In vitro and in silico toxicity estimations

Get Complete Project Material File(s) Now! »

Context and molecules

The concept of contaminants of emerging concern

Water management is one of the most pressing societal challenges of the 21st century. Clean water is essential for human life, agriculture, and a healthy ecosystem. The fol-lowing part contains a short overview regarding contaminants of emerging concern, their number, nature, and the pursuit for their regulations, focusing on the aspects relevant to this project. Increased human consumption, overuse of aquifers, natural calamities, and climate change are reasons behind water scarcity. It is essential to value the existing water resources and recycle wastewater to attain sustainability goals.
Contaminants of emerging concern are de ned as anthropogenic chemicals or substances of natural origin, which are currently not regulated but might be submitted to future regulations because they can pose risks to human health or the environment, as their persistence or toxicity might alter the metabolism of a living being [1]. These are not necessarily new contaminants; emerging data on legacy compounds can also introduce new regulations or make the existing ones stricter. It is an extremely complex eld of study, as 100 million chemicals are registered in the Chemical Abstract Services (CAS) database, and this number increases each year.
The occurrence of emerging pollutants is an important subject of investigation worldwide. Many surveys and studies have been conducted on the topic to better understand their presence and environmental impact [2{6].
More than one-third of Earth’s accessible renewable freshwater is exploited for agricul-tural, industrial, and domestic use, these activities leading to water pollution. Every year around 300 million tons of synthetic compounds of industrial origin and consumer prod-ucts end up in natural waters. This number is 140 million tons for agricultural chemicals mones, steroids, per uorinated chemicals, disinfection by-products, ame retardants, ar-ti cial sweeteners, nanomaterials, pesticides, veterinary products, industrial compounds and by-products, food additives, UV lters… Their sources can be either point-sources (spatially discrete, constrained spatial extent: industrial e uents, municipal sewage treat-ment plants, and combined sewage- storm-water over ows, resource extraction, waste dis-posal sites, buried septic tanks, etc.) or di use sources of pollution (poorly de ned, broad geographical scale: agricultural runo from bio-solids and manure sources, storm-water and urban runo , leakage from reticulated urban sewerage systems, di use aerial deposi-tion, etc.). Usually, the concentrations of emerging pollutants are not too high, although in some cases, it can be signi cant (% 100 ng/l) [11, 12].
The large number and variety of CECs are challenging in terms of regulations, as there is a need for prioritization which itself raises many questions: what kind of quality criteria should be followed – especially in the cases where only limited information is available, which compounds should be targeted, how to take into account spatiotemporal factors and so on. There are \true or really new » contaminants that recently appeared in the scienti c literature, contaminants of emerging interest, previously described but whose environmental impact is not fully understood, and emerging issues appearing about ex-isting chemicals. CECs emerged as epidemiology has improved, and harmful e ects have been demonstrated at lower concentration levels, leading to a dynamically changing eld [13]. For instance, organophosphate pesticides have been replaced by glyphosate, neoni-cotinoid, and pyrethroid pesticides in the early 2000s. Since then, neonicotinoid pesticides have been banned in the EU due to their harmful e ect on pollinators, particularly honey bees. The scienti c community is extensively studying the toxic e ects of glyphosate; re-strictions or bans were recommended and implemented against its use in some European countries [14{16]. When a compound starts raising concerns, data is acquired on its en-vironmental e ects, ecotoxicity, and human toxicity. As su cient evidence and data are accumulated about harmful e ects, governments establish or re-evaluate guidelines, cri-teria, or regulations. CECs should remain emerging until there is not su cient scienti c data and documentation about potential problems. Not all the CECs will be harmful, but the aim is to gather conclusive data to accurately evaluate and assess risks. Richardson et al. publish biennial reviews titled \Emerging Contaminants and Current Issues. » These reviews cover the emerging contaminants, regulations, toxicological data, and advances in analytical chemistry; the last one covers the period between 2017-2019. At the beginning of the 2000s, pesticides were the most studied organic environmental contaminants and, despite regulations, continue to pose a relevant risk due to their em-ployment in large quantities [17]. There are more than 3000 pharmaceutical substances, such as painkillers, antibiotics, lipid regulators, antidepressants, and a substantial amount are entering the water bodies [18]. The testing of their speci c environmental e ects is still limited due to their high number, but estrogenic e ects [19], renal alterations [20], and behavioral changes [21] in aquatic animals were reported in the literature. An unintended consequence of eliminating pathogens from drinking water is the formation of disinfection byproducts, bearing potential harmful e ects for human health, such as carcinogenicity and inducing miscarriage [22]. Every year more and more contaminants emerge as data is becoming available, antibiotics leading to the formation of antibiotic-resistant genes being the most recent addition. It is indisputable that the eld of contaminants of emerging con-cern is extremely dynamic, caused by the continuous increase in chemical manufacturing, together with the advances in highly sensitive, accurate instrumentation and toxicological assessment methodologies, and the expanding availability of monitoring data [23].

Guidelines and regulations on water quality

In the following section, a brief introduction to the timeline of European regulations regarding water quality and contaminants of emerging concern is presented, as it is of particular interest for the AQUAlity project. National agencies, such as the USA Envi-ronmental Protection Agency (EPA), and global organizations, for example, the United Nations Environment Programme (UNEP) and the World Health Organization (WHO), exist to provide guidelines and regulations regarding water quality. European regulations are generally more advanced and complex, taking into account contaminants of emerg-ing concern, despite the fact that their detection and removal requires state-of-the-art technologies and are costly.
EU water framework directive was established in 2000. Under the directive 2000/60/EC, di erent goals were set as a summary of previously held seminars, communications, writ-ten policies to ensure the water quality and avoid long-term deterioration of the European water bodies. The aim is sustainable management and protection of freshwater sources. The primary approach should be preventive in nature. Early and long-term protective measures should be implemented, considering their economic viability and the achieve-ment of the environmental quality standards. Scienti c and technical environmental data should be used for monitoring, risk assessment, elimination of priority hazardous sub-stances, and ultimately ensuring the progressive reduction of pollutants. Hazardous sub-stances are de ned as ‘substances or groups of substances that are toxic, persistent and liable to bio-accumulate, and other substances or groups of substances which give rise to an equivalent level of concern’ [24].
Decision No 2455/2001/EC of the European Parliament and of the Council provided the rst list of 33 priority substances based on combined monitoring-based and modeling-based priority setting (COMMPS) scheme, introduced in Directive 2000/60/EC. This method takes into account the intrinsic hazard of the compounds (ecotoxicity, human toxicity), presence in the environment, and other factors such as production and con-sumption. The list includes both contaminants of anthropogenic origins, like pesticides, ame-retardants, industrial precursors, and naturally occurring compounds, such as met-als and polycyclic aromatic hydrocarbons (PAHs). Emissions, discharges, and losses into water bodies should be phased out for the priority substances originating from human activities. For the naturally occurring substances, background values should be achieved [25].
The Directive 2008/105/EC endorsing and amending the previous Directives gives a higher focus on pollution control, environmental quality standards (EQS), and the e ects of chemical pollution, which should be halted at the source. Acute and chronic toxicity to aquatic organisms, biodiversity loss, accumulation of pollutants in the ecosystem, and hu-man health risk are considered. In Annex II of the directive, EQS are listed for 33 priority substances, including their annual average and maximum allowable concentrations. The need is recognized for high-quality monitoring data on the contaminants in the European Union’s aquatic environment [26].
The directive 2013/39/EU highlights the high cost of wastewater treatment, indicating that the development and implementation of innovative treatment technologies should be supported. In a review of the priority substances, new substances were identi ed, for which the EQS values were set, and some of the existing EQS values were revised; pesticides and per uorooctane sulfonic acid and its derivatives were added to the list. Pharmaceuticals in water and soils were considered as contaminants of emerging concern. The minimum performance criteria for analytical instruments are discussed to obtain relevant environ-mental data. Persistent, bioaccumulative, and toxic substances (PBTs) are among the priority substances, as they are present at levels of signi cant risk, capable of long-range transport, and ubiquitous in the environment. Additional monitoring of substances with PBT-like behavior is encouraged, including spatial and temporal parameters in the mon-itoring. High-quality monitoring data and toxicity tests are required for the prioritization of suspect substances [27]. Under the directive of 2013, a dynamic watch-list was pro-posed to facilitate future prioritization that should include between 10 and 14 substances or groups of substances and be updated every 24 months. Diclofenac, 17-beta-estradiol (E2), and 17-alpha-ethinylestradiol (EE2) were selected for the rst list together with 7 other pollutants or classes of pollutants; the description of their identi cation procedure was presented in a Joint Research Center technical report. This procedure includes data collection, predicted no-e ect concentration calculations, and risk characterization. The selected substances pose a signi cant risk to the aquatic environment (hazard and ex-posure) and lack monitoring data. Compounds belonging to the classes of neonicotinoid and carbamate pesticides, fungicides, re retardants, and antibiotics were identi ed as potentially hazardous [28].
According to Article 8c of the Priority Substances Directive (2008/105/EC), as amended by Directive (2013/39/EU), the European Commission proposes a strategic approach on microbial resistance caused by pharmaceutical substances. This is similar to the com-mitment made in 2017 by the Assembly of the United Nations and WHO. It refers to pharmacovigilance legislation to assess the extent of the water and soil pollution problem regarding pharmaceuticals and personal care products (PPCPs) [29].
Another noteworthy regulation of the European Union, REACH (Registration, Evalu-ation, Authorization, and Restriction of Chemicals), entered into force in 2007, aiming at lowering the risks posed by chemicals for human health and the environment [30]. It encompasses industrial chemicals but also substances used in households. REACH estab-lishes protocols for risk assessment by collecting information on the properties and hazards of substances. Their databases contain valuable information on many compounds. This will in uence future regulations to improve water quality, preserve biota, and protect human health.
The French regulations on water quality for human consumption and raw water are gov-erned by the public health code CSP (Articles L. 1321-1 to 1321-10 and R. 1321-1 to 1321-68). France applies European directives into its national law regarding the water and sanitation eld. The quality limits of water intended for human consumption are the same as those proposed in the council directive 98/83/EC. As an example, the directive indicates an individual pesticide quality limit value of 0.1 µg/L. Exceptions are aldrin, dieldrin, heptachlor, and heptachlor epoxide, with a lower parametric value of 0.03 µg/L. For the sum of pesticides and their relevant metabolites, the quality limit is 0.5 µg/L [31].
Financed by the European Commission, the NORMAN network was established in 2005, being a network of reference laboratories, research centers and related organisations for monitoring of emerging environmental substances. The network aims to improve data collection, enhance information exchange on emerging substances, and standardize mea-surement methods. Problem-oriented research has to be ensured with a focus on tailored solutions for speci c needs. INERIS is a partner of the AQUAlity project and is part of the NORMAN network. In the framework of the 3rd AQUAlity winterschool and meeting, INERIS organized a workshop on substance prioritization. As a result of this fruitful event, a report was drafted to identify challenges and solutions, titled \Contaminants of Emerg-ing Concern in Urban Wastewater Joint NORMAN and Water Europe Position Paper » and an intensi ed interest from stakeholders. To achieve the water quality goals, provide clean water to all, and protect the environment, large-scale collaborations are essential.

READ  In vitro methane production of Eragrostis hay treated with graded levels of urea or nitrate

Compounds selected for the present study

The modeling approach consists of performing in-lab photodegradation reactions on in-dividual compounds or on a limited number of molecules (e.g., a marketed product con-taining the active ingredient), therefore a few compounds had to be selected from the myriad of existing contaminants of emerging concern. For this reason, the NORMAN list of CECs was examined; it contains 1036 compounds, including the most frequently discussed emerging substances and pollutants.
In the framework of the AQUAlity project, 41 contaminants of emerging concern were selected to study their presence in the environment, photochemistry, potential advanced elimination technologies, and toxicity. These contaminants were chosen based on a priori-tization performed by INERIS. This was a simpli ed version of the NORMAN approach; the substances were ranked using a score calculated from key indicators such as expo-sure, hazard, persistency, bioaccumulation, and mobility [33, 34]. Furthermore, during the Workshop on the Prioritization of CECs in Urban Wastewaters, organized within the AQUAlity project, additional CECs were suggested to be studied. In the framework of this PhD project, the studied compounds from the proposed NORMAN list were: per uorooc-tanoic acid, per uorononanoic acid, imidacloprid, acetamiprid, and maprotiline. Other studied CECs were selected based on their relevant environmental concentrations, risk or toxic e ects: naproxen, benzisothiazolinone, enro oxacin, amitryptiline, and gem brozil. Five contaminants were selected for detailed photochemical studies and are introduced below. Extensive information and literature review about benzisothiazolinone, naproxen, and maprotiline is presented in the results chapter within the published articles.
In addition to their relevance in terms of environmental presence and potentially harmful e ects, these molecules were also selected to increase our group’s level of expertise in pho-todegradation mechanism elucidation. The compounds were selected to represent di erent classes of compounds with a variety of functional groups. Understanding the formation of the photoproducts was challenging and helped us deepen our knowledge in photochem-istry and interpretation of MS fragmentation patterns. They also show di erent kinds of biological activity, therefore adequate toxicity testing approaches were essential.
Benzisothiazolinone (Figure 1.1.1), a preservative and antimicrobial agent, was the rst compound selected, as it is of high interest for our working group due to an obvious lack of information regarding its photodegradation mechanism. The LCM plans future works on benzisothiazolinone photodegradation in mixture with other pesticides, as this compound is often marketed in solution with active substances. Being in a mixture could alter the photodegradation kinetics of the present compounds or the nature of the photodegradation products.
Naproxen (Figure 1.1.2), another candidate molecule on the NORMAN list of emerg-ing pollutants, is a nonsteroidal anti-in ammatory drug. This compound was selected due to its extensive consumption and high detection frequency in the environment. The photodegradation of naproxen was previously studied, although only a simpli ed mecha-nistic pathway was suggested for the reaction that was mainly focused on the degradation kinetics, without in-depth structural elucidation of photoproducts [35].

Table of contents :

1 Bibliography 
1.1 Context and molecules
1.1.1 The concept of contaminants of emerging concern
1.1.2 Guidelines and regulations on water quality
1.1.3 Compounds selected for the present study
1.2 Photochemistry
1.2.1 Generalities on photochemistry
1.2.2 Photolysis of contaminants
1.2.3 Advanced oxidation processes
1.2.4 Applications and limitations of AOPs
2 Materials and methods 
2.1 Photodegradation set-ups and parameters
2.2 In vitro and in silico toxicity estimations
2.3 HRMS and non-targeted analysis
2.4 ESI and FT-ICR MS
2.5 Direct infusion mass spectrometry (DI{MS)
2.6 Data processing
3 Modeling approach – Results on direct photochemical reactions 
3.1 General approach
3.2 Photolysis of benzisothiazolinone
3.3 Photolysis of naproxen
4 Modeling approach – Results on indirect photochemical experiments 
4.1 General approach
4.2 Photodegradation experiments on per uorooctanoic acid
4.3 Photoinduced transformation of maprotiline
4.4 Role of iron in the photodegradation of enro oxacin
4.5 Photodegradation of enro oxacin in a pilot plant
5 Untargeted approach using SPIX 
5.1 Introduction of Spix
5.2 SPIX software
5.3 Latest developments of SPIX – 3D approach


Related Posts