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Article

Combined Effects of Polyethylene and Bordeaux Mixture on the Soil–Plant System: Phytotoxicity, Copper Accumulation and Changes in Microbial Abundance

by
Silvia Romeo-Río
1,2,
Huguette Meta Foguieng
2,3,
Antía Gómez-Armesto
1,2,*,
Manuel Conde-Cid
1,2,
David Fernández-Calviño
1,2 and
Andrés Rodríguez-Seijo
1,2
1
Área de Edafoloxía e Química Agrícola, Departamento de Bioloxía Vexetal e Ciencia do Solo, Facultade de Ciencias, Universidade de Vigo, As Lagoas s/n, 32004 Ourense, Spain
2
Instituto de Agroecoloxía e Alimentación (IAA), Universidade de Vigo, Campus Auga, 32004 Ourense, Spain
3
Departamento de Educación y Divulgación Ambiental, Facultad de MedioAmbiente, Universidad Nacional de Guinea Ecuatorial (UNGE), Malabo 661, Equatorial Guinea
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(15), 1657; https://doi.org/10.3390/agriculture15151657
Submission received: 30 June 2025 / Revised: 18 July 2025 / Accepted: 29 July 2025 / Published: 1 August 2025
(This article belongs to the Special Issue Impacts of Emerging Agricultural Pollutants on Environmental Health)
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Abstract

Greenhouses have positively impacted plant production by allowing the cultivation of different crops per year. However, the accumulation of agricultural plastics, potentially contaminated with agrochemicals, raises environmental concerns. This work evaluates the combined effect of Bordeaux mixture and low-density polyethylene (LDPE) microplastics (<5 mm) on the growth of lettuce (Lactuca sativa L.) and soil microbial communities. Different levels of Bordeaux mixture (0, 100 and 500 mg kg−1), equivalent to Cu(II) concentrations (0, 17 and 83 mg kg−1), LDPE microplastics (0, 1% and 5%) and their combination were selected. After 28 days of growth, biometric and photosynthetic parameters, Cu uptake, and soil microbial responses were evaluated. Plant germination and growth were not significantly affected by the combination of Cu and plastics. However, individual Cu treatments influenced root and shoot length and biomass. Chlorophyll and carotenoid concentrations increased with Cu addition, although the differences were not statistically significant. Phospholipid fatty acid (PLFA) analysis revealed a reduction in microbial biomass at the highest Cu dose, whereas LDPE alone showed limited effects and may reduce Cu bioavailability. These results suggest that even at the highest concentration added, Cu can act as a plant nutrient, while the combination of Cu–plastics showed varying effects on plant growth and soil microbial communities.

1. Introduction

Plastic pollution in terrestrial ecosystems has been a growing concern over the last decade due to its widespread distribution across all terrestrial ecosystems. Once plastics have lost their usefulness, they can easily reach terrestrial and aquatic ecosystems, where they are exposed to different physicochemical and biological factors (e.g., UV radiation, mechanical abrasion, temperature changes, microbial degradation, etc.), facilitating their degradation [1]. These processes can take centuries, although under certain conditions, plastic waste can degrade more rapidly, leading to the formation of meso- or macroplastics (>5 mm), which eventually break down into fragments of irregular shapes and smaller sizes (<5 mm), known as microplastics (MPs) and nanoplastics (NPs) (<0.1 µm). These MPs are the most common form of plastic debris in different ecosystems, mainly found as fibres, films, or pellets, and they come in diverse sizes and shapes. Once they reach the soil and start to degrade, they can impact it in multiple ways, both affecting soil physicochemical properties and organisms, with long-term effects on ecosystem functions [1,2,3,4,5].
Once plastics reach the soil, they are not chemically inert. As they degrade, they can become pollutants due to the release into the environment of additives added during their production to improve their properties or to catalyse their degradation. Due to their porous nature, they can also absorb different contaminants, act as carriers of contaminants, and release these attached contaminants into the medium, potentially causing adverse effects on biota [5,6,7,8]. This issue is of particular concern in agricultural ecosystems due to the widespread use of plastics in agriculture and animal husbandry (greenhouse covers, mulch, silage plastic, irrigation systems, etc.) and the short life cycle of plastics, ranging from a few months (e.g., mulch) to 3–5 years, as in the case of greenhouse covers. Additionally, plastic debris can break down after use (through breakage, fragmentation, abrasion, etc.) and be easily incorporated into the soil, so their recovery and recycling are not always possible. In addition, they also present other problems as they can be contaminated by different agrochemicals used as fungicides, pesticides, antibiotics, or hydrocarbons, etc., making their reuse and recycling impossible [7,9,10].
In this sense, MPs’ impact is highly dependent on shape (greater as fibres than as film or pellet), polymer type (e.g., polypropylene has a greater adverse effect than polyethylene), concentration (greater impact at concentrations above 3%), or target organisms as in the case of crops (e.g., greater impact on monocotyledons than on dicotyledons). In any case, the effects of microplastics can vary across different physical, chemical, and biological properties of soils, as well as on plant growth [5,11,12,13]. Regarding plants, some contradictory effects have been reported, depending on the type of polymer, size and concentration used, with variability in the impact on root growth, the aerial part, and the level of oxidative stress [14]. It is not expected that MPs (<5 mm) can be taken up by roots, although they can be taken up in sizes less than 100 µm. The fact that they impact soil porosity or increase water-holding capacity may favour plants by providing a greater amount of water and promoting root development. However, the presence of MPs in soils can also negatively affect nutrient uptake and/or bioavailability of existing soil contaminants since MPs in soils can affect the bioavailability of potentially toxic elements, modify metal bioaccumulation or impact on soil microbiota [12,13,15,16,17,18,19].
Microplastic impacts on soil enzymatic activities (increase or decrease) are often contradictory, while the richness and diversity of soil bacterial and fungal communities generally decrease [20,21,22,23]. Furthermore, some works have indicated that plastic contamination can impact microbial communities, as studied through the analysis of phospholipid fatty acids (PLFAs) [24,25,26], a common technique used to detect changes in soil microbial communities with increasing levels of inorganic and organic contaminants [27,28].
Bordeaux mixture (BM) is a widely used agrochemical due to its fungicidal properties in different crops, including those cultivated in greenhouses. However, its mismanagement has several impacts on soil and water ecosystems due to wide and indiscriminate application. Spraying BM on plants can also reach soil or agricultural plastics, such as mulching films used in agriculture. For these reasons, we conducted an experiment with microplastics and Cu applied as BM to assess a potential synergistic impact on soil microbiota and higher plants (lettuce). This study aims to elucidate how polyethylene MPs influence Cu(II) bioavailability, plant growth, Cu uptake and the impact of contamination on the microbial structure.

2. Materials and Methods

2.1. Contaminants and Test Soil

An artificial OECD soil (70% sand, 20% clay and 10% peat; pH 6.0 ± 0.5 and 5% organic matter) [29] was used as the test soil to avoid background Cu or plastic contamination. Before the assay, the soil pH (pHH2O and pHKCl) and electrical conductivity were measured in a 1:5 soil–water suspension. Soil moisture was measured by oven drying at 105 °C until a constant weight. The organic matter content was measured according to the loss-on-ignition method (450 °C, 4 h) to meet the OECD soil specifications [30].
White low-density polyethylene (LDPE) [(C2H4)n] (specific density = 0.92 g cm−3) was used due to its wide use in agriculture, especially in mulch film applications for preventing weed growth and preserving soil moisture [31,32]. The plastic was purchased as a film from a commercial hardware store and left in the field for three months to undergo partial weathering and simulate more realistic conditions. Once in the laboratory, the plastic film was manually cut with scissors to obtain fragments with different morphologies and diameters less than 5 mm [6]. Three concentrations were used: 0% (no microplastics added), 1% to simulate a low concentration, and 5% to simulate a medium concentration of microplastics in soils [33].
Different concentrations of LDPE and Cu as Bordeaux mixture (BM) were used individually or in combination to assess their effects on plant growth. Therefore, LDPE was applied at three concentrations (0%, 1% and 5% MP), whereas Bordeaux mixture (BM) was added at concentrations of 0 mg kg−1, 100 mg kg−1 and 500 mg kg−1. The concentrations of BM—used in the form of CuSO4·3Ca(OH)2 [31]—were equivalent to 0, 17 and 80 mg kg−1 of Cu(II), respectively, which are typical concentrations reported by different works on soils with continued application of BM [16,34,35,36].

2.2. Soil Contamination

Bordeaux mixture was diluted in distilled water, which was then sprayed and mixed into the soil to obtain concentrations of 100 and 500 mg kg−1. The Cu-contaminated soils were maintained at 25 ± 2 °C for 7 days to allow uniform distribution in the soil. Microplastics were also incorporated into the soil and mixed, then stored for an additional 7 days under the same conditions. Before plastic incorporation into the soil, plastics were decontaminated with deionised water and 70% ethanol and were dried overnight on aluminium foil at 40 °C [6]. In all cases, soil moisture was regulated daily. A multifactorial trial was conducted, evaluating nine experimental conditions that tested different combinations of BM and LDPE: Control (0 BM and 0 LDPE), BM (100 and 500 mg kg−1), LDPE (1 and 5% w/w), and the interaction of both contaminants (Table 1).

2.3. Seed Germination and Plant Growth Tests

To test the potential toxicity effects of Cu and MPs on plants (lettuces) and to assess metal bioaccumulation and phytotoxicity, a seedling emergence and growth test (Lactuca sativa L. cv Merveille de Quatre Saisons supplied by Semillas Clemente S.A., Vitoria, Spain) was performed in 2023, following the OECD 208 guidelines [29]. Before the beginning of the assay, seeds purchased from a local supplier were surface sterilised [10 min in 70% (v/v) ethanol; 7 min in 20% (v/v) sodium hypochlorite (5% (v/v) active chlorine) containing 0.05% (w/v) Tween-20] and washed thoroughly with several rinses of deionised water before planting. After disinfection, ten seeds were placed in each experimental pot (1 L) with approximately 800 g of soil for the nine experimental treatments described in Section 2.2. The amount of deionised water required to adjust the water-holding capacity (WHC) of the soil to 45% was used to moisten the soil at the start of the test. Three replicates were performed per experimental condition (3 × 9). Each test vessel had different holes for water drainage. The water level in each test vessel was adjusted as needed to maintain soil moisture. At the beginning of the test, a commercial fertiliser [N: 0.7%; P2O5: 0.5%; K2O: 0.6%; B (17 ppm), Cu (45 ppm), Fe (300 ppm), Mn (85 ppm), Mo (2.5 ppm) and Zn (100 ppm)] was added to each test vessel and was previously diluted according to the manufacturer’s recommendation. The pots were maintained under constant conditions of temperature (20 ± 2 °C), photoperiod (16 h Light: 8 h Dark), humidity (60%) and light intensity (180 µmol m−2 s−1). The trial started when at least 50% of the seeds of the control treatment germinated [30]. To avoid intraspecific competition, only the first three germinated seeds were left to grow in each pot; the remaining ones were counted and harvested. During the exposure period (28 days), the water level was adjusted every two days with deionised water to guarantee the necessary soil moisture conditions. At the end of the experiment, plants were used for the evaluation of plant growth parameters (biomass, root, and shoot length).

2.4. Post-Harvest Treatment: Determination of Soil Properties and Plant Growth Parameters

At the end of the exposure period, the soil on which the plants were grown was air-dried for two weeks and sieved through a 2 mm sieve. The soil pH (pHH2O and pHKCl), moisture, and organic matter content were then measured according to the methods mentioned above. Additionally, a DTPA (diethylenetriaminepentaacetic acid) soil extraction method (0.005 M DTPA + 0.1 M triethanolamine + 0.01 M CaCl2 at pH 7.3) was used as a predictor of metal bioavailability and ecotoxicity (1/2 soil-to-extractant ratio, 2 h shaking, 120 rpm) [37]. The DTPA-extractable Cu contents were measured by ICP-OES (iCAP PRO XP Duo, Thermo Scientific, Waltham, MA, USA) at CACTI (Ourense, Universidade de Vigo, Spain).
Plants were thoroughly washed several times, first with tap water and then with distilled water, to remove as many soil particles as possible from their surface. After that, root and shoot lengths were recorded for each treatment in each replicate. Afterwards, roots and leaves from each plant in three experimental replicates were dried in an oven at 60 °C until a constant mass was reached. The dry roots and shoots were weighed to calculate dry biomass, and the root–shoot ratios for dry biomass were also calculated.

2.5. Cu Bioaccumulation

After weighing the dry biomass, the Cu bioaccumulated in roots and shoots was determined by acid extraction. Specifically, 0.2 g of each plant part was weighed and added to Pyrex tubes with 7 mL of HNO3 (65% v/v). Then, the digestion was carried out on a hot plate (105 °C) by two heating cycles, and once the digestion process was completed, the sample was filtered (0.45 µm syringe filters), transferred, and diluted to a final volume of 25 mL in a volumetric flask. Cu contents were determined by ICP-OES. The transfer factor between the Cu content in shoots (Cshoots) and roots (Croots) was calculated as TF = Cshoots/Croots, where TF > 1 indicates effective translocation of elements from the root to the shoot [38].

2.6. Quantification of Photosynthetic Pigments

The extraction and quantification of photosynthetic pigments (total chlorophylls and carotenoids) were performed in frozen leaf aliquots (approximately 200 mg) based on the protocol of Lichtenthaler [39]. The results were expressed in mg of chlorophyll a, b, total chlorophylls and carotenoids per g−1 fresh mass (f.m.) [30]. The ratios of chl a:b, chl a–carotenoids and chl–carotenoids were also calculated as indicators of plant response to stress [40,41,42].

2.7. Microbial Community Abundance

The abundance of microbial groups was estimated by phospholipid fatty acid (PLFA) analysis [43]. Briefly, lipids were extracted from soils by weighing 2 g (dry weight) of soil with a chloroform/methanol/citrate buffer mixture (1:2:0.8 v/v/v) and separated into neutral lipids, glycolipids, and phospholipids using a prepacked silica column. Phospholipids were then subjected to mild alkaline methanolysis, and the resulting fatty acid methyl esters were identified by gas chromatography. A total of 32 different PLFAs were identified and quantified. The PLFAs were designated in terms of the total number of carbon atoms and double bonds, followed by the position of the double bond from the methyl end of the molecule. Furthermore, cis and trans configurations are indicated by “c” and “t”, respectively. The prefixes “a” and “i” indicate anteiso- and iso-branching positions, “br” indicates unknown methyl-branching positions, “Me” indicates a methyl group on the tenth carbon atom from the carboxyl end of the molecule, and “cy” refers to cyclopropane fatty acids. The abundance of different microbial groups was estimated following Joergensen [44]: Firmicutes: i14:0, i15:0, i16:0a, i17:0, i18, a15:0, a16:0, a17:0, a18:0, a19:0; Actinobacteria: 10Me16:0, 10Me17:0, 10Me18:0; Gram-positive (G+) bacteria: Firmicutes + Actinobacteria; Gram-negative (G-) bacteria: cy17:0, cy19:0, 16:1ω7, 16:1ω9, 17:1ω8, 18:1ω7; Bacteria: G+ plus G-; Arbuscular mycorrhiza fungi (AMF): 16:1ω5c; Zygomycota: 18:1ω9c; Ascomycota and Basidiomycota: 18:2ω6c; Unspecific fungal PLFA: 18:3ω6,9,12; Fungi: AMF + Zygomycota + Ascomycota and Basidiomycota + unspecific fungal; Unspecific microbial PLFA: 14:0, 15:0, 16:0, 17:0, 18:0, 20:0, 20:4ω6,9,12,15; Total microbial PLFA: bacterial + fungal + unspecific microbial.

2.8. Statistical Analysis

All analyses are presented as the mean ± standard deviation (SD) based on three experimental replicates (n = 3). Data were subjected to a one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test when significant differences were found (p < 0.05). All statistical analyses were performed with GraphPad Prism 8 (GraphPad software, San Diego, CA, USA).

3. Results and Discussion

3.1. Impact of BM and MPs Contamination on Soil Properties After Plant Growth

Figure 1a–d show the evolution of soil properties after plant growth. No significant differences in pHH2O or pHKCl were observed between treatments (Figure 1a,b), nor in organic matter content. However, soil moisture was significantly reduced in all contaminated soils compared with the control treatment (p < 0.05). There were no significant differences between the contaminated soil samples (with BM and/or plastics).
It is widely known that MPs can affect soil moisture content, especially at larger sizes (>3–5 mm), as MPs increase the soil water evaporation, soil drying, and cracking [4,12,45]. In our case, both contaminants exhibited similar behaviour in terms of soil moisture, with a significant reduction compared to the control treatment (p < 0.05), which aligns with [46] for lettuce pots exposed to polyethylene microplastics (1–10%).

3.2. Impact of BM and MPs Contamination on Plant Growth Parameters

Plant germination was significantly reduced for all treatments where BM was added individually or in combination with 1% and 5% of plastic (p < 0.05) (Figure 2a). When plastics were added individually, at 1% and 5%, reduced germination was observed, but differences were not significant (p > 0.05). Root length was not significantly affected in any contaminated soil compared to the control, but was higher under the 5%LDPE + BM100 treatment compared to the BM100 treatment (Figure 2b). Shoot length was significantly reduced under all contaminated treatments, whether applied individually or in combination, compared with the control (no addition) (Figure 2c), but no significant differences were observed among them. The same pattern was observed for root dry biomass (Figure 2d). When assessing the shoot dry biomass, all treatments had significant differences with the control treatment except for the BM500 treatment (Figure 2e).
The mean R:S ratio based on dry weight was below 1 for all treatments, and lower than the control in all contaminated treatments (in most cases, statistical differences were found). The lower values were observed for Cu and LDPE when individually added (BM100, BM500 and 5%LDPE), and for combined application of 1%LDPE + BM100 and 5%LDPE + BM500 (Figure 3a). Regarding the R:S ratio based on length, statistical differences between treatments were also recorded, with higher ratios being found at 1%LDPE, followed by 5%LDPE + BM100, 1%LDPE + BM100 and 5%LDPE + BM500 (Figure 3b).
The observed results for plant growth were similar to those reported by other studies for Cu and microplastics. Although Cu is an essential element for plants, it can have adverse effects on plant growth at relatively high concentrations, including negative impacts on germination, root growth and aboveground length and biomass [47,48]. Similar results have also been reported for microplastics, as germination can be delayed or reduced, although not completely inhibited [49,50,51,52]. Regarding plant growth, some authors reported differences between polymer types, from potential increased biomass when exposed to LDPE, Polypropylene (PP) or Polyvinyl chloride (PVC) [53,54], to significant reduction when exposed to other polymers [50,52].
Some studies have shown that microplastic contamination could improve plant growth, especially at the root level, mainly due to a reduction in bulk density and favouring root penetration. Li et al. [53] observed that roots from lettuce exposed to 0–2% PVC were significantly increased, both in length and root volume. A similar pattern was also observed by [46] for lettuce exposed to 1–10% LDPE.
Plants exposed to both contaminants showed reduced R:S by dry weight (p < 0.5) (Figure 3a), but high R:S by length (>2) (Figure 3b), indicating that roots are relatively long but thin compared to the shoot system. The root–shoot (R:S) ratio represents the plants’ ability to compensate for limiting resources in the environment, and thus to survive and succeed. In recent decades, it has been suggested as a sensitive indicator of plant stress induced by physical and chemical agents, and it can be estimated in the form of dry weight (dw) (Rdw/Sdw ratio) or by length (Rlength/Slength ratio) [55]. A root–shoot ratio (R:S) between 1 and 2 is considered optimal, while high ratios indicate high absorption and water storage as a conservative plant hydration system by reducing evaporative leaf area, while the root system is highly developed to maintain plant water storage [56,57]. Poorter et al. [58] explained that plants with a low nutrient supply have an increased allocation to roots, resulting in higher R:S ratios as a plant’s growth strategy under the different stressors, to maximise surface area and absorb water or nutrients. This parameter has not been widely studied for microplastics, and there is no clear tendency. Some authors [3,59,60] also reported an increase in the R:S ratio by biomass under plastic contamination, and especially under water-stress conditions. However, [50,61] also indicated that under plastic contamination, root elongation was promoted, increasing plant biomass by reducing the R:S biomass ratio, as reported in our results. In fact, there are no differences between control treatment and combined contamination with 1%LDPE, 5%LDPE and Cu, which suggests that plastics in soils mitigate the potential stress induced by Cu contamination (Figure 3a). Overall, the shoot biomass, and therefore crop yield, was significantly affected by LDPE fragments, both individually and combined with Cu; these results are aligned with those reported by [62,63].

3.3. Impact of BM and MPs on Metal Bioavailability and Plant Bioaccumulation

As observed in Figure 4a, Cu(II) bioavailability increased with higher BM doses. Interestingly, Cu in soil appears to be more bioavailable when a higher amount of plastic is added. Regarding the behaviour of Cu in plant roots, it was highly taken up when BM was applied individually (Figure 4b), followed by combined treatments of 5% plastic and BM (both at 100 and 500 mg kg−1). The Cu content in shoots followed a similar pattern (Figure 4c) with higher Cu contents at the highest BM dose (BM500 mg kg−1) and under higher plastic dose (5%LDPE and BM500 mg kg−1). However, Cu(II) was highly translocated under lower doses of BM and plastics, followed by 1%LDPE + BM500 (Figure 4d).
In general, the observed results align with those reported by different authors. Polyethylene in soils can increase the bioavailability of heavy metals (e.g., Cu, Cd, etc.), and enhance their accumulation by soil organisms, such as earthworms [64] and plants [65,66,67]. Microplastics can interact with heavy metals in soils, increasing plant uptake from the soil due to their hydrophobic properties and large specific surface area. Moreover, they can promote metal mobility in soils due to changes in soil pH, and therefore, influence their bioavailability in the soil–plant system [65,68]. This is also consistent with the DTPA-extractable Cu(II) concentrations observed at the highest plastic doses, which are similar to those reported by [68] for Cd and wheat.

3.4. Impact of BM and MPs Contamination on Plant Photosynthetic Performance

Individual or combined exposure of Cu and plastics led to a significant reduction in chlorophylls (-a, -b and total content) and carotenoids (Figure 5a–d). In both chlorophylls and carotenoids, BM applied alone had less impact on photosynthetic performance than plastics, with the 5%LDPE treatment showing the lowest content of chlorophylls and carotenoids. Interestingly, when both contaminants were combined, a potential mitigating effect was observed, as indicated by an increase in pigment content at BM 100 mg kg−1 combined with 1% and 5% LDPE, especially in the 1%LDPE + BM100 and 5%LDPE + BM500 treatments. However, this pattern was not observed with the lower plastic dose and the highest Cu concentration (1%LDPE + BM500), where the adverse effects were similar to those of the highest plastic dose alone (5%LDPE).
The chlorophyll a:b (Figure 5e), chlorophyll a–carotenoids (Figure 5f) and chlorophyll–carotenoid (Figure 5g) ratios were also determined. These ratios are widely used as indicators of plant acclimation to environmental stress. Under non-stressed conditions, chlorophyll a usually dominates, resulting in a higher chl a:b ratio [69], while a low chlorophyll to carotenoid ratio can indicate excess light and/or oxidative damage, such as from contaminants or nutrient starvation [70]. In our case, the highest ratios were always observed when Cu was added individually (BM100 followed by BM500 mg kg−1) or combined (5%LDPE + BM100), suggesting a potential enhancement of chlorophyll production. However, in general, no significant differences were observed between treatments and the control (Figure 5e–g).
The obtained results align with the existing literature. Although Cu is an essential nutrient for plants, increased Cu levels in soils and plant tissue can adversely affect photosynthetic pigment biosynthesis by reducing chlorophyll and carotenoid content [71,72]. In fact, the reduction in Chl under Cu stress could be attributed to the increased activity of photosynthetic enzymes and the instability of the pigment–protein complex, as Cu(II) can replace Mg(II) in chlorophyll molecules [73]. In this sense, pigment levels decreased as BM concentrations increased. Interestingly, the reduction in Chl-b under BM500 cannot be fully explained, as Chl-b is less directly involved in electron transfer than Chl-a, the primary light-harvesting pigment [74].
The impact of microplastics on photosynthetic pigments ranges from no effects [53,75] to significant reductions [52,63,75]. In general, plastic contamination affects photosynthesis through direct and indirect mechanisms. When nanoplastics are taken up by roots, oxidative stress effects are expected; in contrast, larger particles like those used in this study are more likely to cause indirect impacts via soil structure changes, reduced water and nutrient uptake due to pore clogging, or alterations in microbial communities [76]. In our case, a reduction in Chl-a and Chl-b was observed when plastics were incorporated alone (Figure 5a–c). According to [77], Chl-b production is inhibited under plastic-induced stress.
When both contaminants were added, a slight increase in photosynthetic parameters was observed, especially under treatments with BM100 and BM500 (17 mg Cu(II) kg−1 and 80 mg Cu(II) kg−1, respectively) and the highest dose of plastics (5%), suggesting a potential protective effect of plastics that could modulate the potential Cu toxicity, possibly by adsorbing Cu and controlling its release to plants, as plastics can act as metal carriers [78]. However, the mechanisms by which MPs induce toxicity in plant growth and development, including oxidative stress pathways, remain unclear, and further research is needed [79].
The ratio of total chlorophyll (Chl) and total carotenoids is used to assess plant senescence. Typically, Chl a:b ratios range from 2.6 to 3.3 in C3 plants such as L. sativa, which matches our findings. When exposed to 100 mg kg−1 BM, the highest ratio observed suggests that Cu may be acting as a nutrient, balancing pigment production [40,41]. Trifunović-Momčilov et al. [73] indicate that a total Chl–carotenoids ratio below 4.2 in sunlight-exposed plants may indicate damage to the photosynthetic apparatus, which is expressed by a faster breakdown of Chl than carotenoids. Although values were slightly lower (around 4) across all treatments, even in the control, they remained above 3.5, indicating senescence. In our case, a slight increase in this ratio was observed under mixed contamination (Figure 5e), suggesting that Cu may alleviate MP-induced stress in plants, particularly when Chl-b is inhibited by MP stress [79]. In any case, Jia et al. [79] also reported no clear trend regarding microplastics’ impact on the chl–carotenoids ratio and recommend measuring oxidative stress parameters (e.g., SOD, catalase, GSH, MDA) to obtain a holistic understanding of combined contaminant effects on plants.

3.5. Impact of BM and MPs Contamination on Soil Microbial Structure

Phospholipid fatty acids (PLFAs) were used in the present work as indicators of microbial biomass and for assessing the microbial community structure. Since PLFAs provide a general picture of the living microorganisms at the moment of sampling, they are a useful indicator for detecting rapid changes in soil environmental quality and health [43,80,81]. Here, we assessed the synergistic effects on soil microorganisms resulting from the addition of both Cu and LDPE to agricultural soil where lettuce cultivation was carried on. In this context, numerous studies have evaluated the individual responses of microbial communities to heavy metals through PLFAs [82,83,84,85] and to microplastics [86,87,88,89]. However, to the best of our knowledge, no previous study has investigated changes in soil microbial composition via PLFAs associated with the combined effect of both contaminants.
In the present work, we obtained a total microbial biomass in the control, measured as total PLFA content, of 59.3 ± 11.2 nmol g−1 (Figure 6). We did not find significant differences compared to the different metal–MPs treatments, except for the BM500 dose, where the total PLFA content significantly decreased compared to the control (41.4 ± 1.4 nmol g−1). In this sense, the toxicity of Cu to microorganisms, especially Fungi, has been widely recognised because of its fungicidal properties [85,90]. Beyond Cu, the most influential variable on microbial communities is soil pH. However, no significant changes were observed in pH across the different metal–MP doses, indicating that almost all the variation in PLFAs at the BM500 dose could be attributed to Cu toxicity. Furthermore, the available Cu fraction is more relevant for microbial toxicity than total Cu content [85]. In the case of the BM500 dose, a high proportion of copper was in the available form, as indicated by the DTPA extraction (about half of the total Cu(II) added as Bordeaux mixture; see Figure 4a).
We did not find significant differences between the control and BM100 dose (17 mg Cu(II) kg−1), which is in accordance with some studies reporting considerable microbial biomass reductions at concentrations higher than 20–60 mg kg−1 [91]. On the contrary, other authors identified Cu toxicity threshold values as high as 1000 mg kg−1 based on PLFA profiles [92]. In fact, [90] reported a stimulatory effect of Cu on microbial biomass at a concentration of 177 mg kg−1. This demonstrates that Cu toxicity depends not only on dose but also on other soil properties such as pH, organic matter content, or C/N ratio.
In terms of microbial group-specific PLFAs, we also observed a significant reduction across all groups at the BM500 dose, as shown in Figure 7 (bacterial groups: Firmicutes, Actinobacteria, Gram-positive and Gram-negative) and Figure 8 (fungal groups: Arbuscular Mycorrhizal Fungi, Zygomycota, Ascomycota and Basidiomycota). Regarding Firmicutes, there were significant increases in PLFA content in the 5%LDPE and 5%LDPE + BM500 treatments. These results, although somewhat unexpected, could be explained by a protective effect of LDPE microplastics against Cu toxicity. In this sense, Cu could be adsorbed onto the negatively charged MPs surfaces, thereby limiting its bioavailability and toxicity to microorganisms [93].
In general, as reported in multiple studies, the sole effect of MPs over soil microorganisms tends to have no impact or slightly increases PLFA values [86,87,88,94]. Some authors associate the increase in PLFAs upon LDPE addition with an increase in available C, which could serve as a substrate for microorganisms [86]. However, according to [95], it seems unlikely that microorganisms can use the C supplied by LDPE due to its resistance to microbial attack, especially when an external source of available C is present in the soil, as for agricultural soils [96]. Therefore, other abiotic environmental factors such as temperature, UV light, changes in soil bulk density, water content, or evapotranspiration might stimulate microbial activity [3,97]. Since our experiment was conducted under controlled external conditions (temperature, photoperiod, moisture and light intensity), we can rule these out as influencing microbial growth.
Regarding the fungal to bacterial ratio (F/B) (Figure 9a), higher ratios are indicative of a more stable and favourable environment for microbial life [98]. In our study, the F/B was significantly higher in all treatments containing LDPE, irrespective of Cu presence (Figure 9b). These findings suggest a slight improvement in soil conditions favourable for microbial growth and reinforce the previously mentioned results. In this sense, microplastics could act as new microbial habitats, since MP–soil aggregates provide growth sites, together with the fact that microorganisms can also promote MPs biodegradation via cracking and void formation [87].
On the other hand, the Gram-positive to Gram-negative ratio (G+/G−) (Figure 9b), an indicator of ecosystem disturbance, decreased slightly but significantly in all treatments except for 5%LDPE. The most pronounced decrease was observed in the 1%LDPE treatment, where Gram-negative bacteria slightly increased with respect to the control (Figure 7d). This fact may indicate that Gram-positive bacteria are more sensitive to low LDPE concentrations, as can be seen from the observed reduction in Figure 7c.
Finally, the individual effects of heavy metals on the structure and biomass of soil microbial communities have been widely studied and are well documented. However, the addition of microplastics seems to have limited or no effect on microbial biomass and composition, consistent with the findings reported by [86,87,88]. Therefore, further research is needed to better understand how microorganisms respond to the combined effects of heavy metals and microplastics, including the role of other important microbial groups such as soil protists [99], which are known to influence both bacteria and Fungi significantly.

4. Conclusions

Plastic pollution, especially when combined with agrochemicals at higher concentrations, can affect the physicochemical properties of the soil. In our work, we observed that the individual or combined application of both Cu and LDPE generates adverse effects on plant growth, including changes in biometric parameters and photosynthetic pigments. At the same time, when combined, these negative effects were not always evident, suggesting that plastics act on the one hand by increasing soil aeration and thus more space for root growth, but also by adsorbing contaminants on their porous surface. As expected, soil microbial abundance was affected when Cu was added alone, especially at higher concentrations. However, this effect was not observed under metal–MP treatments, where in some cases, microbial abundance even increased. Nevertheless, further experiments are needed to confirm these results, including the assessment of oxidative stress parameters in plants and a deeper investigation into the role of plastics as contaminant carriers.

Author Contributions

S.R.-R.: Investigation; Formal analysis; Data curation; Methodology; Writing—original draft; review and editing. H.M.F.: Investigation; Formal analysis; Data curation, Methodology, Validation; Writing—original draft. A.G.-A.: Investigation; Formal analysis, Data curation, Methodology, Writing—review and editing. D.F.-C.: Formal analysis; Conceptualisation; Data curation, Methodology, Validation, Supervision, Writing—review and editing. M.C.-C.: Data curation; Methodology; Supervision, Writing—review and editing. A.R.-S.: Conceptualisation; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Supervision; Validation; Visualisation; Writing—original draft; review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

The authors would like to recognise the financial support of the Consellería de Cultura, Educación e Universidade (Xunta de Galicia, Santiago de Compostela, Spain) through the contract ED431C2021/46-GRC granted to the research group BV1 of the University of Vigo (Ourense, Spain). HMF thanks the Universidade de Vigo and Fundación Mujeres por África for their financial support to carry out her MSc thesis (2022 0000 121D 482.10 and 2023 0000 121D 482.10). A.R.S. was supported by IJC 2020-044197-I funded by MICIU/AEI/10.13039/501100011033 and by European Union NextGenerationEU/PRTR. M.C.C. holds a postdoctoral fellowship (ED481B-2025/055) financed by Xunta de Galicia. This article/publication is based upon work from COST Action CA20101 Plastics monitoRIng detectiOn RemedIaTion recoverY—PRIORITY.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare that they have no conflicts of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Soil pHH2O (a), pHKCl (b), soil moisture (c) and organic matter content (d) after plant growth under different soil treatments with Bordeaux mixture (BM) and microplastics (LDPE). Data represented are mean ± SD (n = 3). Letters above bars denote significant differences between treatments (p < 0.05).
Figure 1. Soil pHH2O (a), pHKCl (b), soil moisture (c) and organic matter content (d) after plant growth under different soil treatments with Bordeaux mixture (BM) and microplastics (LDPE). Data represented are mean ± SD (n = 3). Letters above bars denote significant differences between treatments (p < 0.05).
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Figure 2. Plant germination (a), root and shoot length ((b) and (c), respectively) and shoot fresh and dry weight ((d) and (e), respectively) of L. sativa exposed to contaminated soil with different concentrations of Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n  =  3). Different letters above bars denote significant differences between before and after plant growth (p < 0.05).
Figure 2. Plant germination (a), root and shoot length ((b) and (c), respectively) and shoot fresh and dry weight ((d) and (e), respectively) of L. sativa exposed to contaminated soil with different concentrations of Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n  =  3). Different letters above bars denote significant differences between before and after plant growth (p < 0.05).
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Figure 3. Root–shoot ratios based on dry weight (dw) (Rdw/Sdw ratio) (a) and by length (Rlength/Slength ratio) (b) of L. sativa exposed to contaminated soil with different concentrations of Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars denote significant differences between before and after plant growth (p < 0.05).
Figure 3. Root–shoot ratios based on dry weight (dw) (Rdw/Sdw ratio) (a) and by length (Rlength/Slength ratio) (b) of L. sativa exposed to contaminated soil with different concentrations of Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars denote significant differences between before and after plant growth (p < 0.05).
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Figure 4. Soil DTPA-available Cu(II) content (a), Cu(II) contents in roots (b) and shoots (c), and the translocation factor of Cu(II) from roots to shoots (d) in the contaminated soil with different concentrations of Bordeaux mixture (BM) and microplastics (LDPE). The data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth (p < 0.05).
Figure 4. Soil DTPA-available Cu(II) content (a), Cu(II) contents in roots (b) and shoots (c), and the translocation factor of Cu(II) from roots to shoots (d) in the contaminated soil with different concentrations of Bordeaux mixture (BM) and microplastics (LDPE). The data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth (p < 0.05).
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Figure 5. Total chlorophyll-a (a), chlorophyll-b (b), total chlorophylls (c), total carotenoids (d) contents, chlorophyll a:b (e), chlorophyll a–carotenoids (f) and chlorophyll–carotenoids (g) ratios in L. sativa exposed to contaminated soil with different concentrations of Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth (p < 0.05).
Figure 5. Total chlorophyll-a (a), chlorophyll-b (b), total chlorophylls (c), total carotenoids (d) contents, chlorophyll a:b (e), chlorophyll a–carotenoids (f) and chlorophyll–carotenoids (g) ratios in L. sativa exposed to contaminated soil with different concentrations of Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth (p < 0.05).
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Figure 6. Total PLFA content in soils after combined exposure to both Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth at p < 0.05.
Figure 6. Total PLFA content in soils after combined exposure to both Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth at p < 0.05.
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Figure 7. Bacterial groups: Firmicutes (a), Actinobacteria (b), Gram-positive (c), Gram-negative (d) and Bacteria (e) in soils after combined exposure to both Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth at p < 0.05.
Figure 7. Bacterial groups: Firmicutes (a), Actinobacteria (b), Gram-positive (c), Gram-negative (d) and Bacteria (e) in soils after combined exposure to both Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth at p < 0.05.
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Figure 8. Fungal groups: Arbuscular Mycorrhizal Fungi (AMF) (a), Zygomycota (b), Ascomycota and Basidiomycota (c) and Fungi (d) in soils after combined exposure to both Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth at p < 0.05.
Figure 8. Fungal groups: Arbuscular Mycorrhizal Fungi (AMF) (a), Zygomycota (b), Ascomycota and Basidiomycota (c) and Fungi (d) in soils after combined exposure to both Bordeaux mixture (BM) and microplastics (LDPE). Data presented are mean ± SD (n = 3). Different letters above bars indicate significant differences between treatments after plant growth at p < 0.05.
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Figure 9. Fungal to bacteria ratio (a) and Gram-positive to Gram-negative ratio (b). Data presented are mean ± SD (n = 3) in soils after combined exposure to both Bordeaux mixture (BM) and microplastics (LDPE). Different letters above bars indicate significant differences between treatments after plant growth at p < 0.05.
Figure 9. Fungal to bacteria ratio (a) and Gram-positive to Gram-negative ratio (b). Data presented are mean ± SD (n = 3) in soils after combined exposure to both Bordeaux mixture (BM) and microplastics (LDPE). Different letters above bars indicate significant differences between treatments after plant growth at p < 0.05.
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Table 1. Experimental treatments used in this study.
Table 1. Experimental treatments used in this study.
TreatmentCu(II) [mg kg−1]Polyethylene [% v/v]
Control00
BM10017 mg kg−10
BM50080 mg kg−10
1%LDPE01%
5%LDPE05%
1%LDPE + BM10017 mg kg−11%
5%LDPE + BM10017 mg kg−15%
1%LDPE + BM50080 mg kg−11%
5%LDPE + BM50080 mg kg−15%
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Romeo-Río, S.; Foguieng, H.M.; Gómez-Armesto, A.; Conde-Cid, M.; Fernández-Calviño, D.; Rodríguez-Seijo, A. Combined Effects of Polyethylene and Bordeaux Mixture on the Soil–Plant System: Phytotoxicity, Copper Accumulation and Changes in Microbial Abundance. Agriculture 2025, 15, 1657. https://doi.org/10.3390/agriculture15151657

AMA Style

Romeo-Río S, Foguieng HM, Gómez-Armesto A, Conde-Cid M, Fernández-Calviño D, Rodríguez-Seijo A. Combined Effects of Polyethylene and Bordeaux Mixture on the Soil–Plant System: Phytotoxicity, Copper Accumulation and Changes in Microbial Abundance. Agriculture. 2025; 15(15):1657. https://doi.org/10.3390/agriculture15151657

Chicago/Turabian Style

Romeo-Río, Silvia, Huguette Meta Foguieng, Antía Gómez-Armesto, Manuel Conde-Cid, David Fernández-Calviño, and Andrés Rodríguez-Seijo. 2025. "Combined Effects of Polyethylene and Bordeaux Mixture on the Soil–Plant System: Phytotoxicity, Copper Accumulation and Changes in Microbial Abundance" Agriculture 15, no. 15: 1657. https://doi.org/10.3390/agriculture15151657

APA Style

Romeo-Río, S., Foguieng, H. M., Gómez-Armesto, A., Conde-Cid, M., Fernández-Calviño, D., & Rodríguez-Seijo, A. (2025). Combined Effects of Polyethylene and Bordeaux Mixture on the Soil–Plant System: Phytotoxicity, Copper Accumulation and Changes in Microbial Abundance. Agriculture, 15(15), 1657. https://doi.org/10.3390/agriculture15151657

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