Study of the degradation of uv-filters in chlorinated waters and toxicological assessment

Mariana Maia de Oliveira e Sá Study of the degradation of UV-filters in chlorinated waters and toxicological assessment. Dissertation of candidature for the master degree in Environmental Toxicology and Contamination submitted to Biomedical Science Institute Abel Salazar of University of Porto Adviser: Professor Joaquim Carlos Gomes Esteves da Silva Category: associate professor Department: Department of Chemistry and Biochemistry of Faculty of Sciences of University of Porto Co-adviser: Dra. Margarida da Silva Miranda Category: external collaborator Department: Department of Chemistry and Biochemistry of Faculty of Sciences of University of Porto “Se sábio não julgas ser ainda bem que te conheces. Se julgas tudo saber o teu saber empobreces.” - Alberto Sá (1913-2004) i Acknowledgment To Professor Joaquim Carlos Gomes Esteves da Silva and to Margarida da Silva Miranda for all the guidance and support along this project. To Professor Jorge Miguel de Ascenção Oliveira, Professor Isabel Filipa Martins de Almeida and to Professor José Paulo Cabral de Sousa e Silva from Faculty of Pharmacy of the University of Porto for all the support about the toxicological assays with zebrafish. I also really thank Ana Rocha for all the time spent with me and all the transmitted knowledge. To Dr. Zélia Azevedo for all the help with the LC-DAD-MS analysis. To all my colleagues from the laboratory for all the help and the friendship. A special thanks to Diana Crista for all the support along all the work. To my father for giving me the opportunity to continue with my studies and for encouraging me every day to learn more and more. To all my family: without you, I could never get this far. To Manuela Barros for always being by my side in any situation: your support was crucial and I am eternally grateful. To all my friends: you make life so much more special. To Pedro Fernandes for showing me what was invisible to my eyes: you are a really special person! Thank you! ii iii Abstract Pharmaceuticals and Personal Care Products (PPCPs) is a category of compounds where are included pharmaceutical drugs, cosmetic ingredients, food supplements, shampoos, lotions and sunscreens cosmetics. These products contain a number of organic compounds such as UV-filters, fragrances, antimicrobials, preservatives and insect repellants also collectively referred as PPCPs. PPCPs have been found in surface water such as lakes, rivers and sea, waste water and tap water and also in bathing water and swimming pool water. Disinfection of swimming pool water is essential to inactivate pathogenic microorganisms. However chlorine based disinfectants, the most commonly used, react with the organic matter present in water leading to the formation of disinfection byproducts (DBPs) which are known to be associated to adverse health effects. PPCPs may also suffer transformation/degradation caused by sunlight UV radiation. The present study aims improve the knowledge about the transformations of UV-filters occurred in swimming pools. The present work had two major objectives: (i) the assess of the stability of four UV-filters (benzyl salicylate, phenyl salicylate, 4-methyl benzilydene camphor and octocrylene) in chlorinated water, (a) evaluating their stability in water samples with different pH values and different free chlorine conditions; (b) to determine their half-lives; (c) tentatively identify the major DBPs and (d) to study their stability under time, and (ii) assess the toxicity of another UV-filter (4-methoxy-4`-tertbutyldibenzoylmethane) comparing the toxicity caused by the parental compound to the toxicity of its DBPs acutely exposing larvae of zebrafish (Danio rerio) to several concentrations of 4-methoxy-4`-tert-butyldibenzoylmethane and corresponding DBPs solution. The effect of these compounds regarding mortality and anatomic abnormalities was assessed. The reactions between the UV-filters and chlorine were followed by HPLCMS. Benzyl salicylate and phenyl salicylate reacted with chlorine yielding three DBPs each one. 4-methyl benzilydene camphor and octocrylene were found to be stable under the experimental conditions used. It was not possible to conclude if 4-methoxy-4`-tertbutyldibenzoylmethane and its DBPs at 1 mg/L have impact on the larvae although it was observed some abnormalities at 0.1 mg/L of DBPs. Keywords: UV-filters, personal care products, chlorination, chlorinated byproducts, benzyl salicylate, phenyl salicylate, 4-methyl-benzylidene camphor, 4-methoxy-4`-tertbutyldibenzoylmethane, zebrafish iv v Resumo Produtos farmacêuticos e outros produtos de cuidado corporal (PPCPs, do Inglês Pharmaceuticals and Personal Care Products) são uma categoria de produtos que inclui fármacos, ingredientes de cosméticos, suplementos alimentares, champôs, loções, protectores solares entre outros. Estes produtos contêm vários compostos orgânicos como filtros UV, fragrâncias, compostos com propriedades antimicrobianas, conservantes e repelentes de insecros que se incluem também na categoria de PPCPs. Vários PPCPs têm sido encontrados em águas de superfície como lagos, rios e mar, águas de esgoto, água da torneira, bem como também em águas de piscina. A desinfecção da água das piscinas é fundamental para inactivar os microrganismos patogénicos. No entanto, os desinfectantes à base de cloro, que são os mais usados, reagem com a matéria orgânica presente na água levando à formação de produtos de desinfecção (DBPs, do Inglês Disinfection Byproducts) que podem estar associados a efeitos nefastos para a saúde. PPCPs também podem sofrer transformações resultantes da exposição à radiação UV solar. Este estudo visa aumentar o conhecimento sobre os processos de transformação sofridos pelos filtros UV em piscinas. Os seus dois maiores objectivos passam por (i) avaliar a estabilidade de quatro filtros UV (benzil salicilato, fenil salicilato, 4metilbenzilideno cânfora e octocrileno) em água clorada (a) avaliando a sua estabilidade sob diferentes valores de pH e diferentes concentrações de cloro, (b) determinando os tempos de semi-vida, (c) identificando os principais DBPs (d) estudando a sua estabilidade ao longo do tempo, e (ii) avaliar a toxicidade de um outro filtro UV (4-metoxi4`-tert-butildibenzoilmetano) comparando a toxicidade causada pelo composto parental com a toxicidade causada pelos seus DBPs, expondo de forma aguda larvas de peixezebra (Danio rerio) a várias concentrações de 4-metoxi-4`-tert-butildibenzoilmetano e correspondentes DBPs. Os efeitos destes compostos foram avaliados ao nível da mortalidade e malformações. As reacções entre os filtros UV e o cloro foram seguidas por HPLC-MS. Benzil salicilato e fenil salicilato reagiram com o cloro formando três DBPs cada um. 4-metilbenzilideno cânfora e octocrileno mantiveram-se estáveis sob as condições experimentais usadas. Não foi possível concluir se 4-metoxi-4’-tertbutildibenzoilmetano e os seus DBPs tiveram impacto nas larvas à concentração de 1 mg/L apesar de se ter observado alguma mal formações a concentrações de 0.1 mg/L dos DBPs. xii 33 Chapter III: Study of the transformation of two salicylates used in personal care products in chlorinated water Fig.1. Structural formula of two studied salicylates (a) BzS and (b) PS. 41 Fig.2. Influence oh pH and free chlorine concentration on the stability of BzS. 43 Fig.3. Influence of pH and free chlorine concentration on the stability of PS. 43 Fig.4. ln(C/C0) vs Reaction time for BzS and PS during the reaction with chlorine at pH 7.0, room temperature and in the dark. 44 Fig.5. Proposed structure formula of the detected DBPs of BzS. 44 Fig.6. Time course of some DBPs (pH = 7.0, 10 mg/L of chlorine, 1 mg/L salicylate, dark) for (a) Bzs and (b) PS. 45 Figure S1. HPLC chromatogram of the mixture resulting from the reaction of BzS with chlorine. 48 Figure S2. MS spectra of the mixture resulting from the reaction of BzS with chlorine: BzS. 49 Figure S3. MS spectra of the mixture resulting from the reaction of BzS with chlorine: Cl-BzS (1). 49 Figure S4. MS spectra of the mixture resulting from the reaction of BzS with chlorine: Cl-BzS (2). xiii 50 Figure S5. MS spectra of the mixture resulting from the reaction of BzS with chlorine: Cl2-BzS. 50 Figure S6. HPLC chromatogram of the mixture resulting from the reaction of PS with chlorine. 51 Figure S7. MS spectra of the mixture resulting from the reaction of PS with chlorine: PS. 51 Figure S8. MS spectra of the mixture resulting from the reaction of PS with chlorine: Cl-PS (1). 52 Figure S9. MS spectra of the mixture resulting from the reaction of PS with chlorine: Cl-PS (2). 52 Figure S10. MS spectra of the mixture resulting from the reaction of PS with chlorine: Cl2-PS. 53 Chapter IV: Study of the transformation of 4-MBC and OC by chlorine in chlorinated water and by UV radiation Figure 4.1. Structure formula of 4-MBC. 60 Figure 4.2. Structure formula of OC. 60 Figure 4.3. C/Co(%) vs Reaction time for 4MBC during reaction with chlorine at pH 7.0, room temperature and in the dark. 66 xiv Figure 4.4. C/Co(%) vs Reaction time for OC during reaction with chlorine at pH 7.0, room temperature and in the dark. 66 Figure 4.5. Time profiles of E-form and Z-form of 4-MBC (1 mg/L) during 30 min of artificial UV radiation treatment. 67 Figure 4.6. Time profiles of 4-MBC (10 mg/L) during 30 min of artificial UV radiation treatment. 68 Figure 4.7. Time profiles of E-form and Z-form of 4-MBC of 4-MBC (1 mg/L) during 30 min of natural UV radiation treatment. 69 Figure 4.8. Time profiles of 4-MBC (1 mg/L) alone and in presence of chlorine (10 mg/L) during 30 min of natural UV radiation treatment. 70 Figure 4.9. Time profiles of E-form and Z-form of 4-MBC of 4-MBC (1 mg/L) during 120 min of artificial UV radiation treatment. 70 Figure 4.10. Time profiles of E-form and Z-form of 4-MBC of 4-MBC (1 mg/L) during 360 min of natural UV radiation treatment. 72 Figure 4.11. Time profiles of 4-MBC (1 mg/L) alone and in presence of chlorine (10 mg/L) during 360 min of natural UV radiation treatment. 72 Figure 4.12. Time profiles of OC (1 mg/L) alone and in presence of chlorine (10 mg/L) during 240 min of artificial UV radiation treatment. xv 73 Figure 4.13. Time profiles of OC (1 mg/L) alone and in presence of chlorine (10 mg/L) during 360 min of natural UV radiation treatment. 74 Chapter V: Toxicological assays Figure 5.1. Percentage of mortality at the end of the assay (6 dpf) for the four control solutions (autoclaved water, deionized water enriched with NaCl, deionized water spiked with chlorine to get 0.30 ppm of free chlorine, and deionized water spiked with chlorine to get 0.30 ppm of free chlorine enriched with NaCl), 1 mg/L of BDM and 1 mg/L of DBPs with 0.30 ppm of chlorine. 89 Figure 5.2. Percentage of abnormalities at the end of the assay (6 dpf) for the four control solutions (autoclaved water, deionized water enriched with NaCl, deionized water spiked with chlorine to get 0.30 ppm of free chlorine, and deionized water spiked with chlorine to get 0.30 ppm of free chlorine enriched with NaCl), 1 mg/L of BDM and 1 mg/L of DBPs with 0.30 ppm of chlorine. 89 Figure 5.3. Percentage of abnormalities along the assay for the four control solutions (autoclaved water, deionized water enriched with NaCl, deionized water spiked with chlorine to get 0.30 ppm of free chlorine, and deionized water spiked with chlorine to get 0.30 ppm of free chlorine enriched with NaCl), 1 mg/L of BDM and 1 mg/L of DBPs with 0.30 ppm of chlorine. 90 Figure 5.4. Percentage of abnormalities at the end of the assay (7 dpf) for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 mg/L of BDM and 0.1 mg/L of DBPs. 92 Figure 5.5. Percentage of abnormalities along the assay for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 mg/L of BDM and 0.1 mg/L of DBPs. xvi 92 Figure 5.6. Percentage of mortality at the end of the assay (7 dpf) for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 µg /L, 1 µg/L and 10 µg/L of BDM, and 0.1 µg /L, 1 µg/L and 10 µg/L of DBPs. 94 Figure 5.7. Percentage of abnormalities at the end of the assay (7 dpf) for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 µg /L, 1 µg/L and 10 µg/L of BDM, and 0.1 µg /L, 1 µg/L and 10 µg/L of DBPs. 95 Figure 5.8. Percentage of mortality along the assay for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 µg /L, 1 µg/L and 10 µg/L of BDM, and 0.1 µg /L, 1 µg/L and 10 µg/L of DBPs. 96 xvii Index of tables Chapter II: State of art Table 1: Disinfectant byproducts (DBPs) and corresponding precursors present in body fluids. 17 Chapter III: Study of the transformation of two salicylates used in personal care products in chlorinated water Table 1: HPLC-MS results from the analysis of DBPs of BzS. 43 Table 2: HPLC-MS results from the analysis of DBPs of PS. 45 Chapter V: Toxicological assays Table 5.1: Percentage of positive reactions to the mechanic stimuli applied in the tail and the head of the larvae. Each experimental condition has two replications. 97 xviii xix List of abbreviations 4-MBC: 4-Methyl Benzilydene Camphor dpf: Days post Fertilization hpf: Hours post Fertilization lw: Lipid Weight ppm: Parts per Million vs: Versus ANT: Antipyrine BCF: Bioconcentration Factor BDM: 4-Methoxy-4’-Tert-Butyldibenzoylmethane BP-3,BP3: Benzophenone-3 BP-4,BP4: Benzophenone-4 BP-8,BP8: Benzophenone-8 BzS: Benzyl Salicylate C: Concentration C0:Initial Concentration Ctrl: Control DAD: Diode-Array Detection DBPs: Disinfection Byproducts DOM: Dissolved Organic Matter EHDPABA: 2-Ethylhexyl-p-Dimethylaminobenzoate EHMC: Ethylexyl Methoxycinnamete ES: 2-Ethylhexyl Salicylate GC: Gas Chromatography HAA: Haloacetic Acids HBQs: Halobenzoquinones HPLC: High-Performance Liquid Chromatography HPLC-MS: HPLC with Mass Spectrometry HPLC-UV-DAD: High-Performance Liquid Chromatography with UV-Diode-Array IAMC: Isoamyl Methoxicinnamte LC: Liquid Chromatography MS: Mass Spectrometry NOECs: No Observable Effect Concentrations xx N-DBPs: Nitrogenous-DBPs OC,OCT: Octocrylene OS: Octyl Salicylate PABA: p-Aminobenzoic Acid PBSA: Phenyl Benzimidazole Sulfonic Acid PCPs: Personal Care Products () PPCPs: Pharmaceuticals and Personal Care Products PS: Phenyl Salicylate R: Correlation Coefficients ROS: Rreactive Oxygen Species UV: UltraViolet UV/Vis: UltraViolet-Visible Detection xxi List of oral communications presented at scientific congresses “Degradation of UV-filters benzyl salicylate and phenyl salicylate in chlorinated waters.”, IJUP’14 – 7th meeting Investigação Jovem da Universidade do Porto, February 20014 “Study of the Photostability of UV-filters 4-Methylbenzylidene Camphor and Octocrylene in Chlorinated Water”, 4PYChem – 4th Portuguese Young Chemists Meeting, April 2014 6 1.4. References Allen, J M, Gossett, C J, Allen, S K (1999a) Photochemical formation of singlet molecular oxygen (1O2) in illuminated aqueous solutions of p-aminobenzoic acid (PABA). J. Photochem. Photobiol., B 32(1-2):33–37 Allen, J M, Gossett, C J, Allen, S K (1999b) Photochemical formation of singlet molecular oxygen in illuminated aqueous solutions of several commercially available sunscreen active ingredients. Chem. Res. Toxicol. 9(3).605–609 Buth, J M, Arnold, W A, McNeill, K (2007) Unexpected products and reaction mechanisms of the aqueous chlorination of cimetidine. Environ. Sci. Technol. 41(17):6228-6233 Giokas, D L, Sakkas, V A, Albanis, T A (2004) Determination of residues of UVfilters in natural waters by solid-phase extraction coupled to liquid chromatographyphotodiode array detection and gas chromatography-mass spectrometry. J. Chromatogr. A 1026(1-2):289-293 Hayashi, T, Okamoto, Y, Ueda, K, Kojima, N (2006) Formation of estrogenic products from benzophenone after exposure to sunlight. Toxicol. Lett. 167(1):1–7 Inbaraj, J J, Bilski, P, Chignell, C F (2002) Photophysical and photochemical studies of 2-phenylbenzimidazole and UVB sunscreen 2-phenylbenzimidazole-5-sulfonic acid. Photochem. Photobiol. 75(2):107–116 Kunz, P. Y.; Fent, K. (2006) Estrogenic activity of UV filter mixtures. Toxicol. Appl. Pharmacol. 217(1):86–99 Lakind, J S (2010) The good, the bad, and the volatile: can we have both healthy pools and healthy people? Environ. Sci. Technol. 44(9):3205-3210 Morohoshi, K.; Yamamoto, H.; Kamata, R.; et al. (2005) Estrogenic activity of 37 components of commercial sunscreen lotions evaluated by in vitro assays. Toxicol.in Vitro 19(4): 457–469 Negreira, N, Canosa, P, Rodriguéz, I, et al. (2008) Study of some UV-filters stability in chlorinated water and identification of halogenated by-products by gas chromatography–mass spectrometry. J.Chromatogr. A 1178(1-2):206-214 Onesios, K M, Yu, J T, Bouwer, E J (2009) Biodegradation and removal of pharmaceuticals and personal care products in treatment systems: a review. Biodegradation 20(4):441-466 Poiger, T, Buser, H-R, Balmer, M E, et al. (2004) Occurrence of UV filter compounds from sunscreens in surface waters: regional mass balance in two Swiss lakes. Chemosphere 55(7):951-963. 7 Richardson, S D, DeMarini, D M, Kogevinas, M, et al. (2010) What’s in the pool? A comprehensive identification of disinfection byproducts and assessment of mutagenicity of chlorinated and brominated swimming pool water. Environ. Health Perspect. 118(11):1523-1530 Rodil, R, Moeder, M, Altenburger, R, Schmitt-Jansen, M. (2009) Photostability and phytotoxicity of selected sunscreen agents and their degradation mixtures in water. Anal. Bioanal. Chem. 395(5):1513–1524 Sakkas, V A, Giokas, D L, Lambropoulou, D A et al. (2003) Aqueous photolysis of the sunscreen agent octyl-dimethyl-p-aminobenzoic acid. Formation of disinfection byproducts in chlorinated swimming pool water. J. Chromatogr. A 1016(2):211-222 Salvador, A, Chisvert, A (2005) Sunscreen analysis. A critical survey on UV-filters determination. Anal. Chim. Acta 537(1-2):1-14 Santos, A J, Crista, D M A, Miranda, M S, et al. (2013) Degradation of UV-filters 2ethylhexyl-4-methoxycinnamate and 4-tert-butyl-4’-methoxydibenzoylmethane in chlorinated water. Environ. Chem. 10(2):127-134 Santos, A J, Miranda, M S, Esteves da Silva, J C G (2012) The degradation products of UV-filters in aqueous and chlorinated aqueous solutions. Water Res. 46(10):3167-3176 Shen, R.; Andrews, S.A. 2011. Demonstration of 20 pharmaceuticals and personal care products (PPCPs) as nitrosamine precursors during chloramine disinfection. Water Res. 45(2):944-952 Stevenson, C, Davies, R J H (1999) Photosensitization of guanine specific DNA damage by 2-phenylbenzimidazole and the sunscreen agent 2-phenylbenzimidazole-5sulfonic acid. Chem. Res. Toxicol. 12(1):38–45 Sui, Q, Huang, J, Deng, S, et al. (2011) Seasonal variation in the occurrence and removal of pharmaceuticals and personal care products in different biological wastewater treatment processes. Environ. Sci. Technol. 45(8):3341-334 Westerhoff, P, Yoon, Y, Snyder, S, Wert, E (2005) Fate of endocrine-disruptor, pharmaceutical, and personal care product chemicals during simulated drinking water treatment processes. Environ. Sci. Technol. 39(17):6649-6663 Zhang, S, Chen, X Q, Ge, L et al. (2010) Quantum chemical investigation and experimental verification on the aquatic photochemistry of the sunscreen 2phenylbenzimidazole-5-sulfonic acid. Environ. Sci. Technol. 44(19):7484–7490 8 9 Chapter II State of art 10 11 2.1. UV-filters chlorination The following chapter was written in response to the invitation from Springer International Publishing Switzerland to the group of Professor Joaquim Carlos Gomes Esteves da Silva. The design of the structure and the selection of the topics covered were conducted by the three authors of the publication according to the publisher suggestion to address the topic of “occurrence of personal care products and transformation process in chlorinated waters”. The text was written by the author Mariana M. de Oliveira e Sá. The supervision, revisions and suggestions of improvement were added by Margarida S. Miranda and Joaquim C. G. Esteves da Silva. 12 13 14 15 22 23 24 25 26 27 28 29 2.2. Pharmaceuticals chlorination Besides sunscreens, others PPCPs such as pharmaceuticals may also be present in pool water. For instance, the antimicrobial triclosan is used in hygienic products like soaps and bodywash and can therefore be released into water during swimming activities. When it comes into contact with chlorine, triclosan suffers chlorination yielding several byproducts (Fiss et al., 2007). Chloroform and chlorophenol are the two DBPs formed from triclosan chlorination, however they appear only under specific conditions. The chlorophenol yields are inversely correlated with the chloroform yields. Fiss et al. (Fiss et al., 2007) observed that in unfavorable conditions for chloroform formation, in addition to chlorophenol it is also released intermediate (chlorophenoxy)phenols: 5,6-dichloro-2-(2,4dichlorophenoxy)phenol, 4,5-dichloro-2-(2,4-dichlorophenoxy)phenol and 4,5,6-trichloro-2- (2,4-dichlorophenoxy)phenol). This happens maybe because chloramines are formed due to the presence of another soups’s ingredients, like ammonium salt. Those chloramines react with triclosan producing chlorophenols and (chlorophenoxy)phenols, but have insufficient oxidizing power to cleave the phenol ring of triclosan and produce chloroform. So, chloroform is only yielded when a sufficient excess of free chlorine is present to cleave the phenol ring of triclosan. Again, the soap composition may affect the amount of free chlorine because the other ingredients also suffer chlorination (Fiss et al., 2007). Antipyrine (ANT) is a pharmaceutical and has anti-inflammatory and analgesics properties. It was already found in several aquatic environments because it cannot be removed completely during water and wastewater treatments (Cai et al., 2013a; Cai et al., 2013b). So, ANT appears in drinking water and, subsequently, in pool water. Then, reaction between chlorine and this pharmaceutical can occur. Cai et al. (Cai et al., 2013b) identified sixteen chlorination by-products including a monochlorine substitution product (4-chloro-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one) called ANT-Cl, which results from halogenation by free chlorine attack (Fig.2.1) (Cai et al., 2013a; Cai et al., 2013b). The potential pathways of ANT chlorination were proposed, including halogenations, dealkylations and hydroxylations (Cai et al., 2013b). But pharmaceutical chlorination is also regulated by some factors, such as initial chlorine concentration and pH. For instance, ANT chlorination reaction is faster when pH is lower than 7 (Cai et al., 2013a) and increases with initial chlorine concentration increasing (Cai et al., 2013b). Cimetidine, another pharmaceutical found in the environment, reacts with chlorine (Fig.2.2) during water treatment yielding other DBPs which were already characterized. Given its structure, cimetidine chlorination may be expected to result in minor structural changes such as sulfur oxidation, electrophilic halogenation and N-chlorination of one or 30 more of its amino groups. Excess of free chlorine gives rise to cimetidine sulphoxide (C10H16N6SO) and then to 4-hydroxymethyl-5-methyl-1H-imidazole which forms an ion - 5methyl-4-methylene-1H-imidazol-1-ium – through loss of water (Fig.2.3). From this latter, it can be formed another intermediate - 4-chloro-5-methyl-1H-imidazole – but this requires the breaking of a C-C bound, a transformation not frequently observed in free chlorine reactions of PPCPs. Finally, it is formed a byproduct with chemical structure C5H8N4O2S (Buth et al., 2007). Buth et al. (Buth et al., 2007) failed to distinguish by the mass spectrometric or spectroscopic data if this compound was a β-sultam, N-cyano-N’-methylN’’-β-sultamylguanidine, or a δ-sultam, N-(2-methyl-1,1-dioxide-1,2,4-thiadiazinan-3ylidene)cyanamide. Figure 2.1. In the process of ANT chlorination, free chlorine can attack ANT to bring about halogenation. Adapted from Cai et al., 2013a and Cai et al., 2013b. Cimetidine chlorination is also regulated by pH: the entire reaction pathway shown in Figure 7 with all its steps and intermediates may occur at acidic and neutral pH, whereas the direct conversion of cimetidine to 4-hydroxymethyl-5-methyl-1H-imidazole, 4chloro-5-methyl-1H-imidazole and β-sultam, N-cyano-N’-methyl-N’’-β-sultamylguanidine/δsultam, N-(2-methyl-1,1-dioxide-1,2,4-thiadiazinan-3-ylidene)cyanamide bypassing cimetidine sulfoxide, may occur under basic conditions (Buth et al., 2007). 31 Figure 2.2. Reaction pathways for the cimetidine chlorination through the intermediacy of cimetidine sulfoxide proposed by Buth et al., 2007) Figure 2.3. The detected ion 5-methyl-4-methylene-1H-imidazol-1-ium, resulting from the loss of water from 4-hydroxymethyl-5-methyl-1H-imidazole. The chlorination of diclofenac, bezafibrate, salicylic acid, clofibric acid, naproxen, indomethacine, ketoprofen, ibuprofen and fenoprofen has also already been studied. 38 39 40 41 42 43 44 45 46 47 54 55 Chapter IV Study of the transformation of 4-MBC and OC in chlorinated water and by UV radiation 56 57 4. Study of the transformation of 4-MBC and OC in chlorinated water and by UV radiation 4.1. Photodegradation of UV-filters Besides the reaction between chlorine and UV-filters described above (Chapter II), these compounds may also suffer transformation induced by sunlight. These photochemical reactions are one of the most important abiotic processes determining the aquatic fate of organic compounds in natural waters (Rodil et al., 2009). It is important that UV-filters are stable under sunlight exposure because a high screening efficiency is only guaranteed if the UV-filter has high stability (Santos et al., 2012; Rodil et al., 2009). In a commercial formula of sunscreen, these photostability depends not only on the UV-filter but also of the presence of other UV-filters, other active substances, like antioxidants (Gaspar and Maia Campos, 2007). However, several papers have been published reporting photodegradation of some UV-filters. In 2006, Gaspar and Campos (Gaspar and Maia Campos, 2006) analyzed the photostability of four different UV-filters combinations after UVA and UVB irradiations: EHMC, BP3 and octyl salicylate (OS) (formulation 1); EHMC, BDM and 4-MBC (formulation 2); EHMC, BP3 and OC (formulation 3); and EHMC, BDM and OC (formulation 4). They studied UV-filters formulations instead individual UV-filters because the behavior of sunscreens is not predictable from the photostability of its individual filter but from the behavior of their UVfilters combinations (Gaspar and Maia Campos, 2006). So, they observed that, in terms of EHMC recovery, formulation 3 was the most stable, followed by formulation 4, formulation 1 and formulation 2. In terms of BDM recovery formulation 4 was more stable than formulation 2, and regarding BP3 recovery formulation 3 was more stable than formulation 1. In terms of OC recovery, formulation 3 was more stable than formulation 4. It was observed that BDM and EHMC react with each other during irradiation time forming cycloaddition products and maybe other photoadducts, fact that may explain why formulation 3 was more stable than formulations 4 and 2. In this study, it was also observed that OC and 4-MBC can stabilize BDM, OC is very effective in stabilizing EHMC in presence of BDM and OC is good UV stabilizer, since formulation 3, which contained EHMC, BP3 and OC, was more stable than formulation 1, which contained EHMC, BP3 and OS. Concerning BDM, Huong et al. (Huong et al., 2008) observed that, under irradiation in aqueous solution, the enol form tautomerizes to the keto form and is also fully degraded. Substituted benzoic acids, benzils, dibenzoylmethanes and dibenzoyl ethanes were the photoproducts found by Huong et al. (Huong et al., 2008). 58 In 2003, Sakkas et al. (Sakkas et al., 2003) studied photodegradation of another UV-filter: EHDPABA. In this study, differently to the paper referred above, photodegradation was analyzed in different types of water: distilled, chlorinated pool and seawater. It was observed that reaction rates followed the order: distilled water>swimming pool water>seawater. After 60h of natural irradiation (daylight exposure) 86, 83 and 80% of this UV-filters as degraded in distilled, swimming pool and seawater, respectively. However, simulated solar irradiation was more effective in EHDPABA: only 10h were enough to degrade more than 99, 96 and 92%, respectively. It was also observed that the presence of organic matter like humic acids inhibits the photodegradation rate of EHDPABA. This retardation on the degradation rate occurs maybe due to a competition process between organic matter and the UV-filter for the available photons. Incident light scattered by particulate matter suspended in the water column and a partial binding between organic matter and EHDPABA by hydrophobic partitioning or weak van der Waals forces which causes a fraction that will never be available to photolysis action are another reasons to the retardation on photodegradation rate. In seawater, this is also consistent with •OH scavenging by chloride ions. Rodil et al. (Rodil et al., 2009) analyzed the stability of six UV-filters exposed to artificial sunlight in water: BP3, ethylexyl methoxycinnamete (EHMC), isoamyl methoxicinnamte (IAMC), EHDPABA, OC and 4-MBC. Half-lives and phototransformation products of these UV-filters were identified. And they observed that BP3, OC and 4-MBC are very photostable, while EHMC, IAMC and EHDPABA clearly showed photodegradation. It was also observed a fast Eto Zisomerization of EHMC, IAMC and 4MBC. This isomerization phenomenon was confirmed as a way to reduce the absorber UV energy. Isomerization of EHMC was previously observed by Huong (Huong et al., 2007). Concerning photodegradation products, Rodil et al. (Rodil et al., 2009) found two products resulting from dealkylation of EHDPABA, a photodissociation process whereby the loss of one and two of the methyl groups of EHDPABA occurs, respectively. These photoproducts showed to be stable for several days. Another compound was also observed and it was proposed as a methylated derivate of the parent compound. These photoproducts showed to be stable for several days. On the other hand, no photodegradation product was detected for EHMC and IAMC. Few years later Liu et al. (Liu et al., 2011) reported again the significant photostability of BP3 under both artificial and natural sunlight. However, in this study BP3 was significantly degraded when in contact with another UV-filter (benzotriazole) and with humic acids in solution. In this case, it was detected one photoproduct produced through the loss of hydroxyl and benzoyl functional group. This product was determined and identifies as 2,4-dimethylanisole. 59 The photostability of p-aminobenzoic acid (PABA), EHDPABA, EHMC, BP3, PBSA in different solvents was studied by Serpone et al. (Serpone et al., 2002): water, methanol, acetonitrile and n-hexane, under aerobic and anaerobic conditions. It was observed that in both types of solvents (aqueous and non-aqueous) PABA had an extensive photodegradation: 87%, 65%, 60% and 45% in n-hexane, water, methanol and acetonitrile respectively. On the other hand EHDPABA had a higher degradation in n-hexane (97%) followed by acetonitrile (94%), water (75%) and methanol (15%). Regarding photodegradation of EHMC, 90% of this UV-filter was degraded in water, 45% in acetonitrile and 40% in methanol and in n-hexane. These percentages were obtained after 30 min of UV exposure, however after 2h of UV exposure EHMC degraded in 95% in nhexane yielding several photodegradation products. Similar to Rodil et al. (Rodil et al., 2009) and to Liu et al. (Liu et al., 2011), in this study Serpone et al. (Serpone et al., 2002) also observed a high stability of BP3 in water (20% of degradation after 2h of UV exposure) as well as in acetonitrile and n-hexane (5-10% and 15% of degradation, respectively, after 2h of UV exposure). However, in methanol BP3 was particularly unstable, photodegrading almost completely (90%) after 2 h. Finally, PBSA showed a high insolubility in n-hexane but its degradation was very fast in water (90% after only 10 min of UV exposure). In acetonitrile the degradation was nearly by 50% after only 20 min of UV exposure and 70% complete after 2 h. The degradation was also significant in methanol. Serpone et al. (Serpone et al., 2002) also observed that PABA, EHDPABA, EHMC and BP3 have a faster degradation under aerobic than under anaerobic conditions: 60% vs. 55% after 1h for PABA, 55% vs. 20% after 10 min for EHDPABA, 85% vs. 65% after 20 min for EHMC, and 50% vs. 15% after 260 min for BP3, respectively. This happens because oxygen is converted in some reactive oxygen species. In the case of PABA, this UV-filter is a good photosensitizer when exposed to UV radiation yielding molecular oxygen. PABA is also a good sink for these reactive oxygen species which causes PABA self-destruction. In 2010, Zhang et al. (Zhang et al., 2010) confirmed that PBSA photodegradates under artificial UV radiation in aqueous solution and they identified several photodegration products in pure water: a desulfonated product (2-phenylbenzoimidazole), three products formed from the cleavage of the benzene ring adjacent to the imidazole ring (phenylimidazolecarboxylic acid derivatives) and two compounds produced from the opening of the imidazole ring (benzimidamide and benzamide). The aim of this step was: (i) assess the stability of 4-MBC and OC in chlorinated water, in conditions similar to those existent in swimming pools, and (ii) assess the stability of 4-MBC and OC under artificial and natural UV radiation. 60 4.2. UV-filters under study 4-MBC is an UV-filter used in many PCPs like sunscreens, lotions and shampoos being one of the most commonly used UV-filters (Buser et al., 2005). This UV-filter is one of the authorized UV filters in Europe and Australia and has been submitted for approval in the USA (Scalia et al., 2007). It absorbs in UVB region (290-320 nm) (Giokas et al., 2007; Scalia et al., 2007) and can exist as a cis- (Z) and trans- (E) isomer due to exocyclic carbon-carbon double bound (Fig.4.1). Although only the E-form is present in commercial products, both isomers can be found in environmental samples (Buser et al., 2005). 4MBC has a good photostabilizer effect in the sunscreens formulations. OC is another UV-filter introduced in sunscreens products approximately 14 years ago (Avenel-Audran et al., 2014). This UV-filter is an ester (Fig.4.2) formed by the condensation of a diphenylcyanoacrylate with 2-ethylhexane (Avenel-Audran et al., 2014) and it absorbs in UVB region (390-360 nm) (González et al., 2008). OC has been increasingly used due to its spectrum efficiency covering UVB but also short UVA wavelengths (Avenel-Audran et al., 2014). It is photostable (González et al., 2008) and it is also used as photostabilizer in sunscreens formulations (Palm and O’Donoghue, 2007). Figure 4.1. Structure formula of 4-MBC. Figure 4.2. Structure formula of OC. 61 4.3. Materials and Methods 4.3.1. Reagents 4-MBC, CAS Registry No. [36861-47-9], 98%, (molecular formula C18H22O) was purchased from Sigma-Aldrich. OC, CAS Registry No. [6197-30-4], 98%, (molecular formula C24H27O2) was purchased from Aako. A commercial sodium hypochlorite solution with a chlorine content of <5% (28 mg/L of free chlorine) was used in the chlorination studies. This solution was stored at 4ºC and its free chlorine content was periodically measured by a small compact photometer for chlorine (HANNA Checker Mini Hi 701 series). Stock solutions of the UV-filters were prepared with methanol from Merck. Deionized water (conductivity < 0.1 S cm-1) was used in all experiments. Ascorbic acid (99.7%) used to stop the chlorination reaction was obtained from Merck. The pH of the solutions was adjusted to a pre-determined value with HCl solutions 0.1 mol/L. Eluents for chromatographic analysis (methanol and acetonitrile) were liquid chromatographic grade and were bought from Merck. 4.3.2. Study of the reaction kinetics of 4-MBC and OC in chlorinated water For this study stock solutions of the two UV-filters in methanol (about 100 mg/L) were first prepared. The kinetics of the reactions was evaluated at room temperature: (20.0 ± 1.0)ºC. The reactions were carried out in glass vessels containing 100 mL of deionized water. At first, the deionized water samples were spiked with free chlorine to get the initial concentration of 10 mg/L for the two UV-filters. After that, the pH of the aqueous solution was adjusted to (7.0 ± 0.1) with 0.1 mol/L HCl and the resulting solution was then spiked with the stock solutions of the UV-filters in methanol (experiments were performed separately for each one) to get the concentration of 1 mg/L (ratio UV-filter:chlorine 1:10). These experimental conditions were chosen with the intent to mimic swimming pool conditions and allow the development of pseudo-first order conditions (Deborde and Von Gunten, 2008). These solutions were kept in the dark and were stirred during the whole experiment. At fixed reaction times (every 20 min for 4-MBC; and from 20 to 4162 min for OC) an aliquot of the reaction mixture was taken, the excess of chlorine was quenched with ascorbic acid according the stoichiometric relationship of 2.5 parts of ascorbic acid to 1 part of free chlorine, and the samples were then immediately analyzed by HPLC-UVDAD. A blank solution with only the UV-filter (without chlorine) was also analyzed. The ratios UV-filter:chlorine 1:20 and 0.25:1 were also studied for OC. 62 4.3.3. Study of the degradation induced by UV radiation of 4-MBC and OC In a first stage, to study the photodegradation of 4-MBC, a 30 min assay analyzed by a spectrophotometer Hewlett Packard 8452A Diode Array and also by HPLC-UV-DAD was conducted. In this step stock solutions of 4-MBC in methanol (about 100 mg/L) were first prepared. The reactions were carried out under artificial sunlight UV radiation in glass vessels containing 100 mL of deionized water spiked with the stock solutions of the UVfilter to get the concentration of 1 mg/L. After that, the pH of the aqueous solution was adjusted to (7.0 ± 0.1) with 0.1 mol/L HCl. This solution was kept during the whole experiment under artificial UV radiation provided by a mercury lamp (PHILIPS, HPLR 400W). The distance between the water surface and the lamp was 5.5 cm. The temperature of the solutions increased during the experiment due to the heat released by the lamp (from 23.8 ºC to 26.5 ºC). At fixed reaction times (from 10 to 30 min) an aliquot sample of the reaction mixture was taken and the samples were then immediately analyzed by spectrophotometer and HPLC-UV-DAD. It was also analyzed a blank solution with the UV-filter kept in the dark but suffering the same temperature fluctuation. This 30 min assay was performed also under natural UV radiation. In this case, the reactions were carried out separately in glass vessels containing 100 mL of deionized water spiked with the stock solutions of 4-MBC in methanol to get the concentration of 1 mg/L. After that, the pH of the aqueous solution was adjusted to (7.0 ± 0.1) with 0.1 mol/L HCl. Blank solutions with the UV-filter kept in the dark was also analyzed. At fixed reaction times (from 10 to 30 min, every 10 min) an aliquot of the reaction mixture was taken and the samples were then immediately analyzed by spectrophotometer and HPLC-UV-DAD. To study the behavior of OC under UV radiation, stock solutions of OC in methanol (about 100 mg/L) were first prepared. The reaction was carried out under artificial UV radiation in glass vessels containing 100 mL of deionized water spiked with the stock solutions of OC to get the concentration of 0.5 mg/L. After that, the pH of the aqueous solution was adjusted to (7.0 ± 0.1) with 0.1 mol/L HCl. This solution was kept during 30 min under artificial UV radiation provided by a mercury lamp with (PHILIPS, HPLR 400W). The distance between de water surfaces into the glass vessel and the lamp was 5.5 cm. The temperature of the solutions fluctuated according the increasing of the temperature caused by the heat of the lamp: (24.0 ± 2.0)ºC. After 30 min of reaction an aliquot sample of the reaction mixture was taken and was immediately analyzed by spectrophotometer and HPLC-UV-DAD. In a second step, stock solutions of 4-MBC and OC in methanol (about 100 mg/L) were first prepared. The degradation was evaluated at constant temperature (20.0 ± 2.0)ºC ensured by a glass vessel with double walls with water circulating at room 63 temperature. The reactions were carried out in 100 mL of deionized water. The deionized water samples were spiked with the stock solutions of the UV-filters in methanol (experiments were performed separately for each one) to get the concentration of 1 mg/L. After that, the pH of the aqueous solution was adjusted to (7.0 ± 0.1) with 0.1 mol/L HCl. These solutions were kept under artificial UV radiation provided by a mercury lamp (PHILIPS, HPLR 400W) emitting in the range of UVB (280 to 315 nm) and were stirred during the whole experiment. The distance between de water surfaces into the glass vessel and the lamp was 5.5 cm. At fixed reaction times (from 30 to 150 min, every 30 min for 4-MBC, and from 60 to 240, every 60 min for OC) an aliquot of the reaction mixture was taken and the samples were then immediately analyzed by HPLC-UV-DAD. A blank solution with the UV-filter kept in the dark was also analyzed. In addition to artificial source of UV radiation, studies under natural UV radiation were also conducted. For these studies stock solutions of 4-MBC and OC in methanol (about 100 mg/L) were first prepared. The reactions were carried out in glass vessels containing 100 mL of deionized water spiked with the stock solutions of the UV-filters in methanol (experiments were performed separately for each one) to get the concentration of 1 mg/L. After that, the pH of the aqueous solution was adjusted to (7.0 ± 0.1) with 0.1 mol/L HCl. These solutions were kept during the whole experiment under direct natural UV radiation. In this case, the temperature fluctuated according to the environmental temperature (between 21 and 28 ºC for 4-MBC and between 24 and 31 ºC for OC). At fixed reaction times (from 60 to 360 min, every 60 min for 4-MBC, and from 60 to 480, every 60 min for OC) an aliquot of the reaction mixture was taken and the samples were then immediately analyzed by HPLC-UV-DAD. It was also analyzed a blank solution with the UV-filter kept in the dark but suffering the same temperature fluctuation. There was also tested the effect caused by chlorine in these two steps. In the 30 min assay, chlorine was added only to 4-MBC. The reactions were carried out in glass vessel containing 100 mL of deionized water. The deionized water samples were spiked with free chlorine to get the initial concentration of 10 mg/L. After that, the pH of the aqueous solution was adjusted to (7.0 ± 0.1) with 0.1 mol/L HCl and the resulting solution was then spiked with the stock solutions of 4-MBC in methanol to get the concentration of 1 mg/L (ratio UV-filter:chlorine 1:10). The solutions were kept under artificial UV radiation provided by a mercury lamp (PHILIPS, HPLR 400W) emitting in the range of UVB (280 to 315 nm) during the whole experiment and the temperature of the solutions fluctuated according the increasing of the temperature caused by the heat of the lamp: (24.0 ± 2.0)ºC. The distance between de water surfaces into the glass vessel and the lamp was 5.5 cm. At each 10 min in a reaction time of 30 min an aliquot of the 4-MBC reaction was taken, the excess of chlorine was quenched with ascorbic acid according the 70 maintained under the lamp covered by aluminum foil being that the control sample was taken at 0 min at reaction after a stirring time to ensure a good dissolution. At each 30 min a sample was taken and immediately analyzed by HPLC-UV-DAD. The results showed that 4-MBC suffers a quickly isomerization. The concentration of the E-form decreased along the reaction time and the concentration of the Z-form increased along the reaction time (Fig.4.9). Figure 4.8. Time profiles of 4-MBC (1 mg/L) alone and in presence of chlorine (10 mg/L) during 30 min of natural UV radiation treatment. Concentrations are related to the initial concentration. Results obtained by HPLC-UV-DAD. Figure 4.9. Time profiles of E-form and Z-form of 4-MBC of 4-MBC (1 mg/L) during 120 min of artificial UV radiation treatment. Concentrations are related to the initial concentration. Results obtained by HPLC-UV-DAD. Each point is the mean of two measurements. 0 20 40 60 80 100 120 0 10 20 30 Normalized response (%) Reaction time (min) E-form w/ chlorine Z-form w/ chlorine E-form w/o chlorine Z-fomr w/o chlorine 0 20 40 60 80 100 0 30 60 90 120 Normalized response (%) Reaction time (min) E-form Z-form 71 The photodegradation of 4-MBC under natural UV radiation was also followed during a higher reaction time. A solution with 1 mg/L of 4-MBC was kept under direct natural UV radiation during 360 min and samples were taken at each 60 min. The samples were immediately analyzed by HPLC-UV-DAD. The results showed a quickly isomerization of the filter between the E-form and the Z-form with the concentration of the E-form decreasing and the concentration of the Zform increasing slightly (Fig.4.10). So, it was added chlorine to evaluate if the degradation under natural UV radiation is higher in presence of chlorine and this reaction was followed during 360 min. The reaction was carried out in glass vessels containing 100 mL of deionized water. The deionized water samples were spiked with free chlorine to get the initial concentration of 10 mg/L. After that, the pH of the aqueous solution was adjusted to 7.0 ± 0.1 and the resulting solution was then spiked with 1 mg/L of 4-MBC. This solution was kept under natural UV radiation. In this case, and once 4-MBC did not showed degradation in the solution covered by aluminum foil during the first approach, no control solution was performed being that the control sample was taken at 0 min at reaction after a stirring time to ensure a good dissolution (this control sample did not contained chlorine). At each 60 min a sample of the solution was taken, the excess of chlorine was quenched with ascorbic acid according and immediately analyzed by spectrophotometric and also by HPLC-UV-DAD. The results showed that 4-MBC suffers quickly isomerization. In presence of chlorine, the concentration of the E-form decreased and the concentration of the Z-form increased. Comparing to the results from the reaction without chlorine, the concentration of the E-form was higher than its concentration in presence of chlorine, while the concentration of the Z-form was similar with and without chlorine. (Fig.4.11). Regarding the photodegradation of OC, it was performed a study very similar to the 4-MBC one. At the 30 min assay, a solution with 0.5 mg/L of OC was kept under artificial UV radiation provided by a mercury lamp. After 30 min of reaction a sample of the solution was taken and immediately analyzed by spectrophotometric and also by HPLCUV-DAD. And it was observed that, contrary to 4-MBC, OC doesn’t suffers isomerization. Done this first approach, the photodegradation of OC was followed during a higher reaction time. A solution with 1 mg/L of OC was kept under artificial UV radiation provided by a mercury lamp during 240 min and the temperature was maintained constant. The control sample was taken at 0 min at reaction after a stirring time to ensure a good dissolution. At each 60 min a sample was taken and immediately analyzed by HPLC-UVDAD. The results showed that the concentration of OC maintained relatively constant (Fig.4.12). 72 Figure 4.10. Time profiles of E-form and Z-form of 4-MBC (1 mg/L) during 360 min of natural UV radiation treatment. Concentrations are related to the initial concentration. Results obtained by HPLC-UV-DAD. Figure 4.11. Time profiles of 4-MBC (1 mg/L) alone and in presence of chlorine (10 mg/L) during 360 min of natural UV radiation treatment. Concentrations are related to the initial concentration. Results obtained by HPLC-UV-DAD. So, it was added chlorine to evaluate if the degradation under artificial UV radiation is higher in presence of chlorine and this reaction was followed during 240 min. The reaction was carried out in glass vessels containing 100 mL of deionized water. The 0 20 40 60 80 100 0 60 120 180 240 300 360 Normalized response (%) Reaction time (min) E-form Z-form 0 20 40 60 80 100 0 60 120 180 240 300 360 Normalized response (%) Reaction time (min) E-form w/ chlorine Z-form with chlorine E-form w/o chlorine Z-form w/o chlorine 73 deionized water samples were spiked with free chlorine to get the initial concentration of 10 mg/L. After that, the pH of the aqueous solution was adjusted to 7.0 ± 0.1 and the resulting solution was then spiked with 1 mg/L of OC. This solution was kept under artificial UV radiation. A control solution without chlorine was kept covered by aluminum foil under artificial UV radiation. At each 60 min a sample of the reaction solution was taken, the excess of chlorine was quenched with ascorbic acid according and immediately analyzed also by HPLC-UV-DAD. Fig.4.12 compares the results obtained in this experiment and the results obtained in the reaction with no chlorine kept under artificial UV radiation. Figure 4.12. Time profiles of OC (1 mg/L) alone and in presence of chlorine (10 mg/L) during 240 min of artificial UV radiation treatment. Concentrations are related to the initial concentration. Results obtained by HPLC-UV-DAD. The photodegradation of OC under natural UV radiation was also followed during 360 min. A solution with 1 mg/L of OC was kept under direct natural UV radiation during 360 min and samples were taken at each 60 min. The samples were immediately analyzed by HPLC-UV-DAD. The results showed that its concentration decreased along the reaction time (Fig.4.13). So, it was added chlorine to evaluate if the degradation under natural UV radiation is higher in presence of chlorine and this reaction was followed during 360 min. The reaction was carried out in glass vessels containing 100 mL of deionized water. The deionized water samples were spiked with free chlorine to get the initial concentration of 10 mg/L. After that, the pH of the aqueous solution was adjusted to 7.0 ± 0.1 and the resulting solution was then spiked with 1 mg/L of OC. This solution was kept under natural 0 20 40 60 80 100 120 0 50 100 150 200 250 Normalized response (%) Reaction time (min) w/ chlorine w/o chlorine 74 UV radiation and at each 60 min a sample of the solution was taken, the excess of chlorine was quenched with ascorbic acid according and immediately analyzed by HPLCUV-DAD. Fig.4.13 compares the results obtained in this experiment and the results obtained in the reaction with no chlorine kept under artificial UV radiation. Figure 4.13. Time profiles of OC (1 mg/L) alone and in presence of chlorine (10 mg/L) during 360 min of natural UV radiation treatment. Concentrations are related to the initial concentration. Results obtained by HPLC-UV-DAD. 0 20 40 60 80 100 120 0 100 200 300 400 Normalized response (%) Reaction time (min) w/ chlorine w/o chlorine 75 4.5. References Avenel-Audran, M, Dutartre, H, Goossens, A, et al. (2014) Octocrylene, an emerging photoallergen. Arch Dermatol. 146(7):753-757 Buser, H-R, Mϋller, M D, Balmer, M E, et al. (2005) Stereoisomer composition of the chiral UV filter 4-methylbenzylidene camphor in environmental samples. Environ. Sci. Technol. 39(9):3013-3019 Gaspar, L R, Maia Campos, P M B G (2006) Evaluation of the photostability of different UV filter combinations in a sunscreen. Int. J. Pharm. 307(2):123-128 Gaspar, L R, Maia Campos, P M B G (2007) Photostability and efficacy studies of topical formulations containing UV-filters combination and vitamins A, C and E. Int. J. Pharm. 343(1-2):181-189 Giokas, D L, Salvador, A, Chisvert, A (2007) UV filters: From sunscreens to human body and the environment. Trends Anal. Chem. 26(5):360-374 González, S, Fernandéz-Lorente, M, Gilaberte-Calzada, Y (2008) The latest on skin photoprotection. Clin. Dermatol. 26(6):614-626 Huong, S P, Andrieu, V, Reyner, J-., et al. (2007) The photoisomerization of the sunscreen ethylhexyl p-methoxy cinnamate and its influence on the sun protection factor. J. Photochem. Photobiol. A: Chemistry 186(1):65-70 Huong, S P, Rocher, E, Fourneron, et al. (2008). Photoreactivity of the sunscreen butylmethoxydibenzoylmethane (DBM) under various experimental conditions. J. Photochem. Photobiol. A: Chemistry 196(1):106-112 International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. (2005) ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures, Text and Methodology Q2(R1). Liu, Y-S, Ying, G-G, Shareef, A, Kookana, R S (2011) Photostability of the UV-filter benzophenone-3 and its effect on the photodegradation of benzotriazole in water. Envir. Chem. 8(6):581-588 Palm, M D, O’Donoghue, M N (2007) Update on photoprotection. Dermatol. Ther. 20(5):360-376 Rodil, R, Moeder, M, Altenburger, R, Schmitt-Jansen, M. (2009) Photostability and phytotoxicity of selected sunscreen agents and their degradation mixtures in water. Anal. Bioanal. Chem. 395(5):1513–1524 Sakkas, V A, Giokas, D L, Lambropoulou, D A, Albanis, T A (2003) Aqueous photolysis of the sunscreen agent octyl-dimethyl-p-aminobenzoic acid Formation of 76 disinfection byproducts in chlorinated swimming pool water. J.Chromatogr. A 1016(2):211222 Santos, A J, Miranda, M S, Esteves da Silva, J C G (2012) The degradation products of UV-filters in aqueous and chlorinated aqueous solutions. Water Res. 46(10):3167-3176 Scalia, S, Mezzena, M, Iannuccelli, V (2007) Influence of solid lipid microparticle carriers on skin penetration of the sunscreen agent, 4-methylbenzylidene camphor. J. Pharm. Pharmacol. 59(12):1621-1627 Serpone, N, Salinaro, A, Emeline, A V, et al. (2002) An in vitro systematic spectroscopic examination of the photostabilities of a random set of commercial sunscreen lotions and their chemical UVB/UVA active agents. Photochem. Photobiol. Sci. 1(12):970-981 Zhang, S, Chen, X Q, Ge, L et al., (2010) Quantum chemical investigation and experimental verification on the aquatic photochemistry of the sunscreen 2phenylbenzimidazole-5-sulfonic Acid. Environ. Sci. Technol. 44: 7484–7490 77 Chapter V Toxicological assays 78 79 5. Toxicological assays 5.1. Introduction PPCPs have been found in several water supplies, waste waters and natural aquatic environmental around the world (Gago-Ferrero et al., 2013; Subedi et al., 2012; Sui et al., 2011; Westerhoff et al., 2005). They were already detected in several animal as happened with some UV-filters found in tissue liver of Franciscana dolphin (Pontoporia blaivinelli) from Brazilian coast area (Gago-Ferrero et al., 2013), and antihistamines, antidepressants and musks found in fishes from German Environment Species Bank (Subedi et al., 2012). UV-filters have high lipophilicity (mostly with log Kow 4–8) whereby they have been shown to accumulate in the food chain and in human milk fat. However, at present, there is a scarcity of data on environmental concentrations of UV-filters (Díaz-Cruz et al., 2008; Gago-Ferrero et al., 2013). Moreover, concentrations reported fluctuate significantly as a function of sample location, size of the system under study (e.g., lakes and swimming pools), frequency and type of recreational activities, season of the year and hour of the day. Still, maximum concentrations reported have corresponded to mid-day on warm summer days, as expected (Díaz-Cruz et al., 2008). In natural waters BP3 is the most frequently detected UV-filter with its concentrations ranging from 2 to 125 ng/L. However, is 4-MBC the UV-filter that is detected in the highest concentrations: up to 82 ng/L (DíazCruz and Barceló, 2009) Lakes river waters are the less contaminated aquatic environments. Nevertheless there are several studies describing concentrations of UV-filters in fish from rivers and lakes contaminated with wastewaters: 1.8 mg/Kg lipid (4-MBC), 2 mg/Kg lipid and 0.5 mg/Kg lipid (others UV-filters) (Díaz-Cruz and Barceló, 2009). It can be said that fishes are the primary organisms to monitor the presence of lipophilic compounds such as UVfilters (Díaz-Cruz and Barceló, 2009). OC, which also has a high lipophilicity (Kow 6.88), was also already detected in tissues liver of dolphins (Pontoporia blainvillei) with concentrations in the range 89−782 ng/g lw and there is evidence that maternal transfer may occur through placenta and likely also through breast milk (Subedi et al., 2012). Swimming pool water had the higher levels of UV-filters. 4-MBC and BP3 were already detected in concentrations of 330 ng/L and 400 ng/L respectively (Díaz-Cruz and Barceló, 2009). In swimming pools the chlorine used in disinfection may react with the UVfilters as reacts with natural organic matter of water, yielding DBPs such as chloroform and other trihalomethanes, nitrosamines or haloacetic acids which have toxic effects like carcinogenic effects in animals and human beings (Hrudey, 2009). Now, it is mandatory to 86 content were renewed with freshly prepared exposure solution. Potential anatomic abnormalities were also recorded. 5.2.3.5. Touch response test At the end of the third assay, with the larvae with 7 dpf, it was performed a touch response test. At 96 hpf the larva is freely swimming and is able to change swimming directions spontaneously and to direct its swimming towards targets. Mechanical stimuli near the head of the larva induce a fast escape response (Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva). Tests like this on allow the identification of changes in sensory and locomotor functions (The Developmental Neurotoxicity of Fipronil: Notochord Degeneration and Locomotor Defects in Zebrafish Embryos and Larvae). After the last reading of the third assay, each larva was displaced to a petri dish with autoclaved water. After a little period of habituation and stabilization (around 2 min), it was applied a short mechanical stimulus with a pipette tip in its head and tail. It was recorded the response of the larvae regarding escaping or don’t. 5.2.4. Statistics All the statistical tests were performed in Microsoft Excel 2013. 5.3. Results and discussion Zebrafish (Danio rerio) was used to assess the toxicity caused by another UV-filter already studied: BDM (Santos et al., 2013). Larvae of Zebrafish were acutely exposed to several concentrations of BDM and corresponding DBPs solution resulting from chlorination reaction. The effect of these compounds regarding mortality and anatomic abnormalities was assessed. 5.3.1. First assay Larvae were randomly distributed in 6-well plate (5 larvae per exposure condition; 400 µL solution/well) and kept at (28 ± 1)ºC on a photoperiod 14:10 h (light:dark) 87 throughout the assay. Larvae were continuously exposed to 1 mg/L of BDM and 1 mg/L of its DBPs from 3 dpf to 5 dpf. In this assay, the control solution was deionized water spiked with free chlorine to get the concentration of 10 mg/L and bubbled with air to remove the chlorine. At the beginning of this assay, the chlorine level was 0.04 ppm. The DBPs were obtained by the reaction between BDM and free chlorine (ratio UV-filter:chlorine 1:10 mg/L). This reaction was performed at the beginning of each day whereby the DBPs solutions contained some chlorine. Before all these toxicological tests, the behavior of the DBPs was followed by HPLC-MS and the pikes obtained in the chromatogram were consistent to the pikes obtained by Santos et al. (Santos et al., 2013). After 30 min of reaction BDM is completely transformed and the concentration of its DBPs reaches its maximum. After that, it was observed that the concentration of the DBPs decreases as the time goes by, as well as the concentration of free chlorine. However, at the beginning of this assay, the concentration of chlorine was too high that all the larvae exposed to the solution of DBPs died after the first day of the assay. All the larvae exposed to BDM showed abnormalities and the larvae in the control also showed mortality and some abnormalities So, this first assay was finished after the first day of exposure and the solutions of the exposure conditions were improved to others assays. 5.3.2. Second assay Larvae were randomly distributed in 24-well plate (5 larvae per well, 15 larvae per exposure condition; 500 µL solution/well) and kept at (28 ± 1)ºC on a photoperiod 14:10 h (light:dark) throughout the assay. Larvae were continuously exposed to 1 mg/L of BDM and 1 mg/L of its DBPs from 3 dpf to 6 dpf. In this assay, two different solutions of DBPs were tested. In the first one, similar to the first assay, the DBPs were obtained by the reaction between BDM and free chlorine (ratio UV-filter:chlorine 1:10 mg/L) and this reaction was performed at the beginning of each day. So, these DBPs solutions contained high levels of free chlorine. In the second one, and since the behavior of the DBPs and free chlorine concentration along the reaction time was followed, the DBPs were obtained also by the reaction between BDM and free chlorine (ratio UV-filter:chlorine 1:10 mg/L) but it was applied to the larvae only after 3 days, when the concentration of free chlorine is lower (0.30 ppm). In the first assay, the higher mortality observed may be due to the fact that the tested solutions did not contain any nutrients necessary to a correct development of the larvae since the tested solutions were prepared with deionized water. So, to overcome 88 this question, at the beginning of the assay the tested solutions were applied with the double of the concentration diluted in the zebrafish medium (50:50 of tested solution:zebrafish medium). In the next days, the tested solutions were enriched with NaCl to provide the nutritional requirement of the larvae. So, the control solutions were autoclaved water (which was the zebrafish medium), deionized water with free chlorine ate 0.30 ppm, deionized water with NaCl and deionized water with free chlorine (0.30 ppm) enriched with NaCl. In the end of this assay (larvae at 6 dpf) the larvae exposed to BDM showed neither mortality (Fig.5.1) nor abnormalities (Fig.5.2). However, the larvae exposed to the BDM solution with 0.30 ppm of chlorine showed some abnormalities (Fig.5.2) at the end of the assay. These abnormalities consisted in skeletal defects (curved spine) but these results don’t have statistical significance (p>0.05). All the larvae exposed to BDM solution with 10 mg/L of chlorine died after the first day of the assay. Since the lack of nutrients may be the reason to the high mortality during the first assay, here it was compared two control solutions: autoclaved water and deionized water enriched with NaCl. No mortality was observed in these two tested conditions (Fig.5.1) however, at the end of this assay (larvae at 6 dpf), the larvae in autoclaved water showed skeletal defects (Fig.5.2) which don’t have statistical significance (p>0.05). To assess the effect the 0.30 ppm of free chlorine, it was compared two others control solutions: deionized water with free chlorine ate 0.30 ppm and deionized water with free chlorine (0.30 ppm) enriched with NaCl. No mortality was recorded in any of these two tested conditions (Fig.5.1). However, at the end of this assay (larvae at 6 dpf), the larvae exposed to the solution with free chlorine enriched with NaCl showed skeletal defects (Fig.5.2) which don’t have statistical significance (p>0.05). Still, at the third day of the assay (larvae at 5 dpf) the number of larvae exposed to the solution with free chlorine enriched with NaCl with skeletal defects was higher than the number recorded at the end of the assay (larvae at 6 dpf) (Fig.5.3), whereby the larvae showed some kind of regeneration. Similar to the previous result, these effects don’t have statistical significance (p>0.05). 89 Figure 5.1. Percentage of mortality at the end of the assay (6 dpf) for the four control solutions (autoclaved water, deionized water enriched with NaCl, deionized water spiked with chlorine to get 0.30 ppm of free chlorine, and deionized water spiked with chlorine to get 0.30 ppm of free chlorine enriched with NaCl), 1 mg/L of BDM and 1 mg/L of DBPs with 0.30 ppm of chlorine. Figure 5.2. Percentage of abnormalities at the end of the assay (6 dpf) for the four control solutions (autoclaved water, deionized water enriched with NaCl, deionized water spiked with chlorine to get 0.30 ppm of free chlorine, and deionized water spiked with chlorine to get 0.30 ppm of free chlorine enriched with NaCl), 1 mg/L of BDM and 1 mg/L of DBPs with 0.30 ppm of chlorine. % mortality Autoclaved water 0 NaCl Ctrl 0 0.30 ppm Cl Ctrl 0 0.30 ppm Cl + NaCl Ctrl 0 1 mg/L BDM 0 1 mg/L DBP w/ 0.30 ppm Cl 6,67 0 20 40 60 80 100 Mortality (%) % abnormalities Autoclaved water 6,7 NaCl Ctrl 0,0 0.30 ppm Cl Ctrl 0,0 0.30 ppm Cl + NaCl Ctrl 6,7 1 mg/L BDM 0,0 1 mg/L DBP w/ 0.30 ppm Cl 13,3 0 20 40 60 80 100 Abnormalities (%) 90 Figure 5.3. Percentage of abnormalities along the assay for the four control solutions (autoclaved water, deionized water enriched with NaCl, deionized water spiked with chlorine to get 0.30 ppm of free chlorine, and deionized water spiked with chlorine to get 0.30 ppm of free chlorine enriched with NaCl), 1 mg/L of BDM and 1 mg/L of DBPs with 0.30 ppm of chlorine. So, due to the lack of statistical relevance of this assay, it cannot be concluded if the parental compound on larvae as different effects than the ones caused by its DBPs. 5.3.3. Third assay Larvae were randomly distributed in 24-well plate (15 larvae per well, 30 larvae per exposure condition; 500 µL solution/well) and kept at (28 ± 1)ºC on a photoperiod 14:10 h (light:dark) throughout the assay. Larvae were continuously exposed to 0.1 mg/L of BDM and 0.1 mg/L of its DBPs from 3 dpf to 7 dpf. In this assay, two different solutions of DBPs were tested. In the first one, similar to the others assay, the DBPs were obtained by the reaction between BDM and free chlorine (ratio UV-filter:chlorine 1:10 mg/L). In this case, this reaction was performed at 12h before the each day of the assay. After 30 min of reaction (this reaction time assures that BDM is totally transformed by chlorine) the solution was concentrated by liquid-liquid extraction with ethyl acetate and the organic solvent was evaporated. These procedure ensured that the chlorine present in the solutions was removed, eliminating this variable in the assay. Nevertheless, the chlorine level of the solution of DBPs was controlled as well as the pH (pH near 7). Whenever 0 20 40 60 80 100 3 4 5 6 Abnormalities (%) days pos fertilization (dpf) Autoclaved water NaCl Ctrl 0.30 ppm Cl Ctrl 0.30 ppm Cl + NaCl Ctrl 1 mg/L BDM 1 mg/L DBP w/ 0.30 ppm Cl 91 there was free chlorine in the solutions, it was added ascorbic acid to neutralize it. The behavior of the DBPs in these solutions was followed by HPLC-UV-DAD and it was observed that the concentration of the DBPs decreased along the time, whereby the solutions of DBPs were prepared daily. To ensure the nutritional requirement of the larvae, it was maintained the same procedure of the second assay: at the beginning of the assay the tested solutions were applied with the double of the concentration diluted in the zebrafish medium (50:50 of tested solution:zebrafish medium). In the next days, the tested solutions were enriched with NaCl to provide the nutritional requirement of the larvae. So, the control solutions were autoclaved water (which was the zebrafish medium), and deionized enriched with NaCl. Once there was the risk that chlorine was not completely removed during the extraction process, a solution of deionized water and ascorbic acid (0.15 ppm, the minimum value of ascorbic acid necessary to neutralize the free chlorine; this value was obtained during preliminary experiments) was also applied as control. In the end of this assay (larvae at 7 dpf) the larvae exposed to BDM showed no mortality but it was recorded some skeletal defects (curved spine): 13.33% of the larvae exposed to this exposure condition (Fig.5.4). However, comparing to the control (deionized water enriched with NaCl) these results don’t have statistical relevance (p>0.05). Similar to the previous assay, at the fourth day of the assay (larvae at 6 dpf) the percentage of larvae exposed to the BDM with skeletal defects (60%) was higher than the percentage recorded at the end of the assay (larvae at 7 dpf) (Fig.5.5), whereby the larvae showed some kind of regeneration. This result was statistical relevant (p<0.05). Regarding the larvae exposed to the DBPs, it was observed no mortality but it was observed skeletal defects (curved spine): 3.33 % of the larvae at the end of the second day of the assay (larvae at 4 dpf) but this result was not statistical relevant (p>0.05) comparing to the control (deionized water with ascorbic acid enriched with NaCl). In the rest of the assay, the larvae apparently recovered from these defects (Fig.5.5). In the controls, it was observed a larva with a hemorrhage in the autoclaved water since the beginning of the assay. This larva survived until the end. In deionized water enriched with NaCl, 3.33 % of the larvae showed skeletal defects (curved spine) since 5 dpf and no effect caused by ascorbic acid in the corresponding control was detected. These differences between these two controls are not statistical relevant (p>0.05) (Fig.5.4). 92 Figure 5.4. Percentage of abnormalities at the end of the assay (7 dpf) for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 mg/L of BDM and 0.1 mg/L of DBPs. Figure 5.5. Percentage of abnormalities along the assay for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 mg/L of BDM and 0.1 mg/L of DBPs. 0 20 40 60 80 100 3 4 5 Abnormalities (%) days pos fertilization (dpf) Autoclaved water NaCl Ctrl Ascorbic acid 0.1 mg/L BDM 0.1 mg/L DBPs % abnormalities Autoclaved water 3,34 NaCl Ctrl 3,34 Ascorbic acid Ctrl 0,00 0.1 mg/L BDM 13,33 0.1 mg/L DBPs 0,00 0 20 40 60 80 100 Abnormalities (%) 93 So, from this assay, it can be concluded that BDM causes abnormalities to the larvae of zebrafish at the concentration of 0.1 mg/L but its DBPs at the same concentration have no effect on the larvae. 5.3.4. Fourth assay Larvae were randomly distributed in 24-well plate (5 larvae per well, 15 larvae per exposure condition; 500 µL solution/well) and kept at (28 ± 1)ºC on a photoperiod 14:10 h (light:dark) throughout the assay. Larvae were continuously exposed to 0.1 µg/L, 1 µg/L and 10 µg/L of BDM and 0.1 µg/L, 1 µg/L and 10 µg/L of its DBPs from 3 dpf to 7 dpf. In this assay, two different solutions of DBPs were tested. In the first one, similar to the others assay, the DBPs were obtained by the reaction between BDM and free chlorine (ratio UV-filter:chlorine 1:10 mg/L). In this case, this reaction was performed several days before the each day of the assay and the concentrated samples were kept at -4ºC. After 30 min of reaction (this reaction time assures that BDM is totally transformed by chlorine) the solution was concentrated by liquid-liquid extraction with ethyl acetate and the organic solvent was evaporated. These procedure ensured that the chlorine present in the solutions was removed, eliminating this variable in the assay. Nevertheless, the chlorine level of the solution of DBPs was controlled as well as the pH (pH near 7). Whenever there was free chlorine in the solutions, it was added ascorbic acid to neutralize it. Still, in this assay it was not necessary add ascorbic acid to the DBPs solutions. The behavior of the DBPs kept in ice was followed by HPLC-UV-DAD and it was observed that the concentration of the DBPs decreased along the time but not so fast as it happens at the temperature room, whereby the solutions of DBPs were prepared several days before the beginning of the assay. However, every day of the assay, new solutions of DBPs were prepared to minimize the decrease of its concentration. To ensure the nutritional requirement of the larvae, it was maintained the same procedure of the second assay: at the beginning of the assay the tested solutions were applied with the double of the concentration diluted in the zebrafish medium (50:50 of tested solution:zebrafish medium). In the next days, the tested solutions were enriched with NaCl to provide the nutritional requirement of the larvae. So, the control solutions were autoclaved water (which was the zebrafish medium), and deionized enriched with NaCl. Once there was the risk that chlorine was not completely removed during the extraction process, a solution of deionized water and ascorbic acid (0.15 ppm, the minimum value of ascorbic acid necessary to neutralize the free chlorine. This value was obtained during preliminary experiments) was also applied as control. 94 Figure 5.6. Percentage of mortality at the end of the assay (7 dpf) for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 µg /L, 1 µg/L and 10 µg/L of BDM, and 0.1 µg /L, 1 µg/L and 10 µg/L of DBPs. In the end of this assay (larvae at 7 dpf) the larvae exposed to 0.1 µg/L of BDM showed 93.3 % of mortality (Fig.5.6). Comparing to the control (deionized water enriched with NaCl) these results have statistical relevance (p<0.05). 6.67 % of these larvae showed an abnormal position swimming with the side facing down (Fig.5.7). However, comparing to the control (deionized water enriched with NaCl) these results don’t have statistical relevance (p>0.05). The larvae exposed to 1 µg/L and to 10 µg/L of BDM showed no mortality and none abnormalities. In the end of this assay (larvae at 7 dpf) the larvae of the three experimental conditions (0.1 µg/L, 1 µg/L and 10 µg/L of DBPs) showed 100 % of mortality (Fig.5.6) and these results have statistical relevance (p<0.05). It was observed that the larvae exposed to 10 µg/L died at the second day of the assay (4 dpf) (Fig.5.8). At this point, the larvae exposed to 1 µg/L of DBPs have already 80 % of mortality (p<0.05) and the larvae exposed to 0.1 µg/L of DBPs don’t have mortality although they showed 33.3 % of some abnormalities: 26.6 % of heart failure, and 6.6 % of the larvae showed an abnormal position swimming with the side facing down. However, comparing to the control % abnormalities Autoclaved water 0,00 NaCl Ctrl 0,00 Ascorbic acid 0,00 0.1 ug/L BDM 6,67 1 ug/L BDM 0,00 10 ug/L BDM 6,67 0.1 ug/L DBPs 0,00 1 ug/L DBPs 0,00 10 ug/L DBPs 0,00 0 20 40 60 80 100 Abnormalities (%) 95 (deionized water enriched with NaCl) these results don’t have statistical relevance (p>0.05). At the third day of the assay (5 dpf), the conditions of the larvae exposed to 1 µg/L of DBPs didn’t suffer any change, while the mortality of the larvae exposed to 0.1 µg/L increased to 33.3 % and 26.6 % showed an abnormal position swimming with the side facing down. The dead larvae were those were with heart failure at 4 dpf. However, these results don’t have statistical relevance (p>0.05) comparing to the control (deionized water enriched with NaCl). At the fourth day of the assay (6 dpf), the mortality of the larvae exposed to 1 µg/L increased to 80 % (p<0.05) and it was observed none abnormalities. The mortality of the larvae exposed to 0.1 µg/L was the same and the percentage of abnormalities increased to 33.3 % (p>0.05). Figure 5.7. Percentage of abnormalities at the end of the assay (7 dpf) for the three control solutions (autoclaved water, deionized water enriched with NaCl, ascorbic acid enriched with NaCl), 0.1 µg /L, 1 µg/L and 10 µg/L of BDM, and 0.1 µg /L, 1 µg/L and 10 µg/L of DBPs. % mortality Autoclaved water 0,00 NaCl Ctrl 0,00 Ascorbic acid 66,67 0.1 ug/L BDM 93,33 1 ug/L BDM 0,00 10 ug/L BDM 0,00 0.1 ug/L DBPs 100,00 1 ug/L DBPs 100,00 10 ug/L DBPs 100,00 0 20 40 60 80 100 Mortality (%) 102 103 6. Conclusions The present work had two major objectives: (i) the assess the stability of four UVfilters commonly used in PCPs: benzyl salicylate (BzS), phenyl salicylate (PS), 4-methyl benzilydene camphor (4-MBC) and octocrylene (OC) in chlorinated water, in conditions similar to those existent in swimming pools, (a) evaluating their stability in water samples with different pH values and different free chlorine conditions; (b) to determine their halflives; (c) tentatively identify major DBPs and (d) to study their stability under time, and (ii) assess the toxicity of an UV-filter whose chlorination reaction already studied by Santos et al. (Santos et al., 2013) (BDM) comparing the toxicity caused by the parental compound (BDM) to the toxicity of its DBPs acutely exposing larvae of zebrafish (Danio rerio) to several concentrations of BDM and corresponding DBPs solution resulting from chlorination reaction. The effect of these compounds regarding mortality and anatomic abnormalities was assessed. 6.1. Study of the reaction kinetics of BzS, PS, 4-MCB and OC in chlorinated water The kinetics of the reactions between the four UV-filters and chlorinated water, the DBPs formation and the effect of sample pH and free chlorine concentration were studied by HPLC-UV-DAD. The studied salicylates (BzS and PS) react with chlorine leading to the formation of DBPs. PS was found to be less stable in chlorine and we think this reactivity difference is due to the possibility of a higher stabilization of the transition state of the electrophilic substitution reaction. Regarding 4-MBC and OC the chlorination reaction occurs but not in useful time. DBPs of the two salicylates have been tentatively identified by HPLC-MS. Two monoand one dichlorinated by-product have been detected for both salicylates. In contrast to what happens to the parental compounds, DBPs of BzS showed a good stability while PS by-products were found to significantly degrade after 20 min reaction. It was observed no DBPs for 4-MBC and OC. To study the chlorination of the four UV-filters, free chlorine concentrations applied were from 1 mg/L and 10 mg/L. These concentrations are acceptable in several places around the world: free chlorine levels of less than 1 mg/L are acceptable in some countries but, in other countries higher levels are considered acceptable. In public and semi-public swimming pools and hot tubes it is acceptable levels of 3 mg/L and 5 mg/L of 104 free chlorine, respectively. However, periodically, in some situations there are applied concentrations up to 20 mg/L as a shock dose to better manage the microbial quality of the water (WHO, 2006). The pH should be maintained between 7.2 and 8.0 (WHO, 2006). So, in order to assess whether the two salicylates react to a significant extent with low chlorine concentrations, such as those used in swimming pools, a set of experiments was designed. The effect of three different pH (6.0, 7.0, 8.0) on the extent of the reaction between the two salicylates and four concentrations of free chlorine (1, 2, 3 and 5 mg/L) was studied separately for each salicylate. These experimental conditions were chosen with the intent to include the conditions used in swimming pool and hot tubs and this study was not applied to 4-MBC neither OC once these two UV-filters didn’t react with chlorine in the previous one. It was possible to conclude that the extent of the reactions between BzS and PS and free chlorine depends on the studied parameters: concentration of chlorine and the pH of the water. It was observed that at pH 6.0 there is a small transformation of both salicylates (maximum about 20%) and is almost independently from free chlorine concentration. At pH 7.0 and 8.0 and for free chlorine concentrations from 2 to 5 mg/L the transformation percentage of BzS and PS increases with the increase of the solution pH. This can be due to the fact that the anionic forms of the salicylates (phenoxide anions) react faster with HOCl than the neutral forms with OCl-[pKHOCl,25ºC = 7.54, (Deborde and Von Gunten, 2008)]. We can also conclude that at higher pH values PS reacts with chlorine in a higher extent than BzS. 6.2. Study of the degradation induced by UV radiation of 4-MBC and OC Once the reaction with chlorine was not observed and to increase the knowledge about the reactions suffered by 4-MBC and OC in swimming pool water, the degradation induced by UV radiation was studied. It was verified that 4-MBC suffers isomerization between the E-form and the Z-form when under both artificial and natural UV radiation. This isomerization was already described by other authors (Giokas et al., 2007; Rodil et al., 2009). The photostability of 4-MBC was already described (Gaspar and Maia Campos, 2006; Rodil et al., 2009) but here it was observed that, in presence of chlorine, the concentration of the E-form was lower than its concentration in absence of chlorine, while the concentration of the Z-form was similar with and without chlorine. OC doesn’t suffers isomerization and, although the photostability of OC was also already described (Giokas et al., 2007; Rodil et al., 2009; (Kockler et al., 2013), here it was observed the slightly decrease of the concentration of OC along the reaction time under natural UV radiation. The effect of chlorine was inconclusive since it was verified a 105 lower concentration of OC in presence of chlorine under artificial UV filter but the concentration under natural UV radiation was similar with and without chlorine. 6.3. Toxicological assays Zebrafish is used as a toxicological model having several advantages for drug screening (McGrath and Li, 2009). This organism is small, cheap to keep in the laboratory and easily bred in large numbers. Larvae of zebrafish can live for seven days in a single well of microplates and their nutritional requirement is almost provided by nutrients stored in yolk sac. Zebrafish allows an easy administration of tested compounds because the larvae can absorb small molecules diluted in the medium through their skin and gills. Zebrafish has advantages comparing to other animal models since it is necessary small amounts of the tested drug, a large number of specimens can be used in each assay, and their organs and tissues (brain, heart, liver, pancreas, kidney, intestine, bones, muscles, nerve system and sensory organs) are similar to their mammalian counterparts regarding anatomical, physiological and molecular levels (McGrath and Li, 2009). The development of the zebrafish embryo is very fast. The larvae start feeding 5 dpf suggesting that most organs acquire a functional state at this time (Yang et al., 2009). Zebrafish was already utilized as animal model to assess the toxicity of OC by Blüthgen et al. (Blüthgen et al., 2014) in a chronic exposure. Here, it can be concluded that the presence of nutrients is essential for larvae survival and the concentration of NaCl used (5.03 mM) was enough to ensure the nutritional requirements. Ascorbic acid was a good way to eliminate free chlorine from the tested solutions and, although it was observed some skeletal deformations in the larvae exposed to the controls of ascorbic acid (fourth assay), these results were not statistically significant. It was not possible to conclude if BDM and its DBPs at 1 mg/L have impact on the larvae (second assay) although it was observed some abnormalities at 0.1 mg/L of DBPs (third assay). On the other hand, BDM at lower concentrations (0.1 µg/L) (fourth assay) caused mortality as well as DBPs at 0.1 µg/L, 1 µg/L and 10 µg/L. Usually, UV-filters have a high lipophilicity (Díaz-Cruz and Barceló, 2009) and it was observed that, after the end of the assays, the wells containing the BDM solutions showed an oily residue what may indicate a poor solubility of BDM in the water. So, this poor solubility may explain the high mortality caused at lower concentrations of BDM since at the higher concentration BDM could not be completely dissolved forming film which is not contacted with the larvae. 106 Regarding the touch response test, it was not observed significant differences between the effects caused by BDM and the effects caused by the DBPs at the end of the assay. Generally, the percentage of positive reactions of the stimuli applied in the tail was higher than the percentage of positive reactions of the stimuli applied in the head (Table 5.1) During the development of the larvae, the movements of the tail appears first than the movements of the head (Saint-Amant and Drapeau, 1998) so, it is normal that the experimental conditions affect the escape response induced by a stimulus near the head more than near the tail. It was also observed that all the larvae with some abnormality have negative responses to these stimuli. The concentrations used in these assays viewed to coincide with the levels of UV-filters usually found in the environmental (Zhang and Lee, 2013). After an optimization process it was possible test concentrations raging µg/L. However, due to the method used to obtain the DBPs, it was not possible ensure that the real concentration of DBPs present in the solutions was the calculated. The fact that the concentration decreases along the time is another reason to not be possible to ensure that the concentrations were the expected. Now, it is important to develop methods to optimize the determination of the real concentrations. In the natural ecosystems, the organisms are exposed to mixtures of many compounds whereby the effects observed may be synergistic even when the individual compounds are present at no observable effect concentrations (NOECs) (Díaz-Cruz and Barceló, 2009). As sunscreens are applied containing several UV-filters, it is critical to assess their activity in mixture combinations (Díaz-Cruz and Barceló, 2009). Future work should investigate the presence of these DBPs in swimming pool water and waste water, address potential risks for human health due to dermal contact and evaluate possible environmental toxic effects. Environmental chemistry studies should also focus on strategies to minimize the formation of these DBPs by the development of new formulations that prevent PPCPs release into chlorinated water and strategies to reduce PPCPs by pool water treatment and by pre-swim showering. From the point of view of the water quality of swimming pools where the PPCPs used by swimmers are released to water this work is quite relevant because these PPCPs and their transformation products accumulate in the bathing water rising human health concerns. Environmental risk can also arise after discharge of the swimming pool effluents into the aquatic systems. 107 6.4. References Blüthgen, N, Meili, N, Chew, G, et al. (2014) Accumulation and effects of the UVfilter octocrylene in adult and embryonic zebrafish (Danio rerio). Sci. Total Environ. 476477:207-217 Deborde, M., von Gunten, U., 2008. Reactions of chlorine with inorganic and organic compounds during water treatment-kinetics and mechanisms: A critical review. Water Res. 42, 13-51. Díaz-Cruz, M S, Barceló, D (2009) Chemical analysis and ecotoxicological effects of organic UV-absorbing compounds in aquatic ecosystems. Trends Anal. Chem. 28(6):708-717 Gaspar, L R, Maia Campos, P M B G (2006) Evaluation of the photostability of different UV filter combinations in a sunscreen. Int. J. Pharm. 307(2):123-128 Giokas, D L, Salvador, A, Chisvert, A (2007) UV filters: From sunscreens to human body and the environment. Trends Anal. Chem. 26(5):360-374 Kockler, J, Motti, C A, Robertson, S, et al. (2013) Method for the simultaneous determination of the UV-filters butyl methoxy dibenzoylmethane and octocrylene in the presence of their photodegradants. Chromatographia 76(23-24):1721-1727 McGrath, P, Li, C-Q (2008) Zebrafish: a predictive model for assessing druginduced toxicity. Drug. Discov. Today 13(9-10):394-401) Rodil, R, Moeder, M, Altenburger, R, Schmitt-Jansen, M. (2009) Photostability and phytotoxicity of selected sunscreen agents and their degradation mixtures in water. Anal. Bioanal. Chem. 395(5):1513–1524 Saint-Amant, L, Drapeau, P (1998) Time course oh the development of motor behaviours in the zebrafish embryo. J Neurobiol. 37(4):622-632 Santos, A J, Crista, D M A, Miranda, M S, et al. (2013) Degradation of UV-filters 2ethylhexyl-4-methoxycinnamate and 4-tert-butyl-4’-methoxydibenzoylmethane in chlorinated water. Environ. Chem. 10:127-134 World Health Organization (WHO), 2006. Guidelines for Safe Recreational Water Environments, Vol. 2: Swimming pools and similar environments, Chapter 4, 60-79. Yang, L, Ho, N Y, Alshut, R, et al. (2009) Zebrafish embryos as models for embryotoxic and teratological effects of chemicals. Reprod. Toxicol. 28(2):245-253 Zhang, Y, Lee, H K (2013) Determination of ultraviolet filters in environmental water samples by temperature-controlled ionic liquid dispersive liquid-phase microextraction. J.Chromatogr. A 1271(1):56-61 108 109 Appendix 110 111 Appendix A.1. Percentage of mortality (left column) and abnormalities (right column) in zebrafish caused by different conditions per well along all the days of the second assay and corresponding results from statistical analysis (One-way ANOVA). BDM BDPs BDM BDPs 1 0 0 0 0 2 0 0 0 0 3 0 20 0 0 BDPs 0.30 ppm Cl Ctrl BDPs 0.30 ppm Cl Ctrl 1 0 0 0 0 2 0 0 0 0 3 20 0 0 0 BDM BDPs BDM BDPs 1 0 0 0 60 2 0 0 0 0 3 0 0 0 0 BDPs 0.30 ppm Cl Ctrl BDPs 0.30 ppm Cl Ctrl 1 0 0 60 20 2 0 0 0 20 3 20 0 0 0 BDM BDPs BDM BDPs 1 0 0 0 40 2 0 0 0 0 3 0 0 0 0 BDPs 0.30 ppm Cl Ctrl BDPs 0.30 ppm Cl Ctrl 1 0 0 40 20 2 0 0 0 0 3 20 0 0 0 Mortality (%) Abnormalinities (%) BDM/BDPs BDM/BDPs BDPs/0.30 ppm Cl Ctrl 2º day 3º day BDPs/0.30 ppm Cl Ctrl 4º day BDM/BDPs BDPs/0.30 ppm Cl Ctrl Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 20 6,666667 133,3333 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 66,66667 1 66,66667 1 0,373901 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 266,6667 4 66,66667 Within Groups 0 4 0 Total 333,3333 5 Total 0 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 20 6,666667 133,3333 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 66,66667 1 66,66667 1 0,373901 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 266,6667 4 66,66667 Within Groups 0 4 0 Total 333,3333 5 Total 0 5 Mortality Abnormalities 2º day BDM/BDP s BDPs/0.30 ppm Cl Ctrl. 118 Appendix A.3. Percentage of mortality (left column) and abnormalities (right column) in zebrafish caused by different conditions per well along all the days of the fourth assay and corresponding results from statistical analysis (One-way ANOVA). 10 ug/L 1 ug/L 0.1 ug/L 10 ug/L 1 ug/L 0.1 ug/L 1 0 0 0 0 0 20 2 0 0 20 0 20 0 3 0 0 20 0 20 20 1 100 80 0 0 0 20 2 100 100 0 0 0 60 3 100 60 0 0 0 20 w/o Ascorbic acid w/ Ascorbic acid w/o Ascorbic acid w/ Ascorbic acid 1 0 60 0 20 2 0 40 0 0 3 0 0 0 20 10 ug/L 1 ug/L 0.1 ug/L 10 ug/L 1 ug/L 0.1 ug/L 1 0 0 100 0 0 0 2 0 0 100 0 0 0 3 0 0 80 0 0 0 1 100 80 0 0 0 20 2 100 100 0 0 0 60 3 100 60 0 0 0 20 w/o Ascorbic acid w/ Ascorbic acid w/o Ascorbic acid w/ Ascorbic acid 1 0 60 0 20 2 0 40 0 0 3 0 0 0 20 10 ug/L 1 ug/L 0.1 ug/L 10 ug/L 1 ug/L 0.1 ug/L 1 0 0 100 20 0 0 2 0 0 100 0 0 0 3 0 0 80 0 0 0 1 100 80 20 0 0 20 2 100 100 60 0 0 60 3 100 60 20 0 0 20 w/o Ascorbic acid w/ Ascorbic acid w/o Ascorbic acid w/ Ascorbic acid 1 0 80 0 0 2 0 60 0 0 3 0 20 20 0 10 ug/L 1 ug/L 0.1 ug/L 10 ug/L 1 ug/L 0.1 ug/L 1 0 0 100 20 0 0 2 0 0 100 0 0 0 3 0 0 80 0 0 20 1 100 100 100 0 0 0 2 100 100 100 0 0 0 3 100 100 100 0 0 0 w/o Ascorbic acid w/ Ascorbic acid w/o Ascorbic acid w/ Ascorbic acid 1 0 0 80 0 2 0 0 100 0 3 0 0 20 0 3º day BDM DBPs Autoclaved water 4º day BDM DBPs Autoclaved water Autoclaved water 2º day Abnormalities (%) DBPs BDM Mortality (%) DBPs Autoclaved water 5º day BDM 119 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 40 13,33333333 133,3333333 Column 3 3 40 13,33333333 133,3333333 Column 3 3 40 13,33333333 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 355,5555556 2 177,7777778 4 0,078717201 5,14325285 Between Groups 355,5555556 2 177,7777778 2 0,216 5,14325285 Within Groups 266,6666667 6 44,44444444 Within Groups 533,3333333 6 88,88888889 Total 622,2222222 8 Total 888,8888889 8 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 240 80 400 Column 2 3 0 0 0 Column 3 3 0 0 0 Column 3 3 100 33,33333333 533,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 16800 2 8400 63 9,39144E-05 5,14325285 Between Groups 2222,222222 2 1111,111111 6,25 0,03411 5,14325285 Within Groups 800 6 133,3333333 Within Groups 1066,666667 6 177,7777778 Total 17600 8 Total 3288,888889 8 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 100 33,33333333 933,3333333 Column 2 3 40 13,33333333 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 1666,666667 1 1666,666667 3,571428571 0,131777567 7,70864742 Between Groups 266,6666667 1 266,6666667 4 0,11612 7,70864742 Within Groups 1866,666667 4 466,6666667 Within Groups 266,6666667 4 66,66666667 Total 3533,333333 5 Total 533,3333333 5 Ascorbic acid Ctrl 2º day BDM DBPs Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 Column 3 3 280 93,33333333 133,3333333 Column 3 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 17422,22222 2 8711,111111 196 3,42614E-06 5,14325285 Between Groups 0 2 0 65535 #DIV/0! 5,14325285 Within Groups 266,6666667 6 44,44444444 Within Groups 0 6 0 Total 17688,88889 8 Total 0 8 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 240 80 400 Column 2 3 0 0 0 Column 3 3 0 0 0 Column 3 3 100 33,33333333 533,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 16800 2 8400 63 9,39144E-05 5,14325285 Between Groups 2222,222222 2 1111,111111 6,25 0,03411 5,14325285 Within Groups 800 6 133,3333333 Within Groups 1066,666667 6 177,7777778 Total 17600 8 Total 3288,888889 8 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 100 33,33333333 933,3333333 Column 2 3 40 13,33333333 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 1666,666667 1 1666,666667 3,571428571 0,131777567 7,70864742 Between Groups 266,6666667 1 266,6666667 4 0,11612 7,70864742 Within Groups 1866,666667 4 466,6666667 Within Groups 266,6666667 4 66,66666667 Total 3533,333333 5 Total 533,3333333 5 3º day BDM DBPs Ascorbic acid Ctrl 120 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 20 6,666666667 133,3333333 Column 2 3 0 0 0 Column 2 3 0 0 0 Column 3 3 280 93,33333333 133,3333333 Column 3 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 17422,22222 2 8711,111111 196 3,42614E-06 5,14325285 Between Groups 88,88888889 2 44,44444444 1 0,42187 5,14325285 Within Groups 266,6666667 6 44,44444444 Within Groups 266,6666667 6 44,44444444 Total 17688,88889 8 Total 355,5555556 8 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 240 80 400 Column 2 3 0 0 0 Column 3 3 100 33,33333333 533,3333333 Column 3 3 100 33,33333333 533,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 7022,222222 2 3511,111111 11,28571429 0,009261 5,14325285 Between Groups 2222,222222 2 1111,111111 6,25 0,03411 5,14325285 Within Groups 1866,666667 6 311,1111111 Within Groups 1066,666667 6 177,7777778 Total 8888,888889 8 Total 3288,888889 8 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 20 6,666666667 133,3333333 Column 2 3 160 53,33333333 933,3333333 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 4266,666667 1 4266,666667 9,142857143 0,039020527 7,70864742 Between Groups 66,66666667 1 66,66666667 1 0,3739 7,70864742 Within Groups 1866,666667 4 466,6666667 Within Groups 266,6666667 4 66,66666667 Total 6133,333333 5 Total 333,3333333 5 4º day BDM DBPs Ascorbic acid Ctrl Anova: factor único Anova: factor único SUMÁRIO SUMÁRIO Grupos Contagem Soma Méday Variância Grupos Contagem Soma Méday Variância Coluna 1 3 0 0 0 Coluna 1 3 20 6,666666667 133,3333333 Coluna 2 3 0 0 0 Coluna 2 3 0 0 0 Coluna 3 3 280 93,33333333 133,3333333 Coluna 3 3 20 6,666666667 133,3333333 ANOVA ANOVA Fonte de variação SQ gl MQ F valor P F crítico Fonte de variação SQ gl MQ F valor P F crítico Entre grupos 17422,22222 2 8711,111111 196 3,42614E-06 5,14325285 Entre grupos 88,88888889 2 44,44444444 0,5 0,62974 5,14325285 Dentro de grupos 266,6666667 6 44,44444444 Dentro de grupos 533,3333333 6 88,88888889 Total 17688,88889 8 Total 622,2222222 8 Anova: factor único Anova: factor único SUMÁRIO SUMÁRIO Grupos Contagem Soma Méday Variância Grupos Contagem Soma Méday Variância Coluna 1 3 300 100 0 Coluna 1 3 20 6,666666667 133,3333333 Coluna 2 3 300 100 0 Coluna 2 3 0 0 0 Coluna 3 3 300 100 0 Coluna 3 3 20 6,666666667 133,3333333 ANOVA ANOVA Fonte de variação SQ gl MQ F valor P F crítico Fonte de variação SQ gl MQ F valor P F crítico Entre grupos 0 2 0 65535 #DIV/0! 5,14325285 Entre grupos 88,88888889 2 44,44444444 0,5 0,62974 5,14325285 Dentro de grupos 0 6 0 Dentro de grupos 533,3333333 6 88,88888889 Total 0 8 Total 622,2222222 8 Anova: factor único Anova: factor único SUMÁRIO SUMÁRIO Grupos Contagem Soma Méday Variância Grupos Contagem Soma Méday Variância Coluna 1 3 0 0 0 Coluna 1 3 200 66,66666667 1733,333333 Coluna 2 3 0 0 0 Coluna 2 3 0 0 0 ANOVA ANOVA Fonte de variação SQ gl MQ F valor P F crítico Fonte de variação SQ gl MQ F valor P F crítico Entre grupos 0 1 0 65535 #DIV/0! 7,70864742 Entre grupos 6666,666667 1 6666,666667 7,692307692 0,05015 7,70864742 Dentro de grupos 0 4 0 Dentro de grupos 3466,666667 4 866,6666667 Total 0 5 Total 10133,33333 5 5º day BDM DBPs Ascorbic acid Ctrl 121 BDM H2O NaCl BDM H2O NaCl 1 0 0 20 0 2 20 0 0 0 3 20 0 20 0 1 0 0 0 0 2 0 0 20 0 3 0 0 20 0 BDM H2O NaCl BDM H2O NaCl 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 BDM H2O NaCl BDM H2O NaCl 1 100 0 0 0 2 100 0 0 0 3 80 0 0 20 1 0 0 0 0 2 0 0 0 0 3 0 0 0 20 BDM H2O NaCl BDM H2O NaCl 1 0 0 0 0 2 0 0 0 0 3 0 0 0 20 BDM H2O NaCl BDM H2O NaCl 1 100 0 0 0 2 100 0 0 0 3 80 0 0 20 1 0 0 0 0 2 0 0 0 0 3 0 0 0 20 BDM H2O NaCl BDM H2O NaCl 1 0 0 20 0 2 0 0 0 0 3 0 0 0 20 BDM H2O NaCl BDM H2O NaCl 1 100 0 0 0 2 100 0 0 0 3 80 0 20 0 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 BDM H2O NaCl BDM H2O NaCl 1 0 0 20 0 2 0 0 0 0 3 0 0 0 0 Mortality (%) Abnormalities (%) 2º day 0,1 mg/L BDM/H2O NaCl 1 mg/L BDM/H2O NaCl 10 mg/L BDM/H2O NaCl 5º day 0,1 mg/L BDM/H2O NaCl 1 mg/L BDM/H2O NaCl 10 mg/L BDM/H2O NaCl 3º day 0,1 mg/L BDM/H2O NaCl 1 mg/L BDM/H2O NaCl 10 mg/L BDM/H2O NaCl 4º day 0,1 mg/L BDM/H2O NaCl 1 mg/L BDM/H2O NaCl 10 mg/L BDM/H2O NaCl Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 40 13,33333 133,3333 Column 1 3 40 13,33333333 133,3333333 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 266,6667 1 266,6667 4 0,116116524 7,708647 Between Groups 266,6667 1 266,6666667 4 0,116117 7,708647 Within Groups 266,6667 4 66,66667 Within Groups 266,6667 4 66,66666667 Total 533,3333 5 Total 533,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 40 13,33333333 133,3333333 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 0 1 0 65535 #DIV/0! 7,708647 Between Groups 266,6667 1 266,6666667 4 0,116117 7,708647 Within Groups 0 4 0 Within Groups 266,6667 4 66,66666667 Total 0 5 Total 533,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 0 1 0 65535 #DIV/0! 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 0 4 0 Within Groups 0 4 0 Total 0 5 Total 0 5 2º day 0,1 mg/L BDM/NaCl Ctrl 1 mg/L BDM/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl 122 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 280 93,33333 133,3333 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 13066,67 1 13066,67 196 0,000151011 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 266,6667 4 66,66667 Within Groups 266,6667 4 66,66666667 Total 13333,33 5 Total 333,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 0 1 0 65535 #DIV/0! 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 0 4 0 Within Groups 266,6667 4 66,66666667 Total 0 5 Total 333,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 0 1 0 65535 #DIV/0! 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 0 4 0 Within Groups 266,6667 4 66,66666667 Total 0 5 Total 333,3333 5 3º day 0,1 mg/L BDM/NaCl Ctrl 1 mg/L BDM/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 280 93,33333 133,3333 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 13066,67 1 13066,67 196 0,000151011 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 266,6667 4 66,66667 Within Groups 266,6667 4 66,66666667 Total 13333,33 5 Total 333,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 0 1 0 65535 #DIV/0! 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 0 4 0 Within Groups 266,6667 4 66,66666667 Total 0 5 Total 333,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 20 6,666666667 133,3333333 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 0 1 0 65535 #DIV/0! 7,708647 Between Groups 0 1 0 0 1 7,708647 Within Groups 0 4 0 Within Groups 533,3333 4 133,3333333 Total 0 5 Total 533,3333 5 4º day 0,1 mg/L BDM/NaCl Ctrl 1 mg/L BDM/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl 123 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 280 93,33333 133,3333 Column 1 3 20 6,666666667 133,3333333 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 13066,67 1 13066,67 196 0,000151011 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 266,6667 4 66,66667 Within Groups 266,6667 4 66,66666667 Total 13333,33 5 Total 333,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 0 1 0 65535 #DIV/0! 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 0 4 0 Within Groups 0 4 0 Total 0 5 Total 0 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 0 0 0 Column 1 3 20 6,666666667 133,3333333 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 0 1 0 65535 #DIV/0! 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 0 4 0 Within Groups 266,6667 4 66,66666667 Total 0 5 Total 333,3333 5 5º day 0,1 mg/L BDM/NaCl Ctrl 1 mg/L BDM/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl DBPs H2O NaCl DBPs H2O NaCl 1 0 0 20 0 2 0 0 60 0 3 0 0 20 0 1 80 0 0 0 2 100 0 0 0 3 60 0 0 0 DBPs H2O NaCl DBPs H2O NaCl 1 100 0 0 0 2 100 0 0 0 3 100 0 0 0 DBPs NaCl Ctrl DBPs NaCl Ctrl 1 20 0 0 0 2 60 0 60 0 3 20 0 20 20 1 80 0 0 0 2 100 0 0 0 3 60 0 0 20 DBPs NaCl Ctrl DBPs NaCl Ctrl 1 100 0 0 0 2 100 0 0 0 3 100 0 0 20 DBPs NaCl Ctrl DBPs NaCl Ctrl 1 20 0 20 0 2 60 0 60 0 3 20 0 20 20 1 80 0 0 0 2 100 0 0 0 3 60 0 0 20 DBPs NaCl Ctrl DBPs NaCl Ctrl 1 100 0 0 0 2 100 0 0 0 3 100 0 0 20 DBPs NaCl Ctrl DBPs NaCl Ctrl 1 100 0 0 0 2 100 0 0 0 3 100 0 0 0 1 100 0 0 0 2 100 0 0 0 3 100 0 0 0 DBPs NaCl Ctrl DBPs NaCl Ctrl 1 100 0 0 0 2 100 0 0 0 3 100 0 0 0 3º day 5º day 0,1 mg/L DBPs/NaCl Ctrl 1 mg/L DBPs/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl Mortality (%) Abnormalities (%) 2º day 0,1 mg/L DBPs/NaCl Ctrl 1 mg/L DBPs/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl 0,1 mg/L DBPs/NaCl Ctrl 1 mg/L DBPs/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl 4º day 0,1 mg/L DBPsNaCl Ctrl 1 mg/L DBPs/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl 124 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 100 33,33333 533,3333 Column 1 3 100 33,33333333 533,3333333 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 1666,667 1 1666,667 6,25 0,066766545 7,708647 Between Groups 1666,667 1 1666,666667 6,25 0,066767 7,708647 Within Groups 1066,667 4 266,6667 Within Groups 1066,667 4 266,6666667 Total 2733,333 5 Total 2733,333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 240 80 400 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 9600 1 9600 48 0,002278426 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 800 4 200 Within Groups 0 4 0 Total 10400 5 Total 0 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 15000 1 15000 65535 #DIV/0! 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 0 4 0 Within Groups 0 4 0 Total 15000 5 Total 0 5 2º day 0,1 mg/L DBPs/NaCl Ctrl 1 mg/L DBPs/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 100 33,33333 533,3333 Column 1 3 80 26,66666667 933,3333333 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 1666,667 1 1666,667 6,25 0,066766545 7,708647 Between Groups 600 1 600 1,125 0,348641 7,708647 Within Groups 1066,667 4 266,6667 Within Groups 2133,333 4 533,3333333 Total 2733,333 5 Total 2733,333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 240 80 400 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 9600 1 9600 48 0,002278426 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 800 4 200 Within Groups 266,6667 4 66,66666667 Total 10400 5 Total 333,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 15000 1 15000 65535 #DIV/0! 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 0 4 0 Within Groups 266,6667 4 66,66666667 Total 15000 5 Total 333,3333 5 3º day 0,1 mg/L DBPs/NaCl Ctrl 1 mg/L DBPs/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl 125 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 100 33,33333 533,3333 Column 1 3 100 33,33333333 533,3333333 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 1666,667 1 1666,667 6,25 0,066766545 7,708647 Between Groups 1066,667 1 1066,666667 3,2 0,148148 7,708647 Within Groups 1066,667 4 266,6667 Within Groups 1333,333 4 333,3333333 Total 2733,333 5 Total 2400 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 240 80 400 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 9600 1 9600 48 0,002278426 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 800 4 200 Within Groups 266,6667 4 66,66666667 Total 10400 5 Total 333,3333 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 20 6,666666667 133,3333333 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 15000 1 15000 65535 #DIV/0! 7,708647 Between Groups 66,66667 1 66,66666667 1 0,373901 7,708647 Within Groups 0 4 0 Within Groups 266,6667 4 66,66666667 Total 15000 5 Total 333,3333 5 4º day 0,1 mg/L DBPs/NaCl Ctrl 1 mg/L DBPs/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 15000 1 15000 65535 #DIV/0! 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 0 4 0 Within Groups 0 4 0 Total 15000 5 Total 0 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 15000 1 15000 65535 #DIV/0! 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 0 4 0 Within Groups 0 4 0 Total 15000 5 Total 0 5 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 3 300 100 0 Column 1 3 0 0 0 Column 2 3 0 0 0 Column 2 3 0 0 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 15000 1 15000 65535 #DIV/0! 7,708647 Between Groups 0 1 0 65535 #DIV/0! 7,708647 Within Groups 0 4 0 Within Groups 0 4 0 Total 15000 5 Total 0 5 5º day 0,1 mg/L DBPs/NaCl Ctrl 1 mg/L DBPs/NaCl Ctrl 10 mg/L DBPs/NaCl Ctrl 126 BDM DBPs BDM DBPs 1 86,67 60 33,33 40 2 66,67 73,33 33,33 60 w/ Ascorbic Acid w/o Ascorbic Acid w/ Ascorbic acid w/o Ascorbic Acid 1 86,67 100 80 33,33 2 100 93,33 100 33,33 BDM/DBPs Autoclaved water Toutch Response Test tail head Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 2 153,34 76,67 200 Column 1 2 66,66 33,33 0 Column 2 2 133,33 66,665 88,84445 Column 2 2 100 50 200 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 100,100025 1 100,100025 0,693106792 0,49269 18,51282051 Between Groups 277,8889 1 277,8889 2,778889 0,237443323 18,51282051 Within Groups 288,84445 2 144,422225 Within Groups 200 2 100 Total 388,944475 3 Total 477,8889 3 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 2 186,67 93,335 88,84445 Column 1 2 180 90 200 Column 2 2 193,33 96,665 22,24445 Column 2 2 66,66 33,33 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 11,0889 1 11,0889 0,199640108 0,698735 18,51282051 Between Groups 3211,4889 1 3211,489 32,11489 0,029755407 18,51282051 Within Groups 111,0889 2 55,54445 Within Groups 200 2 100 Total 122,1778 3 Total 3411,4889 3 Head BDM/DBPs Ascorbic Acid Tail BDM H2O NaCl BDM H2O NaCl 1 86,67 86,67 33,33 80 2 66,67 100 33,33 100 DBPs Ascorbic acid + NaCl Ctrl DBPs Ascorbic acid + NaCl Ctrl 1 60 100 80 33,33 2 73,33 93,33 100 33,33 tail head BDM/NaCl Ctrl DBPs/Ascorbic acid + NaCl Ctrl Appendix A.4. Percentage of positive responses in tail (left column) and in head (right column) in zebrafish durinh the touch response test in zebrafish exposed to different conditions and corresponding results from statistical analysis (One-way ANOVA). 127 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 2 153,34 76,67 200 Column 1 2 66,66 33,33 0 Column 2 2 186,67 93,335 88,84445 Column 2 2 180 90 200 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 277,722225 1 277,722225 1,922988134 0,299868 18,51282051 Between Groups 3211,4889 1 3211,489 32,11489 0,029755407 18,51282051 Within Groups 288,84445 2 144,422225 Within Groups 200 2 100 Total 566,566675 3 Total 3411,4889 3 Anova: Single Factor Anova: Single Factor SUMMARY SUMMARY Groups Count Sum Average Variance Groups Count Sum Average Variance Column 1 2 133,33 66,665 88,84445 Column 1 2 180 90 200 Column 2 2 193,33 96,665 22,24445 Column 2 2 66,66 33,33 0 ANOVA ANOVA Source of Variation SS df MS F P-value F crit Source of Variation SS df MS F P-value F crit Between Groups 900 1 900 16,20323903 0,056533 18,51282051 Between Groups 3211,4889 1 3211,489 32,11489 0,029755407 18,51282051 Within Groups 111,0889 2 55,54445 Within Groups 200 2 100 Total 1011,0889 3 Total 3411,4889 3 BDM/NaCl Ctrl DBPs/Ascorbic acid + NaCl Ctrl Tail Head