Seawater solubility, degradation and leaching of new nature-inspired antifouling compounds

2

_{Cycle Studies - Master Degree in Quality Control}

Seawater solubility, degradation and leaching

of new nature-inspired antifouling compounds

Cátia Sofia da Silva Vilas Boas

July 2017

Advisor:

Professora Doutora Marta Ramos Pinto Correia da Silva

(Professora Auxiliar da Faculdade de Farmácia da Universidade do Porto)

Co-advisor:

Professora Doutora Maria Emília Pereira de Sousa

III IT IS NOT PERMITED TO REPRODUCE ANY PART OF THIS DISSERTATION

DE ACORDO COM A LEGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTA DISSERTAÇÃO.

V ________________________________________________

(Advisor: Professora Doutora Marta Correia da Silva)

________________________________________________ (Co-advisor: Professora Doutora Emília Sousa)

________________________________________________ (Master Course Director: Professora Doutora Beatriz Oliveira)

VII This work was developed in Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia da Universidade do Porto. This research was developed under the project PTDC/AAG-TEC/0739/2014 supported through national funds provided by Fundação da Ciência e Tecnologia (FCT/MCTES, PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE – Programa Operacional Factores de Competitividade (POFC) programme (POCI-01-0145-FEDER-016793) and– Reforçar a Investigação, o Desenvolvimento Tecnológico e a Inovação (RIDTI, Project 9471) and INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR

IX

AUTHOR’S DECLARATION

The results presented in this dissertation are part of the following scientific

communications:

Poster Communications in Nationals Conferences:

Vilas-Boas, Cátia*; Cravo, Sara; Sousa, Emília; Pinto, Madalena; Correia-da-Silva,

Marta. Sea-water solubility and degradation studies of sulfated small molecules. 10

Meeting of Young Researchers of University of Porto (IJUP17), Porto, Portugal,

February 08-10, 2017, P nº 26.

Abstract in National Conferences Proceedings:

Vilas-Boas, Cátia*; Cravo, Sara; Sousa, Emília; Pinto, Madalena; Correia-da-Silva,

Marta. Sea-water solubility and degradation studies of sulfated small molecules. 10

Meeting of Young Researchers of University of Porto (IJUP17); 2017.

Submitted Paper:

Vilas-Boas, C.*; Sousa, E.; Pinto, M.; Correia-da-Silva, M.; An antifouling model

from the sea: 25 years of zosteric acid studies (Appendix I).

Others:

Vilas-Boas, Cátia*. Communication semi-finalist of the Famelab Competition,

“Bioincrustacao Marinha, um problema a combater", Fundação Calouste

Gulbenkian, April 22, 2017.

XI

“Live as if you were to die tomorrow. Learn as if you were to live forever

.”

XIII

GENERAL INDEX

FIGURE INDEX ... XV TABLE INDEX ... XVII GRAPHIC INDEX ... XIX ACKNOWLEDGMENTS ... XXI ABSTRACT ... XXIII RESUMO ... XXV LIST OF ABBREVIATIONS ... XXVII GENERAL INTRODUCTION ... XXIX

1. Introduction ... 3

1.1 Biofouling and antifouling coatings ... 3

1.2 Searching for ecologically AF compounds ... 6

1.3 Important parameters in AF compounds ... 8

1.3.1 Solubility ... 9

1.3.2 Degradation ... 10

1.3.3 Leaching ... 11

2. Aims ... 15

3. Material and Methods ... 19

3.1 Reagents and solvents ... 19

3.2 Quantitative rp-HPLC-DAD assay ... 19

3.2.1 HPLC equipment and methodology ... 19

3.2.2 Method optimization ... 19

3.2.3 Method validation ... 20

3.2.3.1 Specificity/Selectivity ... 20

3.2.3.2 Linearity and range ... 20

3.2.3.3 Detection and quantification limits ... 21

3.2.3.4 Accuracy and precision ... 21

3.2.4 Water solubility... 22

3.2.5 Degradation assays ... 22

3.2.6 Leaching assays... 23

3.3 Statistical analysis ... 24

4. Results and Discussion ... 29

4.1 Quantitative rp-HPLC-DAD assay ... 29

XIV

4.1.2 Method validation ... 31

4.1.2.1 Specificity/Selectivity ... 31

4.1.2.2 Linearity and ranges ... 37

4.1.2.3 LOD and LOQ ... 38

4.1.2.4 Accuracy and precision ... 39

4.1.3 Solubility in Water ... 40 4.1.4 Degradation assays ... 41 4.1.5 Leaching assays ... 50 5. Conclusions ... 59 6. References ... 62 7. Appendix ... 67 7.1 Appendix I ... 102 7.2 Appendix II ... 67

XV

FIGURE INDEX

dispersion. ... 3

Figure 2. Structure of ZA (para-(sulfoxy)-coumaric acid). ... 7 Figure 3. Representative chromatograms of several concentrations of AGS (10, 50, 100, 200 and

500 µM) dissolved in UPW with a chromatographic signal at ± 5 min at 243 nm when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile for degradation assays (38:62 v/v) (Fortis BIOC18 column) was used. ... 32

Figure 4. Representative chromatograms of several concentrations of AGS (10, 50, 100, 200 and

500 µM) dissolved in SW and diluted (1:3) with 60 mM of TBAB before injection, with a chromatographic signal at ± 5 min at 243 nm when a mobile phase containing an aqueous solution with 25 µM of TBAB and acetonitrile for degradation assays (38:62 v/v) (Fortis BIOC18 column) was used. ... 32

Figure 5. Representative chromatograms of several concentrations of AGS (10, 50, 100, 200 and

500 µM) dissolved in UPW with a chromatographic signal at ± 9.5 min at 243 nm when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile for leaching studies (50:50 v/v) (Fortis BIOC18 column) was used. ... 33

Figure 6. Representative Chromatograms of 500 µM of AGS dissolved in UPW before and after

being passed through the cartridge, with a chromatographic signal at of ± 9.5 min and a recovery rate of 77 % at 243 nm when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile for leaching studies (50:50 v/v) (Fortis BIOC18 column) was used. .... 33

Figure 7. Representative chromatograms of several concentrations of 3,4DOHX (5, 10, 25, 50, 100

and 200 µM) with a chromatographic signal at ± 20 min at 236 nm when a mobile phase containing an aqueous solution with 0.1 % of TFA and acetonitrile (80:20 v/v) (Fortis BIOC18 column) was used. ... 34

Figure 8. Representative chromatograms of several concentrations of Econea® _{(5, 10, 25, 50, 100}

and 200 µM) with chromatographic signal at ± 15.6 min at 283 nm when a mobile phase containing an aqueous solution with 0.1 % of TFA and acetonitrile (45:55 v/v) (Fortis BIOC18 column) was used. ... 34

Figure 9. Representative chromatograms of 200 µM of 3,4DHX dissolved in methanol before and

after being passed through the cartridge, with a chromatographic signal at ± 20 min and a recovery rate of 98 % at 236 nm when a mobile phase containing an aqueous solution with 0.1 % of TFA and acetonitrile (80:20 v/v) (Fortis BIOC18 column) was used. ... 35

Figure 10. Representative chromatograms of 50 µM of Econea® _{dissolved in methanol before and}

after being passed through the cartridge, with a chromatographic signal at ± 15.6 min and a recovery rate of 99 % at 283 nm when a mobile phase containing an aqueous solution with 0.1 % of TFA and acetonitrile (45:55 v/v) (Fortis BIOC18 column) was used. ... 36

Figure 11. Chromatograms of several concentrations of ZA (10, 30, 50, 100 and 200 µM) dissolved

in UPW with a chromatographic signal at ± 4.5 min at 250 nm when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile for degradation studies (50:50 v/v) (Fortis BIOC18 column) was used. ... 36

Figure 12. Chromatograms of several concentrations of ZA (5, 15, 25, 50 and 100 µM) dissolved in

SW after diluted with 25 mM of TBAB before injection (1:2), with a chromatographic signal at ± 4.5 min at 250 nm when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile for degradation studies (50:50 v/v) (Fortis BIOC18 column) was used. ... 37

Figure 13. Representative chromatograms of the degradation of AGS in SW after 6 months, when

exposed to several stress conditions. Chromatographic signal of degradation products at ± 3.6, 6.5, and 10.4 min when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile (38:62 v/v) (Fortis BIOC18 column) was used. ... 43

Figure 14. Representative chromatograms of the degradation of ZA after 6 months, when exposed

to several stress conditions. Chromatographic signal of degradation products at ± 5.25 min when a mobile phase containing an aqueous solution with 25 mM of TBAB and acetonitrile (50:50 v/v) (Fortis BIOC18 column) was used. ... 45

XVI

Figure 15. Representative chromatograms of the degradation of 3,4DOHX in aSW after 2 months,

when exposed to several stress conditions. Chromatographic signal of degradation products at ± 5.5, 6.20, and ±8.9 min when a mobile phase containing an aqueous solution with 0.1 % of TFA and acetonitrile (80:20 v/v) (Fortis BIOC18 column) was used. ... 47

Figure 16. Representative chromatograms of the degradation of Econea® _{in SW after 2 months,}

when exposed to several stress conditions. Signals of degradation products at a retention time of 5.6 and 10 min when a mobile phase containing an aqueous solution with 0.1 % of TFA and acetonitrile (45:55 v/v) (Fortis BIOC18 column) was used. ... 49

Figure 17. Representative chromatograms of blank (1_PU) and a sample with a polyurethane matrix

(2_PU) where no sign of the Econea® _{was detected. ... 51}

Figure 18. Representative chromatograms of blank (F1) and a sample with a silicone matrix (F2)

where a sign of Econea® _{was detected. ... 53}

Figure 19. Representative chromatograms of blank (F1) and a sample with a silicone matrix (F5)

where a sign of the AGS was detected. ... 53

Figure 20. Representative chromatograms of blank (BO_8_1) and a sample with a polyurethane

matrix (BO_E4_1) where a sign of the Econea® _{was detected. ... 54}

Figure 21. Representative chromatograms of blank (1_PU) and a sample with a polyurethane matrix

(3_PU) where no sign of the Econea® _{was detected. ... 55}

Figure 22. Representative chromatograms of blank (F1) and a sample with a silicone matrix (F3)

where a sign of the Econea® _{was detected. ... 56}

Figure 23. Representative chromatograms of blank (BO_8_1) and a sample with a polyurethane

XVII

TABLE INDEX

Table 1. Several types of AF coatings... 4

Table 2.Main AF biocides currently used in marine/industrial coatings, as well as some fate parameters [17-22]. ... 5

Table 3. Some synthetic nature-inspired ecological compounds with AF properties [31]... 7

Table 4. USP solubility classification [34]. ... 9

Table 5. Preliminary investigation of several mobile phases for AGS and ZA dissolved in UPW29 Table 6. Optimization of several mobile phases for AGS and ZA dissolved in SW. ... 29

Table 7. Optimization of several mobile phases for AGS and ZA dissolved in SW and diluted with several concentrations of TBAB. ... 30

Table 8. Mobile phase optimization of 3,4DOHX and Econea® _{dissolved in methanol. ... 30}

Table 9. Linearity and range data for 3,4DOHX, Econea®_{, AGS, and ZA. ... 37}

Table 10. LOD and LOQ for all the several compounds in the different matrices. ... 38

Table 11. Accuracy and RSD for all the several compounds in the different matrices. ... 39

Table 12. Results obtained on solubility assay for 3,4DOHX dissolved UPW (pH 5) and aSW (pH 8.3) by HPLC. ... 41

Table 13. Leaching results of 3,4DOHX, AGS and Econea® _{incorporated by direct immobilization in} the several types of coatings. ... 50

XIX

GRAPHIC INDEX

period of 2 (T2M) and 6 months (T6M) and compared with the initial concentration (T0M). p-value statistically significant for p <0.05 (*).Statistical test using two-way ANOVA. ... 42

Graphic 2. Degradation of AGS in SW after being exposed to several stress conditions during a

period of 2 (T2M) and 6 months (T6M) and compared with the initial concentration (T0M). p-value statistically significant for p <0.05 (*); p <0.0001 (****). Statistical test using two-way ANOVA. ... 43

Graphic 3. Degradation of ZA in UPW after being exposed to several stress conditions during a

period of 2 (T2M) and 6 months (T6M) and compared with the initial concentration (T0M). p-value statistically significant for p <0.0001 (****). Statistical test using two-way ANOVA. ... 44

Graphic 4. Degradation of ZA in UPW after being exposed to several stress conditions during a

period of 2 (T2M) and 6 months (T6M) and compared with the initial concentration (T0M). p-value statistically significant for p <0.0001 (****). Statistical test using two-way ANOVA. ... 45

Graphic 5. Degradation of 3,4DOHX in UPW after 2 months (T2M), exposed to several stress

conditions and compared with the initial concentration (T0M). Statistical test using two-way ANOVA. ... 46

Graphic 6. Degradation of 3,4DOHX in aSW after 2 months (T2M), exposed to several stress

conditions and compared with the initial concentration (T0M). p-value statistically significant for p <0.0001 (****).Statistical test using two-way ANOVA. ... 47

Graphic 7. Degradation of Econea® _{in UPW after 2 months (T2M), exposed to several stress}

conditions and compared with the initial concentration (T0M). Statistical test using two-way ANOVA. ... 48

Graphic 8. Degradation of Econea® _{in aSW after 2 months (T2M), exposed to several stress}

conditions and compared with the initial concentration (T0M). P-value statistically significant for p <0.001 (**). Statistical test using two-way ANOVA. ... 49

Graphic 9. Calibration curve of several concentrations of 3,4DOHX (5, 10, 25, 50, 100, 200 µM)

dissolved in methanol with a mobile phase containing acetonitrile and an aqueous solution containing 0.1% of TFA (20:80 v/v). ... 102

Graphic 10. Calibration curve of several concentrations of Econea® _{(5, 10, 25, 50, 100, 200 µM)}

dissolved in methanol with a mobile phase containing acetonitrile and an aqueous solution containing 0.1% of TFA (55:45 v/v). ... 102

Graphic 11. Calibration curve of several concentrations of AGS (10, 50, 100, 200 and 500 µM)

dissolved in UPW with the mobile phase contaning acetonitrile and an aqueous solution containing 25 mM of TBAB for degradation assays (62:38 v/v). ... 103

Graphic 12. Calibration curve of several concentrations of AGS (3.3, 16.7, 33.3, 66.7 and 166.6 µM)

dissolved in SW and diluted (1:3) with 60 mM of TBAB before injection, with the mobile phase contaning acetonitrile and an aqueous solution containing 25 mM of TBAB for degradation assays (62:38 v/v). ... 103

Graphic 13. Calibration curve of several concentrations of AGS (10, 50, 100, 200 and 500 µM)

dissolved in UPW, with the mobile phase contaning acetonitrile and an aqueous solution containing 25 mM of TBAB for leaching assays (50:50 v/v). ... 104

Graphic 14. Calibration curve of several concentrations of ZA (10, 30, 50, 100 and 200 µM)

dissolved in UPW, with the mobile phase contaning acetonitrile and an aqueous solution containing 25 mM of TBAB for leaching assays (50:50 v/v). ... 104

Graphic 15. Calibration curve of several concentrations of AGS (5, 15, 25 ,50, 100 µM) dissolved in

SW and diluted (1:2) with 25 mM of TBAB before injection, with the mobile phase contaning acetonitrile and an aqueous solution containing 25 mM of TBAB for leaching assays (50:50 v/v). ... 105

XXI

ACKNOWLEDGMENTS

Este trabalho é o resultado de um longo caminho percorrido através da ajuda de pessoas maravilhosas a quem quero muito agradecer. Através desta experiência aprendi que o que vale na vida não é o ponto de partida mas sim a caminhada, e que caminhando com as pessoas certas tudo se torna mais fácil e prazeroso. Portanto a todos vocês o meu muito obrigado.

Em primeiro lugar, agradeço à minha orientadora, Professora Doutora Marta Correia da Silva, por me ter acolhido nesta aventura, aceitando-me como sou. Obrigada por ter acreditado e investido em mim todo o seu conhecimento, entusiamo e dedicação. Obrigada por ver sempre mais além das minhas dificuldades, e nunca parar de reforçar as minhas virtudes. Consigo aprendi que quando nós fazemos a nossa parte, a vida acaba sempre por nos recompensar e no fim, as dificuldades não passarão apenas de degraus que nos ajudam a chegar mais longe. Obrigada por todo o conhecimento transmitido, mas acima de tudo, pelo seu exemplo de dedicação e persistência.

À Professora Doutora Emília Sousa, minha coorientadora, também o meu muito obrigado. Consigo aprendi que muitas das vezes o caminho certo está apenas à distância de um pensamento “outside the box”. É impossível não reconhecer as suas intervenções certeiras, pois através delas a caminhada tornou-se muito mais fácil e motivadora.

Agradeço à Professora Doutora Madalena Pinto, por me conceder a oportunidade de trabalhar neste grupo de investigação, onde aprendi tanto. Consigo aprendi que a diferença entre o sucesso e o fracasso está apenas na nossa vontade de o alcançar.

À Drª Sara Cravo também o meu muito obrigado por todo o apoio prestado mas acima de tudo, por todo o conhecimento e sabedoria transmitidos. Obrigada por todo o carinho que demonstrou comigo, mas acima de tudo por toda a paciência. Não tenho palavras para descrever o quão precisosa foi a sua ajuda ao longo deste percurso.

À Gisela e à Liliana quero agradecer todo o apoio técnico mas acima de tudo, quero agradecer todo o carinho por mim demonstrado. Obrigada por me terem recebido tão bem nesta enorme família.

À Ana Rita, Francisca e Catarina quero agradecer toda a ajuda e apoio, tanto no laboratório como fora dele.

XXII

Ao João, agradeço todo o conhecimento transmitido sobre estatística.

Aos meus colegas do LQOF o meu sincero obrigado por todos os momentos de diversão e de interajuda. Olhando para vocês, agradeço um dos presentes mais especiais que esta experiência me trouxe, amigos que irei sempre guardar no coração.

Aos meus amigos, o meu muito obrigada, pois quando tudo parecia tão cinzento conseguiram trazer cor e alegria a cada dia desta caminhada. Foram a minha rocha segura nesta aventura.

Ao Gonçalo quero agradecer todo o apoio e carinho. Obrigada por tudo o que compartilhamos e por todos os momentos em que alegraste o meu dia com a tua boa disposição contagiante.

À minha família, mas em especial aos meus pais, um especial obrigado. Sem o vosso amor, educação e incentivo, nada disto teria sido possível. Apoiaram-me em todas as caminhadas da vida e incentivaram-me a continuar independentemente das dificuldades e a nunca desistir. Convosco aprendi a ver sempre mais além e a querer alcançar sempre mais.

A Deus.

"Quem caminha sozinho pode até chegar mais rápido, mas aquele que vai acompanhado, com certeza vai mais longe."

XXIII

ABSTRACT

Marine biofouling is a natural process resulting from colonization and subsequent accumulation of unwanted marine organisms in natural and artificial submerged surfaces. This natural process causes huge anthropological problems, such as in aquaculture, shipping costs, and spread of diseases. To overcome these problems, coatings with incorporated biocides are being used, leading to toxic effects in the environment. As so, there is an increasing demand for environmental friendly antifouling (AF) compounds. Following this, some nature-inspired AF compounds with promising AF activity against macrofouling organism were synthesized in the Laboratory of Organic and Pharmaceutical Chemistry (LQOF). In this work, studies of seawater (SW) solubility and degradation of two of these previously synthesized compounds, sulfated gallic acid (AGS) and 3,4-dihydroxy xanthone (3,4DOHX), as also evaluation of their leaching rate after incorporation in polymeric coatings, were performed by reverse-phase high performance liquid chromatography with diode array detector (rp-HPLC-DAD). First, validation of the analytical method was performed not only for AGS and 3,4DOHX, as well as for Econea®_{, a} commercialized AF biocide, and zosteric acid (ZA), a sulfated marine natural AF product, for comparison purposes. AGS showed the highest solubility value in water, even higher than ZA, and 3,4DOHX exhibited reduced water solubility, however, higher than the biocide Econea®_{. For degradation studies, different stress conditions in aSW/SW and ultra pure} water (UPW) were considered (4 °C, 18 °C, and 25 °C in the dark; 25 °C in natural light). The results showed that degradation in aSW/SW was higher than UPW for the tested compounds. AGS showed lower degradation rate when compared to ZA and 3,4DOHX seems to have higher rate of degradation than Econea®_{. After direct incorporation of AGS} in polyurethane and silicone coatings and 3,4DOHX in polyurethane coatings, these compounds showed lower leaching rate in polyurethane than in silicone coatings. However, the leaching of AGS was much higher than 3,4DOHX. Following, a new chemical immobilization technique was tested for Econea® _{and a considerable decrease in the} leaching rate was achieved in the polymeric coatings.

Keywords: biofouling; antifouling; eco-friendly; synthesis; solubility; degradation;

XXV

RESUMO

A bioincrustação marinha é um processo natural que resulta da colonização e acumulação de diversos organismos marinhos indesejados em superfícies submersas em água. Este processo causa imensos problemas antropológicos tais como impactos na aquicultura, custos na navegação e disseminação de doenças. Para combater este problema, vários processos podem ser utilizados, tais como revestimentos com biocidas nas suas formulações, levando a problemas de ecotoxicidade. Felizmente existe uma procura cada vez maior por produtos ecológicos e por isso alguns compostos anti-incrustantes inspirados na natureza foram sintetizados no laboratório de Química Orgânica e Farmacêutica (LQOF), mostrando uma promissora atividade anti-incrustante contra macro-organismos. Neste trabalho, estudos da solubilidade em água do mar e da degradação de dois desses compostos sintetizados, o ácido galico sulfatado (AGS) e a 3,4-dihidroxi xantona (3,4DOHX), assim como a avaliação das suas taxas de lixiviação após serem incorporados em revestimentos poliméricos, foram realizados por cromatografia líquida de alta eficiência de fase reversa acoplada a um detetor de arranjo de díodos (rp-HPLC-DAD). Em primeiro lugar, foi feita a validação do método analítico, não só para analisar o AGS e a 3,4DOHX, mas também o Econea®_{, um biocida comercial e o ácido zostérico (ZA), um produto} anti-incrustante natural, para fins comparativos. AGS apresentou elevada solubilidade em água, mesmo quando comparado com o ZA. A 3,4DOHX apresentou reduzida solubilidade em água, no entanto o seu valor de solubilidade foi maior do que o Econea®_{. Para os estudos de} degradação, foram consideradas várias condições em água ultra pura e água do mar (4 °C, 18 °C e 25 °C no escuro, e 25 °C com exposição solar). Estes resultados mostraram que a degradação em água do mar foi muito maior do que em água ultra pura para os compostos testados. O AGS mostrou possuir menor taxa de degradação, quando comparado com o ZA e a 3,4DOHX pareceu apresentar maior taxa de degradação que o Econea®_{. Após} incorporação direta do AGS em revestimentos de poliuretano e silicone e da 3,4DOHX em revestimentos de poliuretano, estes apresentaram uma menor taxa de lixiviação em poliuretano. No entanto, a lixiviação do AGS foi muito maior, comparado com a 3,4DOHX. De seguida, uma técnica de imobilização química foi testada para o Econea®_{, sendo} alcançada uma diminuição considerável da taxa de lixiviação nos revestimentos poliméricos testados.

Palavras-chave: bioincrustação; anti-incrustantes; ecológicos; síntese, solubilidade,

XXVII

LIST OF ABBREVIATIONS

3,4DOHX - 3,4Dihydroxy xanthone

ACN - Acetonitrile

AF - Antifouling

AGS - Sulfated gallic acid

ANOVA - Analysis of variance

ASW - Artificial seawater

EC50 - Half maximal effective concentration

HPLC-DAD - High performance liquid chromatography diode array detector

Ip-rp -

k -

LC50 - Half maximal lethal concentration

LC-MS - Liquid chromatography–mass spectrometry

LQOF - Laboratório de Química Orgânica e Farmacêutica

LOD - Limit of detection

Log P - Octanol-water partition coefficient

LOQ - Limit of quantification

MS - Mass spectrometry

SPC - Self polishing copolymer

SW - Seawater

TBAB - Tetrabutylammonium bromide

TFA -

UPW - Ultra pure water

UV-Vis - Ultraviolet- visible

XXIX

GENERAL INTRODUCTION

The present dissertation is structured in seven chapters:

Chapter 1: Introduction

The first chapter includes a brief introduction of biofouling and its problem in several anthropological areas as well approaches for combating this problem. The importance of solubility, degradation and leaching studies are also referred.

Chapter 2: Aims

This chapter includes the aims of this dissertation.

Chapter 3: Experimental

In this chapter, the experimental procedures for the optimization and method validation, as well the solubility, degradation, and leaching studies are described.

Chapter 4: Results and discussion

In this chapter, results obtained from the optimization and method validation, as well the solubility, degradation, and leaching studies are presented and discussed.

Chapter 5: Conclusions

This chapter includes the main conclusions of the developed work.

Chapter 6: References

In this chapter, the references cited throughout the thesis are presented. The main bibliographic research motors were ISI Web of Knowledge, Scopus, PubMed and Google Scholar.

Chapter 7: Appendix

The last chapter contains a non-published mini-review about ZA that is almost celebrating the 25th _{anniversary of its discovery (Appendix I). This mini-review organizes} for the first time all the studies obtained during the past 25 years with the aim of

XXX

understanding “what has been learned during this time?”. All the studies are organized in groups concerning AF screening assays, mechanism of action, environmental fate parameters (solubility, degradation, bioaccumulation, and ecotoxicity) and application in coatings. Synthesis of this natural product is also assembled, as well as the synthesis of ZA analogs that allowed to perform ZA structure-AF activity relationship studies.

This chapter also contains calibration curves for the several analyzed compounds (Appendix II).

1

Chapter 1

3

1. Introduction

1.1

Biofouling and antifouling coatings

Biofouling is a process resulting from colonization and subsequent

accumulation of unwanted marine organisms, such as bacteria, fungi, algae,

mussels, and other marine animals in natural (wood, rocks, other organisms) and

artificial surfaces (hulls, platforms, buoys, wharf) submerged in water [1]. This is

a complex phenomenon that involves a diversity of marine species constituted by

communities whose dynamic growth is managed by biological and physical

processes and can be divided into several stages [2]: i) adsorption of extracellular

polymeric substances, present in the water and produced by microorganisms,

to

a surface; ii) chemical attraction of bacteria through a regulatory system, the

quorum sensing regulation; iii) reversible adsorption of bacteria; iv) irreversible

adsorption of bacteria, involving the fixing of macromolecular fibrils; v)

agglomeration and colonies formation; vi) growth of a secondary bacterial

population, benthic diatoms, protozoa, and simultaneous adhesion of particles.

This stage is called microfouling (figure 1, I) and is considered a precursor step in

the subsequent establishment of macrofouling (figure 1, II) [3]. After this initial

process, a community of macroorganisms such barnacles, bryozoans, sea squirts,

sponges and macroalgaes are developed on the previous community [4].

Figure 1. I. Microfouling formation, divided into three main processes: attachment, growth and propagation. II. Macrofouling formation, after the process of microfouling (adapted from [5]).

4

Although biofouling can be considered a natural process, when grown on

artificial structures, it can lead to several problems and damage for the human

population [1]. The colonization of surfaces, such as ship hulls, fishing nets, water

intake pipes, heat exchangers, membranes, sensors, food stuff, implants, among

others, by fouling organisms become a multifaceted, global-scale problem and

due to the large increase of biofouling in the several areas, this impact can be

divided in three principals interconnected categories [6]: i) impacts on

biodiversity, habitats and ecological processes, as is the case of invasive species

(environmental problems); ii) impacts in aquiculture, coastal infrastructure,

shipping, costs of removal and biofouling control (economic problems) and iii)

spread of diseases (human health problems). Despite biofouling being present in

several anthropological areas, marine biofouling is perhaps the most recognized

form of biofouling and also the most difficult to combat [7]. In this case, fouling

organisms attached to surfaces take advantage of flow by the surface to gain

nutrients and growth [8]. Therefore, AF coatings are perhaps the most common

method of biofouling control, been formulated to protect all the marine structures

(shipping vessels, drilling rigs, production platform, among others) against

corrosion and fouling, acting by chemical, physical and biological processes

(Table 1) [1, 9].

Table 1. Several types of AF coatings.

Types of coatings Mechanism of action Ref.

Metal based coating Toxic copper or silver-coated surfaces [3] Self-polishing copolymer (SPC) Self-polishing paint with copper, tin, zinc or biocides [11] Fouling-release coatings Low surface energy promotes low bioadhesion [10] Hydrogels Hydrophilic surface confuses settlers [12] Biological coating Deterrents include bacteria, algae and invertebrates [11] Enzyme-based coatings Lowers bioadhesion and lyses bacteria [13] Organo-metallic Toxic flexible metal surface [1] Photoactive film Self-cleaning film uses UV or visible radiation [14]

Metal based AF coatings and SPC are perhaps the most used approach and

are mainly constituted by copper metal oxides and broad-spectrum biocides,

causing many problems due to the high accumulation and toxicity in biological

5

systems [15, 16]. In turn, when these toxic compounds are released from coatings,

entering into non-target organisms, disrupting essential life processes [17]. In

Table 2 it is possible to observe some biocides globally used in coatings, as well

as some of their fate parameters [16-22].

Table 2.Main AF biocides currently used in AF coatings, as well as some fate parameters. Main AF biocides

Water solubility (mg/mL)

Log

P Ecotoxicity Degradation Ref.

Irgarol 1051®

0.006 -

0.007 3.27

Acts by inhibition of photosystems II; Highly

toxic to periphyton and macrophytes, possessing a LC50 of 9.734 mg/L for Artemia salina*; t½: 100 - 250 days in SW (persistent in water)

[16-22]

to mammals, birds, moderately toxic to

fishes and highly toxic to aquatic invertebrates, possessing a LC50 of 30.573

mg/L for Artemia salina*;

t½: 14 days in SW by biotic degradation (persistent in water) SeaNine 211® 0.002 2.8 – 4.5

Acts by oxidative stress and perturbation of the mitochondrial respiratory chain; Broad‐spectrum antifoulant, possessing a LC50 of 0.318 mg/L for Artemia salina*; < 24 h in SW by biotic degradation Dichlofuanid 0.0013 3.6 Acts by inhibition of photosystem II; Toxic for

most of biodiversity, possessing a LC50 of 154.944

mg/L for Artemia salina*;

18 h in SW Chlorothalonil 0.0009 2.64-4.38 Acts by inhibition of mitochondrial electron transport; Highly toxic to aquatic species, possessing a

LC50 of 2.683 mg/L for Artemia salina*; 1.8 - 8 days in SW by biotic degradation (Persistent in ecosystem) Copper pyrithione 0.0001 0.97

Acts by multi-site inhibition, responsible for metabolic

processes; Potentially harmful to non-target aquatic organisms, possessing a LC50 of 0.319 mg/L for Artemia salina*;

<24 h in SW by photolysis Econea® 0.00016 – 0.00017 3.5 Acts by uncoupling mitochondrial oxidative phosphorylation; Very toxic

t½: 3 h in SW by hydrolytic

6 Main AF biocides Water solubility (mg/mL) Log

P Ecotoxicity Degradation Ref.

to aquatic life and potentially harmful to non-target

aquatic organisms, possessing a LC50 of 0.9 µg/L

for T. battagliai*;

T½= Half-life; LD50= Median lethal dose; Log P= Octanol-water partition coefficient; *non-target organism

However, the information available on the effects of these agents to the

marine ecosystems is still limited and it is very important to know the risks

associated with the existence of those biocides in the marine environment, as well

as their degradative products [18]. Fortunately, there is an awakening for the

ecological problems and through the appearance of laws (Biocidal Products

Directive 98/8/EC, for example) the course of research is changing, forcing

companies to control the launching of new coatings into the market [23]. As

governments recognize and create legislation against the uses of toxic biocides,

the industry and researchers are actively looking for alternatives. Now, ideal AF

formulations must have some particularities: should permit at least five years of

biofouling life cycle control; should be durable and resistant to damages; should

be repairable; must have low maintenance; should be easy to apply; should be

hydraulically smooth; must be compatible with the existence of anticorrosion

coatings; should be cost effective; must be effective at port and sea; must not be

persistent in the environment and; must be nontoxic to non-target/target species

[24, 25].

1.2

Searching for ecologically AF compounds

To discover environmental friendly and nontoxic AF compounds to be

incorporated in coatings, marine organisms that are usually free of fouling on

their surfaces have been largely investigated and some authors defend the use of

AF compounds inspired in their secondary metabolites to replace toxic

compounds [26]. It was discovered that a large variety of microorganisms like

bacteria, fungi and sessile marine organisms such as sponges, corals, and algae

develop several defenses against biofouling [27] and for that reason, their

7

secondary metabolites have an enormous potential as AF agents [28], as is the

case of ZA (Figure 2), a sulfated phenolic acid produced by the seagrass Zostera

marina (appendix I) [29].

Figure 2. Structure of ZA (para-(sulfoxy)-coumaric acid).

In general, the percentage of natural product antifoulants derived from the

ocean has increased over the previous five years, indicating that the marine

environment is a promising source of “green” AF compounds [30]. The search for

“green” alternatives has led many investigators to focus on the development of

ecological substances with AF properties that are nontoxic, degradable, and

without persistence in the environment [15, 31]. However, only a few can be

considered good candidates due to several reasons [30]: i) the inhibitory effects

are too restricted; ii) the LC50/EC50 ratios are very low; iii) there are difficulties

associated with producing compounds in large quantities; iv) there is limited

information about bioaccumulation and ecotoxicology against target and/or

non-target organisms; v) there is a lack of studies relatively to their mechanism of

action; vi) there is limited information about degradation in SW; and vii) these

compounds need to be compatible with base paint ingredients.

Table 3 presents 4 examples of nature-inspired compounds synthesized by

our research group (LQOF/CIIMAR), with a good LC50/EC50 ratio.

Table 3. Some synthetic nature-inspired compounds with AF properties [31].

Nature-inspired AF compounds EC50a EC50b LD50/ EC50b

3,6-bis(β-D-Glucopyranosyl) xanthone persulfate (XGS)

8

Nature-inspired AF compounds EC50a EC50b LD50/ EC50b

Rutin persulfate (RS)

>1000 μg/mL 36.84 μg/mL >22.13

Gallic acid persulfate (AGS)

> 1000 μg/mL 8.4 μg/mL >26.61

3,4Dihydroxy xanthone (3,4DOHX)

>750 µg/mL 2.63 μg/mL >43.37

EC50= Half maximal effective concentration; LC50= Median lethal concentration; a_Against non-target Vibrio fischeri; b_{Against Mytilus galloprovincialis target.}

All of these sulfated compounds were found to be nontoxic to the

non-target species Artemia salina (< 10 % mortality at 250 µM) and Vibrio fischeri

(LC50 > 1000 μg/mL), as well as 3,4DOHX (< 10 % mortality at 50 µM) and Vibrio

fischeri (LC

nature-inspired molecules to generate new nontoxic AF compounds.

1.3

Important parameters in AF compounds

As mentioned above, although there are several compounds that may be

used against biofouling, several studies should be made in order to understand

their ecological behaviour, as well as the best way to incorporate them into AF

coatings for future commercialization [16]. Anastas and Warner developed twelve

principles of green chemistry that include two concepts: “Less hazardous

9

chemical syntheses” i.e., the generation of substances that possess little or

nontoxicity for human health and the environment; “Design for degradation” i.e.,

compounds should break down into innocuous degradation products, leaving no

harmful footprints at the end of their function [32]. Thus, an ecofriendly AF

compound should take into account its effect in the environment and produce

short-lived nontoxic products that have not impact on non-target species and the

ecosystem [16]. Studies such as solubility evaluation, degradation analysis by

abiotic and biotic processes, and determination of leaching rate to water are very

important and should be considered to understand the behaviour of AF

compounds in coatings and evaluate their persistence after their leaching for the

water.

1.3.1

Solubility

Solubility is described as the maximum amount of solute that can be

dissolved per amount of solvent in order to give a homogeneous solution. It is a

property of all physical states that depends on solvent used, temperature, and

pressure [33]. In general, the determination of solubility consists in the

saturation of a solvent with the solute in accordance with USP, the solubility

range from “very soluble” to “practically insoluble” (Table 4) [34].

Table 4. USP solubility classification [34].

Classification Solubility range _(mg/mL) Solubility assigned _(mg/mL)

Very soluble > 1000 1000

Freely soluble 100 - 1000 100

Soluble 33 - 100 33

Sparingly soluble 10 - 33 10

Slightly soluble 1 - 10 1

Very slightly soluble 0.1 - 1 0.1

Practically insoluble < 0.1 0.01

This parameter can be analyzed by several methods, however, the most used

is the shake flask method. This method consists in adding an excess of compound

to a certain solvent and stir during a predetermined time. After the confirmation

of the saturation, the solution is filtered and analyzed by several analytical tools,

as light scattering/turbidity, ultraviolet (UV) plate reader, HPLC-DAD, and LC–

10

MS [35]. However, it is crucial to understand the limitations of each detection

technique in order to accurately assess the concentration of the saturated solution

[36]. The solubility analysis also allows to understand interactions between

persistence-bioaccumulation-toxicity of the AF compound [37]. For example, low

P and high water solubility suggests that water will retain in solution most of the

AF compound introduced in the ecosystem, expecting that the compound will not

partition appreciably into soil, settling particles in water, sediments or fatty

tissues, having thus a low bioaccumulation potential [38]. On the other hand,

most current commercial fouling-release coatings are based on hydrophobic

polymers, such poly(dimethylsiloxane) for example, and show low adhesion to

polar molecules due to the reduced opportunities for H-binding and polar

interactions [39].

1.3.2

Degradation

Chemical compounds can be protected from biological degradation until

release by the coating polymer matrix, however, for a better understanding of the

behaviour of AF compounds in the ecosystem, studies on degradation should also

be performed in order to determine the half-life of the compounds after being

leached into the oceans. In fact, a long environmental half-life can lead to a high

persistence of compounds into the ecosystem [40]. After release to the water,

degradation can occur by several abiotic processes, such as chemical degradation

(by hydrolysis, pH, temperature, salinity, dissolved organic carbon content,

among others) and photodegradation (by sunlight photolysis), and by biotic

processes, such as biodegradation (gradual breakdown of a material mediated by

a specific biological organism) [41]. In general, the first degradation process is

generated by hydrolytic scission of the chemical bonds, leading to a decrease in

the molecular weight [42]. At both acidic and basic pH, the degradation may

mainly occur by intramolecular transesterification [43]. Temperature stimulate

the removal of AF compounds in SW by exciting the collision between photons

and molecules [44]. In the case of photodegradation, high photon flux can greatly

elevate the probability of collision between active centers and photons and

therefore can generate more free radicals to promote the photoreactions [44].

Analyzing the several degradative processes, photodegradation and

11

biodegradation seem to be the major degradation processes which can naturally

clean up the environment. However, photodegradation is not so simple to occur

due to the poor penetration of sunlight in deep water and the propensity for many

compounds to bind to sediments. Still, all these degradative processes are

variable and dependent on the environmental conditions and compositions [45].

It is important to realize in vitro degradation experiments before incorporating

AF compounds into marine coatings [46]. The degradation solutions can be

subjected to appropriate chemical and physical analysis by HPLC, MS, nuclear

magnetic resonance, among other analytical methods in order to separate,

identify and quantify, if possible, the main degradation products [42].

1.3.3

Leaching

AF marine coatings are mainly constituted by soluble or insoluble polymeric

matrices [47]. In an insoluble matrix, AF compounds can be diffused through the

entire coating layer, being released over time through pores and capillaries

existing on the surface. Therefore, the biocidal compounds decrease

exponentially over time, with consequent decrease of the capability to prevent the

biofouling sources [3]. However, the best insoluble matrices are able to protect

vessels from biofouling for up to 2 to 3 years [3, 25]. In a soluble polymeric matrix,

the leaching rate of AF compounds increase with the movement (situation where

biofouling is less pronounced), constituted a disadvantage of using these

matrices. In order to counteract this issue, these soluble matrices are

manufactured with biocides not bound to the polymer, using for that a slow

release technology [3], which allows protection from biofouling for up to 5 years

[25].

Incorporation of an AF compound on the coating varies according to its

affinity to the matrix. Usually, a typical marine AF coating is based on ingredients

that are less water soluble, and for that reason, hydrophilic compounds can

present difficulties in being incorporated into coatings [48]. Processes of biocide

incorporation on polymeric matrices by direct and chemical immobilization

improve biocide incorporations and thus, reduce the leaching rate of polar

compounds [46, 49].

12

An effective AF compound should be chemically stable, having high

affinity for the interface between water and the hydrophobic paint surface and

also possessing a controlled release into the ecosystem [50]. Environmental

ecofriendly coatings usually exhibit AF functionality between 6 and 24 months,

whereas the desired service life is at least 5 years [51]. Thus, the ideal AF

compounds would be added to coatings in concentrations of µg/g and would be

released at ≤ng/cm/day [8]. Regarding in vitro leaching studies, leached

compounds are usually analyzed after 45 days in contact with water, since

leaching commonly stabilizes after that period [52].

13

Chapter 2

Aims

15

2. Aims

The main aim of this work was evaluate some environmental fate

parameters of two in-house synthesized nature-inspired AF compounds (AGS

and 3,4DOHX) as well for an AF marine natural product (ZA), and a commercial

biocide (Econea

_).

For that, the specific objectives were:

i.

to develop, optimize and validate appropriate analytical methods for

each compound;

ii.

to evaluate the solubility in seawater and ultra pure water for each

also;

iii.

to perform degradation studies using several stress conditions, namely

4 °C, 18 °C, and 25 °C in the dark, and 25 ºC in the presence of natural

light;

iv.

to study the leaching rate of each compound after direct incorporation

in several types of polymeric coatings;

v.

to compare different non-releasing formulations by evaluation of the

leaching rate of Econea

_.

17

Chapter 3

19

3. Material and Methods

3.1

Reagents and solvents

Compounds AGS, 3,4DOHX, and ZA were previously synthesized in LQOF

(purity ≥ 98.0 % for AGS and purity ≥ 99.0 % for 3,4DOHX and ZA) and Econea

was kindly supplied by Dra. Elisabete Geraldes Silva (purity ≥ 98.0 %; FCUL).

Analytical grade UPW was obtained by using a Millipore MilliQ water purification

system. HPLC grade acetonitrile (Biosolve BV, HPLC grade ≥99.9 %) was used

for HPLC analysis. All reagents and solvents were purchased from Carlo Erba

Reagents, VWR Chemicals, Panreac AppliChem, and Sigma Aldrich, and were

used with no further purification process. Artificial seawater (aSW) was kindly

supplied by Dra. Elisabete Silva Geraldes (pH 8.3; FCUL) and collected SW were

kindly supplied by Dra. Joana Rés de Almeida (pH 7; CIIMAR).

3.2

Quantitative

rp-HPLC-DAD

assay

3.2.1

HPLC equipment and methodology

HPLC analyses were performed on Thermo SCIENTIFIC SpectraSYSTEM

equipped with a SpectraSYSTEM UV-8000 DAD detector, a SpectraSYSTEM

P4000 pump and a SpectraSYSTEM AS3000 autosampler and the used software

was ChromQuest 5.0™. The analyses were conducted on a Fortis BIOC18 column

(Fortis BIOC18, Fortis Technologies, 5 µm Fortis BIO C18, 250 x 4.6 mm). After

mixing, mobile phases were filtered through a 0.45 µm filter and degassed before

use by an ultrasonic cleaner (Sonorex Digitec, Bandelin). The flow of the mobile

phase was 1 mL/min and the injection volume was 20 µL.

3.2.2

Method optimization

To analyse AGS, 3,4DOHX, Econea

_{, and ZA in HPLC, several mobile}

phases were tested in order to obtain suitable retention for the several studies of

solubility, degradation, and leaching, by retention factor (k) analysis. This

parameter was calculated according the follow equation: [retention time (T

) -

hold-up volume (T0)] / To. The optimized conditions for AGS in the several

20

studies were an aqueous solution containing 25 mM of TBAB and acetonitrile

(38:62 v/v for degradation assays and 50:50 v/v for leaching assays). For

3,4DOHX and Econea

_{the optimized conditions were an aqueous solution of 0.1}

% trifluoroacetic acid (TFA) and acetonitrile (80:20 v/v and 45:55 v/v,

respectively). For ZA, the optimized condition was an aqueous solution

containing 25 mM of TBAB and acetonitrile (50:50 v/v).

3.2.3

Method validation

The analytical method was validated by determination of the

specificity/selectivity, linearity, range, detection and quantification limits,

precision, and accuracy of each analyte, according to the ICH Guidance for

Industry [53].

3.2.3.1 Specificity/Selectivity

The specificity/selectivity was studied using several concentrations

(standard solutions) for AGS, 3,4DOHX, Econea

_{, and ZA dissolved in several}

matrices (UPW, aSW, SW and methanol), with the mobile phases and the

wavelength established for each compound. To analyze the specificity/selectivity

of the extractive process by OASIS

_{WAX 6cc cartridge for AGS and OASIS}

_HLB

6cc cartridge for 3,4DOHX and Econea

_{, a known concentration of each}

compound in the initial matrix was passed through the cartridges and

subsequently extracted. After extraction and evaporation of the solvent, the dried

compound was resuspended with UPW for AGS and methanol for 3,4DOHX and

Econea

_{to the initial concentrations and injected into the HPLC.}

Chromatograms were compared with the chromatograms resulting from the

injection of the standard solutions before extraction, allowing to infer the

specificity/selectivity of the method. In this way, it was also possible to determine

the recovery rate of the extractive process. This procedure was done in triplicate.

3.2.3.2 Linearity and range

Calibration curves of AGS, 3,4DOHX, Econea

_,

_{and ZA were performed by}

injection of several standard solutions, prepared by dilution of a stock solution of

21

each compound. Stock solutions of AGS (10 mM) were prepared in UPW and SW,

and several standard solutions were made in the concentration range 10-500 µM.

For degradation analysis in SW, each standard solution was diluted (1:3) with 60

mM aqueous solution of TBAB before injection. A stock solution of 3,4DOHX (10

mM) was prepared in methanol and several standard solutions were made in the

concentration range 5-200 µM. A Stock solution of Econea

_{(5 mM) was}

prepared in methanol and several standard solutions were made in the

concentration range 5-200 µM. Stock solutions of ZA (10 mM) were prepared in

UPW and SW, and several standard solutions were made in the concentration

range 10-200 µM. For degradation analysis in SW, each concentration was

diluted (1:2) with 25 mM aqueous solution of TBAB before injection. Two

replicates for each concentration were prepared and each replica was analyzed in

triplicate. The peak area was plotted against the known concentration to obtain

the calibration curve. Coefficients (r

_{) were calculated by least squares linear}

regression analysis.

3.2.3.3 Detection and quantification limits

The limits of detection (LODs) and quantification (LOQs) were determined

by signal/noise ratio (S/N) and the determination of S/N was performed by

comparing measured signals from standard solutions with low concentrations

with signals from blank samples (noise) and establishing the minimum

concentration at which the compound can be detected or quantified, respectively.

The LODs were usually accepted with a 3:1 S/N ratio and LOQs were accepted

with a 10:1 S/N ratio.

3.2.3.4 Accuracy and precision

Accuracy was evaluated using three different concentrations of the standard

solution of each compound. Three replicates for each concentration were

prepared and each replica was analyzed in triplicate. The determination was

made based on the ratio between the concentrations given by the peak area of

each compound in the standard solutions and the nominal concentration.

Precision was evaluated taking into account two factors, repeatability, or relative

standard deviation (RSD), and intermediate precision.

22

3.2.4

Water solubility

To analyze the solubility of AGS in water, 6 μL of UPW and SW were added

to 6 mg of AGS in an eppendorf. The suspension was stirred at 25 °C for 1 h. After

this time, AGS was completely solubilized and the solubility was defined

according to USP [34]. The procedures were made in triplicate. For solubility

quantification in HPLC, UPW and SW were added to 60 mg of 3,4DOHX in an

eppendorf, to a final concentration of 60 mg/mL. This procedure was made in

triplicate. The suspension was stirred at 25 °C for 1 h. After this period, the

suspension of 3,4DOHX was filtered through a 0.20 µm membrane filter with a

syringe. After filtration, 200 µL of each solution of 3,4DOHX was injected in

HPLC and the peak area of each solution was interpolated into the several

calibrations curves. Solutions concentrations were determined from the means

obtained in the different calibration curves.

3.2.5

Degradation assays

To analyse the degradation of AGS in UPW and SW, stock solutions of 10

mM were prepared in each water and 2oo µL were diluted with UPW and SW to

obtain a final concentration of 200 µM in each vial. These procedures were made

in duplicate. After that, 6 of the 8 vials were wrapped in aluminium and stored in

pairs under different conditions for a period of 6 months: 2 vials of UPW and SW

were stored in the refrigerator at a temperature of 4 °C; 2 vials of UPW and SW

were stored in a room at 18 °C; 2 vials of UPW and SW were stored in a room at

25 °C; the other 2 vials were exposed to natural light in a room at 25 °C. The same

procedure was repeated for ZA. Periodically, an aliquot of each vial was directly

injected into the HPLC without any extraction procedure, and the peak area of

AGS and ZA was interpolated into the calibrations curve. The concentration was

determined from the means obtained in calibration curve. In this way it was

possible to analyze the degradation of AGS and ZA over time.

To analyse the degradation of 3,4DOHX in UPW and aSW, stock solutions

of 10 mM were prepared in methanol and 1oo µL were diluted with UPW and aSW

to obtain a final concentration of 100 µM with 1 % of methanol in each vial [54].

23

To analyse the degradation of Econea

_{in UPW and aSW, the same procedure}

was done but with a final concentration of 50 µM in each vial in order to obtain 1

% of methanol solutions [54]. These procedures were done in triplicate. After

that, all the vials of each compound were stored under the same conditions as

AGS and ZA but for a period of 2 months. After this period, each solution was

passed through the SPE columns OASIS

_{HLB 6cc extraction cartridge according}

to the following procedure: condition with 6 mL of methanol; equilibrate the

cartridge with 6 mL of water and 6 mL of water with 10 mmol/L Na2EDTA buffer

in the case of aSW samples [55]; thereafter, the samples were passed through the

columns (aSW samples were previously spiked with 0.8 mg of Na2EDTA). After

drying, each cartridge was washed with 10 mL of methanol. The volume was

reduced to dryness under nitrogen purge by a sample concentrator Stuart

SBHCONC/1 with a block heater Stuart

_{SBH200D/3 at a temperature of 40 °C.}

Then, the residues were dissolved in methanol to a final volume of 10 mL. After

this procedure, each solution was injected in HPLC-DAD, and the peak area of

each solution was interpolated into the several calibrations curves. The

concentration was determined from the averagesobtained in calibration curve.

ASW used in degradation assays of 3,4DOHX and Econea

_{was from FCUL, and}

SW used in degradation assays of AGS and ZA

was from CIIMAR.

3.2.6

Leaching assays

The leaching rate of AGS, 3,4DOHX, and Econea

_{directly and chemically}

immobilized [49] in PVC plates with two types of polymers, polyurethane and

silicone, was evaluated after 45 days submerged in aSW. All the waters were

kindly supplied by Dra. Elisabete Silva Geraldes (FCUL).

The waters 2_PU,

3_PU, 4_PU, F2, F3, BO_E4_1, and BO_EM9_1 were passed through the

OASIS

_{HLB 6cc cartridge according to the following procedure: conditioning}

with 6 mL of methanol; equilibrate the cartridge with 6 mL of water and 6 mL of

water with 10 mmol/L Na2EDTA buffer [65]; thereafter, the water sample was

spiked with 32 mg of Na2EDTA and passed through the columns. After drying,

the cartridge was washed with 10 mL of methanol. The volume was reduced to

dryness under nitrogen purge by a sample concentrator Stuart

_SBHCONC/1

24

with a block heater Stuart® SBH200D/3 at a temperature of 40 °C. Then the

residues were dissolved in 200 µL of methanol in order to concentrate the

vestigial concentrations of 3,4DOHX and Econea

_{present in waters.}

The waters 5_PU and F5 were divided in three portions and passed through

OASIS

_{WAX 6cc cartridge according to the following procedure: conditioning}

with 6 mL of methanol; equilibrate the cartridge with 6 mL of water; thereafter,

the water sample was acidified with 0.5 % of formic acid and passed through the

columns. After drying, each cartridge was conditioned with 10 mL of UPW with

2% of formic acid and 4 mL of methanol, eluted with 30 mL of methanol basified

with 5 % of ammonia and 10 mL of UPW basified with 1 % of ammonia for a glass

balloon. The solvents were evaporated on the rotary evaporator Büchi

Rotavapor R-200 System to concentrate and then, the solutions were transferred

for a vial and the remaining solvent was reduced to dryness under nitrogen purge

by a sample concentrator Stuart

_{SBHCONC/1 with a block heater Stuart}

SBH200D/3 at a temperature of 40 °C. After evaporation of the solvents, the

residues of each vial were dissolved with 200 µL of UPW in order to concentrate

the vestigial concentration of AGS present in both waters. After this procedure,

each solution was injected in HPLC according to their mobile phases, and the

peak area of 3,4DOHX, Econea

_{, and AGS were interpolated into the several}

calibrations curves. The concentration was determined from the averages

obtained in calibration curves and in this way it was possible to determine the

leaching rate of 3,4DOHX, Econea

_{, and AGS}

_{after 45 days in contact with aSW.}

In order to better identify the chromatographic signal of 3,4DOHX, Econea

_{, and}

AGS, waters 1_PU, F1, and BO_8_1 (blank solutions), was also passed through a

cartridge using the same procedure mentioned above and also injected in HPLC

in the same conditions. Leaching rate was calculated according the follow

equation: [leached concentration / incorporated concentration]*100.

3.3

Statistical analysis

The question hypothesized in this study is that different stress conditions

might affect the degradation rate of several compounds dissolved in UPW and

aSW/SW. To test for statistical differences between the different conditions, a

25

two-way analysis of variance (ANOVA) was performed, followed by Tukey’s

multiple comparisons test. To evaluate whether the distribution was normal or

not a Shapiro-Wilk test of normality was performed. Differences were considered

statistically significant for p-values below 0.05.

The results were analyzed using GraphPad Prism version 6 (GraphPad

Software, La Jolla, CA, USA) and Excell 2013 (Microsoft Corporation, Redmond,

WA, Estados Unidos).

27

Chapter 4

29

4. Results and Discussion

4.1

Quantitative

rp-HPLC-DAD

assay

4.1.1

Method optimization

To analyse AGS and ZA in UPW and SW, several mobile phases containing

different proportions of acetonitrile and acidified water (0.1 % acetic acid) were

prepared; however, the retention time were not satisfactory, with overlapping

with the solvent front. As the separation of charged molecules was not being

achieved in regular reversed-phase, mobile phases containing different

proportions of acetonitrile and water with 25 mM of TBAB were used in order to

obtain ion-pairing reversed-phase (Ip-rp) [56]. Ip-rp is a very useful analytical

technique for the separation of charged molecules that are not retain in regular

reversed phase [57]. The retention factor (k) presented in Table 5 (values should

be between 1-10 [58]) allowed mobile phases’ evaluation.

Table 5. Preliminary investigation of several mobile phases for AGS and ZA dissolved in UPW.

Mobile phase (TBAB : ACN) 38 : 62 (V/V) 50 : 50 (V/V)

k of AGS 1 4

k of ZA 0 1

ACN: Acetonitrile; k: Retention factor; TBAB: Tetrabutylammonium bromide;

When AGS and ZA were dissolved in SW, a completely different

chromatographic behaviour than previously demonstrated in UPW was observed

(no retention was obtained), even when the proportions of the mobile phase were

changed (Table 6).

Table 6. Optimization of several mobile phases for AGS and ZA dissolved in SW. Mobile phase

(TBAB : ACN) 38 : 62 (v/v) 50 : 50 (v/v) 65 : 35 (v/v) 70 : 30 (v/v) 80 : 20 (v/v) k of AGS 0 < 0 0 < 0 > 40

k of ZA 0 1

(with tailing) 2 ND ND