博士論文審査報告書

Graduate School of Creative Science and Engineering, Waseda University

博士論文審査報告書

Doctor Thesis Screening Results Report

論 文 題 目

Thesis Theme

Oxidation mechanism and overall removal rates of endocrine disrupting chemicals by aquatic plants

水生植物による内分泌撹乱物質の酸化メカニズム および総括除去速度に関する研究

申 請 者

(Applicant Name)

Andre RODRIGUES DOS REIS

(Major in Civil and Environmental Engineering, Water and Environmental Engineering)

Februar y, 20 13

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Mr. Rodrigues dos Reis’s dissertation presents the oxidation mechanism and overall removal rates of endocrine disrupting chemicals (EDCs) and estrogens by aquatic plants. Literature review results on the current situation of EDCs and estrogens contamination in aquatic environments are shown and discussed. Emerging organic contaminants such as EDCs and estrogens have been detected in aquatic environment all over the world, and their effects on human health and ecological system have received considerable attention. These compounds are released into the aquatic environment mainly via municipal and industrial effluents and agricultural runoff. In addition, natural estrogens excreted by humans or animals and entering aquatic systems are among the most potent endocrine disrupting chemicals. Because of identified and suspected endocrine disrupting effects, effective methods for the removal of these EDCs from wastewater, surface water and contaminated sites are required. The phytoremediation technology is proposed as a cost-effective alternative for the treatment of contaminated sites. Aquatic plants can be a feasible alternative for organic pollutants removal from contaminated aquatic environments because they can bio-accumulate and degrade several types of organic compounds through enzymatic reactions

The thesis consists of six chapters beginning with an introduction of research objectives and a literature review focused on the current situation of EDCs and removals by phytoremediation process. The first chapter provides an overview of the extent and implications of emerging organic pollutants. From literature review, it was reported that different types of aquatic plants are able to remove EDCs; however, mechanisms, primal enzymes, or reactions responsible for oxidation of EDCs have not been well understood. Moreover, most studies on EDCs treatment have been conducted using batch experiments with relatively high concentrations of EDCs (i.e. mg/L level) comparing to observed levels in aquatic environments or wastewaters. Batch experiments could indicate the possibility of removing EDCs, however, continuous performance and its stability have not been studied. Therefore, rational operation and design conditions with information on appropriate plants have not been available.

In order to evaluate the kinetics and the stability of EDCs removals by aquatic plants, long-term continuous treatments were conducted and treatment results were shown in Chapter 2. EDCs used in this study were bisphenol-A (BPA), 2,4-dichlorophenol (2,4-DCP), 4-tert-octylphenol (4-t-OP), nonylphenol (NP), and pentachlorophenol (PCP). These compounds were selected, because they are often detected in rivers, industrial effluents, landfill leachates, municipal wastewater, and sludge treatment plants at concentrations ranging from ng/L to μg/L. Referring to reported detection levels in wastewater and landfill leachates, the feed concentration of each EDC was set at 100 μg/L. From semi-continuous and continuous experiments, it was found that every EDC except PCP was efficiently removed by different aquatic plants. The presence of microorganism in continuous treatment did not show significant interference on EDCs removal. It is considered that these results are new findings, indicating further developments in phytoremediation process or wastewater restoration using aquatic plants.

Chapter 3 describes the oxidation mechanism and primal enzymes responsible for the EDCs removals. In

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order to simulate the degradation of EDCs inside aquatic plants, in vitro batch treatments of BPA, 2,4-DCP, 4-t-OP, NP and PCP, as well as estrogens such as estrone (E1), 17β-estradiol (E2), and ethinylestradiol (EE2) were conducted using crude enzymes (i.e., soluble peroxidase (SPO), ionically cell wall-bound peroxidase (IPO), cell wall-bound peroxidase (CPO), polyphenol oxidase (PPO), laccase (LAC) and gluthathione S-transferase (GST) extracted from aquatic plants. Every EDC except PCP was efficiently degraded through the following reaction catalyzed by peroxidases: EDCs + H2O2 → Products + H2O2. It was considered that POs were primal enzymes in EDCs oxidation due to the high amounts of different POs fractions in aquatic plants.

Peroxidases were able to remove phenolic EDCs in the presence of H2O2 over a wide pH range (from 3 to 9).

Histochemical localization of peroxidases showed that they were located in every part of the root and stem cells, while highly concentrated zones were observed in the epidermis, cell walls and vascular tissues. Furthermore, oxidation of guaiacol was observed mainly in the cell walls without the addition of H₂O₂. These results suggest the presence and locations of cellular endogenous H₂O₂ and the reaction sites of EDCs oxidation in aquatic plants. In addition, it was found that endogenous hydrogen peroxide concentrations and SPO, IPO and CPO activities in aquatic plants were almost kept constants during the continuous treatment of EDCs.

In Chapter 3 it was found that peroxidases and H2O2 were primal enzymes and endogenous key substances on EDCs oxidation in aquatic plants, respectively. In plant cells, chloroplasts are considerable sources of reactive oxygen species (ROS) including superoxide radicals, hydroxyl or hydrogen peroxide. As it was reported that photosynthetic active chloroplasts are the main suppliers of H2O2, the effects of nutrients and enzymes on the photosynthesis were studied. Chapter 3 also describes experimental results indicating nitrogen fertilization and nitrate reductases were important factors in photosynthesis of plant. In addition, it was observed that FeSOD and MnSOD, superoxide dismutase isoenzymes localized in the chloroplast were mainly sources for generation of H2O2 especially in nitrogen deficient plants.

In Chapters 2 and 3, it was shown that PCP was not effectively removed by different types of aquatic plants or enzymes. As PCP is a recalcitrant compound, it is considered that a more powerful oxidation reaction is needed for the treatment of PCP. In Chapter 3, it was observed that different types of aquatic plants were able to produce stably endogenous H2O2 in plant tissues even under long-term continuous treatment of phenolic EDCs.

These results mean that H₂O₂ is being produced stably in plants themselves through photochemical reaction or respiration process. Chapter 4 describes a new treatment method for the complete decomposition of PCP in water, utilizing endogenous hydrogen peroxide in aquatic plants in the presence of ferrous iron to proceed the biological Fenton reaction. It is known that Fenton’s reaction generates hydroxyl radicals, which can oxidize almost every class of organic compounds. In order to know the optimum ferrous iron concentration to proceed a Bio-Fenton reaction using aquatic plants, a batch experiment was conducted with different concentrations of Fe²⁺

(0, 0.28, 1.4, 2.8, 14 and 28 mM). From experiments, it was found that ferrous iron concentration around 2.8 mM or less seems to be suitable for the PCP treatment. There was a rapid removal of PCP with consumption of endogenous hydrogen peroxide as well as production of chloride ion. When the initial PCP concentration was high (5 mg/L), more than 80% of PCP was removed within two days. These results demonstrated that PCP was

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oxidized through a biological Fenton reaction, and hydrogen peroxide in aquatic plants was a key endogenous substance in treatment of refractory toxic pollutants. It can be recognized that Biological Fenton is a novel process which can be applied to almost all refractory environmental pollutants.

Chapter 5 describes the mass transfer rates of EDCs using an electrochemical method developed based on the limiting current theory. An electro-conductive polymer was sprayed on the surface of aquatic plants and was emerged in reactor with Cu anode. The limiting current is obtained from measurements of the electric current as a function of the potential applied between the cathode and the anode. The electrochemical experiment is based on the deposition of Cu²⁺ ions on the cathode surface (aquatic plants), and is a conventional procedure used to measure the mass transfer coefficients. These measurements are usually performed with a supporting H₂SO₄ electrolyte solution. The electric current was increased in small increments until a well-defined decrease in the oxidation reduction potential (ORP) was observed. At this point, the limiting current was reached, and the liquid film thickness of aquatic plants and the mass transfer coefficient of EDCs were analyzed. The liquid film thickness of analyzed aquatic plants was in the range from 195 to 427 μm.

The specific removal rates of EDCs by the primal enzymes in Chapter 3 or by the biological Fenton reaction in Chapter 4 were much larger than those by aquatic plants. Based on these differences, a mathematical model was developed based on an assumption that overall removal rates of EDCs are controlled by the mass transfer rates in liquid film on the surface of aquatic plants, and comparisons of calculated and observed results were made to evaluate the net removal rates of EDCs by aquatic plants. Observed specific removal rates of BPA, 2,4-DCP, 4-t-OP, NP, E1, E2 and EE2 by aquatic plants as well as PCP by biological Fenton oxidation were in reasonably good agreement with calculated specific removal rates by the mathematical model. Based on these results, it can be expected that the removal performance of EDCs and pharmaceuticals in constructed wetland or phytoremediation processes would be enhanced if plants possessing high specific surface area, larger amounts of POs, and higher concentration of endogenous H2O₂ are selected and cultivated.

Finally, the conclusions of this study and recommendation for future works are given in Chapter 6.

His dissertation gives useful information on oxidation mechanisms and EDCs removal using aquatic plants.

The referees recognize that his paper devotes the development of the water and environmental engineering and meets given requirements of a doctorate (Doctor of Engineering).

February 2013

Principal Referee:

Prof. of Waseda Univ. Dr. of Eng. (Nagoya University) Yutaka Sakakibara

Sub Referee

Prof. of Waseda Univ. Dr. of Eng. (University of Tokyo) Tomoya Shibayama Prof. of Waseda Univ. Dr. of Eng. (Waseda University) Hirokazu Akagi Prof. of Waseda Univ. Dr. of Eng. (Waseda University) Masato Sekine