The impact of fillers on the biodegradation of plasticized PVC-materials exposed to soil microorganisms

. Due to the growing use of polymer materials, in particular in construction sphere, the issue of their recycling at the end of their service life is particularly relevant. Being the base for flooring, plasticized PVC-compositions, are utilized by soil microbial flora, as the plasticizer EDOS has a positive effect on the microbiocenosis of the soil. It was shown that for aqueous extracts of samples of PVC-compositions, there is no toxicity for two test objects - infusoria Paramecium caudatum and crustacean Daphnia magna . Filling plasticized PVC-materials with natural wollastonite Miwoll 10-97 inhibits the processes of soil respiration, and rice husk ash and synthetic wollastonite on its base, contribute to its preservation. At the same time, the use of these fillers reduces the degree of utilization of plasticized PVC-compositions by soil microbial flora, since the plasticizer EDOS is adsorbed on their surface and becomes less accessible for soil microorganisms. This was proved by infrared spectroscopy and occurs due to the formation of hydrogen bonds between EDOS and rice husk ash.


Introduction
The use of plasticizers and natural fillers in a mixture with a synthetic polymer is one of possible directions for development of polymer materials that retain their operational properties over a long period in the process of consumption, and then are subjected to physical, chemical and biological transformations under the environmental impact [1]. When exposed to soil microorganisms, these materials experience accelerated biodegradation of a natural filler and/or a plasticizer, resulting in destruction of a polymer matrix [2].
In this connection, the study of the impact of the fillers based on rice husk ash on soil microbiocenosis, which contribute to improving the performance characteristics of PVC flooring, is of practical and scientific interest [3].
The following fillers were used in the study: ash obtained by burning rice husk at 500°C (RHA); synthetic wollastonite (SW) obtained by solid-phase method on the base of RHA at 900ºC for 3 hours; calcium oxide from limestone was used as the second component [5,6], and natural wollastonite Miwoll 10-97 -calcium metasilicate CaSiO3 (Technical specifications 577-006-40705684-2003), with a characteristic length to diameter ratio of grains 5:1.
The interaction of PVC with the plasticizer EDOS was studied by IR spectroscopy on a Nicolet iN10 spectrometer (Thermo Scientific, USA), in the spectral range of 675-4000 cm -1 . Spectra were obtained on a germanium crystal in ATR mode by accumulating 128 scans with a resolution of 4 cm -1 at room temperature. The spectra were processed (transformed into optical density mode with automatic baseline correction) by means of Omnic 9 Software Complex (Thermo Scientific, USA).
To establish the level of toxicity of filled, plasticized PVC after the end of products' service life, the method of biotesting of aqueous extracts of samples on infusoria and crustaceans was used [7].
Toxicity of polymer materials is determined by the effect on test organisms of aqueous extracts obtained after mixing prepared samples of PVC-compositions with distilled water in the ratio of 1:10, mechanical shaking or stirring, settling for 7 days at room temperature and filtration. The technique involving the use crustaceans is based on determining the survival rate of daphnia when exposed to substances contained in the aqueous extract compared to the reference sample. Acute toxicity is determined by the survival rate of daphnia within 48 hours of exposure [7].
To conduct the experiment with Paramecium caudatum test objects, the commonly used method was used. The method is based on the cultivation of tests objects for 1 hour in the presence of an aqueous polymer extract and counting the number of dead (immobilized) individuals.
The second method for determining the toxicity of PVC-compositions of different compositions comprised the assessment of dehydrogenase activity [8] of bacterial culture Bacillus subtillus when cultured in the presence of 1% solution of triphenyl tetrazolium chloride (TTC), glucose solution and phosphate buffer and adding 1 ml of water extract of PVC composition. In the case of the control sample, 1 ml of the nutrient medium was added to the experimental system. The mixtures were shaken and incubated for 24 hours at 28˚C, then centrifuged at 4000 revolutions per minute with the addition of 95% ethyl alcohol followed by colorimetric analysis of the supernatant at 480 nm. The experiments were repeated three times. To control the activity of the bacterial culture, dehydrogenase activity was additionally evaluated in the absence of an aqueous extract of the PVC sample.
To assess the toxicity, the relative activity (Arel) of Bacillus subtillus culture was calculated by the formula: • 100 (1) where Fca -the average concentration of formazan in the sample when assessing the activity of the culture; Frca -the averaged concentration of formazan in the reference sample when assessing the activity of the culture; Fsample -the averaged concentration of formazan in the sample with PVC extraction; Frsample -the averaged concentration of formazan in the reference sample.
To analyze the bioaccessibility of PVC-materials and components of polymer compositions for soil microorganisms, the modified Sturm method based on the incubation of soil samples in closed vessels in the presence of samples was used. The carbon dioxide released during the experiment was adsorbed by alkali and quantified by titration [9]. In the reference test, a soil suspension containing no polymer was investigated.
Gray forest soil, as one of the most common soil types in the Republic of Tatarstan, sampled in the Zelenodolsky municipal district, was used in the experimental studies. Phosphorus content in the soil in recalculation to P2O5 was on average 255 mg/kg soil, potassium content in recalculation to K2O -140 mg/kg soil.

Results and discussions
It was found that during the biotesting of the aqueous extract of PVC basic composition, no immobilization of infusoria Paramecium caudatum was observed within 1 hour at 22-24˚С. The percentage of daphnias' death in 48 hours in the tested solution was 5% compared to the reference sample, for which no death of individuals was noted.
Similar results were obtained for PVC-compositions containing the studied silicate fillers. Thus, the toxicity of aqueous extracts of PVC-samples of all studied compositions on Paramecium caudatum and Daphnia magna is absent.
At the second stage of the research, the dehydrogenase activity of Bacillus subtillus was analyzed (Table 1) in the presence of aqueous extracts of polymeric PVC-materials and plasticizer EDOS used in their compositions. EDOS, with colloidal solubility in water [10], was found to have a positive effect on the enzyme activity of the test culture under study. For this plasticizer, the maximum increase in dehydrogenase activity (DHA) of the culture Bacillus subtillus was recorded. Effective biodegradation of EDOS is associated with the presence of complex-ether bonds and alcohol groups in its composition, which are easily biodegraded [11].
For aqueous extracts of PVC-samples modified with synthetic and natural wollastonite, an increase in DHA, as compared to the basic formulation (Table 1) was noted. This effect is higher in the case of synthetic wollastonite.
Rice husk ash increases the dehydrogenase activity of the studied cculture even more than calcium silicates. This can be attributed to the presence of an organic phase in its composition [12].
Thus, plasticizer and cellulose and lignin [13] included in the organic part of rice husk ash are used by microorganisms as an organic substrate. Moreover, EDOS is mostly utilized by soil microflora.
Based on the obtained results, we can conclude that the plasticized PVC-compositions have no negative impact on both the enzyme systems of microorganisms and the activity of protozoa.
The results of studies implemented by the Sturm method of the effect of fillers and polymeric materials filled with them on the respirometric activity of soil microbiocenosis are presented in Tables 2 and 3. The table 2 shows that natural wollastonite Miwoll 10-97 inhibits the processes of soil respiration, as evidenced by zero values of carbon dioxide concentration.
It was found that synthetic wollastonite and RHA contribute to the preservation of soil respiration and this effect is stronger for rice husk ash ( Table 2). In the presence of polymer material's sample, the biological activity of soil microbiota is manifested in an increase in the intensity of CO2 release (table 3), which indicates the availability of the polymer for the microbial community, causing its biodegradation.
It should be noted that the high values of respirometric activity of the soil obtained in the experiment with PVC of the basic formulation indicate the possibility of microbiological utilization of the plasticizer contained in its composition.
Less activity of soil microbiocenosis is observed during the process of incubation of polymer samples filled with natural and synthetic wollastonite in soil.
All investigated disperse fillers decrease the respirometric activity of soil in comparison with the basic composition. We assumed that this may be associated with the adsorption of plasticizer EDOS on the surface of calcium oxides and silicates, which reduces its accessibility to microorganisms.
To confirm this assumption, we studied the IR spectra of dispersed fillers (in the example of RHA) before (red spectrum) and after (blue spectrum) their incubation in saturated vapors of the plasticizer EDOS at 70 ˚C 48 hours (Fig. 1).
The comparison of spectra has shown that having conducted the sorption processes, on the filler's surface characteristic bands of valence (3600-3100 cm -1 ) and strain (1596 cm -1 ) vibrations of hydroxyl groups as well as valence (3000-2800 cm -1 ) and strain (1420-1330 cm -1 ) vibrations of the hydrocarbon skeleton appeared.
At the same time, in the infrared spectrum of the initial RHA a broad, but low-intensity band of valent vibrations of OH-groups was determined. It is associated with the presence of silanol groups in its composition [14]. In the process of plasticizer sorption, the number of hydroxyl groups in the IR spectrum of RHA increased.
For comparison, by means of mathematical aliasing of IR spectra of initial RHA and the plasticizer EDOS, an IR spectrum of their mixture (green in Fig. 1) illustrating the result of the sorption process was obtained.
In the range of 3000-2800 and 1420-1330 cm -1 , the initial spectrum correlates well with the spectrum of RHA after sorption of the plasticizer. However, an additional band has appeared at 1596 cm -1 . According to [15], the appearance of this band is often associated with the formation of hydrogen bonds between hydroxyl groups. Changes in the intensity and broadening of the 795 cm -1 band, characteristic of strain vibrations of Si-O, together with the appearance of the 1596 cm -1 band and higher intensity of bands in the region of 3600-3100 cm -1 , in the spectrum of ash after sorption with EDOS, compared to the original spectrum, suggests that the plasticizer adsorbed on the ash surface.
In this case, hydrogen bonds are formed between the hydroxyl groups of the EDOS side substituents and the hydroxyl-containing groups of the RHA.
Natural and synthetic wollastonite produce the similar results. The IR spectra of those after sorption of the plasticizer, compared to the original, indicate the formation of stable hydrogen bonds between these fillers and EDOS.
Soil respirometric activity is slightly higher for PVC-materials filled with ash than for wollastonite, which indicates greater biodegradability of materials exposed to soil microorganisms.
This correlates with the results of toxicity studies on Bacillus subtillus culture of PVC aqueous extract samples filled with rice husk ash (Table 1).
When analyzing the obtained data, it should be taken into account that the plasticizer EDOS in the PVC-compositions has a positive effect on soil microbiocenosis during decomposition. This follows from the results of the study of its aqueous extracts on the enzymatic activity (Table 1).
Therefore, plasticized PVC-compositions are effectively utilized by soil microflora. This indicates the possibility of their accelerated biodegradation when they get to landfills after the end of their service life.

Conclusions
In the experiments, three different methods were used to evaluate the effect of fillers on the biodegradation of PVC-materials by soil microorganisms.
It was shown that there is no toxic impact of aqueous extracts of samples of PVCcompositions of all studied compositions on Paramecium caudatum and Daphnia magna, and they do not have a negative effect on the enzyme systems of microorganisms and on the activity of protozoa.
It was found that the filler Miwoll 10-97 inhibits the processes of soil respiration, and synthetic wollastonite and ash of rice husk contribute to its preservation.
The presence of plasticizer EDOS in PVC-compositions has a positive effect on soil microbiocenosis. This correlates with the data on its effect on the enzymatic activity of aqueous extracts.
Due to the high content of silica in rice husk ash, it can be used as an effective adsorbent of many types of pollutants from aqueous solutions.
The application of the investigated silicate fillers reduces the degree of utilization of plasticized PVC compositions by soil microflora, since the plasticizer EDOS is adsorbed on their surface and becomes less accessible for microorganisms. This was proved by IRspectroscopy on the example of formation of hydrogen bonds between EDOS and rice husk ash.