The influence of lead ions, cationic synthetic surfactants and their combinations on the pigment composition of tissues of plant organisms in water bodies Ceratophyllum demersum and Egeria densa

. The mass of man-made chemical elements entering water bodies along with wastewater is gradually increasing, causing a lot of negative consequences. Plant organisms of water bodies are among the first to experience the effects of any anthropogenic pollutants and especially heavy metals and surfactants, the amount of which increases in the water of rivers and lakes. Changes in the ratio of the pool of green and yellow enzymes in aquatic plants are considered as a reliable diagnostic indicator of the development of disorders in plant cells due to technogenic influences. Changes in the content of photosynthetic pigments in plants of two species ( Ceratophyllum demersum and Egeria densa ) were monitored when they were exposed to very common technogenic substances (lead ions and a solution of cationic synthetic surfactants). The changes found in the quantitative content of photosynthetic pigments in aquatic plants are caused by the appearance and development of stress and some adaptation to it. Under these conditions, the structures of proteins and lipids of thylakoid membranes change, the level of ATP decreases, free radical processes are activated and the intensity of photosynthesis is inhibited. Thus, the action of lead ions, a solution of cationic synthetic surfactants, and especially their combination have a very negative effect on the tissues of Ceratophyllum demersum and Egeria densa , which is expressed in a change in the content of photosynthetic pigments and their ratio.


Introduction
Throughout individual development, the body continuously experiences the dynamics of its many parameters [1].The development of an organism always occurs under the influence of various environmental factors [2].These are chemical, physical, mechanical factors [3].Their influence provides a number of changes in the body, some of which have a positive effect, and some of which have a clearly negative effect.These effects have been monitored in humans [4], in productive animals [5] and in non-productive living organisms [6], as well as in different cell populations [7].
In recent years, the level of environmental pollution by chemicals of anthropogenic origin has increased significantly [8].The amount of chemical elements entering the environment as a result of technogenesis, in some cases, significantly exceeds the level of their natural intake.Involving in natural migration cycles, anthropogenic flows lead to the rapid spread of pollutants in the natural components of the urban landscape.The volumes of pollutants containing heavy metals increase annually and disrupt the ecological balance [9].
In international documents on environmental pollution, more than 10 heavy metals are recognized as dangerous to living organisms, and the most toxic of them are mercury, lead and cadmium [10].
In the last two decades, interest in the environmental aspects of water pollution by synthetic surfactants obtained from petroleum hydrocarbons has increased significantly.This is due, on the one hand, to the increasing scale of production and volumes of use of these compounds in various industries and agriculture, including in the production of synthetic detergents, and on the other hand, to the extremely wide range of negative effects of synthetic surfactants on aquatic ecosystems , and on the human body, as well as their resistance to biodegradation [11].Cationic synthetic surfactants are the most resistant to biodegradation and pose a greater danger to living organisms compared to anionic and amphoteric synthetic surfactants [11,12].
Plant organisms of water bodies are among the first to experience the effects of anthropogenic factors, because Both heavy metals and surfactants are found in significant quantities in the water of reservoirs.Many aquatic plants have a fairly high resistance to pollutants.The ecological plasticity of plants is determined by a complex of morphophysiological adaptations.One of these adaptation mechanisms is the restructuring and change in the structure of chloroplasts.As a result, the ratio of pigments in the system changes, and quantitative changes: the ratio of the pool of green and yellow enzymes can be a reliable diagnostic indicator of a violation of the state of the plant cell.It is against this background that the present study was planned and carried out.
Purpose: to study the influence of lead ions (100 µmol/l), cationic synthetic surfactants (1%), their combination on the pigment composition of the tissues of higher aquatic plants Ceratophyllum demersum and Egeria densa.

Materials and methods
Research was conducted in strict accordance with ethical principles established by the European Convent on protection of the vertebrata used for experimental and other scientific purposes (adopted in Strasbourg March 18, 1986, and confirmed in Strasbourg June 15,2006) and approved by the local ethic committee of Samara University (Record №11, dated December 4, 2019).
The experiment was carried out in laboratory conditions at the same intensity and regularity of the light flux, as well as at a constant temperature (20°C).During the experiment, the plants were divided into groups differing in their growing environment (Table 1).Based on works devoted to aspects of plant stress [13,14], our preliminary studies identified experimental control points that correspond to the phases of the stress response.The condition of the plants was monitored after 1, 2, 4, 12 and 72 hours of being in the test environment.Since 1, 2, 4 and 12 hours of influence correspond to the primary inductive stress reaction, the third day (72 hours of influence) reflects the picture of the second phase of stress.
The content of photosynthetic pigments in plant tissues was studied (quantitative determination of pigments is based on determining the optical density of pigments at wavelengths that are their absorption maxima) [15].Acetone was used to extract pigments from plant tissues.To do this, each plant specimen, ground to a pulp, was placed in identical flasks containing 10 ml of pre-cooled solvent.The mixture was thoroughly mixed and filtered.The filtrate containing the isolated pigments was diluted to 10 ml.The optical density of the finished solution was analyzed on a standard KFK-3 photometer (Russia) at different wavelengths (440.5;644; 662 nm).Pigment concentrations were calculated using formulas corresponding to the methodology.Statistical processing of the research results was carried out by calculating the Student's test.

Results and discussion
In the course of our studies, it was revealed that C. demersum plants experience stress in response to the action of lead ions at a concentration of 100 µmol/l.The plants showed signs of chlorosis after 12 hours of incubation in the pollutant medium and increased with increasing exposure.The dynamics of the content of photosynthetic pigments is shown in Table 2. * -degree of significance of differences with the control level p<0.05.
In the phase of primary stress induction, corresponding to the first 12 hours of incubation in a medium with the addition of 100 µmol/l of lead ions, the content of pigments in the tissues of plants of the experimental group was lower than that of the control group of plants: the content of chlorophyll a by 20-35%, chlorophyll b by 32% lower, carotenoids -30%.After 72 hours of incubation in a pollutant environment, the content of photosynthetic pigments in the experimental group of plants exceeded those of the control group (the content of chlorophyll a by 1.7 times, chlorophyll b by 1.4 times), while the content of carotenoids in the tissues of plants of the experimental group was 40% lower than the control group of plants.
The content of photosynthetic pigments in the leaf is directly related to the phenomena of stress physiology; the relative concentration of pigments can change under the influence of various abiotic factors [16].According to the literature, heavy metal ions cause changes in many structural and functional parameters of photosynthesis: inhibition of a number of enzymes [17], various active centers of photosystem II and the enzyme of chlorophyll synthesis -γ-aminolevulenate dehydratase [18], damage to chloroplast granules and stroma [19], disturbances absorption of elements necessary for them, including manganese and iron, damage to the photosynthetic apparatus or destruction of chlorophyll through an increase in chlorophyllase activity [20], as well as slowing down plastocyanin reactions [21].These effects of heavy metals led to a decrease in the content of photosynthetic pigments during the primary stress induction phase.
The degree of impact of a stress factor can be determined by changes in the chlorophyll a/b ratio.As the index increases, the function of the antenna complexes of photosystem II decreases [22].According to earlier studies [23], even at high concentrations of heavy metal ions, the chlorophyll a∕b ratio remains virtually unchanged, which indicates the invariance of the stoichiometric ratio between the complexes of the reaction centers of photosystems I and II and the light-harvesting complex of photosystem II.As a result of the studies, it was revealed that the chlorophyll a/b ratio exceeded the control group only during the period of rehabilitation from one-hour exposure to lead ions, which indicates the relative resistance of the C. demersum pigment complex to the effects of heavy metals.
The toxic effect of lead ions in the phase of primary inductive stress led to a decrease in the content of photosynthetic pigments (chlorophyll a and b) and an increase in the content of carotenoids.Presumably, this is due to the unfavorable effect of blue ions on photosynthesis, causing disruption of the ultrastructure of chloroplasts, inhibiting the synthesis of pigments and enzymes of the Calvin cycle.A reduction in chlorophyll content is possible due to lead inhibition of the enzyme of chlorophyll synthesis -γaminolevulenate dehydratase [18], damage to the grana and stroma of C. demersum chloroplasts [19], disruption of the absorption of essential elements such as manganese and iron, damage to the photosynthetic apparatus or destruction of chlorophyll through increase in chlorophyllase activity [20].The increased content of carotenoids is explained by the fact that these pigments are the most common and active metabolite of living organisms, participating in the system of protecting cells from the effects of environmental factors [24].
It is known from literary sources that during the adaptation phase, membranes are stabilized [25] with subsequent restoration of ion transport, synthesis processes are enhanced, the activity of mitochondria, chloroplasts and the level of energy supply increase.At the same time, the generation of reactive oxygen species decreases [26].According to the data obtained, the synthesis processes led to an increase in the content of photosynthetic pigments in the tissues of plants of the experimental group.
The dynamics of pigment content in the tissues of C. demersum under the influence of a 1% solution of cationic synthetic surfactants is presented in Table 3. * -degree of significance of differences with the control level p<0.05.During the first 12 hours of incubation in the pollutant medium, corresponding to the phase of primary stress induction, the content of chlorophyll a was 1.5-2.2times higher in the plant tissues of the experimental group, chlorophyll b was 1.7-2 times higher, carotenoids -2.4 times higher.The chlorophyll a/b ratio was 2.2 times higher than in the control group of plants, which indicates the strength of the stress factor and damage to the antenna complexes of photosystem II in the tissues of C. demersum [22].
The dynamics of pigment content in the tissues of C. Demersum under the influence of a combination of pollutants is reflected in Table 4. * -degree of significance of differences with the control level p<0.05.
The phase of primary stress induction as a result of the influence of 100 µmol/l of lead ions and 1 solution of cationic synthetic surfactants proceeded similarly to the phase of primary stress induction as a result of the influence of only lead ions.The content of chlorophyll a was 1.5-2 times lower than that of the control group, the content of chlorophyll b was 2-3.2 times lower, as a result, the chlorophyll a/b ratio of the experimental group was 1.3 times higher than that of the control group of plants.
The adaptation phase to the action of a combination of pollutants turned out to be similar to that to the action of a 1% solution of cationic synthetic surfactants.The content of chlorophyll a and b in the tissues of C. demersum of the experimental group was 2 times lower than that of the control group, while the content of carotenoids was 4.7 times higher.
During the study, it was found that the content of chlorophylls and carotenoids in the studied plants was different.The absolute amount of pigments is a hereditarily determined value, which is associated with the structural and anatomical features of the leaves of a particular species.However, the trend in the dynamics of pigment content in the tissues of Egeria densa was at some points similar to that in the tissues of Ceratophyllum demersum.
Thus, when Egeria densa was incubated in a medium containing lead ions at a concentration of 100 µmol/l, the plants also experienced slight chlorosis, but after 4 hours of incubation in the pollutant medium.The dynamics of the content of photosynthetic pigments of Egeria densa during the observation is reflected in Table 5. * -degree of significance of differences with the control level p<0.05.
In the phase of primary stress induction, corresponding to the first 12 hours of incubation in a medium with the addition of 100 µmol/l lead ions, the content of pigments in the tissues of Egeria densa of the experimental group was higher than that in the tissues of the control group of plants.However, by the same time, the proportion of carotenoids increased and the ratio of chlorophylls to carotenoids increased to approximately the level of 4 (in the control, this indicator corresponded to 6).The increased content of carotenoids, which was noted during the 12-hour exposure period, is explained by the fact that these pigments are the most active metabolites involved in the system of protecting cells from the influence of environmental factors, including the appearance of heavy metals in the environment [24].
According to some authors [22], the degree of impact of a stress factor is determined by changes in the chlorophyll a/b ratio.If the value of this indicator increases, then this means a decrease in the function of the antenna complexes of photosystem II.In the case of Egeria densa, an increase in the chlorophyll ratio by 12 hours of exposure was observed by 45% compared to the same indicators for the Egeria densa control group.
After 72 hours of incubation in a pollutant medium, the content of photosynthetic pigments in the experimental group of plants tended to exceed that of the control group.The content of carotenoids in plant tissues of this experimental group of Egeria densa was 40% lower than this indicator in the control group of plants.
The dynamics of pigment content in Egeria densa tissues under the influence of a 1% solution of cationic synthetic surfactants is presented in Table 6.* -degree of significance of differences with the control level p<0.05.After just one hour of action of the surfactant, the content of chlorophyll a in the tissues of Egeria densa increased by 65%.During the first 12 hours of being in the pollutant environment, which corresponded to the phase of primary stress induction in the plant tissues of the experimental group, the amount of chlorophyll gradually decreased and by 12 hours exceeded the value of the same indicator in the control group by only 28%.The chlorophyll a/b ratio after an hour of exposure to a surfactant compound was 3.8 times higher than that of the control group of plants, which indicates the strength of the stress factor and the development of damage to the antenna complexes of photosystem II in the tissues of Egeria densa [22].
The dynamics of pigment content in Egeria densa tissues under the influence of a combination of pollutants is reflected in Table 7. * -degree of significance of differences with the control level p<0.05.When analyzing data on the content of photosynthetic pigments in the tissues of Egeria densa under the influence of combined factors (100 µmol/l lead ions and 1 solution of cationic synthetic surfactants), it was found that the response of plants to the action of pollutants was similar to that when exposed only to blue ions.During the beginning of the primary stress induction phase (hour of exposure), the content of both chlorophyll a and chlorophyll b (by 34%) was lower than the values of this indicator in the control group of Egeria densa.
The maximum value of the chlorophyll a/b ratio of the experimental group was found 12 hours after exposure and was almost 3 times higher than that of control plants.
Apparently, the identified trends in the content of photosynthetic pigments in plants of two species (Ceratophyllum demersum and Egeria densa) and under the influence of various pollutant factors (100 µmol/l of lead ions and 1 solution of cationic synthetic surfactants) characterize the patterns of response of aquatic plants to anthropogenic impacts.The duration of the influence of the factors corresponded to the transition of plants from a normal state to a state of stress and the subsequent possible adaptation of plants to the action of these pollutants.Such changes in the quantitative content of photosynthetic pigments are characteristic of the phases of emergence, development and some adaptation to stress influences.
During the first phase, the permeability of membranes increases as a result of changes in the molecular composition of their components, and the membranes gradually depolarize, which causes inhibition of H+-ATPase.A decrease in the activity of the latter leads to a decrease in the pH of the cytoplasm and activation of hydrolases, which enhance the processes of biopolymer decomposition.Under these conditions, the rate of hormonal metabolism decreases, acetylcholine, catecholamine, histamine, and serotonin are synthesized, which interact with phytohormones.At the same time, at this stage, the expression of repressed genes and the synthesis of a number of stress proteins occur.Also, under these conditions, the assembly of cytoskeletal elements is activated, which leads to an increase in the viscosity of the cytoplasm [27].The intensity of photosynthesis is inhibited due to changes in the structure of proteins and lipids of thylakoid membranes.Respiration in plant cells is initially activated, but then inhibited as photosynthesis weakens and the level of ATP decreases.The situation is aggravated by the activation of free radical processes in plant cells [28].

Conclusion
In the last two decades, interest in the environmental aspects of water pollution by synthetic surfactants obtained from petroleum hydrocarbons has increased significantly.This is due to the increasing scale of production and volumes of use of these compounds in various industries and the extremely wide range of negative effects of synthetic surfactants on aquatic ecosystems and the human body.Plant organisms of water bodies are among the first to experience the effects of anthropogenic factors, because Both heavy metals and surfactants are found very early and in significant quantities in the water of rivers and lakes.Many aquatic plants have a fairly high resistance to pollutants.Their high ecological plasticity is associated with a complex of their morpho-physiological adaptations.One of these adaptation mechanisms is the restructuring and change in the structure of chloroplasts.The work revealed changes in the content of photosynthetic pigments in plants of two species (Ceratophyllum demersum and Egeria densa) and under the influence of lead ions and a solution of cationic synthetic surfactants, considered as a response of aquatic plants to anthropogenic impacts.As the duration of the influence of these toxic factors in plants increased, a transition occurred from a normal state to a state of stress and subsequent adaptation of plants to the negative influence.It can be assumed that as a result of the action of pollutants (lead ions, cationic synthetic surfactants and their combinations) they have a very negative effect on the tissues of aquatic plants.In this regard, Ceratophyllum demersum and Egeria densa can be considered reliable objects for assessing environmental distress due to the assessment of the level of photosynthetic pigments in them and their ratio.

Table 2 .
Effect of presence of 100 µmol/l of lead ions in a medium on the content of chlorophyll and carotenoids in the tissues of Ceratophyllum demersum under experimental conditions

Table 3 .
Effect of 1% content of cationic synthetic surfactants in the medium on the content of chlorophyll and carotenoids in the tissues of Ceratophyllum demersum under experimental conditions

Table 4 .
Effect of 100 µmol/l of lead ions and 1 solution of cationic synthetic surfactants on the content of chlorophyll and carotenoids in the tissues of Ceratophyllum demersum during the experiment

Table 5 .
Effect of 100 µmol/l lead ions on the content of chlorophyll and carotenoids in Egeria densa tissues under experimental conditions

Table 6 .
Effect of a 1% solution of cationic synthetic surfactants on the content of chlorophyll and carotenoids in Egeria densa tissues under experimental conditions

Table 7 .
Effect of 100 µmol/l of lead ions and 1 solution of cationic synthetic surfactants on the content of chlorophyll and carotenoids in the tissues of Egeria densa under experimental conditions