3.1. Analysis of Soils
Pollutants emitted by poultry farms end up in water, air, and soil. They can infiltrate soils from the air, but improper manure storage or application is more often the source of contamination. During animal production, tons of animal waste are generated in each breeding cycle [
20]. Manure management strategies in the European Union assume its use in the surrounding agricultural fields as an organic fertilizer [
22], but the amount of manure produced usually exceeds the amount needed to fertilize the surrounding fields, which means that it needs to be stored [
13]. To minimize the risk of contaminants from manure getting into the soil or waters, it should be processed and stored in special buildings, closed or open lagoons, or places with impermeable pads [
40]. The spreading of contaminated manure to the fields causes the penetration of micropollutants into the soil, thus affecting changes in the properties and deterioration of soil quality. Leachate and runoff from improperly stored animal manure also pose a threat to the surrounding fields. Soil contamination is one of the greatest threats to sustainable agriculture [
41]; therefore, the soil quality in areas affected by intensive poultry farms should be constantly monitored. Two soils collected from farmlands located near chicken farms were analyzed and characterized (
Table 1). Soil S1 is regularly fertilized with manure, while soil 2 (S2) is taken from the field closest to the manure heap (at a distance of approx. 3 m; see map 1 (
Scheme 1)).
Both soils tested were characterized by low content of total organic carbon (TOC = 9.6 g × kg
−1 and 12.8 g × kg
−1 for soil 1 and soil 2, respectively), which reflected the general characteristics of the majority of Polish agricultural soils [
42] and was slightly below the optimal threshold level for light soils in Europe [
43]. Furthermore, in both soils, the TC/TN ratio was below 10; such low TC/TN values were previously reported by Ukalska-Jaruga et al. [
44] for arable soils and suggested the predominance of soil organic carbon decomposition processes in the soil. Another important threat to soil quality is soil acidification, which was identified as a serious land degradation problem [
45].
The pHKCl of soil S1 was equal to 5.7 (
Table 1), while soil S2 was more acidic (pHKCl = 4.9). Acidification of the soil (pH < 5.5) significantly reduces crop yields (e.g., peas) and the abundance and activity of bacteria involved in the transformation of organic compounds in soils; it inhibits many soil beneficial processes, e.g., nitrification or the fixation of free nitrogen. In acidic soils, rhizobium bacteria involved in atmospheric nitrogen fixation by the roots of legumes are reduced [
46]. Under low pH conditions, the uptake of nutrients by plants is reduced (N, P, K, Ca); at the same time, the availability of many micronutrients (Zn, Mn, Fe) is increased, reaching even toxic levels [
43] The high concentration of Fe
2+, Mn
2+ as well as Al
3+ and H
+ ions in very acidic and acidic soils also has toxic effects on the roots of the cultivated plants, resulting in a shortening of roots and a reduction in the surface area of the root system. These changes reduce the efficiency of water and nutrient uptake and consequently reduce plant growth [
43,
46]. Acidic soils are also characterized by increased mobilization and bioavailability of heavy metals [
45]. Other inorganic soil pollutants include nitrates and phosphates. Although these compounds are plant nutrients and are not considered toxic, their high concentrations can cause environmental problems. Phosphorus extractability was rather high; available P in S1 was equal to the average content of this nutrient in Polish soils (16.7 mg P
2O
5 × 100 g
−1, [
47]), the amount of available P in S2 was almost 2.5-fold higher and reached a value of 40.4 mg P
2O
5 × 100 g
−1). It is highly probable that this large difference resulted from the phosphate leakage from the manure heap. Soils significantly differed in mineral nitrogen content (sum of N-NO
3 and N-NH
4): 9.27 mg × kg
−1 and 46.46 mg × kg
−1 in soil S1 and S2, respectively. Soil 1 had similar levels of ammonium and nitrate N, while soil S2 was dominated by N-NH
4 (34.97 N-NH
4 mg × kg
−1). During rain, the nitrogen and phosphorus compounds present in manure are leached directly into the soil. Excessive amounts of phosphate in soils limit the uptake of iron and zinc by plants ([
48], which results in symptoms of deficiency of these elements. Plants are the primary source of zinc for humans, and a deficiency of this element in plants can lead to a corresponding deficiency in humans. This problem has already been known and considered widespread and significant for 60 years [
49]. Nowadays, in the post-pandemic era, it is particularly important as zinc increases human resistance to bacteria and viruses.
The largest group of soil pollutants, however, are organic pollutants. Due to their persistence, potential for bioaccumulation, and mobility in the environment, they are considered a global environmental problem [
50]. The largest group of organic pollutants emitted by intensive poultry farming are pharmaceuticals. Although the use of antibiotics in poultry farms in Poland is subject to legal regulations, the scale of their use is not fully known. Consumption reports are based on the value of sales obtained from pharmaceutical companies [
9]. Therefore, there is a need to monitor the presence of pharmaceuticals in the soil surrounding poultry houses. Soil S1 and soil S2 were screened for the presence of pharmaceuticals. No pharmaceutical residues were detected in the soil regularly fertilized with manure. In the soil taken from a field located near a heap of manure, the presence of various classes of pharmaceuticals was found (
Table 2). Enrofloxacin, ciprofloxacin, and trimethoprim, found in soil S2, are broad-spectrum antibiotics. They are given to fight bacterial infections in chickens [
51]. Although the concentrations of the compounds detected were low, their presence in the soil indicates the routine use of these antibiotics on farms. Moreover, the detection of pharmaceuticals in soil located near manure storage piles suggests that manure is the source of these compounds in the environment. Pharmaceuticals present in the soil can be taken up by plants and thus enter the food chain. Additionally, pharmaceuticals present in the environment, even at low concentrations, contribute to the spread of antibiotic resistance, thus posing a risk to human health [
52]. In Poland, 26, 52, 36% of
Salmonella spp. strains isolated from farm animals in 2020 showed resistance to ampicillin, ciprofloxacin, and tetracycline, respectively. Among
E. coli strains, 78, 73 and 68% of strains, respectively, showed resistance to these antibiotics [
53]. Antibiotics entering the environment as contaminants of manure, even at low concentrations, can affect the composition of the soil and plant microbiome [
52]. Changes in the composition of the soil microbiota, resulting from significant environmental contamination with antibiotics, may cause disturbances in the species composition of the phyllosphere of plants growing on contaminated soils. Soto-Giron et al. [
54] indicate that agricultural practices have the greatest impact on the formation of the plant phyllosphere. However, the plant phyllosphere is usually represented by those bacteria that develop best in a given environment. The presence of antibiotics in the environment causes selection pressure, which results in the growth and development of pathogenic and antibiotic-resistant bacteria while inhibiting the development of commensal bacteria [
55]. It can, therefore, be assumed that the phyllosphere of plants growing on soils containing antibiotics will be rich in pathogenic and antibiotic-resistant strains, which poses a risk to consumers’ health. In addition to antibiotics, residues of metoclopramide, an antiemetic drug that improves peristalsis in the digestive system, were also found in soil S2. This compound is given to chickens to stop the defecation process and keep the chicken houses more hygienic
poultrydvm.com (accessed on 12 April 2023). In soil S2, residues of carbamazepine
—a derivative of dibenzazepine, used as an anxiolytic and sedative
—were also determined. In veterinary medicine, dibenzoazepines are administered to reduce stress in animals during transport and to eliminate behavioral problems [
12]. However, the environmental consequences of the presence of these compounds are still unknown.
Pesticide and herbicide residues were also detected in soil S2 (
Table 3). Their presence in the soil was not the result of the emission of pollutants from chicken houses but was probably due to the wide use of these compounds in crops in the surrounding fields. Particularly alarming is the presence of the
p,p′DDE compound in the soil—a decomposition product of the organochlorine pesticide DDT. WHO indicates DDT as a carcinogenic compound for humans [
56]) and chronic exposure to this compound causes lung, liver, breast, and kidney cancer [
56]. Despite the ban on its use since the 1980s, residues of DDT and its decomposition products are still detected in most agricultural soils in Poland. DDT level in Polish soils is, on average, 0.064 mg/kg
−1, sometimes reaching 0.120 mg × kg
−1 [
57].
Organochlorine pesticides, as a result of deposition from the atmosphere and as runoff from agricultural fields, also pollute surface waters [
58], posing a threat to aquatic organisms. DDT is lipophilic and easily accumulates in the cuticle [
59], making aquatic plants with a lipid cuticle particularly vulnerable.
3.2. Analysis of Groundwater Collected from the Vicinity of the Farm
Groundwater is the main source of fresh water for people around the world. One-third of the world’s population uses groundwater as a source of drinking water [
60]. Intensive agriculture is one of the factors deteriorating the quality of groundwater, thus contributing to the deepening of the water crisis [
61], and pollutants from chicken houses emitted to soils (including medicines) may also pollute groundwater. Pharmaceuticals present in the soil undergo various transformations, like degradation, adsorption to soil particles, and transport to surface and groundwater [
62] as a result of runoff and leaching from soils and manure. The degree of adsorption of pharmaceuticals in soil depends on the molecular structure of the compound, the polarity of the compound, the soil organic matter content, and the type of soil [
62]. The soils in our study were characterized by a low organic carbon content (<1.2 g·kg
−1), resulting in a low capacity of the soil to retain contaminants and their leaching into the deeper layers of the soil profile and/or groundwater. In addition, in soils with different pH (the case of this research), pharmaceuticals can occur in different ionic forms [
63], characterized by different adsorption abilities. Weather conditions play an important role in the transfer of pharmaceuticals into the soil [
64].
Rainwater that penetrates surface layers of soil can carry pharmaceuticals to deeper layers and contaminate groundwater. This contaminated water can be discharged into surface waters and seep into deep waters, which are often used as a source of drinking water [
64]. The presence of pharmaceuticals in drinking water raises concerns about their safety [
65], particularly in areas where pharmaceuticals are routinely used on farms. Therefore, it is important to monitor groundwater around these farms. Groundwater samples collected at a depth of 26 m have revealed the presence of various classes of pharmaceuticals, including fluoroquinolones such as ciprofloxacin and enrofloxacin. Ciprofloxacin is the most commonly detected antibiotic in European groundwater, often found in high concentrations near pig farms and manure-spreading sites [
66]. In fact, farms have been identified as the main source of ciprofloxacin and enrofloxacin in water [
67]. Ciprofloxacin is highly soluble in water and mobile due to its hydrophilic nature. However, its degradation rate slows down significantly under anaerobic conditions in groundwater, and it remains largely unchanged [
26]. Given its widespread occurrence in groundwater, it is important to monitor its concentrations in drinking water as well [
68].
Small concentrations of trimethoprim were also determined in the groundwater from the vicinity of the farms (
Table 4). This compound is often detected in groundwaters in Europe [
66], including those located in the vicinity of agricultural soils fertilized with manure. Burke et al. [
69] showed the presence of trimethoprim in groundwater in a manure-spreading area. This compound was detected in 11 wells, and the determined concentrations ranged from 5 to 12 ng × L
−1.
The presence of lincomycin was also detected in the groundwater (
Table 4). This compound has not been previously detected in soil samples. However, its presence in groundwater in the vicinity of farms indicates the use of these compounds in chicken farming. Kuchta et al. [
70] showed that the accumulation of lincomycin in soil is transient. Lincomycin residues have been detected in groundwater samples by other researchers [
71], suggesting a high leaching potential of this compound.
Tetracycline residues were also detected in the groundwater (
Table 4) but not in soils collected from the vicinity of chicken farms (
Table 2). Tetracyclines are one of the most commonly used classes of veterinary antibiotics, and approximately 20–70% of the antibiotic dose enters the environment in unchanged form in urine and feces [
72]. They are hydrophilic compounds characterized by very low desorption capacity. As soil pH increases, the ability of tetracyclines to adsorb to soil decreases [
72]. In alkaline soils, the risk of tetracycline leaching into groundwater is, therefore, greater. Tetracyclines are regularly determined in groundwater samples. The highest concentration of tetracycline was determined in groundwater in the USA (500 ng × L
−1), collected from the vicinity of pig farms [
73]. Additionally, Szekeres et al. [
74] showed the presence of tetracycline in groundwater from the vicinity of farms and suggested improper management of animal waste as a source of this. Antibiotics taken up with drinking water enter the trophic chains. Chronic exposure, even to low concentrations of antibiotics, can cause health problems. Sub-therapeutic doses of antibiotics in food and water are especially dangerous for children [
75]. Studies have shown that exposure to antibiotics in the early stages of life is associated with obesity in school-aged children [
76]. Constant intake of antibiotics also contributes to disturbances in the composition of the intestinal microbiota. Disturbances in the composition of the digestive system microbiota are a risk factor for IBS, type II diabetes [
77], and Crohn’s disease [
78]. Additionally, even low concentrations of antibiotics in groundwater pose a risk of antibiotic-resistant genes and strains being selected [
26]. Andrade et al. [
79] showed that 80% of strains isolated from groundwater show multidrug resistance, which is a threat to human health and life around the world.
In addition to antibiotics, residues of carbamazepine and metoclopramide were determined in groundwater as well as in soils collected from the vicinity of the farms. Carbamazepine is a persistent environmental pollutant that is resistant to degradation [
80]. Therefore, its concentrations are very often determined in groundwater. Loos et al. [
81] found that carbamazepine was detected in 42% of samples taken from 164 locations in 23 European countries, with a maximum concentration of 390 ng × L
−1. However, most studies on carbamazepine concentrations in groundwater have been carried out in urban areas. Carbamazepine concentrations in the vicinity of intensive poultry farms have not been reported so far. Additionally, metoclopramide has not been previously determined in the area affected by intensive farms. However, the presence of these compounds in groundwater indicates their wide use in animal husbandry.
3.3. Analysis of Morphological and Biochemical Parameters of Common Pea Pisum sativum L.
The stem length of peas grown in manure-treated soil did not change from the control (
Figure 1A). A decrease in stem length was observed in peas grown on soil collected from the vicinity of the manure heap (a decrease of 15% compared to the control) (
Figure 1A). Similar results were obtained for the length of the main root (27% decrease compared to control) (
Figure 1B), the number of lateral roots (26.5% decrease compared to control) (
Figure 1C), and second leaf area (24.5% decrease % compared to control) (
Figure 1D). These results suggest that the pollutants present in the soil are taken up by plants and induce a stress reaction in them, thus impairing plant growth. Although the concentrations of micropollutants (pharmaceuticals and pesticides) in the soil collected from the vicinity of the manure heap were low (
Table 2 and
Table 3), their synergistic effect is toxic to plants.
Pollutants emitted by intensive poultry farming can accumulate in plant tissues, negatively affecting the morphological and biochemical parameters of plants. Due to human intervention, the number of stress factors to which plants are exposed is significantly increasing [
82]. Groups of pollutants toxic to plants include pesticides, heavy metals, and pharmaceuticals residues. Increasingly, it is indicated that environmental stress factors negatively affect the plant microbiome [
83], which determines the health and productivity of plants [
84]. Intensive agricultural production results in many plants being deprived of microorganisms responsible for the production of key metabolites and vitamins [
85]. Sustainable management of environmental resources increasingly relies, therefore, on our understanding of the interactions between various pollutants present in the soil [
86] and the synergistic effects of various anthropogenic stress factors. This issue is rapidly gaining attention in the field of plant physiology research [
83]. In order to assess the potentially toxic effects of groups of pollutants generated during intensive poultry farming on plants, the morphological and biochemical parameters of pea
Pisum sativum L. growing on soil regularly fertilized with chicken manure (S1) and located in close proximity to a heap of manure (S2) were assessed. The control was peas grown on perlite (S3). Environmental stress factors cause oxidative stress in plants, which is the result of the accumulation of reactive oxygen species (ROS) in plant tissues. The increase in the level of ROS in plant cells affects many different physiological and biochemical functions, which results in a decrease in plant growth and yield ([
87]. Thus, disturbances in the morphological parameters of plants may be considered a visible biomarker of oxidative stress in plants.
Chlorophyll is one of the key factors determining proper plant growth. Reduction in its concentration in a plant is a good stress biomarker [
88]. The concentration of chlorophyll in plants grown on the soil collected from the vicinity of the manure heap decreased by 26% compared to the control (
Table 5). A decrease in the content of chlorophyll in plants exposed to anthropogenic environmental factors has been repeatedly observed. The reduction in chlorophyll content in plants treated with antibiotics was demonstrated by Rydzyński et al. [
31], Margas et al. [
89] as well as Krupka et al. [
38]. However, in most studies assessing the impact of anthropogenic stress factors on chlorophyll, high concentrations of toxic compounds are used. In the soil collected from the vicinity of manure heaps, the concentrations of drugs and pesticides were not determined; however, their presence was verified, and the reduction in chlorophyll content in plants was found.
Chlorophyll degradation may be the result of stress factors already at the stage of its biosynthesis [
90,
91]. The fundamental reaction of the chlorophyll biosynthesis pathway is the condensation of two molecules of δ-aminolevulinic acid (ALA) to porphobilinogen. This reaction is catalyzed by the enzyme ALAD (aminolevulinic acid dehydrogenase) [
92]. Jiao et al. [
33] indicated that the activity of the ALAD enzyme is a factor determining the concentration of chlorophyll in the plant [
33]. In plants grown in soil regularly fertilized with manure, ALAD activity decreased by 24% compared to the control (
Figure 2A). On the other hand, in plants growing on soil taken from the vicinity of the manure, the activity of the ALAD enzyme decreased by 48% (
Figure 2A). The decrease in ALAD activity was correlated with the decrease in ALA content (
Figure 2B). Therefore, it can be unequivocally stated that even small concentrations of pollutants present in the soil affect chlorophyll already at the stage of its synthesis, thus causing a decrease in its concentration in plants. Reducing the activity of the ALAD enzyme in plants has been demonstrated under the influence of bisphenol [
33] and heavy metals [
92]. On the other hand, a decrease in ALA content has been demonstrated under the influence of ciprofloxacin, erythromycin, and sulfamethoxazole [
93]. So far, however, no studies have been conducted on ALAD activity and ALA content in plants exposed to several stress factors simultaneously.
Malonaldehyde (MDA) content is a commonly used parameter for measuring lipid peroxidation in cell membranes. Its concentration increases under conditions of oxidative stress [
94]. Lipid peroxidation is a damaging process that affects membrane properties, causes protein degradation, and reduces ion transport capacity, ultimately leading to cell death [
95]. Increased levels of MDA have previously been demonstrated in plants treated with antibiotics [
38], p,p′-DDE [
96], and heavy metals. In peas grown on soil taken from the vicinity of the manure heap, the concentration of MDA increased 4-fold (
Figure 3A). The contaminants present in the soil caused, therefore, the destruction of cell membranes. Similar results were obtained with mitochondrial damage (
Figure 3B). Mitochondria are organelles that are particularly vulnerable to organic pollutants, including pharmaceuticals [
38]. Pesticides also damage the mitochondria. However, most of the studies on the effects of pesticide residues on mitochondria have been carried out in animals. The effect of these compounds on plant mitochondria has been known very poorly [
97]; it is possible, however, that the decrease in mitochondrial viability may turn out to be an important indicator of biochemical changes in plants caused by the synergistic effect of pollutants emitted by intensive poultry farms.
HSP70 proteins are also essential components of the plant’s response to stress. Their main role is to protect cells from oxidative stress by stabilizing membrane proteins and removing abnormal and damaged proteins [
98]. Although they accumulate in the greatest amount during heat stress, HSP70 proteins are a biomarker of various stress reactions, including those induced by anthropogenic pollution [
38]. An increase in the content of these proteins was observed both in plants growing on soil regularly fertilized with manure and growing on soil collected from the vicinity of manure heaps (
Figure 3C). These results suggest that in plants growing on soil regularly fertilized with manure, a stress reaction was also triggered, although no residues of toxic factors were determined in this soil. These plants showed no changes at the morphological and biochemical level; therefore, the increase in the content of HSP70 proteins seems to be an effective mechanism for protecting plant cells from damage.
3.4. Analysis of Morphological and Biochemical Parameters of the Small Duckweed Lemna minor L.
In order to assess the impact of soil pollutants on the condition of surface waters, the soil was washed with redistilled water, and duckweed was grown in this effluent (see
Section 2.4). Surface water—including rivers, ponds, and lakes—is still the source of drinking water for 159 million people in the world. Polluted water contributes to the deaths of 842,000 people a year [
99]. Intensive agriculture is indicated as the main source of surface water pollution [
1]. Groups of pollutants emitted to surface waters include, e.g., nitrates and phosphates, released from soils fertilized with excessive amounts of manure [
1]. Consumption of water rich in nitrates contributes to the occurrence of methemoglobinemia, a fatal disease mainly affecting children under 6 months of age [
100]. In addition to the direct consequences for human health, nitrates and phosphates present in water cause cyanobacterial blooms, which have negative effects on aquatic ecosystems [
101]. In Europe, 50–80% of released nitrates and phosphates reach surface waters as a result of agricultural practices. Pharmaceuticals and their metabolites also pose a serious threat to the quality and safety of surface waters. Poor manure management practices and excessive use of manure in fields result in drugs entering the surrounding surface waters as a result of runoff and leachate. Park et al. [
102] showed the presence of lincomycin and sulfamethoxazole in a stream near a manure heap. The presence of lincomycin, trimethoprim, and sulfamethoxazole has also been demonstrated in the nearby river [
102]. Antibiotics have also been identified in manure leachate in an experiment simulating rainfall [
102]. A study by Barrios et al. [
103] also showed the presence of antibiotics in runoff from soil fertilized with manure. Tetracycline was also determined in the surface runoff from soil fertilized with manure, and its concentration was 2.79–35.97 µg × L
−1 [
104]. Meng et al. [
105] showed the presence of 23 antibiotics in rivers from rural areas, which are a source of drinking water for local residents. In addition to antibiotic residues, antibiotic resistance genes were also detected in environmental samples [
103]. Pesticides are another large group of surface water pollutants. Although their presence in surface waters may not be a direct result of their use in livestock farming, odors from chicken houses attract insects that can infest crops grown in the surrounding fields [
106]. Therefore, in areas affected by intensive poultry farming, it becomes necessary to use pesticides in the fields. Pesticides are detected in waters in Europe, China, and the USA [
107] as well as in Africa and South America [
108]. Pollution of the aquatic environment with pesticides is, therefore, a global problem. Particularly alarming is the presence of organochlorine pesticides (including DDT) in water reservoirs, characterized by high biomagnification capabilities. DDT, as a lipophilic compound, is easily accumulated in lipid structures [
59]. Aquatic plants covered with a lipid cuticle [
59] and aquatic animals that accumulate DDT in adipose tissue [
109] are particularly vulnerable to its effects. To monitor the quality of surface waters and study the impact of pollutants present in water on living organisms, toxicity tests for duckweed
Lemna minor L. are used [
110]. The analysis of morphological and biochemical parameters of duckweed is used to assess both the presence and toxicity of micropollutants in surface waters.
In order to demonstrate that pollutants emitted as a result of intensive poultry farming penetrate into surface waters and affect aquatic organisms, the morphological and biochemical parameters of duckweed
Lemna minor L. growing on filtrates of soils regularly fertilized with manure (S1) and collected from the vicinity of manure heaps were assessed (S2). MS medium (S3) was used as a control. The OECD recommends the use of morphological parameters to assess the toxicity of micropollutants present in water [
30]. The number of plants growing on filtrate 2 decreased by 50% (
Figure 4A). Similar results were obtained by Krupka et al. [
38]. Tetracycline at 2.5 mM resulted in a 48% reduction in plant numbers. Despite the fact that a high concentration of tetracycline was used in that study, the results described coincide with our current results. Various water contaminants can act synergistically, leading to stress symptoms in plants comparable to those caused by higher concentrations of compounds acting alone. Contaminant-caused disturbances were also demonstrated by comparing the area of duckweed fronds. Plants growing on filtrate number one had a 59% larger frond area compared to the control (
Figure 4B). Park et al. [
111] determined high concentrations of phosphorus in aqueous extracts of chicken manure. Li et al. [
112] indicate that 69% of the phosphorus present in manure is soluble in water and thus easily gets into the environment. Phosphorus is an element that stimulates root growth, thus improving plant growth [
13]. The root length of plants growing on filtrate 1 averaged 21 cm, while the plants grown on MS medium had an average root length of 7.8 mm (
Figure 4C). Duckweed growing on filtrate 2 was characterized by both a smaller frond area (
Figure 4B) and root length (
Figure 4C) compared to the control, which indicates the influence of toxic factors present in the water on plant growth.
Another parameter of the
Lemna minor L. toxicity test is the measurement of the chlorophyll content. Sackey et al. [
113] showed low concentrations of chlorophyll in duckweed growing on a medium with the addition of leachate from landfills contaminated with various groups of micropollutants. Similar results were obtained for duckweed grown on soil filtrate taken from the vicinity of manure. In these plants, the concentration of chlorophyll decreased by 33% (
Table 6); in addition, chlorosis of the fronds was observed (
Figure 5).
The decrease in chlorophyll content was also correlated with decreased ALAD enzyme activity (2-fold decrease in activity) (
Figure 5A) and lower ALA concentration (
Figure 5B). Most studies on ALAD activity and ALA content concern the impact of single stress factors. Our results indicate that these parameters can also be used in the assessment of the toxicity of many water pollutants to
Lemna minor L.
Krupka et al. [
35] indicated that MDA content, HSP70 protein concentration, and mitochondrial damage are important parameters in assessing the toxicity of micropollutants present in water towards
Lemna minor L. Plants growing on filtrate number 2 showed a 4-fold increase in MDA content (
Figure 6A), suggesting damaged cell membranes in these plants. Similar results were obtained for peas grown on soil collected from manure heaps (
Figure 3A). Mitochondria are organelles sensitive to micropollutants (especially antibiotics). Duckweed cells growing on filtrate number 2 showed 7% mitochondrial damage (
Figure 6B), and duckweed cells growing on filtrate number 1 showed 5% mitochondrial damage (
Figure 6B), which is consistent with the results obtained for peas. Both plants growing on filtrate 1 and 2 showed induction of the HSP70 proteins (
Figure 6C), indicating that these plants were initiating a stress response.
Lemna minor L. toxicity tests are particularly useful in testing water samples in which the content of micropollutants has not been determined [
100]. The physiological response of the duckweed is evidence of the presence of toxic components in these tests.