SUMMARY OF PROFESSIONAL ACCOMPLISHMENTS

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1 WROCŁAW UNIVERSITY OF SCIENCE AND TECHNOLOGY FACULTY OF ENVIRONMENTAL ENGINEERING SUMMARY OF PROFESSIONAL ACCOMPLISHMENTS Konrad Matyja, M.Sc., Eng. Promoter: Kazimierz Grabas, D.Sc., PhD, Eng. Wrocław, 2016

2 Name and surname: Konrad Matyja Education: Information about the Author ; PhD student, Wrocław University of Science And Technology, Faculty of Environmental Engineering; ; M.Sc. in Environmental Protection at Silesian University of Technology, Faculty of Environmental Engineering and Energetics, ; B.Sc. in Biotechnology at Silesian University of Technology, Faculty of Environmental Engineering and Energetics ; B.Sc. in Environmental Protection at Silesian University of Technology, Faculty of Environmental Engineering and Energetics, Professional experience: since Research Assistant, Wroclaw University of Science and Technology, Faculty of Chemistry, Division of Bioprocess and Biomedical Engineering The main publications: Cuske M., Karczewska A., Gałka B., Matyja K., 2017, Would forest litter cause a risk of increased copper solubility and toxicity in polluted soils remediated after phytostabilization? Polish Journal of Environmental Studies, 1/26 Matyja K., Małachowska-Jutsz A., Mazur A., Grabas K., 2016, Assessment of toxicity using dehydrogenases activity and mathematical modeling. Ecotoxicology 25/5, Cuske M., Matyja K., Karczewska A., Gałka B., The dynamics of seeds germination inhibition of Sinapis alba caused application of soil solutions extracted from soil contaminated with heavy metals. Episteme 26/ 1, Małachowska-Jutsz A., Matyja K., Ziembińska A., 2011, Cadmium and Copper Toxicity Assessment in Activated Sludge Using TTC Bioassay. Archives of Environmental Protection 37/4, Małachowska-Jutsz A., Matyja K., 2011, Wpływ wybranych soli kadmu na aktywność dehydrogenaz mikroorganizmów osadu czynnego. Przemysł Chemiczny 90/8, , Małachowska-Jutsz A., Matyja K., 2010, Wpływ wybranych soli miedzi na aktywność dehydrogenaz mikroorganizmów osadu czynnego. Przemysł Chemiczny 89/8, , IF(2010): 0.290; IF (5-letni): 0.356; Punkty Ministerialne (2010): 13, (2012):15

3 Description of the dissertation entitled: Modeling of the chemical compounds mixtures toxic effects on the energy budgets of organisms 1. The subject of the work Toxicity testing is carried out based on very well known standard protocols in certain, defined laboratory conditions. Therefore laboratory bioassays have limited applications especially in contects of ecosystem-level testing. Generally all tests and each single replication should be perforemed in stable temperature, ph and the concentration of toxin. Additinaly different external factors for example theintensity of the illumination and purity of tested substances should be kept as same levels, very strictly. Toxicity testing methods have grown in recent years but very often critical factors such as role of exposure time was reduced to an absolute minimum. Ecotoxicology methodology often is limited to several measurements at certain time endpoints. Assessment of changes in parameters of the process is often overlooked. Most likely this is due to lack of appropriate equations that could describe the process under the influence of external factors. However, the conditions in the environment are never fixed, and the number of factors that may influence the process is much greater than in the laboratory. Accordingly, the use of traditional methods does not allow fully understand the phenomenon and gives little information about the potential risk that can be caused by chosen toxic compound. In addition, chemicals can potentially interact each other in different wayss. These interactions can affect the toxic degree - increasing or decreasing it. The first option may pose high an unpredictable risk for organisms. Therefore, from the beginning of the twentieth century some reserchers started to develop methods for evaluating the toxicity of mixtures. Two models that have gained the greatest popularity is the model of Loewe, called the Concentration Addition (CA) and the Bliss model, called Independent Action (IA). These models are used today in medical toxicology, pharmacy and ecotoxicology. They could be linked with more complex models are used to developnew onces. Unlikely, most of models for assessing the toxicity of mixtures is based solely on them.

4 In recent years, there appeared a new trend in science, which main idea is broader look at the reality that surrounds us. This broad-minded approach comes down to considering certain processes in their full complexity and including interactions whith surroundings. Application of these new approaches to biological systems is critical due to extremely complex nature of life organisms and existing boundaries. Their mathematical description is very difficult. Difficult mainly because features that combine reactions on the molecular level and the processes observed at the individual level do not exist or have not been formulated in a mechanistic way, despite the fact that their relationship often seems to be obvious. This problem is particularly manifested in ecotoxicology. There are some models that describe processes such as growth, reproduction, or changes in population size. In many cases, these models are empirical. They differ from the mechanistic models, constructed on the basis of theoretical considerations. The existence of such a function of which parameters changes could be measured, allow to eliminate the tendency to describe the toxicity effect only as occurring at specified exposure time. If these functions were also a mechanistic models and would describe a relationship with the speed of a chemical reaction and activation energy, one could find a relationship between temperature and the rate at which the process takes place. Finally, it is likely that the use of such models in the assessment of toxicity, would provide the foundation for creating a mechanistic model wich equations would describe the effect of several or more components of the mixture of chemical compounds at the same time. Such functions may be derived from the fundamental chemical and physical laws. These laws should also apply to things common to everything that exists - namely energy and its transformations. They can be related to the phenomena of chemical reactions, biochemical, physical processes, such as change of state, heat flow, etc. Talking about energy allows us to see the essence of all kinds of transformation and clearly define the relationship between them and the environment.

5 2. Aim of the work Accordnig to the issues and problems described above following questions need to be answered How to formulate equations that could be used to evaluate the toxicity of mixtures of chemicals? These equations should be expressed in the form of simple laws and relationships between them. A relationship with the possible to measure physical, chemical and biological quantities should be defined. On the other hand, would enhance the continuity and the complexity of the process and its interaction with the environment. These laws also should apply to the the values which are common to everything that exists - energy and mass and their transformations. Therefore it is possible to write a research hypothesis: Energy budgets of organisms and their associated dependence can be used for evaluation of the toxicity of mixtures of chemical compounds Confirmation or rejection of the hypothesis can only come from attempts to derive the new model, therefore, the main aim of the work is: Building a mechanistic model based on energy budgets of organisms which can be used to evaluate the toxicity of mixtures. Below list presents milesones needed to achieve main objetive: to find and formulate the relationship between Dynamic Energy Budget (DEB) model s equations and the equations used in thermodynamics network; derivation of equations describing the changes in DEB model parameters depending on the concentration of the individual compound, a consideration of the various mechanisms of toxicity; derivation of equations describing the change in DEB model parameters depending on the number and concentration of the total active components in the mixture; derivation of equations describing dependencies between mass and energy streams (to describe the indirect effects of the toxins on the energy and mass fluxes); demonstrate the utility of derived model on an example.

6 3. The methods used A great tool that finds use to describe many different types of processes is network thermodynamics. Network analysis allows the use of thermodynamic laws governing electrical circuits to describe any process in which observed flux is driven by some difference of potentials. Understanding of the system in this way leads us directly to the thermodynamics of nonequilibrium systems, the description of subsequent reactions and the theory of activation energy. This allows us to relate changes of system with the basic state functions. Linking the concept with a living organism comes from the biological sciences. Dynamic Energy Budget Theory - DEB combines the energy budgets of living organism with measurable endpoints quantities, such as their length, volume or number of offspring. This property of DEB theory and model in association with the network thermodynamics allows for the construction of the function describing the characteristics of an organism so that we can measure changes of parameters under various kinds of stress. 4. Results of the work and the range of solutions Based on DEB theory and network thermodynamics which have not yet been compiled and linked, new model equations has been derived binding changes at the individual level to the molecular properties of elementary processes. Derivation of these equations from the fundamental physical and chemical forms is the backbone of this work. Some presented relationships are universal. They may be used to describe the flow of the mixture through the membrane, electric current flow or a series of biochemical reactions. This property of the model appears to be very useful and increase its flexibility. The solutions presented in this paper are an alternative to the currently existing models used for assessment of the toxicity of mixtures. However, there are some common features for them and for traditional methods. At the same time it is an alternative which has a good mechanistic rationale. Its great advantage is the fact that the individual parameters of the model have their specific chemical and physical meaning. This eliminates the need to estimate them on the basis of the toxicological experiments. They may be directly measured or determined on the basis of the results of other experiments. Accordingly, the number of parameters to be determined at the same time is significantly lower. This significantly improves the quality and reliability of the results. In addition, the estimated parameters can be verified. Verification may be done by measuring their valuse with another alternative method.

7 Derived model can be used to describe changes in the organisms of any species. Differences between species could be represente mainly by different arrangement of mass and energy streams, and in the values of DEB model parameters (eg. dry egg weight, shape correction coefficient, etc.). The amount and type of toxins affecting the orgnism also does not limit the applicability of the model. However, the mechanism of the effect on each stream must be known. The biggest limitation of the possible application of this model is the period of data is collection. It is required to cover the entire life cycle of the organism or a substantial part of it. Another limitation resulting from the previous is the inability to use the model to assess the acute toxicity. The model can be adapted to describe mortality and changes in population, but only in cases when it is possible to examine the changes in its parameters. When the death of the organism caused by toxin action occurs in a very short exposure time model can not be used.

8 5. The derived model Presented model can by classyfied as a synthetic model. It is built based on the elements which are universal and reflect basic right of physics or chemistry, or has been properly grounded in theory DEB. Fig. 1. DEBkiss model scheme Where: J X, J A, J R, J M, J V - mass fluxes denoting successively: food intake, assimilation, reproduction (or puberty), maintenace and growth; W V, W R and W B stand for the mass of body structures, the weight allocated to offspring production, and the source of mass and energy for the embryo; kappa (κ) - the proportion between the two main streams in theory DEB - flux sustaining the maintenace and growth and flux for the maturation and reproduction Each flux in DEB model (Fig. 1) or other mass and energy balance, prepared for the living organism can be characterized by three values: flow, potential difference, resistance. The toxins can have an impact on each of these values. Changes in one flux may in turn translate into changes in the balance of other fluxes. The first type of impact can be defined as a direct, the other as an indirect influence on the fluxes. Using the basic laws of chemistry and physics leads to new description how temperature, toxin or mixture of toxins can affect the parameters of the equations of these fluxes. It is the mechanistic approach and different from

9 that used in DEB theory, in which the equations are more empirical (equations stress factor "s" and its impact on the parameters of the model DEB). As it turns out it gives a more complete and better substantiated picture of how stress can affect the energy budgets of organisms. Network thermodynamics has been successfully applied to describe many phenomena, eg various biochemical reactions, complex biochemical pathways, and the flow through the membrane. To our knowladge in this work, for the first time, network thermodynamics is applied to simplify the description of such a complex phenomenon that is life and development of the organism. Organism in which thousands of biochemical reactions occur and also the processes of transport and transformation of energy. For this purpose linear approximation described by Ohm's law was used in combination with the DEB theory. This large approximation, however, can bring many benefits, especially in terms of assessing the toxicity of mixtures. Compilation of DEB model and network thermodynamics lies in that each flux occurring in the DEB model can be equated to Ohm s law. According to Ohm's law, each flux can be described as the ratio of the potential difference and resistance. This principle applies to each flux in DEB theory and any other energy budget of organism. For assimilation processes, maintenace, growth and reproduction following equations are valid: J A = fj a Am L 2 = ΔU A r A J M = J M v L 3 = ΔU M r M J V = y VA (κj A J M ) = ΔU V r V J R = (1 κ)j A = ΔU R r R The potential difference is the driving force of the flux, the resistance is a factor that inhibits it. As mentioned earlier, in the orgnism occur thousands of reaction. Some of them are chemical reactions, other biochemical reactions catalyzed by enzymes, others occur under the influence of the electrical potential difference or rely on transport of particles across cell membranes. They all make the organism maintains life, growth and reproduction. The individual reactions may be described as reversible when ther are considered separtly, but when they are considered in the context of the budget they have to be described as irreversible

10 processes. Each flux of energy budget can be divided into separate reactions or processes known as an elementary processes. Many processes inside the organism may run in parallel to each other, but all can be reduced to elementary processes. In this sense they may often take the form of a purely abstract or represent specific biochemical reactions. Each of these elementary processes is supplied by a potential difference, eg. potential difference on both sides of the semi-permeable membrane, the electric potential difference or difference in chemical potentials. Regardless of its type, potential differences can be added directly to each other. According to the definition of the Gibbs potential, the direction of the processes depends only on the potential difference between the end-products and initial substrates regardless of the number (and type) of intermediate processes. This law also applies to organisms and allows to describe all of life processes using one or several potential differences referenced to the individual fluxes of mass and energy budgets. The irreversibility of the subsequent reactions to describe it by an analogy to the series-connected resistors. Kirchhoff's voltage law indicates that it is possible to define the resistance of resistors connected in series as a sum of elementary resistances. Analogously we can proceed in the case of an infinite or a very large number of chemical reactions, biochemical and elementary processes discussed herein. This feature allows to describe the processes of life by using just a few foster sustained resistances of the basic fluxes of DEB model. The toxic substances may affect the reactants and products of reaction, and hence the difference in potential. They can affect also enzymatic catalysts, and therfore, resistance of the process. The impact of toxins on flow is also possible. It can manifest through the emergence of new fluxes in the system that do not directly alter the resistance and the potential differences. New fuxes can change the intensity of the other through the dissipation of mass and energy. All of these properties can be varied independently of each other but can also be associated which can be described by the following equation J sumy = 1+ m i=1 [T i]s Ui 1+ n [T i ] i=1 s ri J 0 + k i=1 [T i ]s Utoxi RT p i e ki The equation may be used to assess the effect of toxic substances, and mixtures on the parameters of mass and energy budgets. It represents the essence of this work. It is a new mechanistic model, derived from fundamental laws of physics and chemistry.

11 The derived model combines the advantages of the DEB theory and network thermodynamics. The main advantages of the model DEB include: mathematically defined relationship between mass and energy budget of the orgnism and their measurable characteristics - the length, volume, density, mass, number of offspring. extremely meticulously described and verified in many instances the relationship between the variables of the model. The main dissadvantages of the model are: lack of connection between parameters that describe the fluxes of energy budget with the paramters of chemical and biochemical reactions, limited opportunities for expansion of the budget. The equations used in network thermodynamics: are closely related to parameters of chemical and biochemical reactions, they can be used to describe a very complex metabolic pathways. The disadvantages of this approach are: no specific links between the parameters of equations and characteristics of organisms measurable at higher than the molecular levels of the organization. The disadvantages of a single approach is countered by the use of a second. Such a combination, of so far two parallel developing theories seems to be extremely effective. Such compilation is presented for the first time in this work.

12 6. The practical use of the model To illustrate how the model can be used results from a specially prepared experiment where assessed. The aim of the study was to assess the effect of cadmium and copper ions individually and in mixtures, on water flea Daphnia magna. Toxicity test was carried out in glass bakers. In each of these five juveniles (<24h) were placed in 50 ml of standard medium (ISO Standard Freshwater, ISO 6341) including selected toxic substances - cadmium sulfate, copper chloride or one of their mixtures. A control sample was uncontaminated. During the experiment the size of daphnids and the number of neonates were measured. Body size of exposed animals and reproduction were affected by the action of toxic metal ions. We tested the effect of the two mechanisms of the individual substances on fluxes of DEB model, and various combinations of these mechanisms which may be used to assess toxicity of the prepared mixtures. Parameters which described the changes observed in the experiment in the best way were chosen for futher analysis. Based on experiments results and calculations, the mechanism of combined action of cadmium and copper on DEB model fluxes can be proposed. In this experiment mechanism 1 was selected and improved by experimental results and can be written as follows: J A = (1+[Cd]s UCd ) fj (1+[Cd]s rcd ) Am a L 2 J M = (1+[Cu]s UCu+[Cd]s UCd ) (1+[Cu]s rcu +[Cd]s rcd ) J M v L 3 + [Cu]s jtox L 3 Model fit to measured data, prepared exclusively on the basis of the results of the experiment on individual substances is shown in Fig. 2. The results of the estimation of the model parameters desribed by mechanism 1 are shown in Figure 3. Comparison of the results of the above mentioned procedures, can bring us to the conclusion that the chosen mechanism may be successfully applied to predict the adverse effects of copper and cadmium ions mixtures on body size and reproduction of water flea Daphnia magna. The model can be successfully applied for different exposures concentrations and proportions of the toxic components. Its predictions can be based on the results of toxicity tests on individual substances, which could not be obtained by traditional methods. Once the mechanism of combined action of the two components was determined it can be used in the assessment of the toxicity of the more complex mixtures.

13 Fig. 2. Changes in the body length of Daphnia magna and the number of their offspring exposed to the mixtures of cadmium and copper - mechanism 1 (no estimation). Concentrations are given in the legend of µmol/dm 3 Fig. 3. Changes in the body length of Daphnia magna and the number of their offspring exposed to mixtures of cadmium and copper - mechanism 1 (the estimation). Concentrations are given in the legend of µmol/dm 3

14 7. The importance of the work and main conclusions In this study we constructed a mechanistic model based on mass and energy balance which can be used to evaluate the toxicity of mixtures. Thus research hypothesis presented at the beginning of this work was confirmed and all of the intermediate objectives were achieved. Therfore there is a possibility of using network thermodynamics and energy budgets of organisms to assess the toxic effects caused by single substances and their mixtures. This possibility leads to a better understanding of these phenomena. It gives oppurtuninty to describe it with such basic and common to everything that exists the quantity - which is an energy. The model derived in this work may be used to assess the toxic effects of the individual compounds and mixtures. Type of tested chemicals, which are components of the mixture, is not a limitation for the use of the model. Toxicity of any substance that may influence the potential difference, or resistance of energy and mass flows, can be assessed. Similarly, the choice of species of the organism, which can be used to assess the toxicity has no limitation. The mass and energy budget can be built for any organism. For this purpose both DEB models and their alternatives can be used. Budgets can be modified and extended, but derived equations describing changes in the potential difference and resistance, and the relationship between the fluxes associated with Kirchhoff's laws will still be true. Some difficulty can occur during the determination of all parameters of the budgets - eg. in DEB model. There are many parameters and there is no way to estimate the value of them from one sample. The fact that the model is mechanistic, allows to determine the values of some parameters in different experiments and their use in the following. Using DEB models brings many benefits, because the value of some model parameters are already available in the literature. This allows to focus on the most important of them and estimate a small number of parameters, so that the obtained values are more reliable. The great advantage of the presented approach is the ability to create a database with parameter values designated for different toxins and various species. Some of these values should be fixed and may be used in the future to assess the toxicity of a much more complex mixtures.

15 The number of components of mixtures can be varied. The practical limitation appears to be a need to understand the mechanism of toxicity of each component and the need to know the mechanisms of their combined action. A very important feature of the presented model is no need to know all the elementary constants rates and equilibrium constants for all possible pairs of toxic substance - reagent. This knowledge was essential in complex models describing the metabolic network. The derived model avoids the need for these data by using the analysis of changes in resistance and changes in potential differences, not their absolute values. The derived model, as well as DEBkiss more fully describes the toxic effects than the CA and IA models. Both describes the time factor, which has a very strong impact on the observed changes of characteristics of an organism under the action of the toxic chemicals. In contrast to its original, stress factor desribed in this work have been derived mechanistically. The model is thus determined by the basic laws of physics and chemistry. The mathematical description and the connection between what is happening at the molecular level (equilibrium constant, inhibition of the enzyme), and what is observed at the individual level was obtaind. The model can be used mainly to describe the changes in the rate of growth and reproduction. The effects observed at the molecular level may provide evidence leading to define the mechanisms of changes in the model parameters or provide confirmation of the usefulness of appropriate mechanisms. Mortality can also be described by presented model. However, this requires the expansion and adoption of several additional assumptions. These issues have not been considered in this work.