The biodegradation tests were carried out in a natural field soil and a soil mix (ASTM D 5988-12). The soil mix included equal parts of sand, horse manure, and garden soil, consistent with an approach previously used [7,8]. The soil mix was held for 2 months at 20 ± 3 °C with humidity maintained at 70% by watering and stirring. The film samples were placed vertically in the soil (30 cm depth) and carried out for 20 months with periodical inspections. Differences in appearance, mass, chemical composition, and degradation of the composites were analyzed.

Biodegradation was assessed in three different environments including:

Prepared soil mix under laboratory conditions (constant temperature (23 ± 3 °C) and constant humidity (60%)).

Prepared soil mix under ambient field environmental conditions in Moscow region, (Kubinka, Moscow region, Russia). A natural soil layer was removed (depth of 30 cm) and replaced with the prepared soil mix. There was no barrier between natural soil and the prepared soil mix.

Natural soil (not prepared soil mix) under ambient field environmental conditions in an experimental field at the Universidad Nacional (Heredia, Costa Rica) [30].

Moscow region has continental climate with expressed differences in temperature between summers and winters. The average annual temperature is +6 °C, but in February it is −12 °C and in July +20 °C. About 650 mm of atmospheric precipitation falls in Moscow region per year.

Heredia has a tropical savanna climate. The differences in the temperature between seasons are not clearly expressed (annual average temperature is +22 °C), but the precipitation has significant differences between dry winter and humid summer. The annual atmospheric precipitation is about 2000 mm.

The prepared soil mix (used in the laboratory conditions and field in Moscow region) and the natural Costa Rican soil were analyzed for chemical properties (Table 1). The soil mix was characterized by greater organic matter (138 g kg−1) and nitrate (728 mg kg−1) compared to the Costa Rican field soil (total organic C = 33.4 g kg−1; nitrate = 2 mg kg−1), and pH was similar between them (prepared soil mix = 6.5; Costa Rica soil = 6.2).

Chemical properties of soil analyzed from Russia and Costa Rica.

Optical microscopy (Carl Zeiss Axio Imager Z2M with AxioVision ver. 4.7.1, magnification 50× and 200× in transmitted and reflected light) was employed to find differences between initial samples and the same samples after soil immersion for 20 months.

Mechanical properties of materials before and after burial were investigated according to ISO 527–1:2012 via universal testing machine Devotrans DVT GP UG (Istanbul, Turkey). The samples were stretched to failure at a temperature of (22 ± 2) °C, and crosshead velocity was 0.25 mm/min. The dimensions of the samples: 70 mm × 10 mm × 0.15 mm, effective length—40 mm. The data for 7 samples were averaged. Differences among means were determined using 95% confidence intervals.

Chemical properties of materials before and after burial were studied on a FT-IR spectrometer Perkin Elmer Spectrum 100 (Waltham, MA, USA) at a temperature of (22 ± 2) °C in the range of wave numbers 4600 ≤ ν ≤ 650 cm−1 by a method of frustrated total internal reflection. Intensities of several peaks referring to polymer oxidation process (i.e., carbonyl group), microbiological colonization, and polymer degradation were determined, and its dynamics during biodegradation tests were detected.

All laboratory tests were carried out using scientific equipment at the Center of Shared Usage «New Materials and Technologies» at Emanuel Institute of Biochemical Physics and Joint Research Center at Plekhanov Russian University of Economics.

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