Susana Patrícia Fontes da Costa

Development of automatic bioassays for the

evaluation of ionic liquids’ toxicity and

biodegradability

Tese do 3º Ciclo de Estudos Conducente ao Grau de Doutoramento em Ciências

Farmacêuticas na Especialidade de Química Analítica

Trabalho realizado sob a orientação da Professora Doutora Maria Lúcia Marques Ferreira

de Sousa Saraiva e co-orientação da Doutora Paula Cristina de Azevedo Gomes Pinto

maio de 2017

III

É autorizada a reprodução integral desta tese apenas para efeitos de investigação,

mediante declaração escrita do interessado, que a tal se compromete.

Assinatura do autor,

IV

“Ninguém é tão ignorante que não tenha algo a ensinar.

Ninguém é tão sábio que não tenha algo a aprender.”

(Blaise Pascal)

V

Às pessoas que sempre acreditaram em mim e me fizeram continuar,

Pai,

Mãe,

Victor,

Sandra.

VI

Agradecimentos

Não posso finalizar esta etapa marcante da minha vida sem relembrar e

agradecer a algumas das pessoas que me incentivaram e ajudaram a superar os

percalços desta viagem atribulada chamada “Doutoramento em Ciências Farmacêuticas”.

Ao Professor Doutor José Luís Costa Lima e à Professora Doutora Sallete Reis

agradeço a autorização para realizar o meu Doutoramento no Laboratório de Química

Aplicada. Os seus incentivos e acolhimento durante todo o percurso fizeram-me sentir

uma privilegiada por ter integrado tão prestigiada equipa de investigação.

À Professora Doutora Mª Lúcia Saraiva, orientadora científica desta tese agradeço

a confiança, o apoio e todos os conselhos e críticas feitos ao longo da execução deste

projeto, só assim foi possível a entrega desta tese. Agradeço também a disponibilidade,

simpatia e amizade, sempre demonstradas.

À Professora Paula Pinto, minha co-orientadora, agradeço por todo o que ensinou

não só a nível científico, mas também na forma de estar na vida, com a sua contagiante

simpatia, bem-estar e constante preocupação com os seus orientandos.

Ao Professor Doutor Fábio Rocha e à Professora Doutora Regina Monteiro

agradeço a disponibilidade e orientação que foram essenciais para a concretização do

trabalho realizado no CENA, Brasil.

Agradeço também a todos os colegas do CENA pela calorosa receção e pronta

ajuda durante a estadia. Um agradecimento especial à Joyce, à Gabriela, ao Eduardo e à

Karina pelo apoio e colaboração decisiva no trabalho desenvolvido nesse período.

Agradeço ainda à Vanessa e à Bárbara pela ajuda no desenvolvimento de

experimental de alguns dos trabalhos aqui apresentados.

Aos Professores do Laboratório de Química Aplicada deixo uma palavra de

apreço pela sua recetividade e simpatia. Ao Professor Doutor Rui Lapa e ao Professor

Doutor João Santos deixo um agradecimento particular pela sua boa disposição, ajuda e

constante preocupação.

À D. Manuela, à Patrícia e à Vânia, pela simpatia e disponibilidade que sempre

demonstraram.

À Cláudia, à Sofia, à Edite, à Ana Marta, à Ana Luísa e ao David muito obrigada

pela vossa amizade e apoio ao longo desta jornada, quer nos momentos bons, quer nos

menos bons. A minha vida nunca foi a mesma desde que vos conheci!

A todos os colegas do laboratório obrigada pelo acolhimento, pelo

companheirismo, pela solidariedade e pelo incentivo. Com particular apreço à Marieta por

toda ajuda e contribuição prestados desde que iniciei-me na investigação.

VII

À Lígia, à Ana Cardoso, à Margarida, à Sandra, à Ana Fróis e à Joana que mesmo

sendo amigas outsider sempre me incentivaram e apoiaram a alcançar os meus

objetivos.

Agradeço à Faculdade de Farmácia da Universidade do Porto por me ter admitido

como estudante de Doutoramento, disponibilizando todos os meios necessários para a

realização deste trabalho. Agradeço, também, à Fundação para a Ciência e a Tecnologia

(FCT) pelo suporte financeiro referente à bolsa de Doutoramento com referência

SFRH/BD/86381/2012 e todo o apoio financeiro, no âmbito do “QREN e POPH e

Tipologia 4.1 e Formação Avançada”, co-financiado pelo FSE e pelos fundos nacionais

do MCTES.

Aos meus pais, ao meu irmão Victor e à minha cunhada Sandra muito obrigada

por tudo! Estiveram sempre presentes quando precisei, apoiando sempre as minhas

decisões e encorajando-me ao longo do percurso, que por vezes tornou-se atribulado e

nem sempre fácil, relembrando-me que a meta estava cada vez mais próxima.

VIII

Abstract

The remarkable physic-chemical characteristics of ionic liquids (ILs) combined to the

possibility of tuning them resulted in an increasing interest of scientific and industrial

communities in these compounds and their potentialities as alternative to traditional

organic solvents. Before the use of these compounds in large scale it is necessary to

ensure their quality, efficiency and security. Concerning the security, it is important

evaluate among other parameters the ecotoxicological effects of these molecules, their

degradability and risk of persistence in the environment. Therefore, the evaluation of ILs

environmental impact before their release to environment is of primordial importance.

Under the scope of this dissertation were applied methodologies in discrete mode and

based in flow techniques to evaluate ILs toxicity and their (bio)degradability. It was used

sequential injection analysis (SIA), with the aim of associate the potentialities of this type

of technique to the assessment of ILs. The described works had the objective of develop

and implement alternative methods with a good sampling rate, good repeatability, robust

and suitable to evaluate the effect of ILs in living organisms and enzymes, as well as to

determine the risk of permanence in the aquatic field. Furthermore, these methods should

present an effective reduction of costs through the decrease in reagents consumption

combined to a small production of effluents.

The first experimental work presented in this thesis consisted in the assessment of acute

aquatic toxicity of six ILs using the freshwater organisms Daphnia magna, Raphidocelis

subcapitata and Hydra attenuata, with the last one being applied for the first time in

ecotoxicological studies with ILs. The bioassays were performed exposing the organisms

to increasing concentrations of ILs and observation of D. magna immobilization, inhibition

of R. subcapitata growth and the effect in H. attenuata morphology and the mortality

associated.

The aquatic toxicity of ILs was also tested using the bacteria Vibrio fischeri and the effect

in its bioluminescent activity. The assays were performed in a SIA system for contact

periods of 5, 15 and 30 minutes, as performed in the conventional methods. To maximize

the sampling rate of the method were used three mixing chambers and conducted assays

with different stop periods in simultaneous. Additionally, the use of mixing chambers

ensured the creation of optimized conditions for the contact between bacteria and tested

ILs. The results were also compared with the obtained data in the conventional test

Biotox®. The inhibitory profile of each IL for each of the tested stop period was determined

and also calculated the concentration of compound necessary to cause 50% of decrease

IX

in the bacteria bioluminescence. These values were analyzed to verify if there were

significant differences between the times of reaction in study.

The effects of ILs in the enzymatic activity were tested for the enzyme cytochrome c

oxidase with the evaluation of its inhibition. The assay based in monitor the increase in the

absorbance at 550 nm due to inhibition of iron oxidation reaction from the oxidation +2 to

+3 in the molecule ferrocytochrome c, which was catalyzed by the enzyme cytochrome c

oxidase. The inhibitory profile of each IL was determined and calculated the concentration

of compound necessary to cause 50% of decrease in the enzymatic activity.

In the methodology to determine ILs chemical oxygen demand (COD) was implemented a

step of photodegradation by UV light with the objective of increase the degradability of

samples in study. These values of COD for the selected ILs were determined by

fluorimetry to evaluate the reduction of cerium (IV) in the presence of ILs. The method

was calibrated resorting to a solution of potassium hydrogen phthalate which is

standardized for COD assays.

The developed method for the rapid determination of biochemical oxygen demand (BOD)

was based in the chemiluminescent reaction between luminol and active oxygen species

resulting from quinone oxidation by Baker’s yeast through a redox reaction, and catalyzed

by ferricyanide. The use of a chemiluminometer with two input ports in the cell guaranteed

that the reaction between active oxygen species and luminol occurred inside the

chemiluminescence cell.

Finishing, in the scope of this thesis, it was assessed the toxicological potential of various

ILs in distinct biological systems and identified possible toxicophores. Moreover, it was

evaluated the chemical oxygen demand of these compounds, as well as their sensitivity to

photodegradation. The works also aimed the development of new analytical procedures

suitable to be implemented for these purposes, with the added value of automation.

Keyword: Sequential injection analysis; Automation; (Bio)degradability; (Eco)toxicity;

Ionic liquids.

X

Resumo

As notáveis características físico-químicas dos líquidos iónicos (LIs) combinado com a

possibilidade de moldagem das mesmas resultou num crescente interesse das

comunidades científica e industrial nestes compostos e nas suas potencialidades como

alternativa aos tradicionais solventes orgânicos. Antes do uso destes compostos em larga

escala é necessário assegurar a sua qualidade, eficácia e segurança. No que refere à

segurança, é importante avaliar entre outros parâmetros os efeitos ecotoxicológicos

destas moléculas, a sua degradabilidade e o risco de persistência no ambiente. Assim

sendo, a avaliação do impacto ambiental dos LIs antes da sua libertação para o ambiente

é de primordial importância.

No âmbito desta dissertação foram aplicadas metodologias em modo discreto e baseadas

em técnicas de fluxo para a avaliação toxicológica dos LIs e sua (bio)degradabilidade. Foi

usada a análise por injeção sequencial (SIA), visando associar as potencialidades deste

tipo de técnicas à avaliação dos LIs. Os trabalhos desenvolvidos tiveram como objetivo o

desenvolvimento e implementação de métodos alternativos com bom ritmo de

amostragem, boa repetibilidade, robustos e apropriados para avaliar o efeito dos LIs em

organismos vivos e enzimas, bem como para determinar o risco de permanência em

meios aquáticos. Além disso, apresentaram uma redução efetiva de custos através da

diminuição de consumo de reagentes associada a baixa produção de efluentes.

O primeiro trabalho experimental apresentado nesta tese consistiu na avaliação da

toxicidade aquática aguda de seis LIs usando os organismos de água fresca Daphnia

magna, Raphidocelis subcapitata e Hydra attenuata, sendo que esta última foi pela

primeira vez aplicada em estudos toxicológicos com LIs. Os bioensaios foram realizados

expondo os organismos a concentrações crescentes de LIs e observação da imobilização

da D. magna, da inibição do crescimento da R. subcapitata e dos efeitos na morfologia da

H. attenuata e mortalidade associada.

A toxicidade aquática dos LIs foi também testada recorrendo à bactéria Vibrio fischeri e

ao efeito na sua atividade bioluminescente. Os ensaios foram realizados num sistema

SIA para tempos de contacto de 5, 15 e 30 minutos, tal como é realizado nos métodos

convencionais. Para maximizar o ritmo de amostragem do método foram usadas três

câmaras de mistura e conduzidos ensaios com os diferentes tempos de paragem em

simultâneo. Adicionalmente, o uso de câmaras de mistura assegurou a criação de

condições ótimas para o contacto entre a bactéria e os LIs em estudo. Os resultados

foram ainda comparados com os obtidos no teste convencional Biotox®. O perfil inibitório

de cada LI para cada um dos tempos de paragem estudados foi determinado e foi

XI

também calculada a concentração de composto necessária para causar 50% de

decréscimo na bioluminescência da bactéria. Esses valores foram analisados para

verificar se existiam diferenças significativas entre os tempos de reação em estudo.

O efeito de LIs sobre a atividade enzimática foi testado para a enzima citocromo c

oxidase tendo-se avaliado a sua inibição. O ensaio baseou-se na monitorização do

aumento da absorvência a 550 nm devido à inibição da reação de oxidação do ferro do

estado de oxidação de +2 para +3 na molécula ferrocitocromo c, a qual era catalisada

pela enzima citocromo c oxidase. O perfil inibitório de cada LI foi determinado e calculada

a concentração de composto necessária para causar 50% de decréscimo na atividade

enzimática.

Na metodologia para determinar a carência química de oxigénio (CQO) dos LIs foi

implementada uma etapa de fotodegradação por luz ultravioleta com o objetivo de

aumentar a degradação das amostras em estudo. Os valores de CQO dos LIs

selecionados foram determinados fluorimetricamente por avaliação da redução do cério

(IV) na presença de LIs. O método foi calibrado recorrendo a uma solução de

hidrogenoftalato de potássio padronizada para ensaios de CQO.

O método desenvolvido para a determinação rápida da carência bioquímica de oxigénio

(CBO) fundamentou-se na reação de quimiluminescência do luminol com espécies ativas

de oxigénio resultantes da oxidação da quinona em reações redox com a levedura do

padeiro, e catalisado pela ferricianida. O uso de um quimioluminómetro com duas portas

de entrada na célula garantiu que a reação entre os radicais ativos de oxigénio com o

luminol ocorresse dentro da célula de quimiluminescência.

Finalizando, no âmbito dos objetivos desta tese, foi avaliado o potencial toxicológico de

vários LIs em distintos sistemas biológicos e identificados possíveis toxicóforos. Além

disso, foi avaliada a carência química destes compostos, bem como a sua sensibilidade à

fotodegradação. Os trabalhos visaram também o desenvolvimento de novos

procedimentos analíticos passíveis de implementação para estes fins, com a mais-valia

da automatização.

Palavras-chave: Análise por injeção sequencial; Automatização; (Bio)degradabilidade;

(Eco)toxicidade; Líquidos iónicos.

XII

Index

AGRADECIMENTOS VII

ABSTRACT IX

RESUMO XI

INDEX XII

INDEX OF FIGURES XVIII

INDEX OF TABLES XXII

ABBREVIATIONS XXV

SYMBOLS XXIX

OUTLINE OF THE THESIS XXX

CHAPTER 1.

General Introduction 1

A. Environmental impact of ionic liquids: an overview of recent

(eco)toxicological and (bio)degradability literature 2

Abstract 3

1. Introduction 3

2. Concernings about the greenness of ionic liquids synthesis 5

3. Degradation of ionic liquids 6

3.1. Recent data regarding ionic liquids biodegradability 6

3.1.1. Anions 6

3.1.2. Cations 7

3.1.3. Biodegradation of ionic liquids by elected species 10

3.2. Advanced oxidation processes (AOPs): chemical, photochemical and

electrochemical degradation 12

3.3. Ionic liquids ability to adsorb 14

4. Toxicological impact of ionic liquids 16

4.1. Effects of ionic liquids on organisms with different levels of complexicity 16

4.1.1. Antifungal and antibacterial properties 16

4.1.2. Enzymatic inhibition 18

4.1.3. Cytotoxicity 18

4.1.4. Aquatic toxicity 19

XIII

4.1.5. Phytotoxicity 21

4.1.6. Others species of living organisms 21

4.2. Structural-activity relationships models 22

5. Summary 23

References 24

B. General and specific objectives of the thesis 55

CHAPTER 2.

Experimental 57

1. Introduction 58

2. Reagents and solutions 58

3. Flow apparatus and instrumentation 58

3.1. General aspects of flow analysis systems 58

3.2. Components of flow systems 59

3.2.1. Propulsion/aspiration devices 59

3.2.2. Fluid selection device 60

3.2.3. Tubing and other manifold components 61

3.2.4. Detection systems 62

3.2.5. Computer control and data acquisition 62

3.3. Other instrumentation 63

4. Development and optimization of the flow systems 64

5. Preparation, maintenance and application of aquatic organisms Daphnia

magna, Raphidocelis subcapitata and Hydra attenuata on ecotoxicological

assays

66

5.1. Maintenance of tested organisms 66

5.2. Toxicity tests 67

6. Statistical treatment of results 69

References 71

CHAPTER 3.

The aquatic impact of ionic liquids on freshwater organisms 73

Abstract 74

XIV

1. Introduction 74

2. Materials and methods 75

2.1. Test compounds 75

2.2. Maintenance of test organisms 75

2.3. Toxicity tests 76

2.4. Data analysis 77

3. Results and discussion 77

4. Conclusions 78

References 79

CHAPTER 4.

Toxicity assessment of ionic liquids with Vibrio fischeri: An alternative fully

automated methodology 81

Abstract 82

1. Introduction 83

2. Materials and methods 83

2.1. Reagents 83

2.2. Apparatus 84

2.3. Reconstitution of lyophilized V. fischeri 85

2.4. Sequential injection procedure 85

2.5.Microplate reader assay 86

3. Results and discussion 86

3.1. Optimization of the SIA system 86

3.2. Evaluation of ILs (eco)toxicity through automated and batch assays 86

4. Conclusions 87

References 88

CHAPTER 5.

Automated cytochrome c oxidase bioassay developed for ionic liquids’ toxicity

assessment 89

Abstract 90

1. Introduction 91

XV

2. Materials and methods 91

2.1. Reagents 91

2.2. Apparatus 92

2.3. Sequential injection procedure 92

3. Results and discussion 93

3.1. Optimization of the SIA assay 93

3.2. Cytochrome c oxidase inhibition in the presence of ionic liquids 94

4. Conclusions 96

References 96

CHAPTER 6.

Environmental impact of ionic liquids: Automated evaluation of chemical oxygen

demand of photochemical degraded compounds 98

Abstract 99

Introduction 99

Results and discussion 100

SIA assay optimization 100

Application of the automated methodology to evaluate ILs 101

Conclusions 102

Experimental Section 103

Reagents 103

Apparatus 104

Sequential injection procedure 104

Dichromate method 104

References 105

CHAPTER 7.

Microfluidic chemiluminescence system with yeast Saccharomyces cerevisiae

for rapid biochemical oxygen demand measurement 107

Abstract 109

1. Introduction 110

2. Materials and methods 112

XVI

2.1 Reagents 112

2.2 Apparatus 113

2.3 Sequential injection procedure 114

3. Results and Discussion 116

3.1 Optimization of the SIA assay 116

3.2 Applicability of the automated BOD method 119

4. Conclusions 121

References 122

CHAPTER 8.

Final conclusions 126

XVII

Index of figures

CHAPTER 1.

General Introduction

A. Environmental impact of ionic liquids: an overview of recent

5

7

11

12

22

(eco)toxicological and (bio)degradability literature

Figure 1. The potential contamination f ionic liquids.

Scheme 1. Possible products of the hydrolysis of dicyanimide anion detected by MS measurements. The assumed degradation products are highlighted

in gray. Adapted from Ref. [50].

Scheme 2. a) Pathway for aerobic biodegradation of omim. b) Products of anaerobic breakdown of omim, as identified by MS measurements. c)

Major degradation pathways of the omim cation in presence of microbial

community of wastewater from Jeonju city (based on information from Refs.

[75, 76, 78], and [79]).

Figure 2. General guidelines about ionic liquids' biodegradability, considering cation, anion and alkyl side-chain moieties.

Figure 3. Effects of ionic liquids on living organisms at cellular and subcellular levels. Image of cell from ChemDraw.

Figure 4. Some (eco)toxicity trends of ILs and routes to synthesize greener ILs. 24

CHAPTER 2.

Experimental

Figure 1. A. Schematic representation of Valco selection valve components. a)

screws to fix the valve head; b) side ports; c) stator; d) rotor; e) actuator; f) valve

body. Adapted from (9). B. Photography of fluid selection device. 61

Figure 2. Photography of mixing chamber. 62

Figure 3. Photography of computer software. 63

Figure 4. Photography of D. magna at real size and amplified through a 67

XVIII

stereomicroscope.

Figure 5. Photography of Neubauer measuring chamber; and, R. subcapitata

viewed under a microscope at 400x of amplification. 68

Figure 6. Progressive changes in H. attenuata morphology from sublethal to

lethal stages when exposed to xenobiotic agents. Adapted from Santos et al.,

2007 (15). 69

CHAPTER 4.

Toxicity assessment of ionic liquids with Vibrio fischeri: An alternative fully

automated methodology

Figure 1. Chemical structures of the studied ILs: (1) 1-ethyl-3-

methylimidazolium methanesulfonate (emim [Ms]); (2) 1-ethyl-3-

methylimidazolium bis(trifluoromethylsulfonyl)imide (emim [Tf2N]); (3) 1-ethyl-3-

methylimidazolium acetate (emim [Ac]); (4) choline acetate (chol [Ac]); (5) 1-

butyl-4-methylpyridinium chloride (bmpy [Cl]); (6) 1-butyl-4-methylpyridinium

tetrafluoroborate (bmpy [BF4]); (7) tetrabutylammonium chloride (N4,4,4,4 [Cl]). 84

Figure 2. Schematic representation of the SIA manifold. C: carrier (NaCl 2%, pH

7); S: syringe; HC: holding coil; SV: selection valve; D: detector; IL: ionic liquid;

MC: mixing chambers (containing a magnet each). 85

Figure 3. Effect of different inhibitor volumes (10-100 µL) in V. fischeri

bioluminescence. 86

Figure 4. Experimental toxicity data of ILs tested in SIA system, for 5 min of

contact time (average of three replicates). 86

CHAPTER 3.

The aquatic impact of ionic liquids on freshwater organisms

Figure 1. Chemical structures of the studied ILs: (1) bmim [PF6]; (2) bmpyr

[BF4]; (3) N1,1 [N1,1,1OOH]; (4) N4,4,4,4 [BF4]; (5) emim [Tf2N]; (6) (Hex)3(TDec)P [Cl];

(7) acetonitrile; (8) methanol. 76

XIX

CHAPTER 5.

Automated cytochrome c oxidase bioassay developed for ionic liquids’ toxicity

assessment

Figure 1. Chemical structures of the studied ILs. 1. emim [Ms]; 2. emim [BF4]; 3.

bmim [BF4]; 4. bmim [Cl]; 5. bmpyr [Cl]; 6. tbph [Ms]; 7. bmpy [BF4]; 8. hmim [Cl];

9. bmim [Ac]; 10. chol [Ac]; 12. bmpyr [BF4]; 13. emim [Tf2N]; 14. emim [TfMs];

15. N4,4,4,4 [BF4]. 92

Figure 2. Schematic representation of the SIA system applied in the enzymatic

inhibition assays. C: carrier (Tris-HCl buffer 10 mmol L-1 pH 7.0, with KCl 120

mmol L-1); S: syringe; HC: holding coil (2 m); SV: selection valve; RC: reaction

coil (1 m); D: detector; IL: ionic liquid; E: enzyme (CytCox); S: substrate (FeC);

W: waste. 93

Figure 3. Effect of different FeC volumes (20-60 µL) in CytCox activity; and

CytCox volume (20-80 µL) in absorbance signal. 94

Figure 4. Influence of enzyme concentration (10-20 mU) in the FeC reduction. 94

Figure 5. Influence of reaction coil length (60-200 cm) in the CytCox activity. 94

Figure 6. Effect of different times of stop period (0.5-7.5 min) in the reaction coil

in CytCox activity. 94

Figure 7. Experimental toxicity data of ILs tested in the developed SIA system. 95

CHAPTER 6.

Environmental impact of ionic liquids: Automated evaluation of chemical oxygen

demand of photochemical degraded compounds

Figure 1. Effect of Ce(IV) concentration (0.1 to 3 mmol L-1) in the fluorescence

intensity. 101

Figure 2. Effect of aliquots intercalation sequences in the fluorescence intensity. 101

Figure 3. Representation of Ce(IV) aspiration flow rate influence on

fluorescence intensity; and, the effect of propulsion flow rate of the mixture to

the reactor coiled to UV-lamp and to detector on the method sensibility. 101

Figure 4. Experimental data of tested ILs COD values resorting to SIA system. 102

XX

Figure 5. Chemical structure of the studied ILs. 1. emim [Ms]; 2. emim [BF4]; 3.

bmim [BF4]; 4. bmim [Cl]; 5. bmpyr [Cl]; 6. hmim [Cl]; 7. bmim [Ac]; 8. emim [Ac];

9. Chol [Ac]; 10. bmpyr [BF4]; 11. N4,4,4,4 [Cl]; 12. emim [TfMs]. 103

Figure 6. Schematic representation of the SIA system applied in the

determination of COD. C: carrier (ultrapure water); S: syringe (5 mL); HC:

holding coil (2 m); SV: selection valve; RC: reaction coil (1 m); FD: fluorimetric

detector; IL: ionic liquid; B: blank (ultrapure water); S: substrate (Ce(IV)); L:

reactor coiled to UV-lamp; W: waste. 104

CHAPTER 7.

Microfluidic chemiluminescence system with yeast Saccharomyces cerevisiae

for rapid oxygen demand measurement

Figure 1. Schematic representation of the SIA system applied in the

determination of BOD values. C: carrier (phosphate buffer, pH 7.0); S: syringe (5

mL); HC: holding coil (2 m); SV: selection valve; H: hydrogen peroxide; L:

luminol; Fe: potassium hexacyanoferrate (III); N: 1,2-naphthoquinone-4-sulfonic

acid; Y: yeast S. cerevisiae; SS: standard solution (GGA); MC: mixing chamber;

D: detector; W: waste. 114

Figure 2. Representation of the effect of reagents concentration in the

percentage of chemiluminescence intensity increase resulting from differences

between blank and GGA assays. 118

Figure 3. Effect of S. cerevisiae concentration in the percentage of

chemiluminescence intensity increase resulting from differences between blank

and GGA assays. 119

XXI

Index of tables

CHAPTER 1.

General Introduction

A. Environmental impact of ionic liquids: an overview of recent

(eco)toxicological and (bio)degradability literature

Table S1 Supplementary data. Ionic liquids ecotoxicity data since 2010. 29

CHAPTER 2.

Experimental

Table 1. Composition of AAM (11). 66

CHAPTER 3.

The aquatic impact of ionic liquids on freshwater organisms

Table 1. Results of R. subcapitata growth inhibition after 72 h of exposure to the

tested compounds, expressed as IC50 (values in mg L-1) and with respective

95% confidence limits. 77

Table 2. Results of D. magna immobilization after 48 h of exposure to the tested

compounds, expressed as LC50 (values in mg L-1) and with respective 95%

confidence limits. 78

Table 3. Results of H. attenuata morphologic modifications or death after 96 h of

exposure to the tested compounds, expressed as EC50 and LC50 values,

respectively (im mg L-1), and with the respective 95% confidence limits. 78

CHAPTER 4.

Toxicity assessment of ionic liquids with Vibrio fischeri: An alternative fully

automated methodology

XXII

Table 1. Analytical cycle for the evaluation of V. fischeri bioluminescence in the

presence of the tested ILs. 85

Table 2. Results of the inhibition of V. fischeri by ILs expressed as EC50 for 5,

15, and 30 min of contact time in the SIA system. 87

Table 3. Results of the inhibition of V. fischeri by ILs expressed as EC50 for 5,

15, and 30 min contact time in the microplate approach. 87

CHAPTER 5.

Automated cytochrome c oxidase bioassay developed for ionic liquids’ toxicity

assessment

Table 1. Analytical cycle for the evaluation of CytCox inhibition in the presence

of the tested ILs. 93

Table 2. Results of the optimization of CytCox inhibition assay in the SIA

system. 93

Table 3. Results of the inhibition of CytCox by ILs expressed as EC50. 95

CHAPTER 6.

Environmental impact of ionic liquids: Automated evaluation of chemical oxygen

demand of photochemical degraded compounds

Table 1. Representation of the developed methodology optimized parameters. 100

Table 2. Results of COD values expressed as mg L-1 of O2 in the automated

and standard assays for 12 different ILs. 102

Table 3. Analytical cycle established for monitoring ILs susceptibility to

photodegradation and determination of COD values. 104

CHAPTER 7.

Microfluidic chemiluminescence system with yeast Saccharomyces cerevisiae

for rapid biochemical oxygen demand measurement

Table 1. Analytical cycle applied in SIA system to determine BOD. 115

XXIII

Table 2. Results of the optimization of BOD assay in the flow system. 116

XXIV

Abbreviations

(Hex)3(TDec)P [Cl] trihexyltetradecylphosphonium chloride

[C2F5]3PF3 trifluoridotris(pentafluoroethyl)phosphate

2-HDEAA 2-hydroxydiethanolamine acetate

2-HDEAB 2-hydroxydiethanolamine butanoate

2-HDEAF 2-hydroxydiethanolamine formate

2-HDEAiB 2-hydroxydiethanolamine isobutanoate

2-HDEAPe 2-hydroxydiethanolamine pentanoate

2-HDEAPr 2-hydroxydiethanolamine propionate

2-HEAB 2-hydroxyethanolamine butanoate

2-HEAF 2-hydroxyethanolamine formate

2-HTEAB 2-hydroxytriethanolamine butanoate

2-HTEAPe 2-hydroxytriethanolamine pentanoate

AAM algal assay medium

AC activated carbon

AChE acetylcholinesterase

AOP advanced oxidation processes

APX ascorbate peroxidase

B[CN]4 tetracyanidoboranate

BDD boron-doped diamond

BF4 tetrafluoroborate

bmim [Ac] 1-butyl-3-methylimidazolium acetate

bmim [BF4] 1-butyl-3-methylimidazolium tetrafluoroborate

bmim [Br] 1-butyl-3-methylimidazolium bromide

bmim [Cl] 1-butyl-3-methylimidazolium chloride

bmim [PF6] 1-butyl-3-methylimidazolium hexafluorophosphate

bmpy [BF4] 1-butyl-4-methylpyridinium tetrafluoroborate

bmpy [Br] 1-butyl-4-methylpyridinium bromide

bmpy [Cl] 1-butyl-4-methylpyridinium chloride

bmpy [dca] 1-butyl-4-methylpyridinium dicyanamide

bmpyr [BF4] 1-butyl-1-methylpyrrolidinum tetrafluoroborate

bmpyr [BF4] 1-butyl-1methylpyrrolidinium tetrafluoroborate

bmpyr [Cl] 1-butyl-1-methylpyrrolidinium chloride

BOD biochemical oxygen demand

C carrier

XXV

C[CN]3 tricyanmethanide

C10mim [Br] 1-decyl-3-methylimidazolium bromide

CAT catalase

CCO channel catfish ovary cells

Ce(IV) cerium (IV)

CHO chinese hamster ovary cells

chol [AA] cholinium amino acids

chol [Ac] choline acetate

Cl chloride

COD chemical oxygen demand

CytCox cytochrome c oxidase

D detector

DABCO 1,4-diazabicyclo[2.2.2]octane

DESs deep eutectic solvents

DNA deoxyribonucleic acid

DOC dissolved organic carbon

EC50 median effective concentration

emim [Ac] 1-ethyl-3-methylimidazolium acetate

emim [BF4] 1-ethyl-3-methylimidazolium tetrafluoroborate

emim [Ms] 1-ethyl-3-methylimidazolium methanesulfonate

emim [Tf2N] 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide

emim [TfMs] 1-ethyl-3-methylimidazolium trifluoromethanesulfonate

epy [BF4] 1-ethylpyridinium tetrafluoroborate

epy [CF3COO] 1-ethylpyridinium trifluoroacetate

ETA extended topochemical atom

FD fluorimetric detector

FeC ferrocytochrome c

FIA flow injection analysis

GC gas chromatography

GGA glutamic acid and glucose

GPX guaiacol peroxidase

GSH glutathione content

GST glutathione S-transferase

HC holding coil

hmim [Cl] 1-hexyl-3-methylimidazolium chloride

hmpy [Br] 1-hexyl-4-methylpyridinium bromide

XXVI

hmpy [Tf2N] 1-hexyl-4-methylpyridinium bis(trifluoromethylsulfonyl)imide

HPLC high performance liquid chromatography

IC50 median inhibitory concentration

ILs ionic liquids

ISO International Organization for Standardization

L reactor coiled to UV-lamp

LC50 median lethal concentration

LD50 median lethal dose

LDH lactic dehydrogenase

m(oe)2mim [Cl] 1-(2-(2-methoxy-ethoxy)-ethyl)-3-methylimidazolium chloride

m-2-HEAA N-methyl-2-hydroxyethylammonium acetate

m-2-HEAB N-methyl-2-hydroxyethylammonium butyrate

m-2-HEAP N-methyl-2-hydroxyethylammonium pentanoate

m-2-HEAPr N-methyl-2-hydroxyethylammonium propionate

MBC minimal bactericidal concentration

MC mixing chamber

MD coarse-grained molecular dynamics

MDA malondialdehyde

MIC minimal inhibitory concentration

moemim [Cl] 1-methoxyethyl-3-methylimidazolium chloride

MS mass spectrometry

N[CN]2 dicyanimide

N1,1 [N1,1,1OOH] dimethylammonium dimethylcarbamate

N1,1,1OOH dimethylcarbamate

N4,4,4,4 [BF4] tetrabutylammonium tetrafluoroborate

NADES deep eutectic solvents from natural sources

OECD Organization of Economical Co-Operation and Development

OH heme oxidase

omim [Br] 1-octyl-3-methylimidazolium chloride

ompy [Br] 1-octyl-4-methylpyridinium bromide

ompyr [Cl] 1-butyl-1methylpyrrolidinium chloride

PAHs polycyclic aromatic hydrocarbons

PE particles electrodes

PF6 hexafluorophosphate

POD peroxidase

PTFE polytetrafluoroethylene

XXVII

QSAR Structure-activity relationships

QSPR Structure-property relationships

RC reaction coil

REACH Registration, Evaluation, Authorization, and Restriction of

chemicals

ROS reactive oxygen species

rsd relative standard deviation

S syringe

S substrate

SEM scanning electron microscopy

SIA sequential injection analysis

SOD superoxide dismutase

SP spectrophotometer

SV selection valve

TAAILs tunable aryl alkyl ionic liquids

tbph [Ms] tetrabutylphosphonium methanesulfonate

TES N-tris(hydroxymethyl)methyl-1-(2-aminoethanesulphonic acid)

Tf2N bis(trifluoromethylsulfonyl)imide

ThCO2 theoretical carbon dioxide

ThOD theoretical oxygen demand

TOC total organic carbon

UV ultraviolet

W waste

WWTP wastewater treatment plant

ZVI/AC zero-valent iron activated carbon

XXVIII

Symbols

T Temperature

t Time

V Volume

Wavelength

2 Reduced chi squared

XXIX

Outline of the thesis

This thesis is organized in eight main chapters:

Chapter 1. Introduction

This chapter provides a theoretical background of the thesis theme and introduces the

main and the specific objectives of the developed work.

Chapter 2. Experimental

This chapter includes the general procedures for the preparation of solutions and the

devices applied in the research activities carried out under the scope of this thesis. The

description of instruments and the experimental methods used in the developed works are

also described in chapter 2.

Chapter 3-7. Original research

These chapters describe the developed work under the scope of the thesis. The

experimental studies were designed to accomplish the general and specific objectives of

the thesis. From that resulted five original manuscripts, four of which were already

published (chapter 3, 4, 5 and 6) and one is in final stretch to be submit for publication.

Chapter 8. Final conclusions

This chapter contains the general conclusions of this thesis, concerning the

methodologies developed and ionic liquids toxicity and (bio)degradability based on the

anions and cation cores.

XXX

CHAPTER 1

General Introduction

1

Environmental Impact of Ionic Liquids: Recent Advances in(Eco)toxicology and (Bio)degradabilitySusana P. F. Costa,[a] Ana M. O. Azevedo,[a] Paula C. A. G. Pinto,[a, b] andM. Lfflcia M. F. S. Saraiva*[a]

ChemSusChem 2017, 10, 2321 – 2347 T 2017 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim2321

ReviewsDOI: 10.1002/cssc.201700261

2

http://orcid.org/0000-0003-2866-1591http://orcid.org/0000-0003-2866-1591http://orcid.org/0000-0003-2866-1591http://orcid.org/0000-0003-2866-1591http://orcid.org/0000-0002-4133-079Xhttp://orcid.org/0000-0002-4133-079Xhttp://orcid.org/0000-0002-4133-079Xhttp://orcid.org/0000-0002-4133-079Xhttp://orcid.org/0000-0002-1259-3873http://orcid.org/0000-0002-1259-3873http://orcid.org/0000-0002-1259-3873http://orcid.org/0000-0002-1259-3873http://orcid.org/0000-0002-1259-3873http://orcid.org/0000-0001-6539-1741http://orcid.org/0000-0001-6539-1741http://orcid.org/0000-0001-6539-1741http://orcid.org/0000-0001-6539-1741http://orcid.org/0000-0001-6539-1741http://orcid.org/0000-0001-6539-1741

1. Introduction

The industry is in constant development of faster, more effi-

cient, and more economical processes for the manufacture ofnew products. Minimization of the hazard potential of a proce-

dure is important during the design and optimization of tech-

nical performance, as it contributes to sustainable develop-ment in accord with the ideals of green chemistry. In this con-

text, a large area of research in the development of greentechnologies is dedicated to the design of new environmental-

ly friendly solvents with the potential to substitute traditionalvolatile organic solvents. Ionic liquids (ILs) are molten salts

composed of organic cations and organic/inorganic anions

with melting points below 100 8C. With regard to the structureof ILs, cations are usually composed of organic cores, such as

imidazolium, pyridinium, phosphonium, pyrrolidinium, piperidi-nium, morpholinium, and cholinium ions. Typical anions in-

clude halides, acetate, fluorine, and cyano derivatives. The nu-merous structural modifications possible on the anionic and/or

cationic moieties enable the nature of the ILs to be modulated,

that is, the synthesis of a considerable number of compoundswith fine-tuned characteristics.[1] Then, the physical, chemical,

and biological properties of ILs can be tailored to the require-ments of a designated process, for example, volatility, stability,

density, flammability, solubility, and antimicrobial ability of theIL.[2] These noteworthy benefits have led to a remarkable in-

crease in the number of publications in the last two decades

involving the use of ILs in different areas, including catalysis,biocatalysis, electrochemistry, and the food and pharmaceuticalindustries.[3] In 2000, in the meeting entitled “Green IndustrialApplications of Ionic liquids”[4] several criteria were set thatshould be met before the development of ILs for industrial ap-plications. Currently, around two dozen companies already use

ILs at the industrial scale. BASF,[5] Degussa, Linde, Pionics, and

IoLiTec/Wanders[3b,6] are just some of the examples of industrialcompanies that commonly use ILs in their industrial processes.

ILs have reduced vapor pressure relative to organic solvents,

near-zero flammability, and are recycle as a result of their ex-cellent stability, and these factors have contributed to ILs

being preliminary designated as “green solvents”. To prove this

premise, several studies have been conducted to evaluate their“greenness” relative to conventional organic solvents. Ideally,

to assess the potential environmental impact of an IL, its lifecycle should be considered, that is, from its starting materials

to its release into the environment. In fact, the synthesis of ILsis not always very clean, and improvements at different levels,

including an increase in energy efficiency, the use of renewable

sources, and the avoidance of large quantities of reagents, es-pecially organic solvents, are required.[7] Additionally, some

concerns about the implications to the environment after ILshave served their purpose have also been put forward by re-

search academia. In 2006, Earle et al.[8] demonstrated thatsome ILs could be distilled at low pressure without decomposi-

tion. Therefore, the possibility of atmospheric contamination

cannot be discarded, at least if ILs are used at elevated tem-peratures. The capacity of these salts to disperse in water

raises doubts about their fate if released into the environmentthrough wastewater discharge or leaching of landfills. Then,

the risk assessment of ILs taking into account potential con-tamination pathways must be performed before they can in-

discriminately be used. Several studies have been conducted

to evaluate the ecotoxicity, biodegradability, and bioaccumula-tion of ILs. The biodegradability assessment of ILs is mainly

based on standardized assays implemented by the Organiza-tion of Economical Co-Operation and Development (OECD)[9]

and the International Organization for Standardization (ISO),[10]

which evaluate the biodegradability of chemical compounds

under biotic conditions. The ILs that do not pass these testshave a greater potential to bioaccumulate in the environment,which is a distressing concern, as the available data show that

various ILs cannot be classified as readily biodegradable.[11] Thechemical structure of ILs influences their biodegradability. The

reduced mineralization of various ILs by microbial communitiesincorporated on usual activated sludge highlights the potential

risk of these salts to persist in wastewater and, consequently,

to contaminate the aquatic and soil systems and living organ-isms.

The toxicity of ILs has been evaluated by resorting to biolog-ical models of different levels of complexity, namely, unicellular

organisms, such as bacteria[12] and fungi,[13] invertebrates,[14]

fish,[15] algae,[16] plants,[17] and mammalian cell lines.[18] These or-

[a] S. P. F. Costa, A. M. O. Azevedo, Dr. P. C. A. G. Pinto, Dr. M. L. M. F. S. SaraivaLAQV, Requimte, Departamento de CiÞncias Qu&micas, Laboratjrio de Qu&-mica AplicadaFaculdade de Farm#cia, Universidade do PortoRua Jorge Viterbo Ferreira 228, 4050-313 Porto (Portugal)Fax: (+351)226-093-483E-mail : lsaraiva@ff.up.pt

[b] Dr. P. C. A. G. PintoA3D-Association for Drug Discovery and DevelopmentRua do Baixeiro n8 38 Aveiro (Portugal)

Supporting Information, containing the toxicity data of ionic liquids re-ported since 2010, and the ORCID identification number(s) for the au-thor(s) of this article can be found under https://doi.org/10.1002/cssc.201700261.

This Review aims to integrate the most recent and pertinentdata available on the (bio)degradability and toxicity of ionic

liquids for global and critical analysis and on the conscious useof these compounds on a large scale thereafter. The integrated

data will enable focus on the recognition of toxicophores andon the way the community has been dealing with them, withthe aim to obtain greener and safer ionic liquids. Also, an

update of the most recent biotic and abiotic methods devel-oped to overcome some of these challenging issues will be

presented. The review structure aims to present a potential se-quence of events that can occur upon discharging ionic liquids

into the environment and the potential long-term consequen-ces.

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https://doi.org/10.1002/cssc.201700261https://doi.org/10.1002/cssc.201700261http://www.chemsuschem.org

ganisms carry distinct characteristics, and in the presence ofILs, they can present different susceptibilities and behaviors.[19]

The extensively reported data has demonstrated trends interms of structural features.[15,20] Additionally, the assay condi-

tions also contribute to differences in the toxicities of ILs.[21] Be-sides (eco)toxicological tests with biological organisms, in silico

models have also been developed to predict the environmen-tal impact of ILs.[22] Jastorff and co-workers were the first touse a mathematical model to predict the environmental

impact of ILs on the basis of five distinct indicators: release,spatiotemporal range, bioaccumulation, biological activity, anduncertainly.[23] It is widely accepted that the head group andthe length of the alkyl side chain are the main contributors to

the effects of ILs on living organisms. The anionic moiety alsoplays an essential role in molecular toxicity ;[24] however, less lit-

erature is available on this subject.

Considering the potential negative impact of ILs on the envi-ronment, various approaches have been suggested to over-

come these drawbacks. For example, the design of safer andreadily biodegradable ILs without neglecting chemical per-

formance, preferably synthesized from natural sources, such ascholine[25] and amino acids.[26] Additionally, to improve the de-

gradability of ILs and their removal from wastewater, other

strategies have been developed, namely, (photo)(electro)chem-ical oxidation processes and methods based on sorption and

enrichment of microbial-community efficiency. Some strains,such as Sphingomonas paucimobilis, Rhodococcus sp. , Brevibac-

terium sanguinis, and Kocuria palustris, show promising resultsfor compounds of recognized low biodegradability in the stan-

dard method.[27] Various chemical degradation processes havebeen reported in recent years, based mainly on Fenton-like

systems,[28] ultrasonic irradiation,[29] and boron-doped diamondelectrodes.[30]

Considering the importance of the environmental impact of

ILs (Figure 1), as would be expected, the topic has alreadybeen reviewed.[31] Hence, herein we focus on some aspects notdeeply covered in other reviews, and in addition, we providean update of the recently reported data on the ecotoxicity and

biodegradability of ILs, more specifically since 2010. In thisReview, lifecycle analysis of ILs and its importance to assess

the environmental impact of these compounds is briefly ap-

proached. Moreover, particular relevance is given to newly de-veloped strategies to improve the degradability of ILs and

their removal from wastewater and soil. Concerning the eco-toxicity of ILs, besides the median inhibitory concentration

(IC50), median effective concentration (EC50), median lethal dose(LD50), and minimal inhibitory concentration (MIC) values and

trends related to possible toxicophores of ILs, the effects of ILs

on living organisms, cell lines, enzymes, and others at the cel-lular and subcellular levels are also described. Additionally,

Susana Costa received her M.Sc.

degree in Pharmaceutical Sciences in

2012 from the Faculty of Pharmacy,

University of Porto. She is currently in

the final year of her Ph.D. program in

Pharmaceutical Sciences at LAQV, RE-

QUIMTE, Faculty of Pharmacy, Porto

University, under the supervision of

Prof. M. Lfflcia Saraiva. Her research in-

terests are focused on the develop-

ment of sustainable automated bioas-

says to evaluate the biological activity,

(eco)toxicity and biodegradability of ionic liquids and the predic-

tion of their environmental impact.

Ana Azevedo is an Invited Assistant at

the Faculty of Pharmacy, University of

Porto, and a Researcher at the Associ-

ate Laboratory LAQV, REQUIMTE. She

obtained her Master’s degree in Phar-

maceutical Sciences from the Faculty

of Pharmacy, University of Porto, in

2013. Her current research interests

focus on evaluating the toxicity of

ionic liquids through automatic enzy-

matic methods. She was awarded the

Best Short Oral Communication at the

20th International Conference on Flow Injection Analysis and Relat-

ed Techniques.

Paula Pinto is currently a Project Man-

ager at A3D. She worked as an Assis-

tant Researcher at the associate labo-

ratory LAQV, REQUIMTE, Faculty of

Pharmacy, Porto University, and is now

an external collaborator of this institu-

tion. She obtained her Ph.D. degree in

Analytical Chemistry (2006) from the

Faculty of Pharmacy, Porto University.

Her research interests focus on se-

quential injection analysis, specifically

on biocatalysis in aqueous and ionic

liquid media. She is now dedicated to the development of post-

graduate training in the field of Pharmaceutical Medicine.

M. Lfflcia Saraiva (Ph.D. degree in

Analytical Chemistry, 2000) is an

Assistant Professor in the Faculty of

Pharmacy, University of Porto, and

a researcher at LAQV, REQUIMTE Asso-

ciate Laboratory. Her main area of

interest is the development of auto-

mated (bio)analytical tools. She now

focuses on the use of ionic liquids

as a replacement for organic solvents

and on studying the impact of ionic

liquids on the environment and

humans by enzymatic screening methods and whole-cell toxicity

assays. She is also involved in studying the potential of deep eu-

tectic solvents.

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new alternative procedures developed to evaluate the ecotox-

icity of ILs and their biodegradability are also discussed in this

Review.

2. Concerns about the Greenness of IonicLiquid Synthesis

In the last years, ILs have emerged as greener alternatives toconventional solvents in a diverse range of chemical transfor-

mations. Their unique properties, including good performanceas reaction media, negligible volatility, and low flammability,

have contributed to this fact. Furthermore, the idea of greencompounds with low toxicity, biodegradability, and recyclabili-

ty has also promoted their widespread use by researchers in

the laboratory and in industry. In addition to currently knowingthat these concepts are not true at least for some ILs, it is nec-

essary to consider that there are many other factors that influ-ence their greenness. In a simplified way, all the stages of

a compound’s life can contribute to its potential environmen-tal impact, starting from its preparation, going through its ap-

plications, and finally ending with its effects on ecosystems

after use. The lifecycle assessment (LCA) method has been ap-plied to assess the potential environmental impact of a com-

pound on the basis of its life cycle, from acquisition of the rawmaterials, through production and use, and finally to treat-

ment, recycling, and final disposal.[32] In the particular case ofILs, their synthesis does not always meet the principles of

green chemistry; for example, some reactions use large quanti-ties of reagents, including organic solvents, which results in

the production of large quantities of waste during the proce-dures, most of it with a negative impact on humans and theenvironment. The assessment of the greenness of ionic liquids

at the synthesis stage is based on a series of green-chemistrymetrics of different categories; among them, the most often

used are atom economy[33] and the environmental factor (E-factor).[34] For each reaction, atom economy provides the ratio

between the mass of the atoms making up the final product(s)

and the mass of the atoms that are incorporated in the start-ing materials. The preparation of 1-alkyl-3-methylimidazolium

halide salts using quaternization, for example, is 100% atomefficient, whereas the ILs prepared by one-pot or metathesis

reactions are

to form a eutectic mixture without the need for any additionalsolvents.[37c,42] Additionally, ILs have the advantage of solvent

recovery and reducing waste generation and emission to theenvironment.[43]

For future processes, there is still room for improvement ingreener procedures both in the synthesis of ILs and in their ap-

plication and treatment before their release to wastewater. Fur-thermore, the design of ILs should be oriented for the synthe-sis of nontoxic, biocompatible, and task-specific ILs. Additional-

ly, for the development of easy and cleaner synthesis methods,the selection of raw materials, preferably from natural sources

and harmless, is also recommended. Consideration of the ef-fects of ILs on living organisms, the risk of their persistence in

the environment, and strategies to overcome inherent prob-lems will be presented in the following sections.

3. Degradation of Ionic Liquids

In recent years, various groups of investigators have beencommitted to evaluating the greenness of ILs. Fundamental

studies have been applied to determine the ecotoxicity, biode-

gradability, and risk of bioaccumulation of ILs in the environ-ment. The sorption of ILs on soils and their biodegradability

seem to be important factors that contribute to soil and waterpollution. Therefore, several researchers have evaluated the

biodegradability of ILs and the risk of their persistence inaquatic and soil fields, and dissimilar results have been ob-

tained for different groups of ILs. Recently, extensive researchon the development of alternative techniques for the removal

of ILs has been performed. In this section, the breakdown of

ILs by microbial cultures, newly developed chemical and elec-trochemical pretreatment methods, and the ability of ILs to

sorb onto soil will be discussed.

3.1. Recent data regarding the biodegradability of ionicliquids

According to the OECD and ISO, there are a number of stand-ardized assays that can be applied to determine the biode-

gradability of a compound. The most commonly used are thedissolved organic carbon (DOC) die-away test (OECD 301A),

modified Sturm (OECD 301B), closed-bottle test (OECD 301D),and CO2 headspace test (ISO 14593). For most of the tests, the

compounds are designated as readily biodegradable if theyreach a biodegradation level of 60% of the theoretical oxygendemand (ThOD) or theoretical carbon dioxide (ThCO2) (70% of

DOC) within 28 days. The pass levels have to be reached ina 10 day window after the degree of biodegradation has

reached 10% of ThOD, ThCO2, or DOC. These methods havebeen applied in the assessment of the ready biodegradability

of ILs and their subsequent risk of persistence in the environ-

ment. The accessible data needs to be interpreted with cautionand used consciously in the future to design new biodegrad-

able ILs, and the results should not be assessed exclusively be-cause the IL ion pair either passes or fails the biodegradation

test. If a compound fails the pass level, this does not necessari-ly means that it will not degrade in the environment. Further

tests would be necessary to explore the initial suspicion of per-sistency. Additionally, the interpretation of biodegradation

tests, especially if parameters such as CO2 evolution or bio-chemical oxygen demand (BOD) are used, can lead to misinter-

pretation. In general, the results are expressed as “percent bio-degradation” on the basis of the total carbon content of the

whole molecule, that is, the carbon content of both the cationand anion comprising the IL. Then, an IL, for example, 1-(pen-

toxycarbonyl)-3-methylimidzolium octylsulfate, can be classi-

fied as readily biodegradable, as it passes the 60% markthrough the oxidizable C content of the side chain and the

anion, but it still contains a non-biodegradable core.[44]

Considering that ILs are formed from a combination of

a large variety of cations and anions, the influence of theirchemical structure on molecular biodegradability has been

evaluated. Recent studies have also been focused on elucidat-

ing the possible biodegradation pathways of ILs and detectionof the resulting metabolites. Additionally, strategies to improve

the biodegradability of ILs have been developed, and this willbe briefly discussed at the end of this section.

3.1.1. Anions

Halides and pseudohalides (e.g. , BF4 and PF6) are usually select-ed to be incorporated into ILs. These anions are not essential

in biodegradation tests based on the measurement of oxidiza-ble carbon, and then the biodegradation level is fundamentally

dependent on the organic cation. The metabolization of these

anions occurs mainly under anaerobic conditions. Conversely, ifan IL comprises organic cations and anions, both contribute to

the carbon source. Therefore, it cannot be assumed that a bio-degradable cation is the same as a biodegradable ion pair. In

fact, the first studies reporting the biodegradability of theanionic part demonstrated differences even for anions of the

same family. For example, the anion diethyl phosphate was de-

gradable in the BOD test,[45] whereas dibutyl phosphate wasnot significantly degraded in the CO2 headspace test (OECD

310).[46] Anyway, more studies are needed to determine if thedifferences are only related to the composition of the anionsor to other factors that also contribute to the observed differ-ences, such as the assay conditions.

Carboxylate anions, such as formates and acetates, if com-bined with 2-hydroxy(di)ethanolamine cations, in general pres-

ent biodegradation superior to 60% after the 28 day assay (86and 69%, respectively). Also, organic linear alkyl sulfates, suchas octyl sulfate, are often selected as the anionic components

of ILs, as these anions exhibit higher biodegradability thanothers anions (25 vs. 5% for analogues with other anionic moi-

eties).[47] The biodegradability of tetrabutylammonium-basedILs seems to improve slightly if combined with natural organic

acids such as succinate, l-tartrate, l-lactate, l-malate, pyruvate,d-glucoronate, and d-galacturonate (9.9–22.8% for natural or-ganic derivatives vs. 0–7.9% non-natural organic deriva-

tives).[11a] However, none of the compounds can be classified asreadily biodegradable [closed-bottle test (OECD 301D)] , which

suggests that the cation is recalcitrant to biodegradation. Thebiodegradability of 18 amino acids combined with the cholini-

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um cation has been investigated by Hou et al.[48] Asexpected, considering the starting materials, all of

the synthesized ILs pass the 60% biodegradability(62–87%) mark in the closed-bottle test and the CO2headspace test. The amino acids with carboxy andamido groups are more susceptible to microbial

degradation. The amino acids with branched sidechains are more resistant to breakdown than un-branched side chains. The low toxicity and high bio-

degradability of cholinium amino acids make thesecompounds interesting for use on a large scale as al-

ternative environmentally friendly solvents.The study of Neumann et al.[49] was one of the few

to evaluate the biodegradation behavior of fluoroor-ganic anions {e.g. , bis(trifluoromethylsulfonyl)amide

(also called bistriflimide) (NTf2), trifluoridotris(penta-

fluoroethyl)phosphate ([C2F5]3PF3)} and cyano deriva-tives {e.g. , dicyanimide (N[CN]2), tricyanmethanide

(C[CN]3), tetracyanidoboranate (B[CN]4)} (80%). However, phenyl-,

cyano-, and nitro-substituted imidazoles are poorly mineralized(

zation, as does the cation with a hydroxyethyl group incorpo-rated in the imidazolium core.[55] Stolte and co-workers[56] have

synthesized a new class of ILs that present a charge distribu-tion completely different from that of traditional imidazolium-

based ILs. Unfortunately, six of the developed tunable aryl alkylionic liquids (TAAILs) show 0% primary biodegradation (OECD

301). Only the 4-carboxy-substituted compounds reveal com-plete primary biodegradation within 28 days. Others modifica-

tions to the imidazolium cation have been attempted with the

incorporation of four and five substituents in the ring; howev-er, these compounds also show low biodegradability (<

10%).[57] The metabolism pathway of 1-hexyl-2H-3-methyl-4,5-dimethylimidazolium iodide has been assessed, and the results

suggest b-oxidation of the side chain with the formation oftwo main products : a cation with an aldehyde group in the

side chain, which results from oxidization of the terminal

methyl group, and a pentyl group instead of the initial hexylgroup. The fusion of aromatic rings in the benzimidazolium

cation improves molecular stability and, consequently, reducesits biodegradability relative to the corresponding imidazolium

cation.[54] Besides modification to the imidazolium core, evalua-tion of the influence of the imidazolium alkyl side chain on

biodegradability has also been studied. For instance, the incor-

poration of a long linear alkyl side chain, such as an octyl,decyl, and dodecyl chain, improves the biodegradation of tet-

rakis(imidazolium)- and tetrakis(benzimidazolium)-based ILs,with biodegradabilities of approximately 10% for the IL with

an ethyl side chain and almost 30% for the IL with a decylchain.[58] According to Docherty,[52] elongation of the alkyl side

chain favors an increase in degradation as a result of two main

factors :

1) The chance of any structural hindrance between the cation-ic ring and the potential binding site is eliminated; thus,

the side chain of the IL becomes readily accessible to bac-terial enzymatic digestion

2) The hydrophobicity of IL increases because of the longer

alkyl side chains, and this might influence its toxicity,namely, increase it ; this may lead to selective grow of the

only species capable of metabolizing the IL

ILs with amino acid residues in the side chain[12b] significantlyimprove the biodegradability of imidazolium derivatives. ILs

containing one or two phenylalanine residues are more than60% biodegraded (61 and 64%, respectively) and can be clas-sified as readily biodegradable under aerobic conditions (CO2headspace test).

The pyridinium cation and its derivatives are commonly

used, as they can be applied differently, for example, in analyti-cal processes and in biocatalysis.[3a, c] Early generations of pyri-

dinium ILs have higher biodegradability than imidazolium

ILs;[52] nonetheless, not all pyridinium-based ILs can be consid-ered readily biodegradable.[55,59] Docherty et al.[59] have as-

sessed the biodegradability of three pyridinium-based ILs {i.e. ,1-butyl-3-methylpyridinium bromide ([bmpy]Br), 1-hexyl-3-

methylpyridinium bromide ([hmpy]Br), and 1-octyl-3-methyl-pyridinium bromide [ompy]Br} through a modified OECD DOC

die-away test. Elongation of the alkyl side chain, as observedfor other families of ILs, increases the biodegradability of the

molecule. In fact, [bmpy]Br and [hmpy]Br are fully degraded bythe activated microbial community in 41 days, whereas

[ompy]Br requires less time (26 days). The [ompy]Br IL is theonly IL to reach the criteria to be classified as readily biode-gradable. The DOC concentration profiles indicate that the lim-iting step to full biodegradation is the cleavage of the pyridini-um ring. The degradation products can be examined by HPLC–

MS and MS–MS methods, with the limitation that only metabo-lites containing the pyridinium ring can be detected. Differentbiodegradation pathways are observed for the studied ILs de-pending on the length of the alkyl chain. All ILs show unsatu-ration of the side chains, but the location of the hydroxylationis dissimilar. Hydroxylation of the aromatic ring occurs for the

bmpy and hmpy cations, whereas hydroxylation of the sidechain occurs in the hmpy and ompy cations. The metabolitesare less toxic to Daphnia magna than the initial compounds,

which suggests that biodegradation of these compounds, andperhaps others, in an aquatic environment contributes to a de-

crease in the toxicity associated with the initial compound. Thebiodegradability of functionalized pyridinium-based ILs has

been evaluated through the CO2 headspace test (ISO 14593).[55]

ILs containing 1-(2-hydroxylethyl) or ester groups on the sidechain generally present good biodegradation rates (62–71%).

In fact, hydroxylated pyridinium derivatives demonstratehigher levels of biodegradability than N-alkylpyridinium-based

ILs.[60] Pyridinium derivatives with a methyl ether group in the3-position and a linear alkyl substituent in the 1-position, on

the other hand, are only moderately degraded under the same

conditions (31–36%). The combination of the bis(trifluorome-thanesulfonyl)imide anion to these compounds reduces the

biodegradability levels even more to 1%. The bis(pyridinium)salts linked by an acetal group also show a very low minerali-

zation percentage at the end of the test period (,5%), whichmight be associated with the inability to hydrolyze the acetal

function. A carbamate derivative has also been evaluated, and

it presents very poor biodegradability (3%).[55] Ester functionali-ties at the 3-position of the pyridinium side chain have beensynthesized by Harjani and co-workers,[38] and the obtained 3-(alkoxycarbonyl)-1-methylpyridinium ILs, also designated as

nicotinium-based ILs, pass the level and can be classified asreadily biodegradable (68–72% in the CO2 headspace test).

Gore et al.[11c] have determined the influence of an aromaticring on the biodegradability levels. Benzylpyridinium bromideshows only 2% biodegradation within 28 days in the CO2headspace test, and it is proposed that the benzyl moiety doesnot participate in breakdown of the molecule or the pyridini-

um ring.Data on the biodegradability of the pyrrolidinium cation is

still scarce, and as far as we are aware, only two groups have

evaluated this type of cation.[27c,60, 61] Stolte et al.[61] have investi-gated the biodegradability of pyrrolidonium methylsulfate

through the CO2 evolution test (OECD 301B) and the mano-metric respirometry test (OECD 301F), and this IL is recalcitrant

to biodegradation (

anions has been evaluated by Neumann et al.[60] As expected,elongation of the side chain improves the biodegradability,

and 1-octyl-1-methylpyrrolidinium [ompyr]Cl meets the criteriato be classified as readily biodegradable (100% primary bio-

degradation and 69% relative oxygen demand BOD/ThOD).Moreover, an N-substituted propyl alcohol IL provides 69%

biodegradation, but the corresponding N-substituted ethyl al-cohol is biodegraded to less than 10%. The ethyl ester deriva-

tive only achieves 34% biodegradation in 28 days, but 10 days

after that period the biodegradability reaches 60%, and it isthus classified as inherently biodegradable. The introduction of

a cyano group decreases the biodegradability of the com-pound (2% of relative oxygen demand BOD/ThOD). The inves-

tigators have also evaluated the biodegradability of other headgroups, pyridinium and imidazolium already discussed aboveand morpholinium and piperidinium, which will be discussed

next.Among the seven morpholinium-based ILs investigated by

Neumann, none show significant biodegradability. The best re-sults are obtained for the N-substituted propyl alcohol IL,which reaches 30% biodegradation in the BOD test. The cyanoderivative show 0% biodegradability, although biotic hydroly-

sis of the cyano group is observed. The three ILs with an ether

group in the side chain do not show any primary biodegrada-tion. These results are in accordance with the obtained data

for other ILs in which the introduction of an ether group re-duces the biodegradability of the compound.[60] N-Alkyl-N-

methylmorpholinium and N-alkyl-substituted 1,4-diazabicy-clo[2.2.2]octane (DABCO) cations show slight differences on

biodegradability (3–30 and 22–40%, respectively, to CO2 head-

space test).[62] In any case, both the DABCO- and morpholini-um-based ILs present low biodegradability, and none of the

studied compounds can be classified as readily biodegradable.The investigators note for both groups that there is an unex-

pected decrease in the biodegradation rate upon elongationof the alkyl side chain. They speculate that heteroatoms incor-

porated into the cation ring can be easily attacked by mono-

oxygenases in ILs with short alkyl chains. Additional informa-tion is necessary to validate this assumption and to understand

why these two groups do not follow the same trend as theother cations. Pernak et al.[63] have synthesized various ILs withmorpholinium cations combined with different organic and in-organic anions. Although the developed compounds show re-

duced toxicity in general, they do not meet the criteria to beclassified as readily biodegradable.

The biodegradability of piperidinium-based ILs has alsobeen assessed by the Neumann group. Among the seventested compounds, none can be classified as readily biode-

gradable (OECD 301F). However, the N-substituted propyl alco-hol derivative is classified as inherently/ultimately biodegrad-

able, as it requires more than 28 days to overpass the stipulat-ed value. The N-substituted ethyl alcohol derivative is classifiedas inherently biodegradable, and it shows a decrease of 85%

in oxygen demand in 60 days. The remaining ILs show elevat-ed recalcitrance to biodegradation (,5%). The ether andcyano groups present trends similar to those observed for themorpholinium group.[60]

Among the linear ammonium cations, the cholinium and tet-rabutylammonium cations are the most commonly used. Con-

cerning the biodegradability of tetrabutylammonium-basedILs, Ferlin et al.[11a] have observed no evidence of biodegrada-

bility for the closed-bottle test (,23%). Furthermore, the ob-tained data suggest that only the anion is degraded, although

detailed information about metabolites is necessary to confirmthis hypothesis. In a follow-up study, the same investigators

have assessed the biodegradability of three chiral tetrabuty-

lammonium ILs: tetrabutylammonium (S)-prolinate, tetrabuty-lammonium (R)-prolinate, and tetrabutylammonium trans-4-hy-

droxy-(S)-prolinate, which also show disappointing levels ofbiodegradation (

pounds, only the ester group is hydrolyzed, which results inthe formation of an amino acid, whereas the remaining com-

pounds are cleaved at the amide bond. Products resultingfrom cleavage of the ester group are non-biodegradable or are

only slightly biodegradable, whereas products resulting fromcleavage of the amide bond show complete degradation of

the phenylalanine ethyl ester. Both degradation pathwaysresult in the formation of persistent transformation products,

with the exception of pyridinium-substituted phenylalanine-de-

rived ILs and the non-ionic derivative. The Stolte group[47a] hasevaluated the toxicity and biodegradability of a new family ofILs, that is, protic ionic liquids, which differ in their structurerelative to “classic”/aprotic ILs. The protic ILs are derived fromaliphatic amines (e.g. , monoethanolamine, diethanolamine,and triethanolamine) and are combined with organic acids

anions (e.g. , formic acid, propionic acid, butanoic acid, isobuta-

noic acid, and pentanoic acid). This study demonstrates thatselected protic ILs can be considered environmentally safer

than aprotic ILs; hence, these compounds have a higher min-eralization level and lower toxicity. Most of the protic ILs show

biodegradability levels greater than 60% or are at least nearthis level. After 28 days of assay, only the cationic 2-hydroxy-

diethanolamine moiety is detectable in the extracts, which in-

dicates that all of the protic ILs, except 2-hydroxydiethanola-mine pentanoate, are completely degraded within 28 days.

Steudte has studied the influence of dicationic structures onecotoxicity and biodegradability. Although they are less toxic

than monocationic ILs, the results are still disappointing, asnone of the dicationic ILs can be considered readily biodegrad-

able under the experimental conditions (

bacteria can effectively be grown as a biofilm in any potentialIL bioremediation reactor, which is an asset for potential exper-

imental applications. The biodegradability efficiency of otherstrains of Rhodococcus[27a] has also been investigated with pyri-

dinium-, pyrrolidinium-, and ammonium-based ILs. All of theevaluated compounds (except [hmpy][Tf2N]) can be readily bio-

degraded in the presence of R. rhodochrous (80–100% biodeg-radation). Additionally, activated sludge shows lower biodegra-dations rates for most of the tested compounds under the ap-

plied conditions. The chemical structures of the ILs influencethe biodegradability efficiency of Rhodococcus ; for example,

pyridinium-based ILs are degraded at a slower rate. Further-more, the introduction of an ester group into the side chain ofthe cation increases both the rate and the extent of biodegra-dation.

Deng and co-workers[53] have examined the biodegradability

of six 1-alkyl-3-methylimidazolium-based ILs with and withoutoxygenated functional groups (e.g. , hydroxy, ester, and ether)

in the presence of four strains (i.e. , R. rhodochrous, Pseudomo-nas viridiflava, Nocardia asteroı̈des, and Candida parapsilosis).

The results, in general, are similar for the four strains. As ob-served in others works, the presence of the ester function in-

creases the biodegradability of the compounds, but its prod-ucts show a tendency to accumulate. Additional studies are

necessary to compare the effectiveness to biodegrade ILs ofthe selected species with standard procedures.

A microbial community in a wastewater treatment plant inJeonju City is able to completely degrade omim derivative

within 24 days.[75] Three degradation pathways are identifiedon the basis of the initial oxidation: one, attack occurs in thealkyl side chains, more specifically, at the fifth carbon atom of

the alkyl chain; two, attack occurs in the end of the long sidechain; three, attack occurs in the end of the short side chain

(Scheme 2). According to the obtained metabolized products,the imidazolium ring does not undergo breakdown. It wouldbe interesting to perform additional studies to evaluate thepotential of the microbial community to biodegrade other

families of ILs and to identify the strains involved in the bio-degradation of ILs.

The primary anaerobic biodegradability of different imidazo-lium-, pyridinium-, and dimethylaminopyridinium-based ILs cat-ions has also been tested, but with poor results.[76] Over

a period of 11 months, the concentrations of the tested com-pounds remain stable, except for the ILs with the 1-(8-hydrox-

Scheme 2. a) Pathway for aerobic biodegradation of omim. b) Products of anaerobic breakdown of omim, as identified by MS measurements. c) Major path-ways for degradation of the omim cation in the presence of the microbial community of wastewater from Jeonju City (based on information fromRefs. [75,76, 78] , and [79]).

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yoctyl)-3-methylimidazolium cation. A recent study performedby Gotvajn et al.[77] demonstrates opposite results for imidazoli-

um- and pyridinium-based ILs. The [bmpy][dca] (dca=dicyana-mide) and [bmim][BF4] ILs have lower toxicity and are more

biodegradable under anaerobic conditions. The [bmim][BF4] ILis more resistant to biodegradation under denitrification condi-

tions than [bmpy][dca] . Further data is necessary to assess thebiotreatability of ILs under anaerobic conditions, which is per-haps an important factor contributing to the mineralization of

ILs in the environment and is not always considered in biode-gradability assays.

The composition of the media also influences the efficiencyof the biodegradability of ILs, as demonstrated by Marckiewicz

through supplementation of vessels containing activatedsewage sludge with glucose (modified OECD 301A DOC die-

away test).[79] The vessels supplemented with glucose, which

provides an easily available source of organic carbon, showslower primary degradation of [omim]Cl than vessels without

glucose. According to the results, the presence of other nu-trients in the sewage or in the environmental media seems to

influence the biodegradability of the ILs. Therefore, it is possi-ble that xenobiotics containing structures that are poorly bio-

degradable will not be metabolized if microorganisms have

other accessible sources of carbon. Docherty et al.[80] have de-termined that variations in the location and time of year influ-

ence the composition of a community’s wastewater treatmentplant (WWTP) and consequently can change the mineralization

rate of ILs.The effect of ILs on microbial strains from soil and their abili-

ty to degrade these strains have been assessed by some re-

searchers.[12a, 81] Fungal strains seem to be more resistant tolong-term exposure to ILs than bacteria, as the fungal strains

show a higher growth rate in presence of the tested com-pounds.[81a] In terms of the cation core, cholinium-based ILs are

only partially degraded by selected soil microbial cultureswithin 2 months, whereas the imidazolium cation is not de-gradable. Short-chain anions, such as ethanoate, lactate, ethyl-

sulfonate, and ethylsulfate, are degraded, even if only partially.The biodegradability of isolated strains of soil bacteria and

fungi has been assessed by the Francis group.[12a, 81b] For exam-ple, Corynebacterium sp.[81b] is able to mineralize completely N-

substituted pyridinium compounds without any alkyl sidechain on the ring within 40 h. The ring is cleaved between C2

and C3 and two main metabolites have been identified byMS–MS (ESI) (i.e. , ethyl [(1Z)-4-oxobut-1-en-1-ylcarbamic acid

or (3Z)-4-[ethyl (formyl)amino]but-3-enoic acid and 4-(carbox-yamino)but-3-enoic acid). The bmim-based IL is not degraded

by the tested bacterium, as Deive has observed for other se-

lected soil microbial cultures.[81a] Additional data detailing theeffects of ILs on soil are discussed in Sections 3.1.1. and 3.1.2.

Figure 2 summarizes the factors affecting the biodegradabil-ity of ILs in terms of the cation, anion, and alkyl side chains.

3.2. Advanced oxidation processes (AOPs): Chemical, photo-chemical, and electrochemical degradation

As discussed in previous sections, various ILs are poorly biode-gradable, and this requires modifications to standard methods,

including analysis of the compositions of microbial communi-ties and identification of strains essential for biological degra-

dation process or selection of microorganisms with the poten-tial to biodegrade ILs efficiently. Moreover, the design of ILswith structural elements that favor biodegradation, such as

ester or alcohol functional groups or cholinium and amino-acidresidues, to enhance the biodegradability of ILs has been con-

sidered. In the last years, other solutions based on photochem-ical, electrochemical, and chemical oxidation processes haveemerged, and they will be described throughout this section.

Stepnoswki and Zaleska[82] were among the first to work on

implementing oxidative processes to degrade ILs. They have

compared three common advanced oxidation processes (i.e. ,UV, UV/H2O2, and UV/TiO2) and note a higher photodegrada-

tion efficiency of imidazolium-based ILs with the UV/H2O2system. A decrease of more than 60% in the 1-hexyl-3-methyli-

midazolium (hmim) cation is observed within 60 min of start-ing the reaction, and only 10% of the initial compound re-

mains for 0.5% H2O2.This promising method shows that the

length of the alkyl chain, as in the biodegradation process, in-

Figure 2. General guidelines about ionic liquids’ biodegradability, considering cation, anion, and alkyl side-chain moieties.

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fluences the photodegradability rate, which follows the orderbmim

of time, and the preparation of the electrode is complex.Therefore, it is necessary that additional research be undertak-

en to find other economical, stable, and efficient materials thatcan be used in electrolysis as electrodes or the BDD electrode

may be modified to generate an electrochemical BDD systemthat integrates the three abovementioned important character-

istics.[30] The results of the electrochemical oxidation of ILs inthe presence of the BDD electrode[90] demonstrate that theanionic group influences the electrochemical process; for ex-

ample, the chloride anion enhances the degradation of thecation of ILs. The anionic 4-toluenesulfonate group, in contrast,competes with the imidazolium cation for the anodically pro-duced HOC radicals. The decay rates of the tested anions followthe order chloride>bromide& tetrafluoroborate&hexafluoro-phosphate& trifluoromethanesulfonate>4-toluenesulfonate.Pieczyńska et al.[89b] have also studied the influence of the

chemical composition of ILs on the degradation efficiency byBDD, namely, the structural elements of the side chain and

head group. The results suggest that at the end of the electrol-ysis the pyridinium salts are decomposed to a greater extent

than imidazolium salts, which highlights the superior electro-chemical stability of the latter cation. In general, the incorpora-

tion of side chains into imidazolium and pyridinium cations is

not essential to the decomposition rate, as observed by Zhoufor the ultrasonic irradiation method. A different tendency is

observed by the same group for the PbO2 system,[89c] in which

the composition of the side chain affects the electrooxidation

of ILs. Elongation of the n-alkyl chain from a butyl chain to anoctyl chain, as well as its substitution (but not with hydroxy

groups), leads to increased stability. The intermediate reaction

products of the electrochemical BDD process for imidazolium-and pyridinium-based ILs result from the same primary path-

way: oxidation of the side chain of the cation, elimination ofthe side chain from the cation, and reaction between the aro-

matic head group and O2C@ radicals. Moreover, the toxicityvalues in general decrease after electrochemical treatment ofthe ILs. However, the results for the [bmim]Cl/NaCl mixture

show that the metabolites are not as harmless as the untreat-ed IL. In fact, the investigators observe a considerable increasein the growth inhibition of the Scenedesmus vacuolatus andLemna minor species after electrolytic treatment.[89b] Recently,AuPd/Fe3O4 nanoparticles have been used as particle electro-des (PEs) in the electrochemical degradation of ILs.[91] The de-

veloped system shows promising results for [bmim][PF6] , thecomplete degradation of which is observed within 90 minunder optimized conditions. The main intermediates observed

by GC–MS are the same as those observed by Zhou in the ZVI/AC microelectrolysis system, that is, 1-butyl-3-methyl-2,4,5-tri-

oxoimidazolidine in the first step, which affords 1-butyl-3-methylurea as an intermediate metabolite, and finally N-butyl-

formamide as the end product.

3.3. The ability of ionic liquids to adsorb

As previously discussed, sorption and biodegradation are the

main processes that are used to remove chemicals from theenvironment during wastewater treatment in activated sludge

systems. Although information on the effects of ILs on soil islimited, the available data suggest the possible persistence of

ILs on soils and adverse effects of ILs on the microbial com-munities of soil. Sun et al. have observed that after 40 days of

contact between soil and [omim][BF4] the concentration of theIL changes by no more than 5%.[92] Ammonium- and phospho-

nium-based ILs also demonstrate the ability to persist on soilfor long periods of time.[93] The length of the alkyl side chain isa major factor affecting the strength of binding of the ILs to

the soil surface.[94] ILs with longer alkyl side chains are ad-sorbed to a higher extent than those with short or/and hy-droxylated side chains in the cation.

Various studies have been developed to promote the remov-al of ILs from aqueous media on the basis of an adsorption–desorption process and the physicochemical properties of the

sorbates. Farooq et al.[95] have studied the adsorption capabili-

ties of different types of activated carbons (ACs) (microporousgranular AC, microporous–mesoporous AC fabric, and AC) pre-

pared from artichoke leaves by using phosphoric acid activa-tion. The efficiency of the adsorption of the IL can be correlat-

ed to the porous structure of the AC, namely, the presence ofoxygen groups promotes electrostatic interactions with the hy-

drophilic bmim cation, which favors its adsorption. As expect-

ed, the structural compositions of the ILs also influence the ad-sorption ability, and cations with longer chains and a pyridini-

um core are more favorably adsorbed than cations witha methylimidazolium core. The Lemus group[96] has also evalu-

ated parameters with the potential to influence the