as small as 1000 mm2 were ana-lyzed by micro-FTIR spectrophotometry (Nexus, Thermo Nicolet)with Continuum microscope. The IR beam entering the spec-trophotometer was focused on a predefined 1000 mm2 zone of thesmall fragment and micro-FTIR spectrum was recorded in thetransmission mode with a resolution of 2 cm1 and absorbancedefined with a 0.001 accuracy. A PP absorption band controlledthe optical path.

    2.6. Size exclusion chromatography (SEC)

    A few milligrams of each sample were put into 4 ml vials, andthen the solvent 1,2,4-trichlorobenzene (from Sigma Aldrich, SaintLouis, Missouri, USA) was added. The solvent was stabilized with0.1% Irganox 1010 (from Ciba/BASF, Basel, Switzerland). A concen-tration of approximately 0.7 mg/ml was obtained. The vials wereplaced into a heating and shaking PLeSP 260VS plate (from Poly-mer Labs, Church Stretton, UK) at 150 C for about 2 h. Once thedissolution had taken place, the vials were transferred into thecarrousel of the chromatograph auto-sampler.

    Molecular weight distributions of polypropylene samples weredetermined with high temperature GPC system Waters 150-C plusat the Basell laboratories in Ferrara, equipped with a PL Olexiscolumn (13 mm internal pore diameter) and Waters Empowersoftware for data acquisition and treatment. The flow rate was1.0 ml/min and the working temperature was 150 C. The injectionvolume was 350 ml.

    Solutions of polystyrene molecular weight standards were usedfor calibration, according to the ISO 16014-2 method.

    2.7. ATP and ADP assays

    ATP Biomass Kit HS by Biothema (Sweden) was used for ATPdetermination. For each determination ATP from entire culture in4 ml vial was extracted. At least three vials were analyzed for eachtime point. ADPwas determined after transformation of ADP to ATPdirectly in the luminometer cuvette. Reaction mixture contained30 ml of the sample extract, 240 ml of diluent B from the ATP kit,amended with 20 mM KCl and 2 mM MgSO4, and 10 ml of solutioncontaining 0.1 M phosphoenolpyruvate and 5mg/ml protein kinase

    Fig. 1. Evolution of carbonyl domain of PPb after photo-oxidation in SEPAP 12.24.

    Fig. 2. Absorbance increase at 1713 cm1 vs exposure duration in SEPAP in PPb. PPbcontrol ( ), PPbePP1 ( ), PPbePP2 ( ), PPbePP3 ( ).

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    in 0.05 M Tris-acetate buffer pH 7.2 [23]. The mixture was incu-bated for 10 min at 37 C, equilibrated to the laboratory tempera-ture for 15 min, and then the light producing reaction was startedby addition of 60 ml ATP reagent HS (Biothema) reconstituted with2.5 ml of ATP free water. Blank experiment was done simulta-neously to correct results for the background signal of the reagents.

    2.8. NMR spectroscopy

    NMR spectra were recorded after filtration of the media through0.2 mm porosity filter.

    NMR samples were prepared as follows: Supernatants (540 ml)issued from biodegradation tests were supplemented with 60 ml ofa 2 mM solution of TSPd4 (sodium tetra deuterated trimethylsilylpropionate, Eurisotop) in D2O (Eurisotop). D2Owas used for lockingand shimming while TSPd4 constituted a reference for chemicalshifts (0 ppm) and quantification. 1H NMR spectra were recorded at400.13 MHz on a Bruker Avance 400 spectrometer at 21 C with5 mm-diameter tubes containing 600 ml of sample. 128 scans werecollected (90 pulse, 6.84 s acquisition time, 1.0 s relaxation delay,4789.272 Hz SW, 65536 data points). Water signal was eliminatedby pre-saturation. An exponential filter was applied before Fouriertransformation, and a baseline correction was performed on spec-tra before integration with Bruker software. Under these condi-tions, the limit of quantification is in the range of 0.05 mM.

    3. Results

    3.1. The abiotic treatment

    As previously observed, to obtain the samples required for theexperiments with micro-organisms, large surfaces of PPs and PPbfilms with additives were exposed for a short duration (20e45 h) inthe Ametek-Sepap 12.24 photo-aging unit and then thermo-oxidation was performed in aerated and ventilated oven at 60 Cfor 336e2712 h. After this period under the action of the pro-oxidants, light and heat, the material expected to be substantiallychemically transformed, and thus more susceptible to a microbialattack.

    At the end of the abiotic treatment, the absorbance increase at1713 cm1 determined by micro-FTIR spectroscopy varied from0.95 to 1.40 for PPs samples with additives, and from 0.61 to 2.02for PPb samples with additives. If we consider that, as established inthe previous study on PE films, the minimum absorbance at1713 cm1 to show some biodegradability is 3/100 (where wasthe film thickness in mm), this limit value could only be reached forPPbePP2 and PPbePP3 films (sample thicknesses as well otherexperimental results are shown in Table 1).

    3.1.1. Control of oxidation extent in fragmented and non fragmentedfilms

    As seen in Table 1 and as expected, films with pro-oxidant areeasily oxidized compared with additive-free PP. The low oxidationlevel obtained for pro-oxidant additive-free PPb probably indicatesthat photo-aging exposure was not long enough to convert allphenolic antioxidant into inactive species. Moreover, PP2 and PP3based films induce a similar degree of oxidation for the PPs and PPbmatrices examined.

    It is recalled that in PP, and most PE films [24e26] 50% loss ofmechanical properties is expected when acidic groups absorbanceincrease at 1713 cm1 and reaches approximately /1000 (0.1 for100 mm). Spontaneous fragmentation, i.e. fragmentation withoutthe application of significant mechanical stress, is expected whenabsorbance increase at 1713 cm1 and reaches/100, depending onthe oxidation mechanisms and on the film process conditions.

    3.1.2. SEC resultsBoth thermal and photochemical aging of PP result in abundant

    chain scissions because of the high level of associated hydroper-oxides generated in the first steps of the oxidation (exampleRefs. [17e22]). Significant amounts of volatile compounds such asmethanol, acetone and acetic acid have also been detected [20,21].

    The average molecular weights of the polymers samples beforeand after abiotic treatments are presented in Table 2. As expected,after the abiotic treatment, both the Mn and Mw values of theoxidized polymers are much lower than those of the initial poly-mers (Mn number average molecular weights values are moresensitive to low molecular weights). Clearly the presence of pro-oxidant additives (which catalyzes the decomposition of hydro-peroxides) enhances the breakage of PP chains under thermo andphoto-oxidation. The reduction of molecular weight of the differentpolymers exposed to abiotic treatment is consistent with the de-gree of oxidation measured (for example the loss in molecularweights is lower in the case of additive-free PP, and lower in PPsthan in PPb). It can be noticed that the reduction of Mw is higher inthe case of PP films compared to PE films [15].

    3.2. Evaluation of the biodegradability of PP films by R. rhodochrousstrain in mineral medium

    All the experiments described were carried out in a mediumcontaining only necessary growth supporting mineral ions andwhere the studied films were the only source of carbon and energypresent: thus, no objection can be raised that the microorganismswere profiting from any media component other than the polymerfilms. In previous studies [12,14e16] the bacterial strainR. rhodochrous ATCC 29672 was found to be most active in terms ofbio-film formation on PE surface and metabolic activity on PE filmsand hence it was again chosen for the evaluation of biodegrad-ability in this PPb and PPs study. Additionally, the Rhodococcusgenus is known to be ubiquitous in the environment, particularly insoils and oceans [27e29].

    3.2.1. PPs and PPb films containing PP1 pro-oxidant (Mn Fe)In order to follow the evolution of the microorganism culture

    and its metabolic activity, ATP content was determined at given

    Table 2Number, weight average molecular weights and polydispersity index of PP samplesbefore and after abiotic and biotic treatments.

    Polymer samples PPsa PPba

    Mn Mw Ip Mn Mw Ip

    P1 Initial 65,620 424,202 6.5 66,654 416,540 6.2After abiotic oxidation 2166 4128 1.9 3462 8782 2.5After biodegradation 2175 4061 1.9 2952 6523 2.2Ref without cells 2106 3990 1.9 3416 8148 2.4

    P2 Initial 60,677 451,625 7.4 6559 224,335 34.2After abiotic oxidation 1577 2265 1.4 2099 3620 1.7After biodegradation 1580 2315 1.5 2011 3445 1.7Ref without cells 1589 2296 1.4 2030 3451 1.7

    P3 Initial 54,141 454,253 8.4 59,649 503,128 8.4After abiotic oxidation 1908 3029 1.6 4611 27,724b 6.0After biodegradation 1943 3146 1.6 3016 6488 2.2Ref without cells 1876 3067 1.6 1380 3736 2.7

    None Initial 60,452 436,789 7.2 59,063 505,264 8.6After abiotic oxidation 4697 13,000 2.8 11,994 66,282 5.5After biodegradation 4813 13,135 2.7 16,572 106,021 6.4Ref without cells 4761 12,162 2.6 15,426 93,761 6.1

    a Basically, as we do not expect any influence of the making films process, initialMn, Mw, Ip values for PPs and PPb should be the same in the limits of the exper-imental error.

    b Too high value due to the unusual presence of high molecular weight (Mwdistribution 300,000e500,000).

    S. Fontanella et al. / Polymer Degradation and Stability 98 (2013) 875e884878

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    time intervals, and the results obtained for the two PP matricestested are presented in Fig. 3a.

    For PPb samples, ATP concentration increased quickly duringthe first 12 days of incubation, and then, after a slight decrease at 30days, it reached a maximum at 11 pmol mL1 (after 60 days). Sta-tionary phase was reached after 120 days, with an ATP concentra-tion of 6 pmol mL1.

    For PPs samples, an increase of ATP concentration was observedafter first 4 days of incubation up to 2.5 pmol mL1. After 120 daysa limited increase of the ATP concentration from 2.5 to 4 pmol mL1

    was observed. Finally, ATP concentration decreased until the end ofincubation (180 days), to reach the level of ATP concentrationobserved with the non additivated films used as reference. It wasobserved that cells incubated in the absence of polymer had muchlower amount of ATP (except for PPs films after 180 days of incu-bation). These results show that R. rhodochrous cells were able touse the oxidized polymer films as carbon source. After 12 days ofincubation, ATP content was 2.5 pmol mL1 with PPs films and9 pmol mL1 with PPb films, which is surprising, since PPb wasabiotically slightly less oxidized than PPs (0.61 against 0.95, seeTable 1). Moreover complementary DSC measurements show thatthe level of crystallinity of PPb (29%) is lower than that of PPs (49%).

    The metabolic state of the microbial population can be bettercharacterized by measuring ADP contents in addition to ATP. TheADP/ATP ratio can be considered as a measure of the energetic stateof the cells, since the lower this ratio, the higher the energetic stateof the cells is. We previously showed [14] that cultures ofR. rhodochrous in rich complete Trypcase Soja medium at the end ofexponential growth phase (and thereforewith a very high energeticstate) had a ratio ADP/ATP 0.25, whereas cultures in the absenceof polymer, with a low level of energy, had a ratio ADP/ATP of about6.0. In this study (Table 1, last column), the ADP/ATP ratios obtainedafter 180 days of incubation with PPs and PPb additivated filmswere 0.7 and 2.0 respectively, confirming a very good metabolicstate of the cells for incubation with PP1-PPb, and a lower one for

    PP1-PPs. To confirm the viability of the cells at the end of the in-cubation, the incubation media was spread on Petri dishes con-taining a rich medium (Tripcase Soja); in both types of films,bacteria could grow actively after 180 days (Table 1, viability test).

    The high metabolic state of the bacteria when incubated withthe different polymers could be explained by the use of substancesissued from the abiotic oxidation of the polymers and highly sol-uble in incubationmedium. To visualize these soluble substrates, 1HNMR spectra of the liquid phase of the incubation media in thepresence or absence of R. rhodochrous with the various polymerswere recorded at regular time intervals. Fig. 4 shows the resultsobtained in case of PPb films containing PP1 additive. Fig. 4a showsthe 1H NMR spectra collected after 0, 90 and 180 days of incubationin the absence of bacteria. The observed NMR signals correspond tosmall molecules eluted from the polymer into the incubation me-dium.Many signals are present between 0.8 and 3.7 ppm, and thesechemical shifts are consistent with substituted or unsubstituted CHand CH2 groups. Other signals are present between 7.21 and8.46 ppm, and they likely correspond to molecules containing C]Cbonds, carboxylic or carbonyl groups. Some specific signals can beeasily assigned as ethanol, resonating at d 3.67 and 1.20 ppm,acetic acid, resonating at d 1.90 ppm, and formic acid, resonatingat d 8.46 ppm. All these small molecules are consistent with ul-timate PP degradation [20,21]. In addition to these very small andmobile molecules presenting sharp NMR resonances, additionalbroad signals resonating between 1 and 2.5 ppm correspondprobably to aliphatic protons of oxidized PP oligomers, or of mol-ecules presenting limited mobility. These last signals are specific toPP polymers and were not detected while studying PE films [15].

    1H NMR spectra were also obtained from the incubation mediain the presence of PPs polymers containing PP1 (Fig. 5a, b), and theywere similar to those of Fig. 4a, b recorded in the presence of PPb.

    In the presence of the bacterial cells, the sharp NMR signalsdisappeared with time (Figs. 4b and 5b), while the broad signals didnot, probably because they correspond to PP oxidized-water

    a PP1 b PP2

    c PP3 d No additive

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    Fig. 3. Evolution of ATP content in Rhodochrous rhodochrous cultures with oxidized PPs (-) and PPb films ( ), and without polymer (B). (a), PPs and PPb films containing PP1additive; (b), PP2 additive; (c) PP3 additive; (d), no additive. All the ATP measurements were made in triplicate and standard deviations are plotted on the graphs. The standarddeviations of all the ATP values do not exceed 10% except in the case of Fig. 3b (PPs t 8, PPb t 90), and Fig. 3d (PPs t 12, PPb t 12) where the standard deviation is around 25%.

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    soluble oligomers that were not rapidly consumed by the cells. Onthe other hand, the bacterial cells did consume the short solublefragments formed from PPb and PPs containing PP1, so maintainingtheir energetic state, but they were unable to use the larger frag-ments, which led with time to an accumulation of these com-pounds in the medium. This observation might be related to thedecrease of ATP concentration with time.

    Finally SEC experiments were performed on polymers at the endof incubation with R. rhodochrous (Table 2). No significant differ-ence was observed for the P1-PPs and P1-PPb after abiotic oxida-tion or after biodegradation. Similar results had also been obtainedwith polyethylene films [15].

    3.2.2. PPs and PPb films containing PP2 pro-oxidant (Co)The change of the ATP content with time for R. rhodochrous cells

    incubated in presence of PPs and PPb films containing PP2 pro-oxidant was quite different from the results obtained with PP1and PP3 (see Sections 3.2.1 and 3.2.3) showing the toxic effect of Co

    (149e157 ppm) for microorganisms. As observed in Fig. 3b, the ATPcontent did not increase during the first days of incubation. A verylow ATP content both for PP2-PPs (3 pmol mL1) as well as for PP2-PPb (0 pmol mL1) was reached, this content being similar to theone observed for incubation without any polymer.

    This lack of energy was confirmed by the high ADP/ATP ratiovalue (4.3 and 17.5 respectively for PPs and PPb films, see Table 1)reflecting a low metabolic state for bacteria. Consequently, no cellpresent in these incubation media was able to grow on Petri dishes(Table 1), indicating the death of the cells at the end of theexperiment.

    Figs. 4c and 5c present the soluble substrates detected by 1HNMR in the incubation media containing PPb and PPs containingPP2, at 0, 90 and 180 days. The NMR fingerprints obtained aresimilar to those obtained with PPb and PPs containing PP1. This isconsistent with the efficiency of oxidation of both polymers due tothe presence of PP1 and PP2. However, although these solublesubstrates are fully available for cell metabolism, the bacteria do

    Fig. 4. 1H NMR spectra recorded on the liquid phase of the incubation of oxidized PPb films additivated with PP1, PP2 or PP3 pro-oxidants, in the presence or absence ofR. rhodochrous cells.

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    not use them as shown in Figs. 4d and 5d. This result confirms thatthe cobalt complex is toxic for the cells, whose metabolism iscompletely inhibited, and explains their low energetic state.

    SEC experiments did not reveal any change before and after thebiotic treatment (Table 2).

    3.2.3. PPs and PPb films containing PP3 pro-oxidant (Mn)The same methodology was used to test the biodegradability of

    PPs and PPb films containing PP3 pro-oxidant. The obtained results(Fig. 3c) were close to the one observed for PPs and PPb containingPP1, with a lower maximum content of ATP (8 against12 pmol mL1 and 3 against 5 pmol mL1 for PPb and PPsrespectively).

    More precise data were obtained by measuring the ADP/ATPratios at the end of the incubations (Table 1). The value of PP3-PPsfilm is higher than PP3-PPb, confirming the lower level of ATPobserved after 180 days for PP3-PPb. The metabolic state of thebacteria was slight better for PPb (ADP/ATP 1.4 for P3-PPb andADP/ATP 1.6 for P3-PPs). This result is also in agreement with thelow state of oxidation reached after abiotic oxidation of PP3-PPs.

    The high metabolic activity of the cells was confirmed by theirviability at the end of the experiments. Growth was observed in thePetri dishes for both PPs and PPb film incubations (Table 1).

    Figs. 4e and 5e present the soluble substrates detected by 1HNMR in the incubation media containing PPb and PPs modifiedwith PP3 after 0, 90 and 180 days. The NMR fingerprints obtainedare similar to those obtained with PPb and PPs containing PP1 andPP2. To maintain their high energetic state the bacteria successfullyuse soluble substrates as shown in Figs. 4f and 5f. However, asobserved for the cases of PPb and PPs containing the PP1 additive,the oligomers presenting broad resonances in 1H NMR spectrawerenotmetabolized efficiently by R. rhodochrous cells. This may explainthe decrease of ATP with time measured during the incubation forPPb and the relatively low ATP concentration measured during theincubation for PPb (Fig. 3c).

    SEC results on PP3-PPs samples were similar after abiotic oxi-dation and after biodegradation (Table 2). On the contrary, withPP3-PPb samples, a very important loss of weight was observed,with values of Mw 27724 after abiotic oxidation and Mw 6488after biodegradation. This difference may be explained by the

    Fig. 5. 1H NMR spectra recorded on the liquid phase of the incubation of oxidized PPs films additivated with PP1, PP2 or PP3 pro-oxidants, in the presence or absence ofR. rhodochrous cells.

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    presence of unusually high molecular weight species that modifythe weight distribution and have a stronger influence on Mw thanon Mn.

    3.2.4. PP films without pro-oxidantIn these experiments the tested films hadmatrices similar to the

    Sections 3.2.1, 3.2.2 and 3.2.3, namely PPs and PPb films, but did notcontain any pro-oxidant additives.

    Fig. 3d shows the evolution with time of the ATP content ofR. rhodochrous cells incubated with the PPs and PPb films. In allcases, the ATP concentration measured was quite low and close tothat observed in the absence of polymer. This result indicates thatthe energetic status of cells remains very low. ADP/ATP ratio weremeasured in spite of a very low content in ATP, and the obtainedvalues of 3.8 for the PPs films and of 1.8 for the PPb films confirmedthis information (Table 1). Bacteria present in the incubation mediacontaining PPs and PPb films was still alive and could grow in Petridishes (Table 1), showing that no toxic compound was present inthe tested PP matrices.

    1H NMR spectra recorded after 0, 90 and 180 days in the liquidphase of the incubation media in the presence of the differentadditive-free polymers with and without R. rhodochrous cells areshown in Fig. 6. It is clear that the bacterial cells are able tometabolize the small soluble substrates eluted from the PPb andPPs polymers, confirming that these polymers are not toxic for thebacteria. The total amount of soluble compounds is lower than inthe case of the polymers with additives (see for comparison Figs. 4and 5), and this result is consistent with the lower degree of oxi-dation of the polymers. This is due to the fact that the bacteria havea limited substrate available, their growth is limited, hence, theirATP content is lower. Interestingly the presence of broad signals is

    not visible in the NMR spectra (Fig. 6), which is consistent witha much lower content of oxidized PP oligomers in the absence ofpro-oxidant additives. As expected, SEC experiments did not showsignificant differences in Mw distribution between samples afterabiotic oxidation and after biodegradation (Table 2).

    4. Discussion and conclusions

    In the present work, we have studied the potential biodegrad-ability of polypropylene (PP) films containing pro-oxidant additivesby using an approach similar to the one we previously applied topolyethylene (PE) films [15]. Two propylene-ethylene copolymers(a statistic-random and a block copolymer) both with high pro-pylene contents were tested in the presence/absence of pro-oxidants based on Mn Fe, Mn and Co.

    Abiotic degradation (photo aging followed by thermal aging incontrolled laboratory conditions), representing one year outdoorweathering followed by a 2e3 years period in soil, was very effi-cient for the three PP formulations with additives, compared withthe additive-free ones (containing only phenolic antioxidant). Botha large increase of carbonyl band (mainly acidic groups) and largedecrease of average molecular weights were consistent with a highdegree of oxidation (carbonyl absorbance close of 3/100, with being the sample thickness in mm) and with the formation ofnumerous chain scissions. It is recalled [20,21] that under oxidizingconditions, PP is well known for generating (more easily than PE)numerous lowmolecular oxidized compounds which should createvery favorable conditions for further biodegradation. Some differ-ences were observed between the oxidation levels reached for thetwo polypropylenes (statistic and block), although exposure con-ditions and sample thicknesses were not exactly the same.

    Fig. 6. 1H NMR spectra recorded on the liquid phase of the incubation of oxidized PPb and PPs films without additives in the presence or absence of R. rhodochrous cells.

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    Moreover, no strong differences were observed in the oxidationlevel reached with the three families of pro-oxidants, in spite ofsignificant differences in metal contents (160e750 ppm).

    The comparison of results in the presence or absence of the Febased photoinductor (PP1) (PP2 and PP3) shows no significantdifferences in the efficiency of abiotic degradation, probablybecause the temperature of the SEPAP accelerated test (60 C) issufficiently high to induce thermal degradation in the presence ofPP2 and PP3 additives. However, in all cases, the photo aging step isnecessary in order to inhibit the effect of phenolic antioxidant.

    Biodegradation in the presence of suitable medium containingno other source of organic substrate and energy than the poly-propylene films was measured both by ATP titration and by ADP/ATP ratio (control of themetabolic state of the bacteria). In addition,the viability of bacteria at the end of the biotest was controlled byculture in Petri dishes. The metabolism of the cells was alsomonitored by 1H NMR spectroscopy performed on the incubationmedia. This technique allows visualizing the soluble substrates is-sued from the oxidized polymers that can be used as source ofcarbon and energy by the bacterial cells.

    PP films containing pro-oxidants based on Mn and Mn Fe givepositive results for the biotest (low ADP/ATP ratio, post-development in Petri dishes). This result was also shown in thecase of PE films containing the same type of metal complexes [15].1H NMR experiments have confirmed the biodegradation of smallsoluble compounds issued from the abiotic oxidation of the poly-mers. However the measured ATP contents tend to be lower in thecase of PP compared to PE polymers [15]. This observation can becorrelated with the accumulation in the incubation media of olig-omers that cannot be metabolized rapidly by the bacterial cells.These compounds are not toxic as shown by the good ADP/ATP ratiobut they are limiting the cell growthwith time. Finally, the behaviorof PP polymers is different from that of PE polymers. In the first stepa large amount of highly oxidizedmolecules of lowerMwwhich arevery volatile and easily biodegradable are produced in the case ofPP polymers (this phenomenon is much less pronounced with PEfilms). In the second step the biodegradation is slowed by thepresence of larger, non mobile molecules which are accumulatingwith time (this is not observed with PE polymers). It can be con-cluded that PP films containing Mn or Mn Fe based pro-oxidantsare biodegraded less efficiently than PE films with the same pro-oxidants. Another point concerns the importance of the polymermatrix. Obviously the PPb films are more easily biodegradable thanthe PPs, although the degree of oxidation is similar in both cases.The lower crystallinity index measured for PPb (33%), comparedwith the one measured for PPs (43%) can explain this, as microor-ganisms are more active in the amorphous phase. In addition, theethylene content measured by IR spectroscopy is slightly higher inPPb films (6%) than in PPs films (4, no development of bacteria in Petri dishes). This toxicitywas also observed in the case of PE polymers containing Co com-plexes [15]. In the case of use of cobalt derivatives, it will benecessary to keep a lower content, checking toxicity and checking if

    the oxidation level obtained under representative conditions issufficient (absorbance at 1713 cm1 3/100). He et al. [30] reportthat Co concentrations in natural soils vary from 5 to 40 ppm inChinese soils and from 10 to 40 ppm in World soils. The Co con-centrations of the PP samples tested in this work exceed thesevalues (Co content 150 ppm): it would be very interesting to testmaterials containing Co concentrations in the range of those foundin natural soils.

    Acknowledgments

    The authors acknowledge Martine Sancelme (ICCF laboratory,Clermont-Ferrand) for her technical help and advice for micro-biology experiments, Mr. Benbakkar, ICP/AES (LMV laboratory,Clermont-Ferrand) for performing the ICP-AESmeasurements, LucaRimessi (GPC laboratory, Basell Ferrara), Wolfgang Buderus (GPClaboratory, Basell Frankfurt), Erwin Schfer (GPC laboratory, BasellFrankfurt), Vinciane Jonnieaux (GPC laboratory, Total Petrochemi-cals Research Feluy) and Marc Van De Water (GPC laboratory, TotalPetrochemicals Research Feluy) for performing measurements. Wealso acknowledge Dr. Giorgio Nadalini (head of the GPC laboratory,Basell Ferrara), Dr. Barbara Gall (head of the GPC laboratory, BasellFrankfurt) and Jean Delrue (head of GPC laboratory, Total Petro-chemicals Research Feluy).

    The authors are also pleased to acknowledge Petrochemicals,Lyondell Basell, Association Neosac and ES3 group for the financialsupport.

    The study was also partially supported by an MSM 7088352101and a GACR 108/10/0200 grants.

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