Effects of biodegradable P3HB on the specific growth rate, root length and chlorophyll content of duckweed, Lemna minor

Heliyon 9 (2023) e23128 Available online 30 November 2023 2405-8440/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Effects of biodegradable P3HB on the specific growth rate, root length and chlorophyll content of duckweed, Lemna minor Petra Proch´ azkov´ a a , * , Sabina M´ acov´ a a , Seçil Aydın b , Helena Zl´ amalov´ a Gargoˇ sov´ a a , Gabriela Kalˇ cíkov´ a c , Jiˇ rí Kuˇ cerík a a Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, Brno University of Technology, Purkyˇ nova 118, 612 00 Brno, Czech Republic b Department of Chemical Engineering, Faculty of Chemistry-Metallurgical, Yıldız Technical University, 34210, Davutpasa Esenler, Istanbul, Turkiye c Faculty of Chemistry and Chemical Technology, University of Ljubljana, 113 Veˇ cna pot, SI-1000, Ljubljana, Slovenia ARTICLE INFO Keywords: Biodegradable plastics Duckweed Ecotoxicity Microbeads Micro-bioplastics P3HB ABSTRACT The extensive production and use of plastics have led to widespread pollution of the environment. As a result, biodegradable polymers (BDPs) are receiving a great deal of attention because they are expected to degrade entirely in the environment. Therefore, in this work, we tested the effect of two fractions (particles <63 μ m and particles from 63 to 125 μ m) of biodegradable poly-3hydroxybutyrate (P3HB) at different concentrations on the specific growth rate, root length, and photosynthetic pigment content of the freshwater plant Lemna minor. Microparticles with similar properties made of polyethylene terephthalate (PET) were also tested for comparison. No adverse effects on the studied parameters were observed for either size fraction; the only effect was the root elongation with increasing P3HB concentration. PET caused statistically significant root elongation only in the highest concentration, but the effect was not as extensive as for P3HB. The development of a biofilm on P3HB particles was observed during the experiment, and the nutrient sorption experiment showed that the sorption capacity of P3HB was greater than PET’s. Therefore, depleting the nutrients from the solution could force the plant to increase the root surface area by their elongation. The results suggest that biodegradable microplastics may cause secondary nutrient problems in the aquatic environment due to their biodegradability. 1. Introduction Due to the growing problem of plastic waste and pollution from conventional plastics, there is an increasing interest in producing environmentally friendly products from degradable materials [1,2]. Biodegradable polymers (BDPs) are polymeric materials that can be decomposed under aerobic conditions into carbon dioxide, water, and biomass by the enzymatic action of microorganisms or under anaerobic conditions to methane [3]. Simply put, they become plastics when mixed with additives, which improve their processability and properties. BDPs are being promoted as a viable alternative to conventional plastics in several industrial sectors, some in medicine, others in textile, personal care products, agriculture, and especially as packaging materials [4,5]. BDPs can be made from petrochemicals or renewable materials; they can be polymers produced by extraction from biomass (polysaccharides, polypeptides, lipids), polyesters made by microorganisms and plants or their mixtures [3,6,7]. * Corresponding author. E-mail address: [email protected] (P. Proch´ azkov´ a). Contents lists available at ScienceDirect Heliyon journal homepage: www.cell.com/heliyon https://doi.org/10.1016/j.heliyon.2023.e23128 Received 30 August 2023; Received in revised form 16 November 2023; Accepted 27 November 2023 Heliyon 9 (2023) e23128 2 Considerable attention is paid to polymers produced from renewable sources, including polyhydroxyalkanoates (PHAs), a group of biodegradable linear polyesters 3, 4, 5, and 6-hydroxy acids, which can be produced under aerobic and anaerobic conditions by various gram-negative and gram-positive bacteria that use them as intracellular reserve materials [8–12]. PHAs were discovered through poly-3-hydroxybutyrate (P3HB) [13,14]. P3HB is a relatively hydrophobic material with a crystallinity greater than 50 %, a high melting point (180 ◦C), and low elasticity. These properties complicate its processing and limit its possible use on an industrial scale [15,16]. The mechanical properties of P3HB can be improved by various additives, either polymers or plasticizers, or by forming copolymers [17]. However, these additives can have a negative effect on the biodegradation rate of the resulting material [18]. Therefore, substances that facilitate biodegradation, such as starch, are also added to these mixtures. Another disadvantage of P3HB is its high production cost, which can be slightly reduced by using waste materials as feedstock, e.g., used rapeseed frying oil [19]. Biomass, waste products of the sugar industry, or coffee bean hydrolysates can also be used for their production [20]. Similar to conventional plastics, biodegradable plastics can break down into small particles under natural conditions, resulting in large quantities of microplastics [17,21]. Despite their biodegradability, these particles can have a very long life because their degradation is influenced by biotic and abiotic environmental factors such as temperature, humidity, pH, biologically active substances, and the presence and activity of microorganisms [22,23]. Biodegradation processes of BDPs often needs specific environmental conditions, and the biodegradation cannot be completed in many natural environments [24]. BDPs mainly showed high degradability in soil and compost environments. However, this is not always the case of the aquatic environment, for example the degree of biodegradation of polyhydroxybutyrate vary from 30 to 99 % depending on the conditions and the type of water [25]. Microplastics from BDPs have also been found to negatively affect freshwater and marine organisms [26]. In the freshwater amphipod Gammarus fossarum, a reduction in biomass gain was observed following exposure to polyhydroxybutyrate compared to the control as a result of their ingestion [27]. The effect of polylactic acid (PLA) microparticles on the marine organism Arenicola marina and microalgal biomass on the sediment surface was investigated by Green et al. [29]. There was no adverse effect of PLA on the viability of A. marina, but there was a significant decrease in microalgal biomass in PLA-containing sediment compared to the control [28]. However, in general, microplastics from BDPs are largely understudied, so understanding their impacts on the nvironmentt is still limited. Due to the gradual acceptance of biodegradable replacements for conventional plastics, increased exposure of environmental components by micro-bioplastics can be expected due to their incomplete biodegradation. For this study, we selected P3HB due to its widespread applications and its growing, extensive use in packaging, agriculture, and biomedicine [29]. Further, the price of P3HB production is decreasing [30,31]. Consequently, it can be expected to be widely used in other branches, mainly as a replacement for polypropylene [32]. For those reasons, this study aimed to evaluate the effect of P3HB microplastics on the freshwater floating plant Lemna minor and compare the observed results to effects caused by PET microplastics with comparable properties. The effects of microplastics on the specific growth rate, photosynthetic pigments content, and root length are investigated. 2. Materials and methods 2.1. Preparation of microparticles The P3HB (ENMAT Y3000) microplastics with a spherical shape were used for ecotoxicological tests with L. minor. They were obtained as a powder from TianAn Biologic Materials Co., Ltd. (Ningbo City, China). A suspension of P3HB microplastics in MilliQ water was sieved on 63 and 125 μ m mesh stainless-steel sieves, and both size fractions (<63 μ m and from 63 to 125 μ m) were subsequently dried in glass beakers at room temperature in a fume hood. The PET microplastics were obtained as a powder produced during mechanical recycling from PETKA CZ a.s. (Brno, Czech Republic), repeatedly washed and then prepared similarly to P3HB to obtain the same size fractions. 2.2. Experimental design The L. minor came from a laboratory culture at the Institute of Chemistry and Technology of Environmental Protection (Faculty of Chemistry, Brno University of Technology, Czech Republic). The plant was cultivated in Steinberg medium [33] under controlled conditions (temperature 23 ±2 ◦C, photoperiod 16/8 h). Due to the lack of standardised protocols for microplastic ecotoxicity testing, there is no accepted procedure for how organisms should be exposed in ecotoxicity tests. Accordingly, we used several approaches to expose L. minor. The effects of (i) suspension (without replacement), (ii) suspension exchange during the test, which used Li et al. [34] procedure in tests with TiO 2 nanoparticles, (iii) leachate and (iv) direct weighing (due to surface tension, some insufficiently wetted P3HB particles remained floating on the surface). Tests were conducted with both size fractions. The suspension (i) was prepared by weighing the desired amount of P3HB and transferring it to a volumetric flask; then, the volumetric flask was filled to the mark with Steinberg medium. The suspension thus prepared was ultrasonicated for 10 min to disperse the particles. In the case of the exchange test medium (ii), the suspension thus prepared was exchanged twice during the test. Leachate (iii) was prepared as follows: a suspension of P3HB in Steinberg medium was prepared and then incubated for seven days under the same conditions as the L. minor test. The medium was then filtered through a 0.8 μ m pore size filter. In direct weighing (iv), P3HB was weighed in the required amount, transferred to a test vessel, and 100 mL of medium was added. Since the results (Section 3) showed no significant difference between the preparation methods, the test was performed with PET in P. Proch´ azkov´ a et al. Heliyon 9 (2023) e23128 3 suspension only (i). Also, leachate from PET was not tested because our previous study showed no effects of PET leachate on various endpoints of L. minor [35]. The experiments were conducted in 150 mL glass beakers, and each was filled with 100 mL of test suspension, leachate, or medium. The concentration of P3HB microparticles was 0, 10, 50, and 100 mg/L, corresponding to 3.20 10 13 particles in 1 g for the fraction <63 μ m and 2.39 10 13 particles in 1 g for the fraction <125 μ m. The number of particles was determined based on their distribution in suspension (figure S1–S4 in Supporting Information). This concentration range was selected based on our initial tests and previous studies on the toxicity of microplastics to L. minor [36]. The PET was used in the same concentrations as P3HB. Each experiment involved three replicates of the same exposure concentrations was repeated three times and was carried out according to the OECD guideline No. 221 [37] and used in many studies when microplastics were tested [36,38–40]. The initial number of leaves was nine, so more than 50 % of the surface area was available for plant growth. Roots were carefully removed from the plants before the start of the test. This experiment was conducted at a temperature of 24 ±1 ◦C, and evaporation of the medium was prevented during the test by covering it with polyethylene foil. A photoperiod of 16/8 h (light/dark) was set at a light intensity of 4300 lx at the plant level, and the duration of the experiment was seven days. Images of P3HB and PET microplastics were taken with an optical microscope and ocular camera, 10 ×/0.25 objective before and after the experiment. At the end of the experiment, the number of leaves was counted (n =9), and the average specific growth was calculated (details are given in section 2.6, Data analysis). 2.3. Measurement of root length L. minor (n =75) were placed on laminated graph paper and photographed with a Nikon D3100 digital camera and an AF-S Micro NIKKOR 40 mm 1:2.8 G lens (Nikon, Japan). Afterward, the root length was evaluated in the ImageJ program. 2.4. Photosynthetic pigment determination Photosynthetic pigments (n =9), chlorophyll a and b, and total carotenoids were determined by a modified Arnon method described by Radi´ c and Pevalek-Kozlina [41]. Approximately 30 mg of fresh plant material from each treatment was used for the photosynthetic pigment analysis. The samples were homogenized in 80 % ice-cold acetone (w/v), and the absorbance of the supernatant was read at 470, 642.8, and 646.2 nm. 2.5. Sorption experiment Both P3HB and PET microplastic fractions were incubated in the Steinberg medium for seven days at the same concentrations (0, 10, 50, and 100 mg/L) and under the same conditions as in the L. minor assay. Media samples were collected at 0, 24, and 168 h and filtered through a 0.45 μ m pore size nylon filter. The nitrate-nitrogen concentration in these samples was determined spectrophotometrically using a commercial kit (Spectroquant®, Millipore, 109713). To evaluate the effects of nutrient depletion of the medium, an additional test was also prepared in which the medium contained 5 %, 10 %, 20 %, 30 %, and 40 % less nutrients than the original Steinberg medium. The ecotoxicity test with L. minor was then performed as described for P3BH. 2.6. Data analysis The average specific growth rate after seven days of exposure was calculated according to ISO 20079 (2005) as follows: μ =ln(Nj)−ln(Ni) t where μ (d −1 ) is the average specific growth rate, N j (/) is the number of leaves at the end of the experiment, N i (/) is the number of leaves at the beginning of the experiment, and t (d) is the exposure time (seven days). %Ir=( μ C− μ T) μ C •100 where %I r is the percentage inhibition of the average growth rate, μ c is the mean growth rate for control, and μ T is the mean μ for specific sample treatment. The content of photosynthetic pigments was calculated according to Lichtenthaler [42]. Results are given in mg of chlorophyll per gram of fresh plant mass. The statistical significance of the differences between control and individual concentrations was determined using Dunnett’s test in the R program, with differences considered significant if p <0.05. P. Proch´ azkov´ a et al. Heliyon 9 (2023) e23128 4 (caption on next page) P. Proch´ azkov´ a et al. Heliyon 9 (2023) e23128 5 3. Results 3.1. Effect of P3HB on the specific growth rate of Lemna minor The results of this study showed that the specific growth rate of L. minor was not affected in the case of suspension test (i), exchange suspension during the test (ii), and direct weighing of P3HB microplastics into the test vessel (iv). Similarly, suspension of PET microplastics did not affect the specific growth rate under those exposure scenarios (Fig. 1). On the other hand, leachate (iii) from both size fractions of P3HB showed a statistically significant difference at the highest concentrations – 100 mg/L for particles <63 μ m, 50 and 100 mg/L for particles from 63 to 125 μ m. The specific growth rate inhibition was concentration dependent: leachates from 10, 50, and 100 mg/L caused 4.72 %, 4.01 %, and 15.95 % for particles <63 μ m, and 2.79 %, 7.61 %, and 17.69 % for particles from 63 to 125 μ m, respectively (Fig. 1). 3.2. Effect of P3HB on chlorophyll content Both fractions of P3HB microplastics in the different treatments ((i) – (iv)) did not have a negative effect on the content of photosynthetic pigments in L. minor leaves (chlorophyll a and b, carotenoids). Similarly, PET did not affect photosynthetic pigment content compared to the control (Figs. 2 and 3). 3.3. Effect on root growth Both P3HB size fractions had statistically significant effects on L. minor root growth in all treatments. The effect was concentrationdependent regardless of the preparation of the suspension ([i], [ii], [iv]); the leachates (iii) of P3HB also caused a statistically significant elongation of the roots of L. minor. Similarly, the length of the roots increased with increasing concentration of PET microplastics, but a statistically significant difference from the control was observed only at the highest concentration (100 mg/L, Fig. 4). 3.4. Sorption experiment To determine the degree to which the presence of microplastics affects the composition of the medium, a series of sorption experiments were conducted on both PET and P3HB. In the presence of microplastic particles <63 μ m, there was up to 30 % reduction of the concentration of N–NO 3 - in the case of P3HB, while there was only 12 % for PET (figure S5). For the second size fraction of microplastics (from 63 to 125 μ m), the depletion of N–NO 3 - in both cases was slightly higher. Thus, sorption occurs for both types of plastics, but a statistically significant effect on root growth was observed only for P3HB and the highest concentration of PET (Fig. 4). In addition, biofilm growth on the settled P3HB and PET was observed at the bottom of the beaker, where filamentous microorganisms occurred (Fig. 5). However, only in the treatments with P3HB did visual inspection indicate other microbial cells attached to particles (Fig. 5B). 3.5. Effect of nutrient depletion Additional experiments with depletion of nutrients in the Steinberg medium (containing 5 %, 10 %, 20 %, 30 %, and 40 % fewer nutrients than the control under the same conditions as the P3HB and PET tests) confirmed that with decreasing nutrient concentrations in the medium, there was a slight inhibition of growth compared to the control (the highest was 6.5 % in the sample with the least amount of nutrients, i.e., 40 % fewer nutrients than in the control. No changes in photosynthetic pigment content were observed. However, a statistically significant increase in root length was observed at all concentrations and increased with decreasing nutrient concentrations in the medium (Fig. 6). 4. Discussion Recent research on microplastics has shown that different types of microplastics can affect growth and development of aquatic plants [43,44]. In contrast, natural particles such as wood or cellulose particles showed no effects [40]. Therefore, we aimed to investigate the effect of bio-based and biodegradable P3HB and compare it with that of non-biodegradable PET. PET was selected for its P3HB-like properties mainly density (1.38 g/cm 3 ) and wettability – the contact angle of PET varies between 66◦and 81◦, that is, it is also poorly wettable compared to P3HB [45]. In fact, both particles belong to the group of polyesters, but P3HB is, unlike, a biodegradable polymer. Although, recent work [46] suggested that PET hydrolysis can also occur under specific conditions, e.g., about 4.5 years is needed for 50 % PET degradation under neutral pH in marine water, it can be concluded that the lifetime of P3HB and PET Fig. 1. Results of specific growth rate: A to E for particles <63 μ m (A – suspension (i), B – exchange of suspension (ii), C – leachate (iii), D – direct weighing (iv) of P3HB, and E −suspension of PET) and F to J for particles from 63 to 125 μ m (F – suspension (i), G – exchange of suspension (ii), H – leachate (iii), I – direct weighing (iv) of P3HB, and J – suspension of PET). Asterisks denote statistically significant differences compared with the control treatment (p-value <0.05). P. Proch´ azkov´ a et al. Heliyon 9 (2023) e23128 6 Fig. 2. Results of average content of total chlorophylls: A to E for particles <63 μ m (A – suspension (i), B – exchange of suspension (ii), C – leachate (iii), D – direct weighing (iv) of P3HB, and E −suspension of PET) and F to J for particles from 63 to 125 μ m (F – suspension (i), G – exchange of suspension (ii), H – leachate (iii), I – direct weighing (iv) of P3HB, and J – suspension of PET). P. Proch´ azkov´ a et al. Heliyon 9 (2023) e23128 7 Fig. 3. Results of average content of carotenoids: A to E for particles <63 μ m (A – suspension (i), B – exchange of suspension (ii), C – leachate (iii), D – direct weighing (iv) of P3HB, and E −suspension of PET) and F to J for particles from 63 to 125 μ m (F – suspension (i), G – exchange of suspension (ii), H – leachate (iii), I – direct weighing (iv) of P3HB, and J – suspension of PET). P. Proch´ azkov´ a et al. Heliyon 9 (2023) e23128 8 (caption on next page) P. Proch´ azkov´ a et al. Heliyon 9 (2023) e23128 9 microparticles in natural systems significantly differ. In general, there are very few studies on the impact of biodegradable microplastics on aquatic organisms; their effects on aquatic ecosystems were not studied until 2015 when Green et al. [28] found that, in some cases, they can negatively affect organisms in the same way as conventional microplastics. Their work compared the effect of PLA, polyethylene (PE), and polyvinyl chloride microplastics on the health and biological activity of lugworms A. marina and the nitrogen cycling and primary productivity of the sediment they inhabit. All three types of microplastics, including biodegradable PLA, significantly reduced the biomass of microalgae in the sediment [28]. Su et al. [47] compared the effect of PLA, polybutylene succinate, PE, and polyamide (PA) microplastics (the sizes of each type of microplastic were 57.4, 53.3, 77.8, and 59.9 μ m) on the marine algae Chlorella vulgaris. PLA showed the most substantial inhibitory effect on the growth of C. vulgaris. Interestingly, in contrast to our study, the microplastics appeared to stimulate the contents of photosynthetic pigments (e.g., chlorophyll a, chlorophyll b, and carotenoids), which was attributed to their ability to act as cellular defences against stress induced by microplastics. The ecotoxicological research on P3BH is limited, but, for example, tests with secondary P3HB nanoparticles by Gonz´ alez-Pleiter et al. [48] demonstrated that with the presence of P3HB nanoparticles (spherical particles with a diameter of around 200 nm, the Fig. 4. Results of measurement of root length: A to E for particles <63 μ m (A – suspension (i), B – exchange of suspension (ii), C – leachate (iii), D – direct weighing (iv) of P3HB, and E −suspension of PET) and F to J for particles from 63 to 125 μ m (F – suspension (i), G – exchange of suspension (ii), H – leachate (iii), I – direct weighing (iv) of P3HB, and J – suspension of PET). Asterisks denote statistically significant differences compared with the control treatment (p-value <0.05). Fig. 5. P3HB particles <63 μ m before the test (A) and after seven days of exposure (B), PET particles <63 μ m before the test (C) and after seven days of exposure (D). The arrows in Figure B indicate the different types of organisms in the biofilm formed on P3HB microplastics versus PET. P. Proch´ azkov´ a et al.