FTIR
The FTIR spectra of dried initial PAAG gel, initial amber, and PAAG -gels with amber are given in Fig. 1. The most informative peaks of all studied materials are in the range of 1800–800 cm− 1. The spectrum of PAAG shows two bands at 3436 and 2924 cm− 1, which correspond with the N–H stretching vibration of the NH2 group and C–H stretching vibrations. The bands at 1645 and 1465 cm− 1 are attributed to the stretching of the C = O group in amide and CH2 scissoring, respectively (Nakanishi 1962). In the FTIR spectrum of initial amber, the wide shoulder of the 1160 cm− 1 peak stretching to 1260 cm− 1 (known as “Baltic shoulder”) corresponds to the high content of succinic acid and other succinate compounds. The intense peak at ~ 1700 cm− 1 is characteristic of the C = O group of carboxylic acids. The bands at 1445 and 1375 cm− 1 are attributed to C–H symmetric and asymmetric stretching vibrations. The 887 cm− 1 band could be assigned to the out-of-plane aromatic C–H bending.(Mänd et al. 2018; Karolina et al. 2022)
In the spectra of polyacrylamide hydrogels with amber, the bands of amide I (1647 cm− 1) and amide II (1605 cm− 1) of acrylamide have the highest intensity. The bands in the range of 3000–3500 cm− 1 correspond with symmetric and asymmetric vibrations of amino groups ν(NH) of polyacrylamide. The maximum at 2938 cm− 1 is attributed to stretching vibrations of the methylene group. The intense maximum ν(C=O) at 1647 cm− 1 (Amide I) corresponds with the amide fragment, which overlaps with the bending vibration maximum δ(NH2) at 1605 cm− 1 (Amide II) with the formation of a broadened doublet. In the "fingerprint" region, a doublet at 1450–1410 cm− 1 caused by bending vibrations of the CH group, as well as a broadened band at ν(CN) = 1350 cm− 1 (Amide III) were noted.(Nakanishi 1962; Boldeskul et al. 2009)
Acute toxicity
Synthetic hydrogels, created by polymerization of highly toxic monomers such as acrylamide, contain unreacted residues that should be removed before using them for medical and biological purposes(Gertsiuk and Samchenko 2007). Acute toxicity of the studied compounds of different washes (from the 1th to the 7th) was determined based on the mortality rate (%) of D. magna. Data on the survival of individuals in each sample during 24 and 48 h of the exposure are presented in Fig. 2, Table 3.
Table 3
Overall risk assessment of toxic compounds present in washing waters on the viability of biomarkers of plant (legume) and animal (D. magna) origin
Washing / AA solution | Conc. AA, mmol/l | Death toll, % |
PAAG | PAAG-A1 | PAAG-A2 |
daphnia | peas / chickpeas | daphnia | peas / chickpeas | daphnia | peas / chickpeas |
AA solution | 1.4 | 100* | 80/80 | 100* | 80/80 | 100* | 80/90 |
AA solution | 0.14 | 100* | 40/70 | 100* | 40/80 | 100* | 50/70 |
AA solution | 0.014 | 100* | 40/70 | 100* | 40/80 | 100* | 50/70 |
1 | 0.00125 | 100* | 10/20 | 100* | 10/20 | 100* | 10/20 |
2 | 0.00096 | 100** | 10 | 28.6** | 10 | 28.6** | 10 |
3 | 0.00054 | 42.8** | 10 | 0 | 10 | 0 | 10 |
4 | 0.00007 | 0 | 0 | 0 | 0 | 0 | 0 |
5 | 0.00003 | 0 | 0 | 0 | 0 | 0 | 0 |
6 | 0.00002 | 0 | 0 | 0 | 0 | 0 | 0 |
7 | 0.00001 | 0 | 0 | 0 | 0 | 0 | 0 |
Control (DV) | | 0 | 0 | 0 | 0 | 0 | 0 |
* - died on the first day of the experiment |
** - died on the second day of the experiment |
The performed experiments indicated that after the first wash of all tested samples, complete death of organisms (100%) was already recorded during 24 and 48 h of the experiment. However, the mortality decreased by 28.6% and 57.2% during 48 h in the second washing water for PAAG-A2 and PAAG-A1, respectively, (Fig. 2). In the third washing water, the mortality of D. magna individuals in the PAAG-A1 and PAAG-A2 samples was absent. No cases of mortality were recorded during 24 and 48 h of the experiment. Based on these results, D. magna was considered as an organism of high sensitivity to washing water composition, which can be used for diagnosis and risk assessment of the hydrogels and unreacted compounds.
The results of biotesting of the toxicity of washing water by the Nelyubov method are depicted in Fig. 3. It was found that 1–3 washes of hydrogels, in which the concentration of acrylamide was from 0.00054 mol/dm3 to 0.00125 mol/dm3 (according to the results of measurements using a UV spectrometer SPECORD M40), were the most toxic for all tested legumes. For peas, 10% of seeds was dead, while for chickpeas, 20%. The results of both biotesting experiments are presented in Table 3. They indicated that the washing water from the 4th wash is safe for both D. magna and legumes. The hydrogels, washed in this way, can be safely used in practice. Tucson et al. also washed PAAG for three days (changing the water every 24 hours), after which the gel was safe for further use as substrates for the study of bacteria (Tuson et al. 2012).
Rheological properties
The structural and mechanical characteristics of hydrogel composites largely determine the application possibilities of these systems. They are related to bioavailability and release of biologically active components from hydrogel materials. Gels are structured systems that demonstrate the structural and mechanical properties of both liquids and solids. Hydrogel, as a dispersed system, acquires the properties of a solid body, that is, shear modulus and elasticity. The most important rheological characteristics of hydrogels include shear stress and viscosity. Effective viscosity is a characteristic of the equilibrium state between the processes of destruction and recovery. Its fluctuation causes a change in the coagulation-crystallization structure of the hydrogel, affecting its performance characteristics. The spatial structure in the hydrogel is determined by measuring the mechanical properties and, in particular, shear deformation under the constant stress. Solids are characterized by a sharp change in the pattern of shear deformation ε depending on the magnitude of the shear stress Р. At rather low stresses (less than the yield strength Pk), a free flow with constant and extremely high viscosity η1 is observed. In this case, the coagulation structure is destroyed, but has time to recover. As the shear stress increases to the yield strength Pk, the viscosity decreases significantly, down to the lowest limit value ηm.
Rheological behaviors observed for agar gel and PAAG are presented in Fig. 4. Non-linear change in effective viscosity values indicates non-Newtonian system properties of studied gels. During the measurement, the destruction of interparticle bonds progresses as the shear rate (γ) increases, which is manifested in the peculiarities of the shape of the flow curves, causing deviations from straight lines. Measuring in the reverse mode indicates recovery of the effective viscosity due to the restoration of the system structure, but the effective viscosity values remain lower than the initial ones. Both dispersed PAAG and agar gel showed thixotropic properties, as evidenced by the hysteresis loops of the dependence of the effective viscosity of gels on the shear rate (Fig. 4a,b).
Table 4 and Fig. 4 show the initial and final values of the effective viscosity at the minimum and maximum shear rates for homopolyacrylamide and agar gels. The initial effective viscosity values are close for both gels. During the measurement. The viscosity values at a shear rate of 2.45 s− 1 at the end of the measurement in the reverse shear rate reduction mode for PAAG and agar are 156.398 and 200.32 Pa∙s, which is 37.0 and 40.2% of the initial value, respectively. This means that PAAG had rheological properties and effective viscosity values similar to agar-agar gel.
Table 4
Effective viscosity at minimum (γ = 2.45 s− 1) and maximum shear rates (γ = 1073 s− 1)
Sample | η ( at γ = 2,45 s− 1), Pа∙s | η ( at γ = 1073 s− 1), Pа∙s |
initial | final | initial | final |
Agar | 498.519 | 200.32 | 0.501 | 0.306 |
PAAG | 422.986 | 156.398 | 0.471 | 0.410 |
Micropropagation
For micropropagation of plants in vitro, all synthesized hydrogels were saturated with a culture medium with a complex of micro- and macronutrients, vitamins, amino acids, growth hormones, etc. This process provided the formation of hydrogel composites with sparingly soluble bioelements, which are localized in the hydrogel pores and can gradually diffuse into the external environment. The experiment showed the acceleration of the rooting time of cuttings on PAAG-A2 gel, which was less than two weeks of incubation. On agar-agar this time equaled three weeks. In addition, an intensification of growth and development of the main shoot was observed on hydrogels: 10 and almost 30% higher on PAAG and PAAG-A2, respectively, compared to the samples grown on agar-agar.
The rooting of the plants on agar medium was 95%, while on hydrogel substrates – higher than 98%. On hydrogel substrates with the addition of amber, there was a 1.7-fold increase in shoot growth intensity, and on substrates without amber – 1.5 times. When the plants remained for 10–15 days in a tall vessel, their height reached 110–130 mm (Fig. 5).
In vitro deposition was carried out with periodic transplantations to the new nutrient medium to avoid drying out and changes in the composition of the environment due to the effect of the products of plant metabolism. After the first cycle, the spent hydrogel material was regenerated – washed in distilled water, sterilized, and used in repeated cultivation cycles. The obtained in vitro rooted plants Cánnabis satíva are suitable for conversion in vivo (Fig. 6).
The increase in the number of the metric indicators of leaf blades were also found. During transferring plants from іn vitro to іn vivo it was found that the dose-dependent effect of plant knees on test subjects persists (Fig. 7, Table 5). In general, using a hydrogel instead of agar stimulated the growth of Cánnabis satíva. Use of hydrogel-amber substrate increased metric indicators of seedling (in comparison to agar): the root length increased by 28%, stem length – by 26.7%, root weight – by 167%, stem weight – by 67%, root and stem length – by 27%, and root and stem weight – by 50%.
Table 5
Metric indicators of Cánnabis satíva seedlings
Experiment techniques | Cánnabis satíva seedlings | Root length (cm) | Stem length (cm) | Root weight (g) | Stem weight (g) | Root and stem lengths (cm) | Root and stem weight (g) |
In vitro | Agar | 3.5 | 7.5 | 0.3 | 0.6 | 11 | 0.9 |
PAAG | 4 | 8 | 0.6 | 0.7 | 12 | 1.3 |
PAAG + A2 | 4.5 | 9.5 | 0.8 | 1 | 14 | 1.8 |
In vivo | Sоil | 15.2 | 21.8 | 1.3 | 1.4 | 37 | 2.7 |
After hydrogel recycling (ten-fold washing with distilled water), all nutrients, succinic acid and products of its transformation were much better absorbed by the plant and stimulated the growth and development of seedlings after transfer in vivo. In addition, the allelopathic activity of extract from biological material in 1:10, 1:50, and 1:100 dilutions was studied. It has been shown that allelopathic stress is dose-dependent when used a typical MS medium. Under 1:10 dilution in vitro, Cucumis sativus L. growth inhibition was observed up to 53%; however growth inhibition was reached 87% (Table 6). Under 1:100 dilution іn vivo "Suppressive" effect almost didn’t fall (95%), which indicates the successful transfer of plants from іn vitro to іn vivo. This pattern can be traced on the example of morphometric parameters of Cánnabis satíva seedlings (Table 6).
Table 6
Allelopathic effect of aqueous extract of aboveground parts of Cánnabis satíva on seedlings of Cucumis sativus L.
Dilution of the extract | In vivo, (%) | In vitro, (%) |
1:10 | 67 | 53 |
1:50 | 75 | 60 |
1:100 | 95 | 87 |
Thus, both in vivo and in vitro a positive effect of amber in the composition of the hydrogel substrate on the main parameters of germination and development of the studied plants was revealed. According to the possible influence mechanism the biologically active components of highly dispersed amber powder affect biological objects at the cellular level, increasing the efficiency of processes in plants, and also take part in forming the microelement balance, i.e., they are bioactive. Highly dispersed amber powder embedded in the copolymer matrix is non-toxic, released gradually, its ionic form quickly includes in biochemical reactions, and therefore, when washing it with MS culture medium, the biologically active amber acid is washed out prolonged and dosed. This fact explains the high bioavailability and biocompatibility of the synthesized nutrient substrate, the possibility of obtaining planting material in a shorter time, accelerated transition of plants from the juvenile to reproductive phase of development, and increased intensity of growth and development of the main shoot.