Introduction

The diversity of polymers and the different factors that control metallic nanoparticles’ properties result in many technical applications for nanocomposites. The current nanocomposites present a relatively easy technology of preparation and application with low cost to produce new materials in substitution of available products [1, 2].

With the emergence of pathogenic bacterial strains resistant to one or more antibiotics, the medicine needs new disinfection systems. Metallic nanoparticle systems are one of the most promising systems for fulfilling this function [3,4,5,6].

Antimicrobial materials are not only used in medical applications, such as catheters [7] and prostheses [8], but also in food packaging [9, 10] and in paint industry [11] among others. One must be aware of the different public sectors where there is the possibility of proliferation of bacteria and fungi, such as hospitals, schools, offices, etc., where infectious diseases can be transmitted through surfaces [12]. In our days, plastics more than ever constitute these surfaces. Among the polyolefins, LDPE has important properties: low (weight, temperature toughness, density, moisture absorption), great mechanical properties, good processability, high stiffness in molded parts, tear-resistance besides being recyclable [13,14,15,16,17,18]. For these properties, LDPE has a wide field of applications in which the biocidal activity can be added based on many examples of polymers cited in the literature. For example, the Polyvinyl butyral (PVB) with different types of nanoparticles showed antibacterial properties. It gained mechanical resistance compared to conventional polymers [19].

Polyacrylonitrile nanocomposite (PAN) films containing silver nanoparticles developed as multifunctional film matrix with has biocidal activity [20]. In parallel, direct surfaces treatment of as for PP (amphiphilic polymer) of PP-g-PEG sheets having embedded metal nanoparticles were synthesized with antibacterial efficiency [6]. Investigation of it on biomaterials, as the literature reports, showed the efficacy in preventing infection of a newly developed PP-g-PEG and Ag-PP-g-PEG polymer-coated shunt catheters. Especially Ag-PP-g-PEG polymer coating was effective to prevent implant-related infections in the central nervous system in rats [21].

Another example is the study of PET-g-MAA fibers coated with AgNPs that had an antimicrobial effect on both bacterial species (E. coli and S. aureus). The synthesized material was obtained easily and cheaply with surface-bound Ag + ions converted to AgNPs (silver nanoparticles) without using any chemical reducing agents and had no cytotoxicity effects on L929 fibroblast cells [22].

Silver nanoparticles coated with poly(styrene-g-soybean oil) graft copolymers using soy oil macroperoxide as a nanosilver carrier. The polyolefin became an efficient bactericide, in addition to producing nanocomposites with fluorescence properties, making them promising materials [11]. PET fabrics knew for not preventing the growth of microorganisms, when PET surfaces were firstly modified by MAA grafting and HMDA attaching, and after modified by silk Sirina-AgNPs immobilization. It was found that the PET S-AgNPs had antimicrobial activity on both gram-negative and gram-positive bacteria [23]. The number of applications for AgNPs increases exponentially, including nanosilver in association with other metallic nanoparticles [24]. With this, the search for new antimicrobial and antiviral materials becomes timely and eminet, including in the fight against Covid 19 [25, 26].

Silver and titanium nanoparticles are known for their biomedical properties in common uses [27]. TiO2 is an antimicrobial agent effective in bacterial cells, fungi, algae, and viruses. Its photodegradation is due to the high oxidizing action and hydroxyl radicals (OH*) species in oxidation systems. The photocatalytic property of TiO2 is manifested under ultraviolet light. The doping of Ag nanoparticles in TiO2 not only increases the photocatalytic activity of titania, but also promotes properties of antibacterial activity for the material in the absence of ultraviolet light [28].

The improved photocatalytic activity of Ag/TiO2 thin film for disinfection can be attributed to the higher production rate of ROS reactive species, compared to TiO2 [29].

The addition of clay-like montmorillonite strengthens the polymeric compound favoring the nucleation of silver nanoparticles. The parallel stacked layered clay structure facilitates the controlled diffusion of silver nanoparticles [30]. The creation of a biocidal material with slow release of silver species can prolong the biocidal activity [31, 32].

At the cellular level, many mechanisms of AgNPs toxicity were reported. This includes the generation of reactive oxidative species (ROS), DNA damage, and cytokine induction during in vitro studies [33]. On the surfaces, the dissolution of AgNPs releases silver ions that trigger the inactivation of respiratory enzymes, interrupt electron transport, and alter the permeability of the bacterial membrane and viral protein wrap, which causes the death of cells [29].

In contrast to the benefits, it can increase the disposal of nanoparticles into the environment. Consequently, it is necessary to be very careful with the application of nanomaterials, as the beneficial properties derived from them can be lost due to inadequate use in biological systems [27] in which cytotoxicity testing [34] and ecotoxicity testing [35] are of great importance.

In this context, the literature also describes the interest in ecotoxicology tests for the evaluation of nanocomposites. The sea urchin Echinometra lucunter, which populates all the Brazilian coast, is used in ecotoxicology tests due to the sensitivity of the early embryonary stages for numerous laboratories of marine coast. For pollutants present in seawater, even in very low concentrations, bioassays routinely use sea urchin larvae embryos to assess water quality [36,37,38].

For these senses, in present work on LDPE nanocomposites proposed the investigation of ecotoxicity of the material in urchin larvae. The innovative aspect of our study is to improve antimicrobial properties to the surface of LDPE nanocomposites by gamma irradiation, as an effective process for totally reducing metallic ions simultaneously to sterilization without chemicals. Evaluation of nanocomposites activity evolving the irradiation for efficient silver nanoparticles on TiO2 carrier reduced the formation of agglomerates, consequently, improved bactericidal efficiency.

Material and methods

Materials

LDPE with a melt flow index—MFI (190 ºC, 2.16 kg) of 0.27 g/10 min and density of 0.92 gcm−3 from Braskem, LLDPE with MFI (190 ºC, 2.16 kg) of 0.80 g/10 min and density 0.92 gcm−3 in the form of pellets provided by Braskem. The silver nitrate (AgNO3) and Polyvinylpyrrolidone (PVP) K30 powder, average molecular weight = 51,000 gmol−1 were supplied by Synth. Titanium (IV) isopropoxide, (C12H28O4Ti) 97%, formula weight = 284.22 gmol−1 and density 0.96 gcm−3 was supplied by Sigma-Aldrich. Antioxidant Irganox® B215 ED from BASF and the clay Cloisite-20A were provided by BYK Additives.

Synthesis of nanoparticles

Synthesis of silver nanoparticles and titanium: PVP solution was prepared in deionized water heated at 60 ºC, followed by AgNO3 solubilization with constant agitation. Subsequently, the solution was followed by addition of titanium (IV) isopropoxide and MMT clay. Sonication was carried out for 30 min to reduce and stabilize the silver under titanium nanoparticles. The equipment used for disruption of agglomerates of silver and titanium nanoparticles in PVP solution was the Eco-Sonics ultrasound, with a working frequency of 60 kHz and output with a maximum intensity of 350 Watts.

Preparation of nanocomposites films

The polyethylene composites (LDPE/LLDPE 90:10 wt%) were processed in a twin-screw extruder (Haake, Model Rheomex PTW 16/25), with the following processing conditions: the temperature profile (feed to die) was 160 to 190 °C, with a speed of 100 rpm. From five different nanocomposites of polyethylene LDPE/LLDPE films, PENC1 to PENC3 were obtained under those conditions containing nanosilver-titanium/MMT in different ratios. The films, PENC4 and PENC5 were, finally, irradiated under air at room temperature, in a 60Co gamma source, at a dose rate of 5 kGy h−1. The total dose absorbed was 25 kGy (monitored by a Harwell Red Perspex 4034 dosimeter), Fig. 1.

Fig.1
figure 1

source irradiation process of nanocomposites

Experimental procedure from synthesis to gamma

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According to the literature, the polyethylene montmorillonite nanocomposites prepared with the addition of powder from sonication process showed improvement of the dispersion compared with non-treated ones [39]. The use of ultrasound is one important tool in nanoparticles research as reported by [38] that confirmed increasing of the surface content of Ag on TiO2 [40].

The formulation of polyethylene nanocomposite (PENC) is shown in Table 1.

Table 1 Formulation of nanocomposites PENC (wt%)
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Simplified Scheme 1 of the preparation process of polyethylene nanocomposite represents the polymer matrix of the polyethylene being irradiated with gamma rays. The distribution of the clay is preferably exfoliated and the AgNPs@TiO2 are anchored in LDPE/LLDPE matrix.

Scheme 1
scheme 1

Model illustrates stabilization and distribution of AgNPs@TiO2 and nanoclay-platelet in film of polyethylene nanocomposite (PENC)

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Characterization of nanocomposites

Ecotoxicity

Specimens of Echinometra lucunter were collected at Santos Bay, SP/Brazil. After the collection, the sea urchins were transported in cool boxes to the UNISANTA—Ecotoxicology Laboratory and placed in 500 L tanks with aerated seawater before testing. The gametes were obtained by KCl (0.5 M) injection according to standard [41]. As soon as the organisms started releasing the gametes, they were targeted for the fertilization procedure. These experiments are internationally accepted as suitable for toxicity, hazard, or risk assessments [36, 37, 41], and have been employed to better understand negative effects on reproduction of an organism broadly recommended for water bioassays.

The short-term chronic toxicity tests with Echinometra lucunter were carried out according to the procedure described by USEPA with adaptations [41], sea urchin embryos are exposed to a specimen of the PENC = Polyethylene-Nanocomposite-Films samples. After the exposure period, the number of larvae that showed normal and anomalous development is evaluated.

The dilution water used in the experiments, both for obtaining gametes and for preparing controls and elutriate samples, was prepared by diluting sea salt (Redsea®) in distilled water, in concentrations of 30 to 36 ppm, followed by filtration in cellulose membrane with 0.22 µm porosity. The experiments were conducted in test tubes containing 10 mL of the test solution.

For each treatment, four replicates were used, where about 300 eggs were inserted, and the set was kept for 36 to 42 h (until observes the Initial formation of the embryos) in an incubator chamber with a constant temperature of (26 ± 1) ºC and a photoperiod of 16 h/8 h (light–dark). After the exposure period, the test was ended by adding of 0.5 mL of borax-buffered formaldehyde to all replicates.

Subsequently, the content of each replica was observed using a Sedgewick-Rafter microscope. The first 100 embryos were counted and their degree of development was assessed. Embryos that reached a well-developed pluteus larva stage were considered normal, while those that presented morphological changes and/or developmental delay were considered affected. The test was considered valid when ≥ 80% of larvae are successfully developed in the control.

Statistical treatment

The results of the chronic toxicity tests were submitted to the variance analysis method (ANOVA). First, the data were analyzed for normality and homogeneity of variance by the Chi-square and Bartlett´s methods, respectively. Subsequently, the data were submitted to the analysis of variance method (ANOVA—p < 0.05) using the Dunnett method.

Antibacterial activity

The cell suspension concentration of the inoculum used for tests was (2.4 × 105 CFUmL−1) for Staphylococcus aureus (ATCC 6538P) and (2.7 × 105 CFUmL−1) for Escherichia coli (ATCC 8739) for each tested step. The procedure was performed separately for each microorganism. Control and test surfaces are inoculated with microorganisms, in triplicate, and then the microbial inoculum is covered with a thin, sterile film. Covering the inoculum spreads it, prevents it from evaporating, and ensures close contact with the antimicrobial surface. The samples of the PENCs films, (40 × 40) mm2, were deposited in a sterile Petri dish inoculated on the surface of 50 mL of a suspension of each organism. All samples were incubated for 24 h at 37 ºC [42].

FESEM-EDX

The specimens were fixed in a sample holder and covered with Au/Pd for test. The acceleration rating used was 10 kV and the equipment was FESEM, JEOL, JSM-6701F-Japan.

SEM–EDX

The samples were fixed in a sample holder and covered with Au/Pd and analyzed using a Hitachi TM3000, coupled with a Bruker Quantax 70 for the collection of energy-dispersive X-ray spectroscopic information. The EDS analysis was carried out at 15 kV and the acquisition period was 90 s.

DSC

The thermal properties of the nanocomposite films were verified in the Mettler Toledo DSC 822e equipment. Samples weighing 10 to 15 mg were placed into an aluminum pan. The samples were heated from − 20 ºC to 220 ºC and kept at 220 ºC for 5 min. After this isothermal process, the samples were cooled to − 20 ºC and then heated to 220 ºC. The heating rate consists of 10 ºC min−1 in a nitrogen atmosphere. The melting temperature (Tm) and degree of crystallinity (Xc) were calculated with the following Eq. (1):

$$ {{Xc}}\; = \;P\; \times \; \frac{{{\triangle{Hf}}}}{\triangle {H0}}\; \times \;100 $$
(1)

The value of ΔHf is melting enthalpy of the sample, ΔH0 was taken for 100% crystalline LDPE, which is assumed to be 280 kJkg−1 [43, 44], and P was the fraction content of LDPE in the sample.

TG

TG curves were acquired in the equipment TGA/SDTA 851—Mettler-Toledo. Samples of 10 to 15 mg were placed into an alumina pan, under a nitrogen atmosphere of 50 mLmin−1, and heated from 25 up to 600 °C, at heating rate of 10°Cmin−1.

SERS

Raman analysis was performed using the Raman laser 785 nm, InPhotonics equipment.

Mechanical properties

The mechanical properties of tensile stress and elongation at rupture followed ASTM-D882-18 [45]. The tests were performed on an Instron 5567 machine at a rate of 50 mm min−1 speed and equipped with pneumatic claws. Dimensions of the test specimens were (70 × 10) mm and 0.08 µm thick. Five specimens were tested for the mechanical properties of each composite formulation.

Results and discussion

Ecotoxicity

Research on the toxicity of NPs can be investigated from two points of view: health and environmental hazard [46]. Morphological properties of the AgNPs influence their biocidal activity, for example: size, shape, and surface coating. It is important to note that same property also influences the cito and ecotoxicity of the AgNPs. These properties of AgNPs, combined with their high production volume, are a stimulus for research on the impact and harmfulness of silver nanoparticles [47, 48].

There is concern about increasing the use of manufactured nanomaterials, for example, TiO2 and AgNPs, and their possible release into the environment via wastewater treatment plants. It poses a potential threat for aquatic organisms, and its effect on the ecosystem is of great importance for environmental risk assessment. Figure 2 shows the larval development of Echinometra lucunter exposed to different polyethylene nanocomposite films.

Fig. 2
figure 2

Results (mean and standard deviation) of Echinometra lucunter embriolarval development for different PENC films; PE0 = control

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Results of the embryo larval bioassay using E. lucunter are shown in Fig. 2. In all nanocomposite polyethylene films containing silver and titanium nanoparticles, satisfactory larval development was observed in the ecotoxicity tests, above the control value sample (PE0). With those results, it was verified that the normal development of E. lucunter larvae was not affected by silver and titanium nanoparticles of the PENC nanocomposite films.

Antibacterial activity

The interesting result illustrated in Fig. 3 is that all nanocomposite films were effective in reducing E. coli and S. aureus.

Fig. 3
figure 3

Antimicrobial activity of PENC films. Obs.: PE0 = Control

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The nanocomposite polyethylene films showed significant results against the bacteria S. aureus and E. coli. The percentage of surviving microorganisms decreased significantly in the PENC1 sample with 5.83% for S. aureus and 8.89% for E. coli, whereas for the PENC2 sample is increased to 21.25% for S. aureus and 26.30% for E. coli, also indicative of a good result. For the PENC3 film, the results were more expressive (S. aureus = 5.42% and E. coli = 7.78%) suggests that increasing silver to 1 wt% has one significant effect on activity against microorganisms.

The PENC4 and 5 films originated, respectively, from PENC1 and PENC3 irradiated at a dose of 25 kGy. The results showed a remarkable efficiency of irradiation in the reduction of ions to nanosilver with consequence of total bacteria death, reaching values very close to zero.

Table 2 reports the antibacterial reduction rate of samples composed of PENC films, using samples in dimensions of (40 × 40) mm2, according to standard test JIS Z 2801 [42].

Table 2 Antibacterial reduction rate, R(%) value, of polyethylene nanocomposite
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Regarding PENC3 compounds with 1 wt% of silver particles and titanium, is remarkable a significant antimicrobial activity against S. aureus with antimicrobial activity values of 1.27 and 1.11 for E. coli, respectively (see Table 2). In addition, the results of the sample irradiated with 25 kGy, PENC4, showed a very significant antimicrobial activity for S. aureus, 3.04, and E. coli, 2.94, and also for the PENC5 sample S. aureus 2.85 and E. coli 2.71.

The results of logarithmic reduction, are very expressive for the irradiated samples PENC4 and PENC5 that presented values 2.85 for S. aureus and 2.71 for E. coli.

The high visible light photocatalytic activity of the Ag/TiO2/MMT is ascribed owing to the increase in surface-active centers and the localized surface plasmon effect of the silver nanoparticles. Under visible light irradiation, the electrons generated on the silver nanoparticles from the plasmon excitation are transferred to the neighboring TiO2 particles, which function as the photocatalytic centers [49].

The release of Ag+ present in the polymer surface depends on the diffusion of water molecules coming from the bacteria medium into the surface of particles [50]. Highly non-polar polymers such as allows the diffusion of those water molecules on interface through holes or micron-scale defects [51]. Gamma irradiation induces ionization and excitation of the water molecules, generating radiolytic molecular and radical species such as solvated electrons, hydroxyl radicals, and hydrogen atoms. The solvated electrons and hydrogen atoms reduce the metal ions to metal atoms, which coalesce to form more nanoparticles [52].

Polymeric nanocomposites irradiated with gamma radiation acquire more comprehensive biocidal properties compared to non-irradiated ones. The nanocomposite is effective against tested pathogenic microorganisms due to its high surface area and reduction in aggregates size due to the gamma rays [53] in surface of the carrier TiO2/MMT to perform a more stable bactericide activity.

FESEM-EDX analysis

It was possible to identify the presence of silver and titanium nanoparticles by EDX analysis. The distribution of silver and titanium in the films surfaces was homogeneous. The micrograph showed in Fig. 4, in nanometric scale indicates the presence of silver and titanium nanoparticles in spherical format and with dimensions from 53.2 to 91.1 nm.

Fig. 4
figure 4

FESEM micrographs of film surface: A PENC1 (scale = 100 nm), B PENC1 (scale = 5 µm) and C EDX Spectrum of PENC1

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Figure 4 shows the FESEM micrographs of the nanocomposite film PENC1.

The deposition of AgNPs on TiO2 enhances the photocatalytic activity of titania and also imparts biocidal properties to the modified material, according to the literature [54]. In other work, the PP-AgNPs showed antibacterial activity toward E. coli and S. aureus via the release of oxygen reactive species and Ag ion diffusion mechanism; thus, the inhibition rates enhanced, potentializes the biocidal effect [55], corroborating with our research work.

SEM analysis

Figures 5 and 6 show the SEM micrographs of the nanocomposite film PENC1 and PENC2.

Fig. 5
figure 5

SEM electron images and SEM–EDX elemental signal maps for PENC1: A PENC1 on the surface, (scale = 4 µm), B SEM–EDX showed blue dots referring to titanium and green dots to the clusters of silver nanoparticles; C EDX and D Semi-quantitative analysis of Ti and Ag

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Fig. 6
figure 6

SEM electron images and SEM–EDX elemental signal maps for PENC2: A PENC2 on surface, (scale = 20 µm), B SEM–EDX—mapping image of showed orange dots referring silicon, blue dots referring titanium and green dots are clusters of silver nanoparticles; C EDX, and D Semi-quantitative analysis (elemental contents) of Si, Ti, and Ag

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Energy-dispersive X-ray spectroscopy (EDX) was used to analyze the elementary constituents of the PENC thin films. Figure 5 displays the spectrum of PENC films obtained by elemental microprobe analysis of EDX. The results showed that titanium at a concentration of 1.27% and silver at 0.17% were the principal elements of PENC films.

EDX analysis also confirmed the presence of elemental silver at a concentration of 0.13%, titanium 0.46%, and silicon 0.16% as measured on thin film surfaces. The metal elements have a uniform distribution which favors the biocidal interaction by contact with bacteria.

Thermal characterization (DSC and TG)

The DSC results concerning on the Tm2 and XC results are presented in Table 3 and refers to the DSC curves, Fig. 7.

Table 3 DSC data of PENC samples during the second run of melting, melt temperature (Tm2), and degree of crystallinity (XC)
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Fig. 7
figure 7

DSC curves of second heating of the polyethylene nanocomposites films—PENCs

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The incorporation of the AgNPs and AgNPs-MMT favored the increase of crystallinity (%) in all PENC samples. This fact suggests that the AgNPs impart a high efficiency to the heterogeneous nucleation of LDPE in addition to samples with AgNPs-MMT as in the PENC3 sample increased the degree of crystallinity (%), compared with the control, of 35.8 to 48.2% (PENC3), indicative of nucleation and possibly release of AgNPs slowly onto the film surface. Benhacine et al. [56] corroborate with that idea and the silver incorporation in MMT plays an important role in the crystallinity degree of PENC that increases when added Ag–MMT. Another important aspect reported by Ghosh et al. [57] is that crystallinity affects the permeability of the nanocomposite films, the permeability decreases with increasing of the crystallinity.

The TG results indicated decomposition (Tonset) of the samples, Table 4.

Table 4 Onset temperature (Tonset) for PENC composites after processing
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From the information obtained in Table 4, it can be seen that the temperature of degradation (Tonset) increased with the addition of inorganic particles such as Ag, TiO2, and MMT, demonstrating a better thermal stability for the nanocomposites.

For PENC4 and PENC5, both irradiated at 25 kGy is verified that radiation contributes to increasing of the thermal stability probable to efficiently reduction of silver avoiding aggregates of nanoparticles Ag/TiO2, and a slight formation of crosslink which corroborates with higher values of Tonset.

Raman spectroscopy

Analyzing the Raman spectrum of polyethylene nanocomposite films PENC, Fig. 8, some results were significant and corroborated with the presence of nanoparticles in the films.

Fig. 8
figure 8

Raman spectrum of polyethylene nanocomposite films PENC

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The broad band with peaks at 385 and 670 cm−1 are associated with two-phonon states in a TiO2 crystal. The 822 cm−1 lines are associated with the Eu(LO)-vibration TiO2 crystal band [58]. Raman spectra measured the Si–Si phonon line position by deconvoluting the spectra into crystalline 520 cm−1 [59].

Studies conducted by identified Ag2O Raman located at 1068 cm−1 [60], 882 cm−1 Si–O stretching [61], and 1436 cm−1 band is associated with amino group [62]. The spectra in 1300 cm−1 CH2-Si wagging modes [63], 968 and 1164 cm−1 ν(SiOSi) [64]. The peak appearing at 659 cm−1 is attributed to the presence of PVP stabilized silver nanoparticles [65]. The broad and intense peak at 1589 cm−1 was attributed to the enhancement of Raman lines by carbon polymeric segments absorbed on silver oxide [66].

Mechanical Properties

Figure 9 shows the recorded tensile strength and tensile strain (elongation) results of the films.

Fig. 9
figure 9

The stress–strain curves of PENC films

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The PE0 and PENC4 samples showed very close tensile stress at yield values, 20 and 17 MPa, but tensile strain at break with little variation 127 and 115%. The other PENC1, 2, 3, and 5 samples showed very significant values of tensile strain at break ranging from 218 to 352%.

Conclusions

These polyethylene nanocomposites films having silver nanoparticles and titanium dioxide synthesized showed antibacterial efficiency. All samples showed expressive values of microorganisms death by contact, using the Escherichia coli and Staphylococcus aureus, mainly in the irradiated samples PENC4 and PENC5. Polyethylene films (LDPE/LLDPE) using Ag/TiO2/MMT nanocomposites obtained via extrusion have biocidal properties and can be promising materials in medical applications, food packaging to prevent bacterial contamination. We highlighted that the films presented very significant elongation values concerning pure LDPE. It is essential to consider that the silver nanoparticles and titanium dioxide used to manufacture of polyethylene films did not interfere as contaminants in the ecotoxicity tests with Echinometry lucunter since the larval development was normal.

The nanocomposites were effective against tested pathogenic microorganisms due to the efficiency of irradiation in the reduction of ions to nanosilver in the TiO2/MMT surface with the consequence of total bacteria death, reaching values close to zero.

Reduction in aggregates size due to the gamma irradiation of the surface films performed a more stable bactericide activity. The obtained materials can be used in various applications with antimicrobial functions.