DYNA

1. Introduction

Biodegradable thermoformed and flexible films can be made from cassava flours and starches by adding substances such as: fique fiber, gelatin, poly(butylene adipate-co-terephthalate), polylactic acid, glycerol, plasticizer, cellulose, pullulan, and natural extracts [1-12]. Nowadays, industries are trying to improve their food container products made from natural compounds [13], such as thermoformed and flexible films obtained from renewable sources (cassava). These constitute a new and environmentally friendly industrial alternative due to their fast and easy degradation and main purpose which is to replace the synthetic plastic that people use regularly [2,4,6,7,10,14,15]. These characteristics greatly benefit different environmental ecosystems.

A typical method for thermoformed manufacture is the compression molding technique, in which the material is placed into an open mold to which pressure and heat are applied. Single screw extrusion is used for the manufacture of biodegradable flexible films, and by turning the screw and applying heat, the material is pushed along and melted [5,12,16].

In developing countries, the accelerated process of industrialization combined with the use of intensive agricultural techniques and the inappropriate management of waste have led to an increase in the levels of substances considered harmful or toxic to living beings, such as heavy metals. These metals can cause serious damage and can enter the human body from emissions in the environment, through contact with industrial fallout as well as agricultural activities such as preparing pesticides, contamination from chemical fertilizers and irrigating with inadequate quality water [17-19]. Materials in contact with foods can cause heavy metal migration, this being a negative interaction between packaging and food. Additionally, heavy metals are not biodegradable, they remain in the environment and accumulate in living organisms over time because they are not metabolized [17,20-23].

Contamination with heavy metals may be produced during the manufacture of biodegradable polymers due to contamination of the raw material used for their processing (cassava flour and starch, fique fiber, polylactic acid, glycerine, etc.) or cross-contamination during manufacture, for example, during petroleum and non-petroleum activities, from pots used for cooking and storage, during drying techniques for moisture content reduction processes, and from utensils or contaminated water [18,24-26]. For this reason, a tracking process during manufacturing is necessary to determine the presence of toxic heavy metals in the film, for the purpose of safeguarding health by limiting exposure [20,27]. Among these metals, lead (Pb) is considered an unsafe and toxic heavy metal that can cause serious health hazards, affecting the cardiovascular, nervous and genitourinary systems, the biosynthesis of hemoglobin and long-term exposure may cause anemia [17,18] and the pathological change of organs. Lead accumulates not only in individual organisms, but also through the use of biodegradable polymers in the packing of dry foods and other degradable products, as it enters the food chain. Excessive Pb accumulation in humans may even cause cancer because lead is considered a “possible human carcinogen” [18,26,28]. Additionally, certain plants can accumulate heavy metals in their tissues and this increases in plants that are grown in zones with soil contamination [22].

Presently, there are no studies reporting the presence of Pb in thermoformed and biodegradable flexible films; there are, however, reports on the presence of Pb in the cortex of cassava tubers being higher than the values recorded in soils [25].

The safety of the materials that come into contact with food is evaluated by the quantity of substances that migrate from the biopolymer into food and fulfil the requirements in the legislation on foods. In the current regulation NTC 4096 [29], a maximum level of Pb of 1 mg/Kg is allowed for plasticizers. Additionally, the World Health Organization (WHO), estimates that the total lead intake from air and water in adults is in the range of 4.0 - 10 µg/day, respectively [26].

Numerous techniques have been used to determine the level of metals in different samples, such as flame atomic absorption spectrometry (F-AAS), and continuous flow micro-extraction combined with graphite furnace-atomic absorption spectrometry (GF-AAS). GF-AAS is a good alternative for the determination of trace elements such as lead due to its high sensitivity, with limits of detection in the order of µg/L [28,30-32]. In this study, sample preparation involving acid digestion was employed.

The goal of the present study is the quantification of lead in samples of thermoformed and biodegradable flexible films and their raw materials, using the standardized technique of GF-AAS after a sample acid digestion treatment. The reason for this is that the thermoformed and flexible films can be used as food containers, and so quality and safety must be ensured in their processing and manufacturing as well as handling and storage, as this can contribute to the intake of lead by the consumer.

2. Materials and methods

3. Results and discussion

Thermoformed samples showed moisture values (after drying at 70ºC in a furnace for 4.5 h) of between 3.71 to 5.80% (RSD lower than 3.4%). Biodegradable flexible films presented higher values of moisture (after drying at 70ºC in a furnace for 4 h) at 7.81-10.35%, with RSD lower than 1.96%, these levels being similar to those reported by Cha et al., 2001 [40].

The graphite furnace heating program was optimized through pyrolysis and atomization curves. Fig. 1A shows the change in absorbance at several pyrolysis temperatures and exhibits a maximum absorbance when the pyrolysis temperature was 700ºC, with an acceptable coefficient of variation of 1.16%. Fig. 1B illustrates absorbance against different temperatures at which atomization was performed; the coefficients of variation ranged from 4.44 to 22.95%. Maximum absorbance was observed when the atomization temperature was 1500°C (Fig. 1B), with an acceptable coefficient of variation of 3.88%.

Figure 1: Optimization of graphite furnace temperature for Pb: (A) Pyrolysis; (B) Atomization

Source: The authors

In order to establish the performance of the method [35] for the accurate quantification of lead in thermoformed and biodegradable flexible films using GF-AAS, the following statistical quality parameters were determined: linear range, precision, sensitivity, LOD and LOQ and accuracy.

To verify the linearity, a calibration curve was built for aqueous standard solutions of lead with concentrations of 2.0-9.0 and 12.0 µg/L. Fig. 2 shows values for the maximum absorbance at 216.9 nm vs concentration of lead. This calibration curve exhibits a linear behavior for different concentration solutions of lead ranging from 2.0 to 12.0 µg/L with relative standard deviations of 1.41-3.71%; the Pearson correlation coefficient was determined to be 0.995.

Figure 2: Calibration curve of lead obtained for the linear range (N= 4).

Source: The authors

Table 1 represents the concentration (in µg/L) of lead for each standard solution prepared and analyzed for intermediate precision and repeatability as follows: a) The calibration curves were analyzed to determine the intermediate precision and were statistically evaluated using the Shapiro-Wilk and Levene tests. They did not show significant differences among them and the dispersion of the data was less than 4.7% (coefficient of variation, CV). This indicates that the proposed method for the determination of lead using GF-AAS provides a good intermediate precision. The average linear equation was y=0.028 (±0.000) x + 0.006 (±0.001) with r² of 0.9989. The Pearson correlation coefficient was determined to be 0.9997.

Table 1: Average absorbance (± standard deviation) of calibration curve for precision: Intermediate precision and Repeatability

Note: Intermediate precision (8 calibration curves) and Repeatability (5 calibration curves

Source: The authors

In all cases, the standard deviation was less than 0.005 µg/L of lead. b) The Shapiro-Wilk and Levene tests showed that each concentration of lead in the five calibration curves was from the same population, presenting a dispersion with a variation less than 4.4%. This indicates that this method has a good repeatability, with an average linear equation of y=0.030 (±0.000) x + 0.019 (±0.002) with r² of 0.9989. The Pearson correlation coefficient was determined to be 0.0095. The precision is adequate according to the acceptance criteria set by INMETRO [41].

The sensitivity of the method was calculated from the calibration curve’s steepest slope with concentrations of lead ranging from 2.0 to 7.0 µg/L. These values correspond to 0.028 and 0.031 for intermediate precision and repeatability, respectively. This method provides an increased sensitivity when the analysis is carried out on the same day, indicating that the proposed method for sample preparation is adequate. Therefore, the sensitivity of the method was 0.030.

The limit of detection (LOD) was 0.618 µg/L (12.4 µg/Kg) and corresponds to the minimum amount of lead derived from the lowest analytical signal that can be detected with reasonable certainly. The limit of quantification (LOQ) was 1.853 µg/L (37.1 µg/Kg) and represents the minimum concentration that can be measured with precision and accuracy. The LOD and LOQ are adequate for the quality control of bio-polymers.

Certified bio-polymers were unavailable, so the validity of the method was evaluated by addition-recovery tests. The accuracy of the method (Table 2) takes into account sample preparation, digestion treatment and analysis of samples using GF-AAS. The percent recovery of lead ranged from 94.79 to 98.52% for the MPER 183 thermoformed material and from 102.75 to 106.50% for the CM 7138-7 pristine flexible film. Comparing percent recoveries of lead in the thermoformed and flexible films with 100% recovery (using a t student test), it was found that some significant differences can be attributed to random errors such as preparation of solutions and analyte loss during the digestion treatment. These values (94.8 and 106.5%) can be accepted as good percent recovery according to the criteria set by INMETRO [41] and the European Commission [42]; therefore, this method exhibits excellent accuracy for the determination of lead in these samples. The values of the standardization parameters studied demonstrated quality assurance when using GF-AAS for the determination of lead in thermoformed and flexible films.

Table 2: Percentages of lead recovery (µg/L) (N=4) in the analysis of thermoformed (MPER 183) and biodegradable flexible films (CM 7138-7). Volume 20 mL.

Source: The authors

The statistical analysis of a solution of 3.0 µg/L of lead, a thermoformed (CM 4574-7) and a pristine flexible film (CM 7138-7), showed high stability over seven consecutive days. For the standard solution, the average concentration was 2.76 µg/L ± 0.06, while the coefficients of variation for the thermoformed and flexible film were less than 4.97%.

Once the analytical technique of GF-AAS was standardized for the determination of lead in bio-polymers, the implementation was performed. Table 3 shows the concentrations of lead in µg/Kg that were quantified in seven thermoformed samples. The concentrations typically ranged from 39.70 to 63.99 µg/Kg. In NATAIMA 31 and HMC 1 samples, traces of lead were found to be under the LOQ (1.853 µg/L), with concentrations of less than 37.2 µg/Kg. This variation in lead may be attributed to the quality of the raw materials used in the elaboration of bio-polymers.

Table 3: Average concentration (± standard deviation) of lead (N = 3) in samples of biodegradable thermoformed material

Source: The authors

Other authors found mercury, arsenic and selenium present in these thermoformed samples. Del Castillo et al., 2012 [43] found mercury ranging from undetectable to 1343.4 µg/Kg, Alvira et al., 2012 [44] found arsenic with concentrations of less than the LOQ (39.2 µg/Kg) and Rada-Mendoza et al., 2014 [45] detected selenium ranging from undetectable to 1240.0 µg/Kg.

As shown in Table 4, for all flexible films in the pristine and hydrolyzed forms, the detected amounts of lead were below the LOQ. This corresponds to a concentration of a trace level of lead of between 37.14 and 37.18 µg/Kg. In these flexible films, mercury was reported to be undetectable to 127.3 µg/Kg [43], arsenic was undetected [44] and selenium was reported to be undetectable to 122.0 µg/Kg [45]. In thermoformed and biodegradable flexible films, the values of heavy metals accumulated were higher for mercury than for Se, Pb and As, suggesting that absorption and bioaccumulation depend upon the availability of metals.

Table 4: Average concentration (± standard deviation) of lead (N = 3) in samples of biodegradable flexible films

Source: The authors

In Europe, Asia, the US and Brazil, there is no legislation for regulating the content of heavy metals in bio-polymers. The Colombian regulation NTC-4096 [29] establishes 1.0 mg/Kg as the maximum level of lead allowed in DOA and DOP plasticizers used in the manufacture of plastics that are in contact with food which indicates that lead concentrations in the range of 39.70 to 63.99 µg/Kg found in the thermoformed material(Table 3), and in lower concentrations than the LOQ found in flexible films (Table 4), are all below the limit established by the regulation. Therefore, it is expected that there is no incidence of this metal in the final product.

However, it is necessary to investigate the source of the lead in these samples. In this case, lead was detected in raw materials (cassava flour and starch and fique fiber) used in both the thermoformed and flexible films. Table 5 shows the concentration in µg/Kg of lead in different samples of the raw materials used in the production of biodegradable thermoformed film. Of these, MBRA 383, CM 523-7 and fique fiber had concentrations in the range of 39.56 to 46.21 µg/Kg of lead, with the fique fiber having the highest concentration of lead. From this, it is evident that this source contributes the most lead to the thermoformed film.

Table 5: Average concentration (± standard deviation) of lead (N = 3) in samples of raw materials used in the production of biodegradable thermoformed material

The concentration of lead in Fique Fiber was 46.21 µg/kg (± 1.94)

Source: The authors

Lead found in the flexible films made from cassava starch (Table 6), the raw material used for producing this bio-polymer, was below the LOQ.

Table 6: Average concentration (± standard deviation) of lead (N = 3) in samples of raw materials used in the production of biodegradable flexible films.

Source: The authors

With the results shown in Tables 3 and 4, it can be inferred that lead found in thermoformed material comes principally from cassava flour and fique fiber, and for flexible films, this heavy metal derives from the cassava starch. The conclusion here is that traces of lead detected in the studied bio-polymers were low, indicating non absorption and contamination from anthropogenic sources, meaning that these materials are not toxic and may be employed as food wrappers and containers, conserving food [1,2,46] and extending its shelf-life, without potential health risks to the consumer.

Lead uptake from soil is highest in plants that are grown in areas with high clay content [22]. It is very important to take precautions when obtaining and handling raw materials, as was mentioned above, because lead is released into the environment and can contaminate the flour, starch and fique fiber through exposure to car fumes, emissions from industrial processes, soils containing heavy metals, and the irrigation of vegetables with contaminated water [21,22,25,38].

Additionally, is important to note that the best way to reduce lead contamination is through the control of raw materials and a thorough understanding of the manufacturing process.

4. Conclusions

This study demonstrates the immense potential of using bio/polymers in packaging and food conservation, and how these materials add value to agricultural activity and help to reduce non/biodegradable plastics in the environment. It is inferred that lead found in these samples comes from the flour, starch and fique fiber at cultivation and shows that lead contamination may be not the result of the manufacturing process.