The influence of toxic substances in the environment on the activity of antioxidant enzymes in the tissues of Ceratophyllum demersum

. In the context of a gradual increase in anthropogenic environmental pollution, detailed environmental studies become relevant. It is very important in this regard to monitor the qualitative and quantitative aspects of the influence of the pollution process on various biological objects that receive the influence of substances entering the environment. Of great practical interest are the toxic effects of technogenic discharges on various hydrobionts and the main mechanisms of their development in macrophyte plants. In this regard, this work studied the influence of heavy metal ions, cationic synthetic surfactants and their combinations on the activity of the main antioxidant enzymes in tissues of Ceratophyllum demersum and Egeria densa. To do this, an experiment was conducted to assess changes in the freshwater macrophytes Ceratophyllum demersum and Egeria densa. The dynamics of the activity of antioxidant enzymes: peroxidase, polyphenol oxidase, ascorbate oxidase and catalase were studied. Their activity was assessed basally and in response to chemical stressors (100 µmol/l lead ions, 1% solution of cationic synthetic surfactants) individually and in combination with different exposures to toxic substances. The identified features of enzymatic activity in the tissues of aquatic macrophytes indicate different levels and power of their antioxidant protection. It becomes clear that the activity of antioxidant enzymes when plants are exposed to certain types of pollutants and their combinations is determined by the chemical nature of the pollutant, the mechanism of its action on the plant organism, the duration of exposure and the localization of these enzymes in cell compartments.


Introduction
Throughout life, the environment has a pronounced influence on the structure and function of living organisms [1].Under the influence of various environmental factors, changes occur in almost all body systems [2].They are adaptive in nature [3] or can lead to the appearance of dysfunctions and pre-pathology [4].For this reason, a detailed clarification of the aspects of environmental influences on the body is very important for assessing the danger of individual negative environmental factors [5] and clarifying the standards for various environmental parameters [6].These studies are of particular practical importance on multicellular organisms [7], as they can reveal their strategies in adapting to various living conditions [8].
With the increase in the number of cities and population density in the world, there is a gradual increase in the amount of transport and the development of the sphere of consumer services, which leads to a steady increase in the amount of man-made emissions into the biosphere.At the same time, in recent years there has been a significant expansion of the chemical composition of pollutants [9,10].
One of the particularly acute environmental problems of our time is the pollution of fresh water bodies with heavy metals and synthetic surfactants.This is because heavy metals and surfactants pose a serious threat to many life forms [11,12].The situation is aggravated by the fact that synthetic surfactants have a pronounced resistance to biodegradation [13,14], and heavy metals are very slowly eliminated from the body [10].
In the context of a widespread increase in anthropogenic pollution of the hydrosphere with heavy metals and synthetic surfactants, a qualitative and quantitative assessment of the degree of environmental pollution by them and a detailed analysis of their biological hazard becomes very relevant [15,16,17].To date, the toxic effects of abiotic stressors on various species of aquatic organisms have been widely studied [18,19,20,21].However, the mechanisms of influence of chemical factors individually and especially when combined on macrophyte plants still require clarification [22,23].In particular, aspects of the responses of submerged macrophyte vegetation need to be clarified.It is capable of reflecting the general state of water bodies and changes in their environmental conditions [24,25] and therefore is a significant object of bioindication of natural waters [26,27].The various reactions of aquatic plants to the appearance of chemical properties with toxic properties in their habitat have been studied to the least extent.
During almost all vital processes of the body, reactive oxygen species are formed in its cells.These compounds are characterized by high chemical activity.Under conditions of negative influences on the body from the environment, the production of reactive oxygen species in its cells is especially pronounced.This ultimately leads to the development of oxidative stress and the appearance of prepathology and pathology [28].During evolution, enzymatic defense mechanisms against the action of reactive oxygen species have been developed, including peroxidase, polyphenol oxidase, ascorbate oxidase and catalase [29].At the same time, the reaction of these enzymes to negative chemical influences on the body from the environment remains poorly understood.Changes in their activity in the tissues of aquatic plants, which can be considered as biological markers of pollution of fresh water bodies with substances of technogenic origin, have been least studied.
Purpose of the work: to study the effects of the release of heavy metal ions and cationic synthetic surfactants into the environment separately in combination on the activity of the main antioxidant enzymes in the tissues of Ceratophyllum demersum and Egeria densa.

Materials and methods
The objects of the study were freshwater macrophytes: submerged hornwort (Ceratophyllum demersum) and Brazilian elodea (Egeria densa).
The experiment was carried out in laboratory conditions at the same sufficient intensity and duration of regular 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 previous work in the field of plant stress response [30,31], experimental control points were determined that corresponded to the phases of its occurrence.Thus, 1, 2, 4 and 12 hours of influence corresponded to the primary inductive stress reaction.In this case, the third day (72 hours of influence) was considered as a manifestation of the second phase of stress.
In plant tissues of Ceratophylum demersum and Egeria densa, the dynamics of the activity of a number of enzymes of the antioxidant system (peroxidase, polyphenol oxidase, ascorbate oxidase and catalase) was studied basally and in response to the action of very common chemical environmental pollutants (100 µmol/l lead ions and 1% solution of cationic synthetic surface -active substances (washing and detergent "Dosya", made in Poland)) individually and in combination.
The activity of the above enzymes was determined using generally accepted methods: the peroxidase activity method [32]; according to the method for catalase activity [33]; according to the method for polyphenoloxidase activity [32]; according to the method for ascorbate oxidase activity [32].Statistical processing of the results was performed using Student's t-test.

Results and discussion
In the course of the studies, it was revealed that the plants Ceratophylum demersum and Egeria densa experience stress in response to the influence of the tested pollutants.The biochemical changes that occurred in the studied plants during the first 12 hours of the experiment reflect the occurrence of the first phase of stress in their tissues -the primary inductive stress reaction [25].However, the dynamics of enzymatic activities are different in the tissues of the studied plants and ambiguous for various enzymes.For both plant species, the most significant differences from control levels and levels in the first 4 hours of exposure to lead ions and a surfactant were noted.
In the tissues of Ceratophylum demersum in the first 4 hours after exposure to lead ions, the activity of catalase and peroxidase decreased by 4 and 2 times, respectively, compared to the control.The activity of ascorbate oxidase and polyphenol oxidase after 4 hours of exposure increased by 43.5% and 28.5%, respectively (Table 2).Similar changes were E3S Web of Conferences 462, 02047 (2023) AFE-2023 https://doi.org/10.1051/e3sconf/202346202047noted for the enzymatic activity of Egeria densa tissues, although the differences were less pronounced (Table 3).Note.Significance of differences between the data in the control and the data during the experiment: * -at the p<0.05 level, ** -at the p<0.01 level.
Analyzing the results obtained, we can say that the appearance of the most significant changes in enzyme activity in the first 4 hours of the toxic effect of lead ions is associated with the development of the primary inductive stress phase in the plant.
In the tissues of Ceratophylum demersum, peroxidase activity after 72 hours (the second phase of stress) of exposure to lead ions remained lower (2 times) than the control values.By the same period, catalase activity almost returned to its original value (control).Ascorbate oxidase and polyphenol oxidase activities, starting from 4 hours of exposure, remained higher than the values of similar control indicators.Thus, after 72 hours of exposure to lead ions, ascorbate oxidase and polyphenol oxidase activities in the tissues of Ceratophylum demersum were almost 2 times higher than the corresponding control values.
Exposure to lead ions on Egeria densa for 72 hours led to an increase (compared to the control) in the activity of all considered enzymes in the tissues of the experimental groups of plants: catalase activity by 1.5 times, peroxidase activity by 39.5%, ascorbate oxidase activity by 1.6 times times, polyphenoloxidase -2 times (Table 3).At the same time, the activity of ascorbate oxidase was significantly different from the control, starting from 4 hours of exposure, and polyphenol oxidase from 2 hours of exposure.Note.Significance of differences between the data in the control and the data during the experiment: * -at the p<0.05 level, ** -at the p<0.01 level.
Analyzing the enzymatic activity in the tissues of Ceratophylum demersum and tissues of Egeria densa under the influence of surfactants, faster and more dramatic changes in the activity of the monitored enzymes were revealed than under the influence of lead ions (Tables 4 and 5).Note.Significance of differences between the data in the control and the data during the experiment: * -at the p<0.05 level, ** -at the p<0.01 level.Thus, after 4 hours of exposure to synthetic surfactants on Ceratophylum demersum, an increase was noted (compared to the control values of similar enzymes) in the activity of: catalase -2.8 times, peroxidase -1.7 times, ascorbate oxidase -1. 4 times, polyphenoloxidases -2.6 times.For the enzymes taken into account, the greatest differences from the level of the control group Ceratophylum demersum were detected after 12 hours of action of synthetic surfactants.Thus, during these observation periods, catalase activity exceeded control values by 4.2 times, peroxidase activity by 4.4 times, ascorbate oxidase activity by 2 times, and polyphenoloxidase activity by 5.3 times.
In the tissues of Egeria densa, when exposed to synthetic surfactants, significant differences were also noted from similar indicators of the control group of plants after 2 hours.Thus, after 2 hours of exposure, catalase activity exceeded control values by 17.8%, peroxidase activity by 36.3%, and ascorbate oxidase activity by 18.5%.However, if for the E3S Web of Conferences 462, 02047 (2023) AFE-2023 https://doi.org/10.1051/e3sconf/202346202047activity of catalase, peroxidase and polyphenoloxidase, as the duration of exposure increased (after 72 hours), their activity increased (1.8 times, 4 times, 3.3 times, respectively), then ascorbate oxidase activity decreased by 72 hours compared to the control by 23.4%.
Apparently, the identified differences in the nature of the response of plants to the action of synthetic surfactants can be explained by the fact that these compounds, once in the aquatic environment, form a film at the phase boundary, which leads to a change in the quality of the flow of abiotic factors: the amount of light penetrated to plants, the gas exchange between the liquid medium and the air deteriorates, leading to a lack of oxygen in the water.Under these conditions, peroxidation processes are intensively launched in plant cells.In response to active free radical oxidative processes in plant cells, the working capabilities of the antioxidant defense system are increased, providing effective deactivation of reactive oxygen species.This was confirmed by an increase in the activity of the enzymes peroxidase and polyphenoloxidase by 72 hours of exposure to synthetic surfactants.
Interesting results were discovered when studying enzymatic activity in the tissues of Ceratophylum demersum and Egeria densa under the influence of both substances under consideration (lead ions -100 µmol/l and 1% solution of a surfactant compound) (Tables 6  and 7).Note.Significance of differences between the data in the control and the data during the experiment: * -at the p<0.05 level, ** -at the p<0.01 level.
In Ceratophylum demersum tissues, catalase activity tended to increase slightly by 72 hours of the experiment.In the tissues of Egeria densa, the level of activity of this enzyme by this time of exposure to toxic substances increased 1.6 times.
As for peroxidase activity, dynamics in the tissues of Ceratophylum demersum were observed similar to those when exposed to lead ions only -a decrease in activity (2.4 times).Dynamics of a similar direction were noted for the activity of peroxidase in Egeria densa (exceeding the control level by 2.3 times).Note.Significance of differences between the data in the control and the data during the experiment: * -at the p<0.05 level, ** -at the p<0.01 level.Analysis of ascorbate oxidase activity in the tissues of Ceratophylum demersum under the combined influence of pollutants showed that, as under conditions of exposure to lead ions only, the activity of this enzyme increased by 72 hours of exposure by 1.7 times compared to the control.In the tissues of Egeria densa, the activity of ascorbate oxidase increased (by 18.3%) throughout the experiment.These data differed from those when Egeria densa was exposed to synthetic surfactants, where a decrease in the activity of this enzyme was observed.

E3S Web of
The results of a study of polyphenol oxidase activity in the tissues of Ceratophylum demersum and Egeria densa showed an increase in the activity of this enzyme, which amounted to 1.8 times for Ceratophylum demersum and 3.2 times for Egeria densa by 72 hours of exposure to both pollutants.
The results obtained can be explained by the genetic characteristics of the organisms affected by the impact taken into account, and their potential for response to external chemical influences [34,35].

Conclusion
The success of an organism's vital activity under any environmental conditions is largely determined by the activity of enzymes in its cells that inhibit the formation of reactive oxygen species and their compounds.In the process of evolution, enzymatic defense pathways against the action of reactive oxygen species have emerged, including peroxidase, polyphenol oxidase, ascorbate oxidase and catalase.In the course of the steady increase in anthropogenic pollution of fresh water bodies, it becomes very important to assess the severity of this process, taking into account their biological danger.As a result of the current situation, elucidating the levels of influence of very common technogenic discharges on the antioxidant protection of aquatic organisms is of particular scientific and practical interest.These man-made factors include heavy metal ions, cationic synthetic surfactants and their combinations.To close the gap in scientific knowledge, an experiment was carried out in laboratory conditions on Ceratophyllum demersum and Egeria densa using various options for their exposure to toxic man-made substances.A study was made of the effect of 100 μmol/l lead ions, a 1% solution of cationic synthetic surfactants, individually and in combination with different exposures on the activity of antioxidant E3S Web of Conferences 462, 02047 (2023) AFE-2023 https://doi.org/10.1051/e3sconf/202346202047enzymes of hydrobionts (peroxidase, polyphenol oxidase, ascorbate oxidase and catalase).The identified features of enzymatic activity in the tissues of aquatic macrophytes indicate different levels and power of their antioxidant protection.The activity of antioxidant enzymes when plants are exposed to individual pollutants and their combinations is determined by the chemical nature of the pollutant itself, the main mechanisms of its action on the plant organism, the duration of exposure and the localization of these enzymes in cell compartments.

Table 1 .
). Experimental groups of Ceratophyllum demersum and Egeria densa in the performed study

Table 2 .
Activity of antioxidant enzymes in the tissues of the plant Ceratophylum demersum located in an environment containing lead ions

Table 3 .
Activity of antioxidant enzymes in the tissues of the Egeria densa plant located in an environment containing lead ions

Table 4 .
Activity of antioxidant enzymes in the tissues of the plant Ceratophylum demersum in a solution of a cationic surfactant compound *Note.Significance of differences between the data in the control and the data during the experiment: * -at the p<0.05 level, ** -at the p<0.01 level.

Table 5 .
Activity of antioxidant enzymes in the tissues of the plant Egeria densa in a solution of a cationic surfactant compound

Table 6 .
Activity of antioxidant enzymes in the tissues of the plant Ceratophylum demersum located in a medium containing lead ions and a cationic surfactant compound

Table 7 .
Activity of antioxidant enzymes in the tissues of the Egeria densa plant located in a medium containing lead ions and a cationic surfactant compound