*3.5.1 Standard stability*

Cadmium standards are prepared (0.8 μg/L) and kept refrigerated while they are analyzed for ten consecutive days. A one-way ANOVA analysis (calculated F < tabulated F, 1,675 < 2,420) indicates that there are no significant differences between the average absorbances during the days analyzed, and therefore this standard can be prepared and stored at a temperature of 4 °C for analysis for up to 10 days [54, 70].

## *3.5.2 Stability of metals in digested samples*

To evaluate the stability of cadmium, a thermoformed sample is subjected to digestion via a dilution of 5 to 100 mL, which is refrigerated and read on the equipment for ten consecutive days. The samples for flexible film were not read, as in our case, they were not detectable.

By applying a Shapiro–Wilk statistical analysis, we were able to conclude that the data come from a normal distribution, since the calculated W is smaller than the tabulated W and the data maintain a homogeneous distribution, since the calculated Levene statistic is smaller than the tabulated one [70].

A one-way ANOVA was applied to determine any significant differences in the average absorbances between the different days analyzed. The results show that cadmium was stable in the thermoformed sample analyzed in our study (CM 4574–7) as the calculated F < tabulated F (1.87 < 2,420) [70].

According to the results obtained, cadmium is stable in the thermoformed digested sample. Thus, these samples can be stored at 4 °C and analyzed on consecutive days [70], allowing the laboratory to establish its analysis times and organize the relevant protocols.

#### **3.6 Determination of cadmium by atomic absorption spectrometry**

### *3.6.1 Thermoformed and flexible films*

To quantify cadmium in the samples, the graphite furnace atomic absorption spectrometer was used under the instrumental conditions shown in **Table 4**. The metal was measured in triplicate in the thermoformed and flexible film samples, as well as in the source materials (cassava flour, cassava starch and fique fiber), in order to determine whether the metal in the samples derives from the raw material used for manufacture.

Cd concentrations in the thermoformed products typically ranged between 4.2 mg/kg and 17.9 mg/kg, which could be the result of the quality of the raw materials used to process the biopolymers. Sample concentrations were significantly different from each other, given that when applying one-way ANOVA, the calculated F value was greater than tabulated F (1507.861 >3.501) at 95% confidence.

Cd was not detected in flexible films, meaning that no contamination was present.

The cadmium concentrations found in the thermoformed products were lower than those established by NTC-40961 (1 mg Cd/kg). Thus, these biopolymers can be used for these purposes, but care needs to be taken as this metal can bioaccumulate, causing serious environmental problems in the long term.


#### **Table 4.**

*Instrumental conditions in the GF-AAS for cadmium determination.*

<sup>1</sup> The standard that regulates the maximum level of heavy metals in samples that are going to be in contact with food.

#### *3.6.2 Raw material*

Once the Cd concentration in the samples of thermoformed products had been determined, we proceeded to analyze the main raw material —cassava flour and fique fiber— in order to identify whether the metal found in the thermoformed products originated in the processing materials.

The results show that the flours presented Cd concentrations of between 3.5–18.2 mg/kg, while Cd concentration in the fique fiber was 7.2 mg/kg, meaning that it contributes the most Cd to the thermoformed film.

Given that the fique fiber and flour are agricultural products, the presence of cadmium in these samples may be due to the fact that this metal is used in herbicides used to control weeds such as Linuron or Dinuron. It can also be present in soil from municipal waste and the incineration of plastic materials, in fertilizers (phosphorous and nitrogenous fertilizers), pesticides and fungicides such as copper oxychloride and carbofurans among others, used to cultivate cassava [4, 10, 42, 72].
