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Zantis, Sam van Loon, Cornelis A.M. van Gestel, Thijs Bosker, Rachel Hurley, Luca Nizzetto, Bartosz Adamczyk, Sannakajsa Velmala, Title: Biodegradable microplastics induce profound changes in lettuce (Lactuca sativa) defense mechanisms and to some extent deteriorate growth traits Year: 2024 Version: Published version Copyright: The Author(s) 2024 Rights: CC BY 4.0 Rights url: https://creativecommons.org/licenses/by/4.0/ Please cite the original version: Sylwia Adamczyk, Laura J. Zantis, Sam van Loon, Cornelis A.M. van Gestel, Thijs Bosker, Rachel Hurley, Luca Nizzetto, Bartosz Adamczyk, Sannakajsa Velmala, Biodegradable microplastics induce profound changes in lettuce (Lactuca sativa) defense mechanisms and to some extent deteriorate growth traits, Environmental Pollution, Volume 363, Part 2, 2024, 125307, https://doi.org/10.1016/j.envpol.2024.125307. https://creativecommons.org/licenses/by/4.0/ Biodegradable microplastics induce profound changes in lettuce (Lactuca sativa) defense mechanisms and to some extent deteriorate growth traits☆ Sylwia Adamczyk a,*, Laura J. Zantis b, Sam van Loon c, Cornelis A.M. van Gestel c, Thijs Bosker b, Rachel Hurley d, Luca Nizzetto d,e, Bartosz Adamczyk a, Sannakajsa Velmala a a Natural Resources Institute Finland (Luke), Latokartanonkaari 9, 00790, Helsinki, Finland b Institute of Environmental Sciences, Leiden University, P.O. Box 9518, 2300, RA Leiden, the Netherlands c Amsterdam Institute for Life and Environment (A-LIFE), Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081, HV Amsterdam, the Netherlands d Norwegian Institute for Water Research (NIVA), Økernveien 94, 0579, Oslo, Norway e Research Centre for Toxic Compounds in the Environment, Masaryk University, 62500, Brno, Czech Republic A R T I C L E I N F O Keywords: PBAT Lipid peroxidation Total phenolic content Salicylic acid CLIMECS system Starch-polybutylene adipate terephthalate A B S T R A C T The development of agricultural technologies has intensified the use of plastic in this sector. Products of plastic degradation, such as microplastics (MPs), potentially threaten living organisms, biodiversity and agricultural ecosystem functioning. Thus, biodegradable plastic materials have been introduced to agriculture. However, the effects of biodegradable plastic substitutes on soil ecosystems are even less known than those of traditional ones. Here, we studied the effects of environmentally relevant concentrations of MPs prepared from a biodegradable plastic (a starch-polybutylene adipate terephthalate blend, PBAT-BD-MPs) on the growth and defense mecha nisms of lettuce (Lactuca sativa) in CLIMECS system (CLImatic Manipulation of ECosystem Samples). PBAT-BD- MPs in the highest concentrations negatively affected some traits of growth, i.e., dry weight percentage, specific leaf area, and both C and N contents. We observed more profound changes in plant physiology and biochemistry, as PBAT-BD-MPs decreased chlorophyll content and triggered a concerted response of plant defense mechanisms against oxidative stress. In conclusion, exposure to PBAT-BD-MPs induced plant oxidative stress and activated plant defense mechanisms, leading to oxidative homeostasis that sustained plant growth and functioning. Our study highlights the need for in-depth understanding of the effect of bioplastics on plants. 1. Introduction For decades, plastics have been seen as biochemically inert to the environment and organisms due to their chemical structure and large molecular size (Teuten et al., 2009). However, under the action of environmental factors, plastic debris may break down into smaller fragments such as micro- and nanoplastics (1 μm-5 mm and <100 nm, respectively) (Helmberger et al., 2020; Hurley et al., 2020). Due to their common use in agriculture, microplastic (MP) concentrations in soils are high, reaching 40 mg kg− 1 (0.004 %) of conventional microplastics and in excess of 300 mg kg− 1 (0.03 %) of mesoplastics in soil treated with mulching films (Li et al., 2020). MPs found in agricultural soil originate, among other sources, from the degradation of agricultural plastics (especially mulching films) and contaminated sewage sludge and biosolid applications (Sajjad et al., 2022), however, wastewater irrigation, pesticides, and atmospheric deposition also contribute to increasing soil MP concentrations (Nizzetto et al., 2016; Zantis et al., 2023a). The potential effects of non-biodegradable MPs on plants include impaired nutrient uptake, alterations in plant root traits (Wang et al., 2020) and seed germination (Hassan et al., 2022). MPs may also cause oxidative stress in plant cells, manifested by an overproduction of reactive oxygen species (ROS), e.g. superoxide anion radical (O2 •− ) and hydrogen peroxide (H2O2), leading to damage in cell structures, including oxidative deterioration of lipids (lipid peroxidation) (Jia et al., 2023; Zantis et al., 2023a). Plant defense mechanisms may respond to MPs with elevated activities of ROS-scavenging enzymes (superoxide dismutase scavenging O2 •− and catalase scavenging H2O2), and increased concentrations of non-enzymatic antioxidants, i.e., phenolics and the stress-related hormone salicylic acid (Jia et al., 2023). ☆ This paper has been recommended for acceptance by Dr. Meththika Vithanage. * Corresponding author. E-mail address: Sylwia.Adamczyk@luke.fi (S. Adamczyk). Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol https://doi.org/10.1016/j.envpol.2024.125307 Received 30 June 2024; Received in revised form 24 October 2024; Accepted 10 November 2024 Environmental Pollution 363 (2024) 125307 Available online 12 November 2024 0269-7491/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). mailto:Sylwia.Adamczyk@luke.fi www.sciencedirect.com/science/journal/02697491 https://www.elsevier.com/locate/envpol https://doi.org/10.1016/j.envpol.2024.125307 https://doi.org/10.1016/j.envpol.2024.125307 http://crossmark.crossref.org/dialog/?doi=10.1016/j.envpol.2024.125307&domain=pdf http://creativecommons.org/licenses/by/4.0/ In recent years, biodegradable materials have been introduced as a substitute for conventional non-biodegradable materials in agricultural plastics, especially in the manufacturing of mulching films. This solution provides an economic benefit for farmers, overcoming the costs of end- of-life and waste management (Hurley et al., 2020). A biodegradable material commonly used in mulching applications is polybutylene adi pate terephthalate (PBAT) blended with biobased materials (such as starch or cellulose) (Fu et al., 2020). Another example of biodegradable plastic is polylactic acid (PLA) (Huerta-Lwanga et al., 2021; Yang et al., 2021). PBAT and PLA blends are degradable in composting conditions and to some extent in soil. During degradation, biodegradable plastics break down, forming biodegradable MPs (BD-MPs) that may progres sively accumulate in soils if the application rates exceed the rate of their degradation. Recent study provided first BD-MPs (PBAT and PLA-based) soil concentrations at the level of up to 500 particles per kg after 2 years of application to soil (Li et al., 2023). However, almost no data on the concentrations of BD-MPs in agricultural soils exists, and their effects on plant physiology and biochemistry are less studied than those of syn thetic MPs (Fan et al., 2022). The limited data available on the effects of BD-MPs on plant growth yields fragmentary and sometimes inconsistent knowledge. Based on available literature, plant response to BD-MPs varies, with some studies showing positive, and others showing negative or no significant effect on plant performance in concentration above 1 % (Chah et al., 2022). For example (Kanwal et al., 2022), showed no toxic effect of degradation products of PBAT on the growth of Chinese cabbage (Brasicca rapa), and (Palsikowski et al., 2018) did not detect any phytotoxic, cytotoxic, or genotoxic effects of PLA and PBAT on meristematic cells of onion (Allium cepa). However, 1 % PBAT-BD-MPs affected rice (Oryza sativa) and buck-horn plantain (Plantago coronopus) growth negatively, but not as much as conventional MPs (Courtene-Jones et al., 2024; Irshad et al., 2024; Yang and Gao, 2022). The mode of action of BD-MPs seems to be similar to that of conventional ones, which includes oxidative stress and negative effects on photosynthesis via a decrease in chlorophyll content (Sun et al., 2023; Yang and Gao, 2022). Inconsistency among the results from different studies may derive from BD-MP dosages, differences in bioplastic physicochemical properties, experimental designs and con ditions, and species-specific effects on plants, as also shown for con ventional MPs (Zantis et al., 2023b). Moreover, we are lacking a holistic view on the effect of MPs and especially their biodegradable substitutes on plant health and crop yield under relevant more natural conditions, as studies concentrate mainly on laboratory conditions. Studies of the effect of PBAT-BD-MPs on plants are rare (just 44 reported articles, Table S1) and often they do not include multiple plant stress endpoints (just 7 reported articles, Table S1). Thus, experiments that resemble realistic, multitrophic environment are needed to disentangle the effect of PBAT-BD-MPs on plant performance and health. In this study we used CLIMECS (CLImatic Manipulation of ECosystem Samples) setup as a model of terrestrial ecosystems con taining plants (lettuce, Lactuca sativa), natural soil, soil microorganisms, and soil invertebrates (including two earthworm species and three springtail species), aims to elucidate how PBAT-BD-MPs affect plants under environmentally relevant conditions. Lettuce was chosen as it is often cultivated with the use of mulch and this species is commonly used in ecotoxicity experiments. The soil of the CLIMECS system was spiked with environmentally relevant concentrations of PBAT-BD-MPs, ranging from 0.025 % to 0.8 % (w/w) of soil dry mass. This range contains the expected level of MPs originating from mulching films degrading on the soil surface and being diluted in the top few cm of the soil (Hurley et al., 2020), including conditions of repeated continuous applications (yearly or multiple times per year). At three harvesting timepoints (4, 8, and 11 weeks), lettuce growth traits and biochemical traits were measured Overall this study adds new knowledge on understudied effect of biodegradable microplastic-induced impacts on plant growth and de fense mechanisms. 2. Materials and methods 2.1. CLIMECS system Cylindrical soil columns measuring 12 cm in depth and 16.6 cm in diameter were constructed and put into the CLIMECS system (CLImatic Manipulation of ECosystem Samples, CLIMECS, Amsterdam, the Netherlands, Ligtelijn et al., 2024) (see Fig. S1 for picture of the CLI MECS). Four concentrations of PBAT-BD-MPs were mixed in with the soil, 0.025 %, 0.05 %, 0.2 %, and 0.8 % (w/w), and for each treatment, including control, we prepared eight replicates. Control did not obtain PBAT-BD-MPs. The columns (in the sum 40) were prepared by filling HDPE cylinders placed on a bottom plate with PBAT-BD-MP-spiked or control soil. The columns were tapped on the ground five times to allow soil compaction, to increase comparability to natural soil structure. After preparing the soil columns, they were inserted into the CLIMECS system and left for two weeks before the introduction of the animals and lettuce, to allow for stabilization of the soil’s natural microbiome. 2.2. Test soil and PBAT-BD-MPs Lufa 2.2 standard natural soil (Lufa Speyer, Germany) was dried for 48 h at 40 ◦C after arrival. Lufa 2.2 soil is a sandy loam soil (soil type USDA). According to the supplier, Lufa 2.2 soil has approximately (mean ± sd) 1.8 ± 0.6 % organic carbon, 0.19 (±0.05) % N, cation exchange capacity of 9.5 (±1.3) meq/100 g, a pH (0.01 M CaCl2) of 5.6 ± 0.3, and a water holding capacity (WHC) of 43 ± 5 %. During the experiment, soil water content was maintained at 22 %, corresponding to 50 % WHC, by watering twice a week with 20 mL of demineralized water, as well as bringing the columns back to their original weight once every two weeks. BD-MPs utilized in this study were obtained from shredding and cryogenically grinding a commercially available PBAT-starch blend based mulching film. The films were first shredded using a benchtop shredder (SHR3D IT, 3DEVO, The Netherlands). Cryogenic grinding was performed using a laboratory-scale 6875 Freezer/Mill High Capacity Cryogenic Grinder (SPEX Sample Prep, USA) at the Norwegian Institute for Water Research. The grinded particles were sieved and adjusted to provide a size distribution that corresponds with field observations of starch-PBAT blend mulching films in real agricultural soils (Hurley et al., 2024). The mean particle size was 131 μm (±161 μm s.d.) and the D50 was 67.0 μm (±225 μm m.a.d.). The size distribution of PBAT-BD-MPs used in this study can be found in Supplementary Table 2, including estimations of the corresponding mass (based on a measured film thickness of 15 μm). The particles were made using a virgin film and were not subject to weathering prior to the generation of test materials. This is due to limitations in homogenously ageing film materials and the physical dimension restrictions of ageing chambers versus producing sufficient amount of test materials (Hurley et al., 2024). PBAT-BD-MPs were mixed in with the soil using a concrete mixer (PROMISCHER PM145L) at 24–29 rpm. PBAT-BD-MPs and the dry soil were mixed for 5 min, after which water was added in batches to bring the soil moisture content to 50 % WHC, but not exceeding this to prevent puddle formation, which would potentially cause PBAT-BD-MPs to float to the surface and coagulate. 2.3. Plants Lettuce (Lactuca sativa L., Zwart Duits) and cress (Lepidium sativum) seeds were obtained from Dutch Garden Seeds (Volendam, the Netherlands). Four patches of 100 cress seeds were sown as shelter for the springtails, one in each quarter of the column, four days prior to the introduction of the animals (t = 0), leaving a 2 cm wide clearing be tween the patches for the lettuce seedlings. Lettuce was sown in pressed peat germination trays (Tuinplus, Heerenveen, the Netherlands), 4 × 4 × 4 cm wide, containing the same experimental soil as used to prepare S. Adamczyk et al. Environmental Pollution 363 (2024) 125307 2 the CLIMECS system. Shortly before the introduction of the animals, five patches of three lettuce seedlings were transferred to the soil column, to provide enough material to sample at different timepoints. 2.4. Earthworms and springtails Earthworms, Aporrectodea caliginosa and Lumbricus rubellus were obtained from Prodigga (Caumont-sur-Durance, France) and Lasebo (Nijkerkerveen, the Netherlands), respectively. In total, four earth worms were added to each soil column: one adult and one subadult individual of each species. Three species of springtails were used in the CLIMECS experiment: Sinella curviseta, Heteromurus nitidus, and Prota phorura fimata. All species were taken from cultures kept at the Vrije Universiteit Amsterdam. Cultures of all species were kept on moist plaster of Paris, containing activated charcoal in a 10:1 ratio, and fed with dry baker’s yeast weekly. Cultures were kept in climate rooms at 75 % relative humidity and a 16:8 light-dark cycle. S. curviseta was kept at 20 ◦C, H. nitidus and P. fimata at 16 ◦C. For each replicate CLIMECS, 125 adult and 125 subadult individuals of each species were collected from the cultures by hand using an aspirator. Adults were separated from subadults by sieving, using a 630 μm (S. curviseta and H. nitidus) or 450 μm mesh width (P. fimata). 2.5. Experimental conditions The CLIMECS were kept at a light-dark regime of 14:10 h. The soil temperature was set at 15 ◦C, with a set daytime heating temperature of 18 ◦C, meaning the air column above the soil was heated by light, until 18 ◦C at a soil depth of 0.5 cm was reached. A set of four temperature sensors (DS28EA00U, Maxim Integrated, 1/16 ◦C resolution and 0.5 ◦C accuracy) monitored the temperature in the soil. Once this temperature was reached, the heating halogen light was switched off, but LEDs were used to maintain proper lighting. Once the earthworms and springtails were introduced, all soil col umns were monitored for an hour to ensure earthworms were alive and well and dug into the soil. If no burrowing activity was observed of an individual earthworm, it was replaced with one from a spare Petri-dish. Two weeks after the introduction of the animals, all cress was cut down to allow the lettuce to develop. At week 4, 8 and 11, lettuce samples were taken for analysis of shoot length, number of leaves, dry weight, and biomarker analysis. 2.6. Seedling sampling and measuring growth traits During each sampling (4, 8, 11 weeks), two replicate plants were taken from each column, and shoot length, shoot fresh weight, and the number of leaves were measured. During the final sampling (week 12), two seedlings were left inside the column. One plant was then used to determine the specific leaf area (SLA, equation [1]) and the necrotic tissue (equation [2]) of the leaves and then dried for 24 h at 60 ◦C to measure dry weight and C and N content with an elemental CN analyzer (LECO, Michigan, USA); analysis was based on ISO 13878 standard. Pictures of the leaves were taken using the camera of an iPhone 13 (Apple, iOS 15, dual 12 MP camera system with ƒ/1.6 aperture) and the total leaf area for the SLA was determined using ImageJ (version 1.53t). The second plant was stored at − 80 ◦C for biochemical analyses. Before analyses, leaves of plants were grounded with liquid N and plant ma terial was divided into specific analyses (see below). Specific leaf area ( cm2 g ) = Total leaf area (cm2) Total leaf dry weight (g) [1] Necrotic tissue (%)= Necrotic changes area (cm2)*100% Total area (cm2) [2] 2.7. Chlorophyll content Chlorophyll a (Chl a) and chlorophyll b (Chl b) concentrations were measured spectrophotometrically (Warren, 2008). To 100 mg of grounded lettuce leaves 1 mL of 100 % methanol was added. Following centrifugation (5 min, 10 000 g) the supernatant was collected, and the pellet was re-extracted with 1 mL of methanol. Combined supernatants (2 mL) were used to measure the absorbance at 663 nm and 645 nm using a microplate reader (BMG Labtech, ClarioStar). Chlorophyll a, b and total chlorophyll were calculated using formulas from Warren (2008). 2.8. Activity of superoxide dismutase (SOD) and catalase (CAT) For enzyme extraction, 300 mg of grounded plant material was ho mogenized in 1 mL of phosphate buffer (50 mM, pH 7.0) with 1 mM EDTA, 1 % PVP and 1 M NaCl. Extraction of SOD included 1 mM sodium ascorbate. After centrifugation (15 min, 5000 g, 4 ◦C), supernatants were used to measure the activity of catalase and superoxide dismutase. Catalase (CAT, EC 1.11.1.6) activity was measured in an assay mixture with phosphate buffer (50 mM, pH 7.0) and 15 mM H2O2 (Dhindsa et al., 1981). Decomposition of hydrogen peroxide was measured at 240 nm, and CAT activity was expressed in μmol H2O2 min− 1 mg− 1 protein. Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured in an assay mixture containing 3 mM EDTA, 73 μM nitrotetrazolium blue (NBT), 13 mM methionine in phosphate buffer (50 mM, pH 6.4) with 60 μM riboflavin (Beauchamp and Fridovich, 1971). Samples were placed under a UV lamp for 10 min for superoxide anion generation and the degree of inhibition of NBT reduction to diformazan by O2 .- was measured at 560 nm. SOD activity was expressed in U mg− 1 protein. Protein concentration was measured with the (Bradford, 1976) method. 2.9. Lipid peroxidation Lipid peroxidation was measured via a lipid peroxidation marker, malondialdehyde (MDA) (Hodges et al., 1999). Grounded plant material (0.25 g) was mixed with 1 mL of 0.1 % trichloroacetic acid (TCA). Following centrifugation, 500 μL of supernatant was mixed with 500 μL 20 % TCA with 0.5 % thiobarbituric acid (TBA). Controls did not have TBA. Samples were incubated at 95 ◦C for 30 min and immediately cooled in an ice bath. The absorbances were measured in the microplate reader (BMG Labtech, ClarioStar) at 532 nm and at 600 nm to subtract non-specific absorption, and at 440 nm to subtract sucrose. The results were expressed as MDA equivalent (nmol g− 1 FW). 2.10. Total phenolic content The measurement of total phenolic content (TPC) followed the pro tocol of (Herald et al., 2012). Grounded plant material (300 mg) was mixed with 80 % methanol (3 mL) and incubated at room temperature for 1 h. Following centrifugation (5 min, 10000 g), 25 μL of supernatant was mixed with 75 μL of water and 25 μL of Folin-Ciocalteu reagent and incubated at room temperature for 6 min. Then, 100 μL 7.5 % Na2CO3 was added, mixed and samples were incubated at room temperature in the dark for 90 min. The absorbance at 765 nm was measured with a microplate reader (BMG Labtech, ClarioStar). Gallic acid was used as a standard. TPC was expressed as μg gallic acid (GA) equivalent g− 1 FW. 2.11. Salicylic acid and salicylic acid glucosides Free salicylic acid (SA) and SA-glucoside concentrations were measured according to (Allasia et al., 2018). Grounded plant material (0.25 g) was mixed with 1 mL 70 % ethanol with 32 μL anisic acid (15.25 ng μL− 1). After centrifugation, the pellet was re-extracted with 1 mL of 90 % methanol. Methanol and ethanol from combined S. Adamczyk et al. Environmental Pollution 363 (2024) 125307 3 supernatants were evaporated in a vacuum concentrator (Speed Vac, 173 2–18 Cdplus, Thermo Fisher) and the pellet was treated with 65 μL 20 % of TCA, and 650 μL of ethyl acetate: cyclohexane (1:1). After centrifugation, the upper phase was collected, and the water phase was re-extracted. The combined upper phase was evaporated in a vacuum concentrator (Speed Vac) to dryness. The pellet was dissolved in 100 μL 10 % methanol containing 0.1 % trifluoroacetic acid (TFA). This extraction provided free SA. The water phase was later mixed with 0.3 mL 12 M HCl, incubated at 80 ◦C for 1 h. After cooling down, 18 μL of anisic acid (15.25 ng μL− 1) was added and samples were extracted twice with 0.9 mL of ethyl acetate: cyclohexane (1:1). Combined upper phases were evaporated to dryness in a vacuum concentrator, dry residue was dissolved in 100 μL 10 % methanol with 0.1 % TFA. This fraction rep resented SA-glucosides. Free and hydrolyzed SA were measured with HPLC (Arc HPLC Waters), with C18 column (Phenomenex, 250 × 4.6 mm, 5 μm) eluted with a methanol gradient from 10 % to 82 % at a flow rate of 1 mL min− 1 at a temperature of 30 ◦C. Eluent contained 0.1 % TFA. Detection of SA and hydrolyzed SA (HSA) was done with a fluo rescence detector (excitation at 305 nm and emission at 407 nm). SA and HSA concentrations were expressed as μg SA g− 1 FW. 2.12. Statistics We used R4.2.2. (R Core Team, 2023) with libraries car (Fox and Weisberg, 2019), multcomp (Hothorn et al., 2008), corrplot (Wei and Simko, 2021), FactoMineR (Lê et al., 2008) and factoextra (Kassambara and Mundt, 2020). We used correlations to study pairwise relationships between quantitative variables at different timepoints at each concen tration of MP and factor analysis of mixed data (FAMD) to explore the associations between both quantitative and qualitative variables. We used two-way-ANOVA with PBAT-BD-MPs treatments and sampling timepoints (4, 8, and 11 weeks) and their interactions as explanatory variables with Tukey’s honest significance test (HSD) post-hoc test. If needed, the response variable was transformed to meet the assumptions of normality (based on Levene’s test). For most response variables, there was a significant interaction of time and treatment, and thus treatment effect was analyzed for all timepoints separately. We adjusted the p-values for multiple comparisons with Holms’s method. Figures were generated with ggplot2 (Wickham, 2016). 3. Results 3.1. Growth traits Shoot height showed a constant declining pattern with increasing MP concentrations over all sampling timepoints (Fig. 1a). Lettuce dry weight after exposure to PBAT-BD-MPs was not significantly different from the control (Fig. 1b–Table 1). Dry weight percentage significantly increased at higher PBAT-BD-MPs after 4 weeks (0.2 % and 0.8 %) by 42 % and 65 % respectively, and after 11 weeks (0.8 %) by 29 % compared to the control (Fig. 1c–Table 1). Specific leaf area (SLA) tended to decrease linearly after 4 and 11 weeks of exposure to increasing concentrations of PBAT-BD-MPs; how ever, this difference was statistically significant only at the highest PBAT-BD-MP concentration (0.8 %) in which 67 % decrease was observed after 4 weeks compared to the control (Fig. 1d). Necrotic tissue levels of lettuce leaves were at a similar level to the control for most PBAT-BD-MP concentrations (Fig. 1e), except for 20 % lower necrotic tissue at the higher PBAT-BD-MP concentrations after 8 and 11 weeks. Number of leaves tended to decrease with increasing concentrations of PBAT-BD-MPs and was 17–29 % lower than the control at 0.8 % PBAT- BD-MPs after 8 and 11 weeks (Fig. 1f–Table 1). Lettuce leaf CN content was measured only at weeks 8 and 11 due to insufficient sample size at week 4. Plant leaf C content showed a con stant declining pattern with increasing PBAT-BD-MP concentrations over all sampling timepoints (Table 1). It was 5 % lower at the highest PBAT-BD-MP concentration (0.8 %) compared to the control (Fig. 2a). Also, N content was decreased by 5–33 % at the higher PBAT-BD-MP concentrations compared to the control after 8 weeks (0.8 %) and 11 weeks (0.2 % and 0.8 %) (Fig. 2b–Table 1). Leaf C/N ratio tended to increase with increasing concentrations of PBAT-BD-MPs after 11 weeks of the experiment and was significantly increased by 50–76 % compared to the control at the highest PBAT-BD-MP concentrations (0.2 % and 0.8 %) (Fig. 2c–Table 1). 3.2. Chlorophyll The concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b) and total chlorophyll in plant leaves were not significantly affected by the PBAT-BD-MPs concentrations after 4 weeks except for elevated Chl a by 9 % at 0.025 % PBAT-BD-MPs (Fig. 3a). However, observations after 8- and 11-weeks showed significant and opposite effects: after 8-weeks Chl a and total chlorophyll concentrations were significantly decreased by 7 % at 0.05 % and 0.2 % PBAT-BD-MPs, and significantly increased by 26 % at 0.8 % PBAT-BD-MPs (Fig. 3a and c, Table 1) compared to the control. After 11 weeks, all chlorophyll concentrations were signifi cantly decreased by 64% at all PBAT-BD-MP concentrations except for 0.05 % and 0.2 % for Chl b. In all cases, the responses did not show a monotonic pattern (Fig. 3). 3.3. Superoxide dismutase (SOD) and catalase (CAT) activities SOD activity in lettuce leaves was significantly affected by PBAT-BD- MPs at all concentrations tested (Fig. 4a, Table 1). Lower PBAT-BD-MP concentrations (0.025 and 0.05 %) increased SOD activity by at least 15 % compared to the control throughout the experiment. After 8 and 11 weeks also 0.2 % PBAT-BD-MPs increased SOD activity by more than 10 %. However, the highest PBAT-BD-MPs concentration (0.8 %) decreased SOD activity by 48 % after 11 weeks. CAT activity in lettuce leaves increased by at least 33 % at the lower PBAT-BD-MP concentrations (0.025 %, 0.05 %) (Fig. 4b), while it also increased by 30 % at 0.2 % PBAT-BD-MPs throughout the whole experimental duration. The highest PBAT-BD-MP concentration (0.8 %) increased CAT activity only after 4 weeks by less than 10 % but did not differ from the control later on. 3.4. Lipid peroxidation (MDA concentration) Lettuce leaf MDA concentration tended to increase with increasing concentrations of PBAT-BD-MPs (Fig. 4c). MDA was significantly increased compared to the control at all PBAT-BD-MP concentrations throughout the whole experiment (Fig. 4c–Table 1), except for the lower PBAT-BD-MP concentrations: 0.025 % and 0.05 % after 4 weeks and 0.025 % after 8 weeks. After 11 weeks all PBAT-BD-MP concentrations significantly increased MDA concentrations in the lettuce leaves by at least 55 %. (Fig. 4c–Table 1). 3.5. Total phenolic content (TPC) After 4 weeks, all PBAT-BD-MP concentrations significantly increased TPC in lettuce leaves by at least 75 % (Fig. 4d–Table 1). However, after 8 weeks only at 0.025 % and 0.2 % PBAT-BD-MP total phenolic content increased by 1000 % compared to the control (Fig. 4d). After 11 weeks, the lower PBAT-BD-MP concentrations (0.025 %, 0.05 %) did not affect TPC while the higher PBAT-BD-MP concentrations (0.2 % and 0.8 %) showed even 21 % decrease of TPC levels compared with the control. 3.6. Salicylic acid (SA) and SA-glycosides (HSA) SA concentration in lettuce leaves was significantly increased by 100 % compared to the control by all PBAT-BD-MP concentrations after 4 S. Adamczyk et al. Environmental Pollution 363 (2024) 125307 4 weeks (Fig. 4e–Table 1). After 8 weeks, the lowest PBAT-BD-MP con centration (0.025 %) still elevated SA concentrations by 24 %, but at the higher PBAT-BD-MP concentrations (0.2 % and 0.8 %) SA levels were 12 % lower than in the control. After 11 weeks, SA concentrations were increased by 16 % at the lower PBAT-BD-MP concentrations (0.025 and 0.05 %), but decreased by 90 % compared to the control at highest PBAT-BD-MPs concentration (0.8 %). The HSA level in the lettuce leaves was significantly reduced by 12%–251 % compared with the control throughout the whole experi ment (Fig. 4f–Table 1) at the highest PBAT-BD-MP concentrations (0.8 %), however, medium PBAT-BD-MPs concentrations increased HSA concentrations by more than 100 % after 11 weeks (Fig. 4f). 3.7. Correlations, principal components and correspondence analysis Studied biomarkers showed both, positive and negative correlations (Fig. S2 in the Supplementary Material). SA positively correlated with enzymatic ROS-scavengers (with SOD after 11 weeks, with CAT after 4 and 11 weeks) for all PBAT-MPs concentrations. When PBAT-BD-MP effects were correlated separately for each concentration, SA corre lated positively with SOD for 0.2 % and 0.8 % PBAT-BD-MPs throughout the whole experiment. SA positively correlated with non-enzymatic ROS-scavengers, TPC (after 4 weeks). However, SA correlated nega tively with lipid peroxidation studied via MDA concentration (after 4 and 8 weeks, but not after 11 weeks) for the lower PBAT-BD-MP con centrations (0.025 % and 0.05 %). TPC correlated negatively with MDA and SOD (after 4 weeks), and with chlorophyll (after 8 weeks). TPC correlated negatively with 0.8 % PBAT-BD-MPs throughout the whole experiment. Associations of both quantitative and qualitative variables with factor analysis of mixed data (FAMD) was conducted to look at all dependent variables together. FAMD analysis separated the control samples, that were not exposed to PBAT-BD-MPs, from samples from the PBAT-BD-MP exposures at all sampling timepoints along the first dimension. After 4 weeks, the first dimension, which separated the high MP treatments from the low MP treatments and the control, was explained by increasing SOD and decreasing TPC content (Supplementary Material Fig. S3). At later growth stages (8 and 11 weeks), the second dimension that separated the control and the lowest PBAT-BD-MP concentrations from the higher ones was explained by increasing lipid peroxidation MDA (Supplementary Material Figs. S3–S5). Moreover, the lowest PBAT-BD-MP concentrations (0.025 % and 0.05 %) were grouped together at week 4, but they separated after 8 and 11 weeks. At week 11, the lowest PBAT-BD-MP concentration was closest to the control. 4. Discussion CLIMECS experiment aimed to elucidate how PBAT-BD-MPs added to soil affect lettuce plants under environmentally relevant conditions as due to limited number of studies the effect of these bioplastics on plant health is not-well known (Table S1). The results revealed that PBAT-BD- MPs altered severely lettuce biochemical traits and, to a lesser extent, several growth traits. The effects were dependent on the time of expo sure and concentration of the PBAT-BD-MPs in soil. The impact on soil invertebrates was less pronounced, with no alterations observed in the springtail community composition and no effects on earthworm survival (caption on next column) Fig. 1. Growth traits of lettuce: a) seedling shoot height, b) seedling dry weight, c) seedling dry weight percentage, d) specific leaf area (SLA, cm2 g− 1), e) necrotic tissue percentage and f) number of leaves (pcs – pieces) following exposure to polybutylene adipate terephthalate biodegradable microplastics (PBAT-BD-MPs) in CLIMECS system. Results are presented as median, inter quartile range and the standard deviation including outliers; Lowercase letters indicate significant differences between treatments (p < 0.05) based on Tukey’s HSD test (Holms adj.p). S. Adamczyk et al. Environmental Pollution 363 (2024) 125307 5 (van Loon et al. unpublished). Though statistically significant effects of PBAT-BD-MPs on the dry weight of the lettuce plants was not observed, some negative effects of the highest PBAT-BD-MPs (0.2, 0.8 %) were observed on the shoot height, number of leaves, specific leaf area, and C and N content. This suggests that PBAT-BD-MPs may deteriorate lettuce growth if present at relatively high concentrations in the soil. In a study using a high PBAT- BD-MP concentration (1 %), Oryza sativa growth was affected negatively (Yang and Gao, 2022). Similarly, 1.5 % PLA-PBAT-BD-MPs severely affected the growth of common beans (Phaseolus vulgaris) (Meng et al., 2021), and dandelion (Taraxacum officinalis) (Xingfan Li et al., 2024b). However, PLA-BD-MPs showed both, negative and positive effects on the growth of plants; for example at 0.1 % PLA-BD-MPs increased, but at 10 % PLA-BD-MPs decreased the growth of maize (Zea mays) (Yang et al., 2021). Thus, BD-MPs may adversely affect the growth of plants and the magnitude of the effect depends on BD-MP properties, concentration, time of exposure, and potentially also plant species. In this study, lettuce physiology and biochemistry seemed to be more sensitive indicators of PBAT-BD-MP stress than growth per se, as confirmed by decreased chlorophyll concentrations, increased oxidative stress, and significant modifications in plant defense mechanisms. A similar effect on chlorophyll was observed also for maize (Zea mays) exposed to a high (10 %) PLA-BD-MP concentration (Sun et al., 2023; F. Wang et al., 2020), common beans exposed to PBAT-PLA-BD-MPs (0.5–5 %) (Meng et al., 2021) and rice at 1 % PBAT-BD-MPs (Yang and Gao, 2022). Decreases in chlorophyll concentrations after exposure to PBAT-BD-MPs in soil may partially emerge from decreased N uptake, and its transportation and assimilation by plants, as revealed by tran scriptomic studies with rice (Yang and Gao, 2022). As chlorophyll is built up from an N-containing porphyrin ring, low plant N status caused by PBAT-BD-MP exposure may well explain the decrease in chlorophyll concentrations. In addition, the N content of plant leaves decreased at higher concentrations of PBAT-BD-MPs, supporting this hypothesis of plant N deficiency. Alternatively, a low plant N status leading to decreased chlorophyll synthesis can emerge from an increased microbial immobilization of N observed after the addition of PBAT-BD-MPs to soil (Reay et al., 2023). Changes in the ratio of Chl a and Chl b may also suggest drought stress due to PBAT-BD-MPs. Under drought stress, the reduction of Chl b is greater than that of Chl a (Ashraf and Harris, 2013), which was observed for lettuce exposed for 11 weeks to 0.8 % PBAT-BD-MPs. Po tential drought stress due to high 0.8 % PBAT-BD-MPs after 11 weeks is also confirmed by the elevated dry weight percentage of the lettuce plants in our study. It is possible that in our experiment, PBAT-BD-MPs accumulated in the vicinity of the lettuce roots, decelerating water up take. It was shown for lettuce that conventional MPs may induce drought effects (Zhang et al., 2023). In higher plants, synthesis and accumulation of chlorophylls may increase due to low levels of stress and decrease in conditions of high levels of stress (Agathokleous et al., 2020). This could partially explain the non-linear response of chlorophylls to PBAT-BD-MPs exposure. Stress in plants is inseparable from oxidative stress, during which reactive oxygen species production is elevated and activity and pro duction of ROS-scavengers are modified accordingly to provide oxida tive homeostasis (Ekner-Grzyb et al., 2022). In line with that, activity of SOD scavenging O2 •− and CAT activity scavenging H2O2 increased after exposure to low PBAT-BD-MP concentrations (0.025–0.05 %). Also in Brassica parachinensis, the activity of SOD and ROS production increased after exposure to high 2 % PBAT-BD-MPs in soil (Xinyang Li et al., 2024a). PBAT-BD-MPs at a concentration as high as 1 % also elevated ROS production in rice (Yang and Gao, 2022). In line with the increases of ROS-scavenging enzymes (SOD, CAT), also lipid peroxidation, measured via concentration of MDA, increased. Similar effects have been observed in other studies with exposure to PBAT-BD-MPs (Liu et al., 2023; Yang and Gao, 2022). In our study, the high MDA con centrations after exposure to 0.8 % PBAT-BD-MPs coincided with lower activities of SOD and CAT. This could emerge from an overproduction of ROS exceeding the catalytic abilities of these enzymes, leading to excessive accumulation of ROS resulting in cell membrane damage (Hodges et al., 1999). Accordingly, another line of plant defense, total phenolic compounds were also elevated by PBAT-BD-MP exposure (4 weeks) to gradually decrease to the control level after 11 weeks. TPC was negatively correlated with SOD activity (4 weeks) underlining its role in scavenging these ROS, which were not effectively removed by the enzymatic line of plant defense. On one hand, TPC levels similar to or even below the control after 11 weeks could point to decreased oxidative stress, on the other hand, the negative correlation between TPC and MDA after 4 weeks could mean that the oxidative stress exceeded the ability of plant defense mechanisms leading to lipid peroxidation and thus to higher MDA concentrations. The decreased level of TPC after 11 Table 1 Analysis of variance (ANOVA) of data on the effects of polybutylene adipate terephthalate biodegradable microplastics (PBAT-BD-MPs) on lettuce growth and biochemical traits. Statistically significant differences were determined based on alpha level P = 0.05. ns - nonsignificant; w4 – week 4, w8 – week 8, w11 – week 11. Chl - chlorophyll, Nr leaves – number of leaves, tot Chl – total chlorophyll, SOD – superoxide dismutase, CAT – catalase, MDA – malondialdehyde, TPC – total phenolic content, SA – salicylic acid, HSA – SA glicosides. Response variable Two-way ANOVA One-way ANOVA explanatory variable Interactiona separate test for each timepoint Endpoint concentration week concentration x week w4 w8 w11 dry weight F4 = 0.6; ns F2 = 537.1; P < 0.001 F8 = 2.0; P < 0.05 ns ns ns shoot height F4 = 3.9; P < 0.01 F2 = 419.7; P < 0.001 F8 = 0.8; ns P < 0.05 ns ns dry weight % F4 = 9.6; P < 0.001 F2 = 39.5; P < 0.001 F8 = 2.3; P < 0.05 P < 0.001 ns P < 0.05 specific leaf area F4 = 7.1; P < 0.001 F2 = 106.2; P < 0.001 F8 = 2.2; P0.05 P < 0.001 ns P < 0.05 necrotic tissue % F4 = 4.8; P < 0.01 F2 = 375.2; P < 0.001 F8 = 1.5; ns ns ns P < 0.05 nr leaves F4 = 11.2; P < 0.001 F2 = 587.4; P < 0.001 F8 = 2.6; P < 0.05 ns P < 0.05 P < 0.001 C F4 = 7.9; P < 0.001 F2 = 3.4; ns F4 = 1.3; ns - P < 0.001 ns N F4 = 19.1; P < 0.001 F2 = 241.7; P < 0.001 F4 = 3.8; P < 0.01 - P < 0.05 P < 0.001 C:N ratio F4 = 25.3; P < 0.001 F2 = 196.0; P < 0.001 F4 = 13.7; P < 0.001 - ns P < 0.001 Chl a F4 = 25.8; P < 0.001 F2 = 799.4; P < 0.001 F8 = 125.5; P < 0.001 P < 0.01 P < 0.001 P < 0.001 Chl b F4 = 3.4; P < 0.05 F2 = 63.2; P < 0.001 F8 = 6.6; P < 0.001 ns P < 0.01 P < 0.001 total Chl F4 = 22.2; P < 0.001 F2 = 622.5; P < 0.001 F8 = 81.6; P < 0.001 P < 0.01 P < 0.001 P < 0.001 SOD F4 = 170.3; P < 0.001 F2 = 68.9; P < 0.001 F8 = 25.5; P < 0.001 P < 0.001 P < 0.001 P < 0.001 CAT F4 = 110.9; P < 0.001 F2 = 1.0; ns F8 = 11.5; P < 0.001 P < 0.001 P < 0.001 P < 0.001 MDA F4 = 27.6; P < 0.001 F2 = 143.7; P < 0.001 F8 = 3.4; P < 0.01 P < 0.001 P < 0.001 P < 0.001 TPC F4 = 372.8; P < 0.001 F2 = 5.3; P < 0.01 F8 = 257.6; P < 0.001 P < 0.001 P < 0.001 P < 0.001 SA F4 = 40.6; P < 0.001 F2 = 546; P < 0.001 F8 = 25.8; P < 0.001 P < 0.001 P < 0.001 P < 0.001 HSA F4 = 257.8; P < 0.001 F2 = 643.4; P < 0.001 F8 = 16.2; P < 0.001 P < 0.001 P < 0.001 P < 0.001 S. Adamczyk et al. Environmental Pollution 363 (2024) 125307 6 Fig. 2. Content of carbon (C) (a) and nitrogen(N) (b), percentage, and C-to-N ratio (c) in lettuce following exposure to polybutylene adipate terephthalate biode gradable microplastics (PBAT-BD-MPs) in CLIMECS system. Results are presented as median, interquartile range and the standard deviation including outliers; Lowercase letters indicate significant differences between treatments (p < 0.05) based on Tukey’s HSD test (Holms adj.p). S. Adamczyk et al. Environmental Pollution 363 (2024) 125307 7 Fig. 3. Concentration of chlorophyll in lettuce following exposure to polybutylene adipate terephthalate biodegradable microplastics (PBAT-BD-MPs) in CLIMECS system: a) chlorophyll a, b) chlorophyll b, and c) total chlorophyll. FW – fresh weight. Results are presented as median, interquartile range and the standard deviation including outliers; Lowercase letters indicate significant differences between treatments (p < 0.05) based on Tukey’s HSD test (Holms adj.p). S. Adamczyk et al. Environmental Pollution 363 (2024) 125307 8 weeks of exposure may also emerge from the downregulation of the phenylpropanoid biosynthesis pathway by the PBAT-BD-MPs (Yang and Gao, 2022). The phenylpropanoid biosynthesis pathway is activated under abiotic stress leading to the production of various phenolic com pounds (Sharma et al., 2019). Salicylic acid was also included to disentangle to effect of PBAT-BD- MPs on lettuce resilience, as it plays a central role in stress signalling (Janda et al., 2020). The SA concentration in the lettuce leaves was initially elevated after 4 weeks of exposure to PBAT-BD-MPs, correlating positively with other elements of plant defense mechanisms (enzymatic and TPC). This is in line with the available literature showing that SA enables plants to cope with stress by regulation of antioxidant enzyme activities (Souza et al., 2017), and the activity of phenylalanine ammonia-lyase, a key enzyme in the biosynthesis of phenolic com pounds (Janda et al., 2020). Taken together, PBAT-BD-MPs significantly affected all studied biomarkers of plant defense clearly underlining that these MPs are not inert to plant health in near-field concentrations. 5. Conclusions This study investigates the effects of PBAT-BD-MPs on lettuce growth, physiology and biochemistry in near-field conditions. Exposure of lettuce to PBAT-BD-MPs induced plant oxidative stress and activated plant defense mechanisms leading to oxidative homeostasis and sus taining plant growth and development, though some negative effects on plant growth were observed, mainly after the longest exposure duration to the highest PBAT-BD-MP concentration (0.8 %) under the conditions of the experiment. To conclude, PBAT-BD-MPs though does not affect lettuce growth much, do affect lettuce metabolism and resilience and these aspects should be further explored. Following steps in under standing the effect of PBAT-BD-MPs on plant health should include extrapolation to field conditions, experiments with a wide set of plant species and experiments with numerous stressors, better reflecting nat ural conditions. CRediT authorship contribution statement Sylwia Adamczyk: Writing – original draft, Methodology, Investi gation, Formal analysis, Data curation, Conceptualization. Laura J. Zantis: Writing – review & editing, Methodology, Investigation, Conceptualization. Sam van Loon: Writing – review & editing, Meth odology, Conceptualization. Cornelis A.M. van Gestel: Writing – re view & editing, Validation, Supervision, Conceptualization. Thijs Bosker: Writing – review & editing, Supervision, Conceptualization. Rachel Hurley: Writing – review & editing, Project administration. Luca Nizzetto: Writing – review & editing, Validation, Funding acqui sition. Bartosz Adamczyk: Writing – review & editing, Methodology, Investigation, Conceptualization. Sannakajsa Velmala: Writing – re view & editing, Visualization, Formal analysis, Conceptualization. Declaration of competing interest No conflict of interest. (caption on next column) Fig. 4. Performance of plant defense systems in lettuce following exposure to polybutylene adipate terephthalate biodegradable microplastics (PBAT-BD- MPs) in CLIMECS system: a) specific activity (U mg − 1 protein) of superoxide dismutase, SOD, b) activity of catalase, CAT, c) concentration of malondialde hyde, MDA, Eq = equivalents d) total phenolic content, TPC, e) salicylic acid, SA, and f) SA-glycosides, HSA. FW = fresh weight. Results are presented as median, interquartile range and the standard deviation including outliers; Lowercase letters indicate significant differences between treatments (p < 0.05) based on Tukey’s HSD test (Holms adj.p). S. Adamczyk et al. Environmental Pollution 363 (2024) 125307 9 Acknowledgements This study is supported by funding from the European Union’s Ho rizon 2020 research and innovation programme under grant agreement No 101000210, project PAPILLONS – Plastic in Agricultural Production: Impacts, Life-cycles and LONg-term Sustainability. Sylwia Adamczyk was funded by The Research Council of Finland (decision nr 360978). The authors thank Henna Pasanen and Tuija Hytönen (Natural Re sources Institute Finland, Luke) for technical support in the laboratory and Prof. Matty Berg and Lotte de Jeu (Vrije Universiteit, The Netherlands) for assistance in running the CLIMECS experiments. We’d also like to thank Dr Nils Borchard for his contribution to the funding of this project. 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Environmental Pollution 363 (2024) 125307 11 https://doi.org/10.1098/rstb.2008.0284 https://doi.org/10.1016/j.chemosphere.2020.126791 https://doi.org/10.1016/j.chemosphere.2020.126791 https://doi.org/10.1080/01904160802135092 https://github.com/taiyun/corrplot http://refhub.elsevier.com/S0269-7491(24)02024-4/sref44 http://refhub.elsevier.com/S0269-7491(24)02024-4/sref44 https://doi.org/10.1016/j.scitotenv.2022.154579 https://doi.org/10.1016/j.scitotenv.2022.154579 https://doi.org/10.1016/j.soilbio.2021.108179 https://doi.org/10.1016/j.soilbio.2021.108179 https://doi.org/10.1016/j.scitotenv.2022.161211 https://doi.org/10.1016/j.envpol.2023.122243 https://doi.org/10.1016/j.envpol.2023.122243 https://doi.org/10.1007/s11368-023-03479-x Sylwia 1-s2.0-S0269749124020244-main Biodegradable microplastics induce profound changes in lettuce (Lactuca sativa) defense mechanisms and to some extent deter ... 1 Introduction 2 Materials and methods 2.1 CLIMECS system 2.2 Test soil and PBAT-BD-MPs 2.3 Plants 2.4 Earthworms and springtails 2.5 Experimental conditions 2.6 Seedling sampling and measuring growth traits 2.7 Chlorophyll content 2.8 Activity of superoxide dismutase (SOD) and catalase (CAT) 2.9 Lipid peroxidation 2.10 Total phenolic content 2.11 Salicylic acid and salicylic acid glucosides 2.12 Statistics 3 Results 3.1 Growth traits 3.2 Chlorophyll 3.3 Superoxide dismutase (SOD) and catalase (CAT) activities 3.4 Lipid peroxidation (MDA concentration) 3.5 Total phenolic content (TPC) 3.6 Salicylic acid (SA) and SA-glycosides (HSA) 3.7 Correlations, principal components and correspondence analysis 4 Discussion 5 Conclusions CRediT authorship contribution statement Declaration of competing interest Acknowledgements Appendix A Supplementary data datalink4 References