IN ST IT U T O D E C IÊ N C IA S B IO M ÉD IC A S A B EL S A LA Z A R C ris tin a B es sa C ru z. SIN G LE A N D C O M B IN ED E FF EC T S O F M IC R O PL A ST IC S A N D C A D M IU M O N I N N A T E I M M U N IT Y A N D A N T IO X ID A N T D EF EN C E I N EU R O PE AN S EABA SS ( D ic en t r a r c h u s la b r a x )
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SINGLE AND COMBINED EFFECTS OF
MICROPLASTICS AND CADMIUM ON
INNATE IMMUNITY AND ANTIOXIDANT
DEFENCE IN EUROPEAN SEABASS
(
D
ICENTRARChUS
LABRAX
)
Cristina Alexandra Bessa Cruz
M
2019M.
ICB
AS
20
19
MESTRADOCRISTINA CRUZ
SINGLE AND COMBINED EFFECTS OF MICROPLASTICS AND
CADMIUM
ON
INNATE
IMMUNITY
AND
ANTIOXIDANT
DEFENCE IN EUROPEAN SEABASS (Dicentrarchus labrax)
Dissertação de Candidatura ao grau de
mestrado em Toxicologia e Contaminação
Ambientais submetida ao Instituto de
Ciências Biomédicas de Abel Salazar da
Universidade do Porto.
Orientador – Doutor Francisco Guardiola
Categoria – Investigador
Afiliação
– Centro Interdisciplinar de
Investigação
Marinha
e
Ambiental
(CIIMAR)
Co-orientador – Benjamín Costas
Categoria – Investigador
Afiliação
– Centro Interdisciplinar de
Investigação
Marinha
e
Ambiental
ii
Acknowledgement
An entire year has passed, and a lot of people helped me to go through it and finish this phase of my life and I can’t go without thanking them.
I want to thank Francisco, for his amazing patience in teaching me, accepting my mistakes and tell me where I was wrong and for calming me down every time I got stressed.
I want to thank Benjamin, for every opinion and support given.
The entire laboratory for accepting me. Marina, thank you for worrying and helping me when I needed an opinion. Bruno, thank you for being there and explain procedures. Lourenço, for every friendly word that helped me to feel more comfortable with all of you. Paulo, thank you for helping me when I needed. Diogo, thank you so much for every advice, help and teaching that you gave me. Carla, thank you so much for being there and listening to me, teaching with all the patience to the most random question and for the walks that helped me calm down.
Thank you Ricardo, Hugo and Tiago from BOGA for all the help and teaching about the bioterio.
I want to thank to the League of Outlaws, for being there from the beginning. For listening to me, nagging and laughing. You guys helped me to stay awake when I needed to work but specially clear my mind when I most needed. Thank you Ferman, Diego, Raquel, Marta and Khang for the vacation that helped me relax and dream. You all are my friends that I will always treasure. Thank you for believing me.
But a special thanks to Ferman, for being there, helping me, listening to me and believing in me, every word was important and helped me to improve myself.
To the girls, Alexandra, Catarina, Inês, Susana, Ana and Inês, for the funny commentaries and dinners shared, that would always help me to relax and laugh.
To my dear friends, Ana, André, Tiago, Gonçalo e Gustavo, there are no words I can use to explain how much I am thankful. You are all my family that helped me growing from the very beginning and are still there when I need.
But specially, I would like to thank my parents, for listening to me with all the patience in the world and giving me always a supportive word.
3 Index Figures Index ... 5 Tables Index ... 7 Abbreviation List ... 8 Resumo ...10 Abstract ...11 1. Introduction ...12 2. State of art ...15 2.1. Microplastics ...15
2.2. Microplastics and metals...16
2.3. Cadmium ...19
2.4. European seabass ...20
3. Objectives ...22
4. Material and Methods ...23
4.1. Chemicals ...23
4.2. Fish and rearing conditions ...23
4.3. Experimental design ...23
4.4. Sample collection...24
4.5. Determination of organo-somatic indexes ...25
4.6. Haematological procedures ...25
4.7. Humoral immune parameters ...25
4.7.1. Lysozyme activity ...25 4.7.2. Peroxidase activity ...26 4.7.3. Protease activity ...26 4.7.4. Antiprotease activity ...26 4.8. Bactericidal activity ...27 4.9. Oxidative Stress ...28 4.9.1. Liver Homogenization ...28 4.9.3. Lipid peroxidation ...28 4.9.4. Catalase ...28 4.10. Statistical analyses ...29 5. Results ...29 5.1. Organo-somatic indexes ...29 5.1. Haematological profile ...29
5.1. Immune parameters measured in plasma ...31
5.1. Immune parameters measured in skin mucus ...31
4
5.1. Hepatic Oxidative Stress...34
6. Discussion ...36
7. Conclusion ...42
References ...43
8. Supplementary results ...53
5
Figures Index
Figure 1. Sources of different plastic size to the aquatic environments (Made in
mindthegraph.com). ... 12
Figure 2. European seabass (Dicentrarchus labrax) (European Commission, 2016). . 21 Figure 3. Experimental design. i) control group (unexposed); ii) Cd group, exposed to Cd
(0.1 mg Cd L-1); iii) MPs group, exposed to MPs (0.25 mg L-1); and iv) MPs-Cd mixture
group, exposed to Cd and MPs (0.1 mg Cd L-1 and 0.25 mg L-1, respectively). ... 24
Figure 4. Spleen (A) and liver (B) organo-somatic indexes (%) of European seabass
specimens unexposed (control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and
MPs-Cd mixture (0.1 mg Cd L-1 and 0.25 mg L-1, respectively). Bars represent the mean ±
SEM (n=8). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). ... 29
Figure 5. Lysozyme (A), peroxidase (B), antiprotease (C) and protease (D) activities
measured in plasma of European seabass specimens unexposed (control) or exposed to
Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1 and 0.25 mg L
-1, respectively). Bars represent the mean ± SEM (n=8). Different letters denote significant
differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks represent the differences between experimental groups. ... 32
Figure 6. Lysozyme (A) and peroxidase (B) activities on skin mucus of European seabass
specimens unexposed (control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and
MPs-Cd mixture (0.1 mg Cd L-1 and 0.25 mg L-1, respectively). Bars represent the mean ±
SEM (n=8). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks represent the differences between experimental groups. ... 32
Figure 7. Protease activities on skin mucus of European seabass specimens unexposed
(control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1
mg Cd L-1 and 0.25 mg L-1, respectively). Bars represent the mean ± SEM (n=8). ... 33
Figure 8. Vibrio harveyi bacteria (A) and Photobacterium damsalae (B) bacteria on
bactericidal activity on plasma of European seabass specimens unexposed (control) or
exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1 and
0.25 mg L-1, respectively). Bars represent the mean ± SEM (n=8). Different letters denote
significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks represent the differences between experimental groups. ... 33
Figure 9. Bactericidal activity of Vibrio harveyi bacteria (A) and Photobacterium damsalae
bacteria (B) on skin mucus of European seabass specimens unexposed (control) or
exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1 and
6 statistically significant differences (P<0.05, Tuckey test). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks represent the differences between experimental groups. ... 34
Figure 10. Lipid peroxidation (A) and catalase (B) on European seabass specimens
unexposed (control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd
mixture (0.1 mg Cd L-1 and 0.25 mg L-1, respectively). Bars represent the mean ± SEM
(n=8). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). ... 35
7
Tables Index
Table 1. Average of the number of microplastics (MPs) per individual and the percentage
of different types of MPs (polyethylene (PE); Polypropylene (PP); Polyethylene terephthalate (PET); Polystyrene (PS)) found in different countries (n=number of individuals). ... 17
Table 2. Concentrations of heavy metals in microplastics and pellets from different sites.
Units are in µg g-1... 18
Table 3. Haematocrit (Ht), haemoglobin (Hb), mean corpuscular volume (MCV), mean
corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cells (RBC) and white blood cells (WBC) in European sea specimens
unexposed (control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd
mixture (0.1 mg Cd L-1 and 0.25 mg L-1, respectively). Values are expressed as means ±
SEM (n=8). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks denote significant differences within each experimental group regarding time (two-way ANOVA; P≤0.05). ns: not significant. ... 30
8
Abbreviation List
AChE- acetylcholinesterase Ag- Silver
Al- Aluminum
BCA- Bicinchoninic acid
BHT- 2,6-Di-tert-butyl-4-methylphenol CAT- Catalase
Cd- Cadmium Cr- Chromium Cu- Copper
DTPA- Diethylenetriaminepentaacetic acid Fe- Iron
GSH- Glutathione HBSS- Hank’s buffer Ht- Hematocrit
KPB- K-phosphate buffer LPO- Lipid peroxidation
MCHC- Mean corpuscular haemoglobin concentration MCV- Mean corpuscular volume
Mn- Manganese MPs- Microplastics MT- Metallothionein NaOH- Sodium hydroxide NPs- Nanoplastics
OSI- Organo-somatic index Pb- Lead
PBS- Sodium phosphate buffer
PBT- Persistent Bioccumulative and Toxic PCP- Personal care products
PE- Polyethylene
9 PMS- Postmitochondrial supernatant fraction
PP- Polypropylene PS- Polystyrene
PTMI- Provisional tolerable monthly intake RBC- Red blood cells
SEM- Standard error of the mean SOD- Superoxide dismutase TBA- 2-Thiobarbituric acid
TBARS- Thiobarbituric acid-reactive substances TCA- Trichloroacetic acid
TMB- 3,3´, 5,5´-tetremethylbenzidine hydrochloride Tris-HCl- Trizma hydrochloride
USEPA- United States Environmental Protection Agency WBC- White blood cells
WHO- World health organization Zn- Zinc
10
Resumo
Microplásticos (MPs) são partículas de plástico (tamanho entre 100 nm e 5 mm)
que podem ser facilmente ingeridas por diferentes organismos. Contudo, uma das preocupações que os MPs trazem, é a sua capacidade de absorver contaminantes pelo que podem alterar a biodisponibilidade e bioacumulação destes contaminantes. Tendo isto em consideração, este estudo tem como objetivo avaliar os efeitos dos MPs e do cadmio (Cd) na resposta imune e na defesa antioxidante em robalo (Dicentrarchus
labrax)após 5 e 10 dias de exposição. Para tal, quatro grupos foram definidos: i) grupo
controlo (não exposto); ii) grupo do Cd, expostos a Cd (0.1 mg Cd L-1); iii) grupo dos MPs,
expostos a MPs (0.25 mg L-1); e iv) MPs-Cd grupo da mistura, expostos a Cd e MPs (0.1
mg Cd L-1 e 0.25 mg L-1). Os resultados mostraram que o índice organo-somático (OSI)
no fígado não se alterou para nenhum peixe exposto aos grupos experimentais. Contrariamente, o baço aumentou para os peixes expostos aos MPs para os 5 dias. Em geral, o perfil heamatológico mostrou não ter muitas alterações, onde estas só foram observadas para o número de glóbulos vermelhos (RBC) e os glóbulos brancos (WBC). Mais especificamente, o número de RBC diminuiu em peixes expostos a MPs comparando com os valores observados em peixes expostos a Cd e mistura MPs-Cd ao dia 5. No caso das WBC, o número destas células aumentou após 5 dias em peixes expostos ao grupo do Cd comparado com os não expostos. No que toca às enzimas medidas no plasma, a atividade da lisozima em peixes expostos à mistura MPs-Cd, diminuiu quando comparado com o grupo do Cd aos 10 dias por outro lado, a atividade da peroxidase aumentou em peixes expostos a Cd em respeito ao controlo aos 10 dias. No muco da pele, a atividade da lisozima diminui em peixes expostos a Cd e MPs comparando com o controlo aos 5 dias, contudo após 10 dias de exposição, houve um aumento da atividade nos peixes expostos ao Cd com respeito ao controlo e a mistura MPs-Cd. A atividade da peroxidase registou um aumento no muco em peixes expostos à mistura MPs-Cd comparado com o grupo controlo aos 10 dias. A atividade bactericida contra a Vibrio harveyi foi reduzida no plasma em peixes expostos a Cd e MPs aos 10 dias. Além disso, o plasma incubado com Photobacterium damsalae, mostrou uma diminuição da atividade em peixes expostos aos MPs aos 10 dias, comparando com o controlo e a mistura MPs-Cd. Contudo, a atividade bactericida no muco contra V. harveyi, exibiu um aumento nos peixes expostos a MPs, comparando com o grupo não exposto comparando com o controlo. Estes resultados sugerem que uma combinação de MPs com o Cd não altera a toxidade do Cd, contudo, uma exposição a MPs e Cd sozinhos afetou o perfil heamatológico, a defesa imune em plasma e muco e a atividade bactericida para ambos plasma e muco.
11
Abstract
Microplastics (MPs) are plastic particles (size between 100 nm and 5 mm) that can be easily ingested by different organisms. However, one of the concerns that these particles have, is their capability of adhering contaminants being able to change the bioavailability and bioaccumulation of these contaminants. Having this in consideration, the present study evaluated the single and combined effects of MPs and cadmium (Cd) on immune response and antioxidant defense in European seabass (Dicentrarchus labrax) after 5 and 10 days of exposure. For this, four experimental groups were established: i)
control group (unexposed); ii) Cd group, exposed to Cd (0.1 mg Cd L-1); iii) MPs group,
exposed to MPs (0.25 mg L-1); and iv) MPs-Cd mixture group, exposed to Cd and MPs
(0.1 mg Cd L-1 and 0.25 mg L-1). Our results showed that the exposure did not caused
alterations in the liver organo-somatic index (OSI) in fish from any experimental group. Contrarily, the OSI in the spleen increased in the fish exposed to MPs at the day 5. In general, the haematological profile was not very affected only were observed alterations in the number of red blood cells (RBC) and white blood cells (WBC). More specifically, the RBC numbers showed a decrease in fish exposed to MPs compared to the values observed in the fish exposed to Cd and MPs-Cd mixture at day 5. In the case of WBC, the cell numbers increased after 5 days in the exposed fish to Cd compared to unexposed ones. Regarding enzymes measured in plasma, the lysozyme activity in fish exposed to MPs-Cd mixture decreased compared to Cd group at the day 10 whilst the peroxidase activity increased in the fish exposed to Cd respect to control and MPs groups at the day 10. Concerning the skin mucus, the lysozyme activity decreased in fish exposed to Cd and MPs comparing to control at day 5, however after 10 days of exposure, this activity increased in fish exposed to Cd respect to control and MPs-Cd mixture groups. Peroxidase activity registered an increase in skin mucus of fish exposed to the MPs-Cd mixture comparing to control group at 10 days. Bactericidal activity against Vibrio harveyi in plasma was reduced in fish exposed to Cd and MPs at 10 days. Additionally, the plasma incubated with Photobacterium damsalae showed a decrease of the bactericidal activity in the fish exposed to MPs at 10 days comparing with control and MPs-Cd mixture groups. However, the bactericidal activity of skin mucus against V. harveyi exhibited an increase in the fish exposed to MPs compared to the unexposed fish. These results suggest that the combination of MPs with Cd do not appear to increase Cd toxicity whilst the exposure to Cd and MPs in a single way affected the hematological profile, immune defense in plasma and skin mucus and bactericidal activity for both plasma and skin mucus.
12
1. Introduction
Plastic consume has been increasing over the years with the mass production of 280 million tons in 2011 (Cole et al., 2011; Yu et al., 2018). The high consumption of plastics has brought severe consequences mainly for the aquatic environment (freshwater and seawater). One of the characteristics of these new pollutants is its durability which makes them resistant to degradation and turns this material into persistent pollutants of the aquatic environment, making 60-80% of all marine debris (Carvalho & Neto, 2016; Cole et al., 2011; Holmes et al., 2012; Lusher et al., 2013; Rochman et al., 2014). As it can be seen in the Figure 1, two sources can be considered: i) sea-based activities, including shipping, fisheries, fishing; and ii) land-based sources as industrial, agriculture or urban discharges or littering by beachgoer (Jasna et al., 2018). However, after reaching the aquatic environment, the plastic will suffer a combination of UV, wind, wave actions, thermal aging, biofilm growth and oxidation and turn them into smaller particles (Détrée & Gallardo-escárate, 2018; Guo & Wang, 2019b) which makes them more dangerous. By turning into small particles, they can be easier ingested by marine organisms and be transported through the food chain (Zhu et al., 2019).
Figure 1. Sources of different plastic size to the aquatic environments (Made in mindthegraph.com).
Plastics can be divided in macro- (size superior to 5 mm), micro- (MPs) (size between 100 nm and 5 mm) and nanoplastics (NPs) (smaller than 100 nm) which can be found in the water and sediment of both marine and freshwater systems where the smaller is the plastic size, the greater can affect a varied range of organisms (Massos & Turner, 2017; Wang et al., 2017) In the case of MPs, depending on the source, the authors have
13 differentiated two categories: i) primary MPs, when the source might come directly from cosmetics or industry; or ii) secondary MPs, when the MPs are originated from the breakdown of larger plastics (Geilfus et al., 2019; He et al., 2019). In the last years, several studies have shown that the MPs have been ingested by a great variety of aquatic organisms such as zooplankton, invertebrates and echinoderm larvae and bigger animals such as mammals, turtles, cetaceans or fish and they have the capacity to be transferred though the food chain (Brennecke et al., 2016; Caruso et al., 2018; Cole et al., 2011).
When exposed, the MPs can affect the growth of primary producers, like phytoplankton and cause physical damages such as oxidative stress and affect the expression of genes involved in certain metabolic pathways to algae cells (Wang et al., 2019b). In fish, several authors also have been reported that the ingestion of MPs can lead to multiple toxic effects in several organs and tissues, such as intestinal damage (Lei et al., 2018), oxidative stress (Yu et al., 2018), neurologic damage (Oliveira et al., 2013) immune response (Ghiasiet al., 2010) and finally reduced growth and survival (Wen et al., 2018), as well as, reducing the predatory performance in fish and the swim velocity (Barboza et al., 2018b; Luís, et al., 2015b). In addition, the MPs can act as vectors and might release chemicals that they were able to absorb when released to the environment such as metals and heavy metals, organic compounds or pharmaceutics allowing those products to enter in their bodies (Rochman et al., 2014; Wen et al., 2018). The interaction between MPs and heavy metals has been studied recently in fish (Barboza et al., 2018a; Barboza et al., 2018b; Lu et al., 2018; Luís et al., 2015a; Wen et al., 2018). This interaction might depend if the MPs are virgin or aged MPs. Beached MPs seem to absorb more because of weather and also organic matter that is absorbed that creates a larger surface (Brennecke et al., 2016; Holmes et al., 2014). Other factors, such as pH, salinity or the size of the particles also seem to have a great influence in the absorption of heavy metals, as for example cadmium (Cd) (Wang et al., 2019a).
The interaction of MPs with heavy metals could cause different consequences which should be the focus of more studies because there is little information about this topic. Recently, some authors have studied the combined effect of the MPs with cadmium, mercury and chromium in different aquatic species such as bivalves (Corbicula fluminea) (Oliveira et al., 2018), European seabass (Dicentrarchus labrax) (Barboza et al., 2018b), discus fish (Symphysodon aequifasciatus) (Wen et al., 2018) and zebrafish (Danio rerio); (Lu et al., 2018);. These authors revealed that the interaction of Cd with MPs seemed to affect Cd accumulation with an increase of Cd accumulation on different tissues (liver, intestine and gills) and caused histological alterations on liver, intestine and gill, as well as, oxidative stress and alterations on gene expressions (oxidative stress, metal
14 detoxification and inflammatory response) in zebrafish and alterations in the innate response and antioxidant response in discus fish (Lu et al., 2018; Wen et al., 2018). In the case of European seabass, an important fish in the Mediterranean aquaculture for human consumption (Bjørndal & Guillen, 2017), few studies have tried to understand the consequences of multi exposure. Barboza et al., (2018a) showed that a multi exposure of Hg and MPs caused neurotoxicity and oxidative damage. Therefore, knowing that plastics have the ability to accumulate through the food chain, this could eventually lead to problems in human health, due to the consumption of fish such as European seabass (Jasna et al., 2018).
15
2. State of art
2.1. Microplastics
Microplastics (MPs) are plastic debris with a size between 100 nm and 5 mm and they are often defined as solid synthetic organic polymers with heterogeneous groups of particles (Lei et al., 2018), differing in size, shape and chemical composition (Jeong et al., 2016). Those can be divided in two categories: i) primary MPs, that are produced for various applications, such as personal care products (PCP) or can it be in the form of preproduction pellets; ii) secondary MPs, that are result from the degradation of macroplastics caused by UV radiation, mechanical abrasion, biological degradation and disintegration (Dris et al., 2015). Regardless of the categories, both types will suffer, through time, degradation by environmental processes, such as solar exposure, thermal aging, bio-film growth and oxidation (Guo & Wang, 2019b). In the case of PCP, these having polyethylene (PE) micro-spheres present in their composition, end up being a source of those particles since they are not captured by wastewater plants (Godoy, et al., 2019). For instance, Godoy et al. (2019) showed that more than 60% of body scrubs and more than 80% of foot scrubs contained PE particles that could reach more than 3,000
particles g-1.
After reaching the environment, the MPs size allows them to be ingested by larger animals but also smaller animals of different species (Wang, 2019b). After being ingested these particles will influence fish performance. For example, a study with common goby (Pomatoschistus microps) from two different estuaries was carried, where they were exposed to different MPs and Artemia nauplii (Sá et al., 2015b). Results, suggested that MPs were mistaken by Artemia and ingested likewise (Sá et al., 2015b). But one estuary had more negative effects in the predatory performance, since this estuary was more polluted indicating that the environment health will cause different harms to the fish. Other trouble is when the MPs are trapped in the gastro-intestinal tract where could release additives or compounds that are previously adhered to these particles (Massos & Turner, 2017). One of those components can be considered to be the dye, which is listed as priority pollutants by United States Environmental Protection Agency (USEPA) (Laskar & Kumar, 2019). However, other chemicals can be found, such as persistent bioaccumulative and toxic (PBTs) compounds, heavy metals, chemical additives, pesticides and organic pollutants (Laskar & Kumar, 2019). These particles could bring different harms on organisms, e.g. animal and human health, inducing physiological harm at scale of populations, or as an individual, tissue, organ, cell and molecular (Barboza et al., 2018a; Yu et al., 2018).
16 In primary producers, like phytoplankton, have been reported that the MPs affect the growth and cause physical damages and cell- and subcellular damage such as, oxidative stress and affect the expression of genes involved in certain metabolic pathways to algae cells (Wang et al., 2019b). However, the damage can be extent to different organisms from the higher levels of the food chain as fish. For instance, a recent study in
European seabass exposed to two different concentrations of MPs (0.26 and 0.69 mg L-1)
for 96 h, reported a reduction of fish swimming velocity and resistance time with an increase of MPs concentration, being able to bring consequences for those species when it comes to escape from a predator or to hunt (Barboza et al., 2018a). MPs were also shown to cause neurotoxicity and oxidative stress, in a similar study with the same conditions, on seabass juveniles by affecting acetylcholinesterase (AChE) levels and lipid peroxidation (LPO) respectively (Barboza et al., 2018b). Regarding the immune system, Wen et al. observed alterations in the activity of several enzymes tested in the kidney [lysozyme (LZM), acid phosphatase and alkaline phosphatase] as well as in the complement 3 in the kidney of discus fish (Symphysodon aequifasciatus) exposed to MPs
(50 and 500 µg L-1) during 30 days (Wen et al., 2018).
Unfortunately, the MPs ability of being able to accumulate on fish bodies and tissues, allows MPs to reach the higher levels in the food chain have been reported (Wang et al., 2019b). For instance, MPs were found in 80% of digestive system of bivalves that were being sold in markets in China and 40% on the wild bivalves (Jin-Feng et al., 2018). Portugal is also in the list of animal contamination by MPs where bivalves, oysters and fish were contaminated (Neves et al., 2015) and they can be found in different commercial species from different countries (Table 1). The extension of this contamination does not just reach living organisms. In fact, MPs were also found in 128 brands of commercial salt from 38 different countries but the concentrations are still low compared to animal resources (Peixoto et al., 2019). MPs can turn into a new vector of contaminants to different species, by being able to absorb different chemicals in which will cause different toxic effects depending of the chemical (Vedolin et al., 2018).
2.2. Microplastics and metals
In the last years, one of the concerns about MPs is their association with toxins, being able to influence the destination of toxic chemicals on the environment and organisms (Barboza., 2018b; Wang et al., 2017). MPs also have the ability to sorb and interact with other contaminants, such as metals, heavy metals, organic compounds, pharmaceutical and also includes components of plastic itself (Barboza et al., 2018a; Holmes et al., 2012). Their large surface area to volume ratio allows MPs to be more susceptible to contaminants (Cole et al., 2011).
17
Table 1. Average of the number of microplastics (MPs) per individual and the percentage of different types of MPs (polyethylene (PE); Polypropylene (PP); Polyethylene terephthalate (PET); Polystyrene (PS)) found in different countries (n=number of individuals).
A various range of different metals can be found in beached pellets and microplastics such as: aluminium (Al), iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), lead (Pb), silver (Ag), cadmium (Cd) and chromium (Cr) (Table 2) (Ashton et al., 2010). The interaction between MPs and metals could allow the transportation of those metals to other different aquatic environments, which could allow them to enter in different food chains having the final consumer the humans (Prunier et al., 2019). The mechanisms in which metal is absorbed by plastic, it is poorly documented but recent studies have suggested that metals can adhere to MPs (Brennecke et al., 2016; Holmes et al., 2012). This assimilation of metals might depend if it is virgin or beached MPs. Beached MPs, absorbs more probably because of the weathering and also, organic matter that is absorbed that creates a larger surface (Brennecke et al., 2016; Holmes et al., 2014). This surface is more reactive and generates anionic active sites for the absorption of metals, making the weathering processes more important for adsorption compared to the plastic type (Brennecke et al., 2016; Holmes et al., 2014). A study showed that aged PE has an altered surface, having a negative charge, due to ketone groups and this creation of functional groups could explain the interaction between MPs, microbes and metals (Fotopoulou & Karapanagioti, 2012).
n #MPs/individual %PE %PP %PET %Acrylic %PS Reference
Greece Digka, et al., 2018 Mytilus galloprovincialis 40 0.8 ± 0.2 75 12.5 - n.d. - Sardina Pilchardus 36 0.8 ± 0.2 65 35 - n.d. - Mullus barbatus 25 0.5 ± 0.2 40 15 15 n.d. 15 Nova Scotia
Mytilus edulis 15 125 n.d. n.d. n.d. n.d. n.d. Mathalon
& Hill, 2014 Mytilus edulis (aquaculture) 15 175 n.d. n.d. n.d. n.d. n.d. England Merlangius merlangus 50 1.75 - n.d. n.d. 55 6.90 Lusher et al., 2013 Aspitrigla cuculus 66 1.95 - - - 54 6.25 n.d.= not defined
18
Table 2. Concentrations of heavy metals in microplastics and pellets from different sites. Units are in µg g-1. Al Fe Mn Cu Zn Pb Cd Cr Reference Pellets Saltram, SW England Beach 55.8 97.8 20.5 0.44 2.68 1.02 0.0016 0.24 Holmes, et al., 2012 Devon, SW England Beach 7.05 25.9 1.58 0.06 0.55 0.15 0.0017 0.02 Ashton et al., 2010 Microplastics Beijiang River - - - 80.9 2414.8 63.03 2.160 - Wang et al., 2017 Arpoador Beach 14 90 2 0.3 1 - - - Vedolin et al., 2018 Persian Gulf, Bushehr 115 531 32.2 3.6 - 4.59 0.035 0.96 Dobaradaran et al., 2018
There is at least three vectors-effects for pollutants by MPs: i)
‘environmental-vector’ where the environment, e.g. currents, take MPs and pollutants to a different place;
ii) ‘organismal-vector’ includes the ingestion of MPs when those are aggregated with
pollutants; iii) ‘cellular-vector’, where plastics with a micro- or nano-size enters the cells, by endocytotic or phagogaocytotic process, creating a way for pollutants to enter the cell (Khan et al., 2015). Jinhui et al., (2019) studied the effects of MPs and heavy metals on seahorse (Hippocampus kuda) where the mixtures showed to have more influence by decreasing body length, body weight, specific growth rate and survival rate compared to MPs alone.
In the case of Cd, similar to other heavy metals, this metal is used as plastics additives (Lu et al., 2018). A recent study carry out by Wang et al., 2019a tested the absorbance of Cd by MPs with different sizes and conditions. The smaller were the particles the higher was the absorption. pH also has influence, where a high pH allows Cd to be absorbed in higher amounts by MPs while an increase of salinity showed to create a high influence by decreasing Cd adoption (Wang et al., 2019a). In other study, Lu et al.,
(2018) reported that the combination of MPs (20 and 200 µg L-1) and Cd (10 µg L-1)
showed a greater accumulation of Cd in different tissues of the fish body compared to Cd alone in zebrafish (Danio rerio) after 3 weeks of exposure. In addition, the MPs-Cd
19 mixture showed to affect some of the biochemical biomarkers tested [glutathione (GSH), superoxide dismutase (SOD) and metallothionein (MT)], as well as, histopathological alterations in liver, intestine and gills and modulation of functional genes related to oxidative stress, inflammatory response and metal detoxification in liver, intestine and gills, compared to fish exposed to Cd alone, allowing to conclude that the MPs have boosted the Cd toxicity (Lu et al., 2018).
2.3. Cadmium
Anthropogenic activities are frequently a source of metals for the aquatic environments (Bielmyer-Fraser et al., 2018). Particularly, heavy metals are harmful pollutants that have a significant influence and can modify both physical and chemical properties of the water (Giri et al., 2016). In the case of Cd, this heavy metal occurs
naturally as a mineral with ranges from 0.1 to 0.5 mg g-1 in earth crust and unpolluted
waters can have this metal present with concentrations below 0.01 µg L-1 (Paul & Small,
2019). However, Cd is a common by-product of the mining industry and can be released in great amounts to the environment and it is also present in fertilizers and pesticides that can lead to coastal and estuaries pollution (Cao et al., 2018; Croizier et al., 2018; Paul & Small, 2019). For example, China had two accidents in Beijang (2005) and in Longjang (2012), as well as in New Zealand rivers, where water reached concentrations levels of
286 µg L-1 to 800 µg L-1 of Cd (Mcrae et al., 2018; Zhao et al., 2018). It is estimated that
Cd released to the environment is approximately 25,000 tons per year, in which half of this value goes to the rivers by weathering of rocks, another part goes to the air by volcano eruptions and fires and the rest is released by human activities (Zhang et al., 2017). This heavy metal is a toxin to different forms of life, including microorganisms, higher plants, animal and man and can biologically accumulate in the mussel, oysters, shrimps, lobsters and fish (Guardiola et al., 2013; Zhang et al., 2017). More particularly, in fish as European seabass, a study showed that muscle and liver accumulate Cd faster compared to Senegalese sole (Solea senegalensis) and the same was observed for depuration (Croizier et al., 2018). Accumulation seems to happen more in the gills compared to other fish organs (Bawuro et al., 2018). Other than that, cadmium exposure showed to affect histology of liver, reduced growth and survival rate and caused oxidative stress in liver in female zebrafish (Yuan et al., 2017)
For humans, the provisional tolerable monthly intake (PTMI) established by World
of Health Organization (WHO) is 25 µg kg-1 body weight where in the drinking water, it’s
not allowed to have more than 3 µg L-1 (World Health Organization, 2010). Cd has a long
biological half-life of 10-35 years and is able to concentrate in the liver and especially on the kidneys (Paul & Small, 2019; World Health Organization, 2010). The accumulation on
20 the kidneys can result in renal tubular dysfunction, causing inability to generate suitably concentrated urine in response to a physiologic stimulus (Gillham, 2007; World Health Organization, 2010). A known disease caused by cadmium pollution released by human activities related to industrialization, in the Jinzu River in Toyama prefecture (Japan), called Itai-Itai, caused osteomalaecia with severe bone pain and renal tubular dysfunction (Nishijo et al., 2017). Although the bone injury is treatable, renal injury is irreversible and worsens terminating in end-stage renal failure in severe cases (Baba et al., 2013).
The ionic form of Cd (Cd2+) is capable of imitating calcium (Ca2+), allowing Cd to
enter in animals, such as fish, via apical epithelial calcium channels (Mcrae et al., 2018). Being a nonessential metal, it is known, that Cd is a genotoxic metal that has been classified as a human carcinogen and toxic to aquatic organisms including microorganisms, higher plants and animals, causing oxidative stress and immunotoxicity (Guardiola et al., 2013; Guo et al., 2017; Paul & Small, 2019; Yuan et al., 2017). The range of effects can also include impacts on vision, olfaction, behaviour, metal homeostasis, reproduction and DNA modification (Guo et al., 2017). Those effects will appear, when the uptake is greater compared to detoxification mechanisms and metabolic ways (Ebenezer & Oladele, 2015).
2.4. European seabass
Aquatic pollution has been one of the biggest concerns for the last decades. To understand the effects of the different sources of pollution, researchers started to use fish as a way to understand the changes on the water quality and the effects that those changes might bring (Ghiasi et al., 2010). European seabass (Figure 2) is a Mediterranean species with a production of 147,000 tonnes in 2013 reaching 191,000 tonnes in 2016 (Bjørndal & Guillen, 2017; European Market Observatory for Fisheries and Aquaculture Products, 2019).
This species is one of the fish that is a favourite for human consume and in the last 25 years it has been grown in aquaculture (Purser & Forteath, 2013). In the wild, this species is more common in shallow waters but it also can be found in coastal waters at 100 m depth (FAO, 2018) and has a size that can reach 1.03 m in length and weight 12 kg (Purser & Forteath, 2013).
European seabass was chosen for this study, because it’s a species easily found in Europe estuaries, and his high consume people with commercial value makes it important for the consequences that might bring. Studies that evaluate the interaction between metals and MPs also used this species, being a good to supplement and compare (Barboza et al., 2018a; Barboza et al., 2018b).
21
22
3. Objectives
Taking into account all these considerations, the aim of the present study was to evaluate the single and combined effects of microplastics and cadmium on innate immunity and antioxidant defense of the European seabass, which is considered one of the most appreciated farmed fish species of Mediterranean area.
23
4. Material and Methods
4.1. Chemicals
Red fluorescent microspheres (1-5 µm diameter) were purchased from Cospheric—Innovations in Microtechnology (USA) and were used as MPs model. The excitation and emission wavelengths of these MPs are 575 nm and 607 nm, respectively,
and have a density of 1.3 g cc-1. A stock suspension of MPs in filtrated salt water, used in
tanks, with a concentration of 1,250 mg L-1 was prepared. Cadmium chloride (CdCl
2,
99.99% purity) was purchased from Sigma (Germany) and used as cadmium source. A
stock solution of cadmium chloride with a Cd concentration of 500 mg L-1 was prepared in
filtrated salt water.
4.2. Fish and rearing conditions
Juvenile European seabass (Dicentrarchus labrax) (64.64 ± 10.09 g body weight
and 18.31 ± 1.12 cm body-length) were obtained from a local fish farm (Cantabria, Spain)
and were kept in seawater aquaria (1,000 L). The water was maintained at 17 ± 1 ºC with
a flow rate of 1,500 L h-1 and a salinity of 35.0 ± 1.0 g L-1. The photoperiod was of 12 h
light: 12 h dark and fish were fed with a commercial pellet diet (Skretting) at a rate of 2%
body weight day-1. Fish were allowed to acclimatize for 15 days before the start of the
experimental trial.
4.3. Experimental design
The trial was conducted in glass aquariums (to minimize plastic exposure) with 60 L of capacity and the fish were acclimated for 15 days before the start of the experimental trial. A total of 64 juvenile animals were randomly assigned to four experimental tanks (50 L) by duplicate and 4 groups were established: i) control group (unexposed); ii) Cd group,
exposed to Cd (0.1 mg Cd L-1); iii) MPs group, exposed to MPs (0.25 mg L-1); and iv)
MPs-Cd mixture group, exposed to MPs-Cd and MPs (0.1 mg MPs-Cd L-1 and 0.25 mg L-1, respectively)
(Figure 3). All experimental tank were exposed daily and therefore for each group, we had a cleaning tank that was used to transfer the fish while the experimental tanks were cleaned (during the acclimatization and experimental periods in order to minimize stress), allowing full renovation of the water, the addition of each contaminant and the decreasing as much as possible the decay of both contaminants (MPs and Cd) that has been reported in different studies (Luís et al., 2015; Oliveira et al., 2018).
To reduce MPs adherence to plastic used on the tubes for aeration, a plastic pipette was used, since it is more inert compared to the plastic tubes. The concentrations chosen for Cd are ecologically relevant (Pedro et al., 2016) as for MPs concentration that were also used in toxicological tests on aquatic organisms (Barboza et al., 2018a).
24 Experimental animals were starved for 24 h prior to sampling and four fish per tank (eight per group) were sacrificed by an overdose of ethylene glycol monophenyl ether (Sigma-Aldrich, 1,000 ppm) and sampled at 5 or 10 days of exposure.
Final solution of Cd was prepared by removing 10 mL of stock solution into glass tubes avoiding any loss of the metal to the wall of the tube. MPs stock solution was exposed to ultra-sound for 5 minutes (Fisherbrand) to avoid the agglomeration of MPs, and 10 mL of the solution would be transferred to glass tubes to avoid absorption.
From the beginning, the water quality was maintained with aeration and daily water changes of 100%. The photoperiod was of 12 h light: 12 h dark and fish fed with a
commercial pellet diet (Skretting, Spain) at a rate of 1.5% body weight day-1. Ammonia
levels in the water were measured every day using commercial kits (Palintest Ltd) and
never exceeded 0.40 mg L-1. The water temperature averaged 17.04 ± 0.5 ºC, salinity was
maintained at 35.0 ± 1.0 g L-1, with a pH of 7.49 ± 0.013 and dissolved oxygen was kept
near saturation (7.0 mg L-1).
This experiment was directed by trained scientists (following FELASA category C recommendations) and conducted according to the European Union Directive 2010/63/EU on the protection of animals for scientific purposes.
Figure 3. Experimental design. i) control group (unexposed); ii) Cd group, exposed to Cd (0.1 mg
Cd L-1); iii) MPs group, exposed to MPs (0.25 mg L-1); and iv) MPs-Cd mixture group, exposed to
Cd and MPs (0.1 mg Cd L-1 and 0.25 mg L-1, respectively).
4.4. Sample collection
Fish were weighted and measured in length, dissected and the liver and spleen weighted. Skin mucus was aseptically collected from specimens using the method described by Guardiola et al. (2018) with slight modifications. Briefly, the skin mucus of each fish was gently scraped by using a cell scraper with enough care to avoid contamination with urogenital and/or intestinal excretions. Collected mucus samples were
25 then centrifuged at 10,000 × g and 4 ºC for 10 min. The supernatant was collected, aliquoted and stored at -80 ºC until further analyses. Following mucus collection, blood samples were withdrawn from the caudal vessel with heparinized syringes, placed in heparinized microtubes and used for the assessment of the haematological profile according to Machado et al. (2015). The remaining blood was used to collect plasma, following centrifugation (10,000 × g, 10 min, 4ºC). Plasma was then frozen in liquid nitrogen and stored at −80 °C until further analysis. Following blood collection, fragments of liver and muscle were obtained, immediately frozen in liquid nitrogen and subsequently stored at −80 °C for later determination of lipid peroxidase (LPO) and catalase (CAT) and Cd accumulation.
4.5. Determination of organo-somatic indexes
Values registered from whole body, spleen and liver weight were used to determinate the organo-somatic indexes (OSI) with the following formula: OSI = (g tissue
g-1 body) x 100.
4.6. Haematological procedures
The haematological profile consisted of total red (RBC) and white (WBC) blood cells counts, haematocrit (Ht) and haemoglobin (Hb; SPINREACT kit, ref. 1001230, Spain). The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were also calculated as follows:
4.7. Humoral immune parameters
4.7.1. Lysozyme activity
Lysozyme activity was measured according to the turbidimetric method described by Welker et al. (2007) with some modifications. Briefly, 20 μl of skin mucus or plasma were placed in flat-bottomed 96-well plates. To each well, 180 µl of freeze-dried
Micrococcus lysodeikticus (0.2 mg ml-1, Sigma) in 40 mM sodium phosphate (pH 6.2) was
added as lysozyme substrate. As blanks, 20 μl of skin mucus or plasma were added to 180 μl of sodium phosphate buffer (PBS). The absorbance at 450 nm was measured after 20 min (endpoint measurement) at 35ºC in a microplate reader (Synergy HT). The amounts of lysozyme present in skin mucus and plasma were obtained from a standard curve made with hen egg white lysozyme (HEWL, Sigma) through serial dilutions in the
above buffer. Skin mucus and plasma lysozyme values are expressed as μg ml-1
26
4.7.2. Peroxidase activity
The peroxidase activity in skin mucus or plasma was measured according to Quade and Roth (1997) with some modifications. Briefly, 120 μl of skin mucus or 15 μl of
plasma were diluted with 30 μl or 135 μl of Hank’s buffer (HBSS) without Ca+2 or Mg+2 in
flat-bottomed 96-well plates, respectively. Fifty μl of 20 mM 3,3´,5,5´-tetremethylbenzidine
hydrochloride (TMB; 10mM) and 5 mM hydrogen peroxide (H2O2) were then added to
each well and serves as substrates. After 2 min the reaction was secured by adding 50 μl of 2 M sulphuric acid and the OD was measured at 450 nm in a microplate reader (Synergy HT). Samples without skin mucus or plasma, respectively, were used as blanks. Absorbance variation more than 1 can be defined as one unit of peroxidase. The final
results were expressed as units ml-1.
4.7.3. Protease activity
Protease activity was measured in skin mucus and plasma samples using the azocasein hydrolysis assay according to Guardiola et al. (2016) with some modifications. Briefly, 100 μl of skin mucus or 30 µl of plasma were diluted with 20 µl or 90 µl of 115 mM PBS (pH 7.0) for skin mucus and plasma samples, respectively, and 125 μl of 2% azocasein (in 60 mM sodium bicarbonate, pH 8.3) were added and the samples incubated for 24 h at 22ºC. Finally, 250 µl of 10% of TCA (trichloroacetic acid) were added and a new incubation for 30 min at 22ºC was done. The mixture was then centrifuged (10,000 x
g, 5 min) being the supernatants transferred to a 96-well plate in triplicate containing 100
µl well-1 of 1 N sodium hydroxide (NaOH), and the OD read at 450 nm using a microplate
reader (Synergy HT). PBS in place of skin mucus, plasma and trypsin served as blank whereas the reference sample was PBS in place of skin mucus and plasma. The percentage of trypsin activity compared to the reference sample was calculated with the following formula:
4.7.4. Antiprotease activity
Total antiprotease activity was determined by the ability of plasma to inhibit trypsin activity with some modifications (Guardiola et al., 2016). Briefly, 30 µl of plasma were
incubated for 10 min at 22ºC with 10 µl of standard trypsin solution (5 mg ml-1, in 60 mM
sodium bicarbonate, pH 8.3). Afterwards, 80 µl of 115 mM PBS (pH 7.0) and 125 μl of 2% azocasein (in 60 mM sodium bicarbonate, pH 8.3) were added and the samples incubated for 60 min at 22ºC. Finally, 250 µl of 10% of TCA were added and a new incubation for 30 min at 22ºC was done. The mixture was then centrifuged (10,000 x g, 5 min) being the
27 NaOH, and the OD read at 450 nm using a microplate reader (Synergy HT). PBS in place of plasma and trypsin served as blank whereas the reference sample was PBS in place of plasma. The percentage inhibition of trypsin activity compared to the reference sample was calculated with the following formula:
In the case of the skin mucus samples, several dilutions were tested but it was not possible to detect antiprotease activity.
4.8. Bactericidal activity
Bactericidal activity was determined using two opportunist marine pathogenic
bacteria: Photobacterium damselae subsp. piscicida (Pdp) and Vibrio harveyi (strains PP3
and ACC6.1, respectively). Strain PP3 was kindly provided by Dr. Ana do Vale (Institute for Molecular and Cell Biology, University of Porto, Portugal) and isolated from yellowtail (Seriola quinqueradiata; Japan) by Dr Andrew C. Barnes (Marine Laboratory, Aberdeen, UK). Bacteria were grown in agar plates (tryptic soy, TSA, BD) at 26ºC for 24 h. Afterwards, fresh single colonies of 1-2 mm were transferred to liquid culture medium (TSB, BD) and cultured as described above on an orbital incubator (250 rpm) until exponential growing at which point bacteria were resuspended in media and adjusted to 1
x 106 CFU ml-1.
Skin mucus and plasma bactericidal activity was then determined according to Machado et al. (2015) with some modifications. Briefly, 20 μl of skin mucus or plasma were added to duplicate wells of a U-shaped 96-well plate. HBSS was added to some
wells instead of the sample and served as positive control. To each well, 20 μl of each
bacterium were added, and plates were incubated for 2.5 h at 25ºC. To each well, 25 μl of
3-(4,5 dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 1 mg ml-1; Sigma-Aldrich)
were added and incubated for 10 min to allow the formation of formazan. Plates were then centrifuged at 2,000 x g for 10 min and the precipitate was dissolved in 200 μl of dimethyl sulfoxide (DMSO, Sigma-Aldrich). The absorbance of the dissolved formazan resulting from the reduction of MTT in direct proportion to the number of viable bacteria present was measured at 560 nm using a microplate reader (Synergy HT). Viable bacteria were expressed as percentage, calculated from the difference between the dissolved formazan
28 in samples and the one formed in the positive controls (100% of live bacteria). The bactericidal activity was calculated as the percentage of non-viable bacteria.
4.9. Oxidative Stress
4.9.1. Liver Homogenization
Livers were weighted between 92-108 mg and placed in a solution of K-phosphate buffer 0.1M (KPB 0.1M) in a reason of 1:10, always working under ice. Tissue samples were homogenized (1:10) in 0.1M phosphate buffer (pH 7.4). Part of the tissue homogenate was used to determine the extent of endogenous LPO by measuring thiobarbituric acid-reactive substances (TBARS) as suggested by Bird and Draper (1984). The remaining tissue homogenate was centrifuged for 20 min at 12,000 x g (4°C) to obtain the postmitochondrial supernatant fraction (PMS). 100 µL of supernatant was removed for CAT and stored at -80ºC.
4.9.2. Total proteins
Total protein content in each intestine homogenates sample was measured using
the bicinchoninic acid (BCA) Protein Assay Kit to express the activities as U mg protein-1.
More details in the supplementary Annex (section 1).
4.9.3. Lipid peroxidation
For lipid peroxidation, 200 µL of homogenized were removed and placed in 2 mL microtube and 4 µL of 4% 2,6-Di-tert-butyl-4-methylphenol (BHT) was added to all the samples and blank. The samples were placed in ice and 100 µL of 100% of TCA was added (including blanks). Then a solution of 2-Thiobarbituric acid (TBA) 0.73%, Trizma hydrochloride (Tris-HCL) and Diethylenetriaminepentaacetic acid (DTPA) was prepared and 1 mL was added to all the samples including blank. To substitute the sample, 200 µL of ultrapure water was added to the blanks. Samples were incubated for 1 hour at 100ºC then centrifuged for 5 minutes at 11,500 x g at room temperature. Supernatant was taken into a 96-well microplate in triplicate and the absorbance read at 535 nm using a microplate reader (Synergy HT).
4.9.4. Catalase
Catalase (CAT) activity was determined in PMS by measuring consumption of the
substrate H2O2 (Clairborne, 1985). For CAT, samples were diluted in KPB 0.1M (1
sample: 0 KPB) and then placed in a UV 96-well microplate in triplicate. Thus, 140 µL
KPB 0.05 M was added to each well. H2O2 was added to the microplates, and results were
read at 240 nm using a microplate reader (Synergy HT), for 2 minutes, with readings
every 15 seconds. Because the fast reaction between H2O2 and the sample, plates were
29
4.10. Statistical analyses
All analyses were conducted in triplicates and the results are expressed as mean ± standard error of the mean (SEM). Data were analysed by Two-way analysis of variance (ANOVA) followed by a Tukey post-hoc test with multiple comparison tests to determine differences between groups and each group respect to time, respectively. Normality of the data was previously assessed using a Shapiro-Wilk test and homogeneity of variance was also verified using the Levene test. Non-normally distributed data were log-transformed to perform parametric tests whilst non-parametric Kruskal-Wallis test, followed by a Dunn's multiple comparison test, was used when data did not meet parametric assumptions. All statistical analyses were conducted using SPSS 24.0 and differences were considered statistically significant when p ≤ 0.05.
5. Results
5.1. Organo-somatic indexes
The spleen-somatic index (SSI) of European seabass increased in the fish exposed to MPs compared to the values found in fish unexposed (control) and exposed to MPs-Cd mixture during 5 days, whereas the index remained unaltered at the end of the trial (10 days of exposure) (Figure 4A). In the case of the hepato-somatic index (HSI), no significant variations were recorded in any group at any sampling point (Figure 4B).
Figure 4. Spleen (A) and liver (B) organo-somatic indexes (%) of European seabass specimens
unexposed (control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1
mg Cd L-1 and 0.25 mg L-1, respectively). Bars represent the mean ± SEM (n=8). Different letters
denote significant differences between experimental groups (two-way ANOVA; P≤0.05).
5.1. Haematological profile
The values of Ht, Hb, MCV, MCH, MCHC, RBC and WBC are presented in table 3. Fish exposed to Cd, MPs or MPs-Cd mixture did not show variations in the values of Ht, Hb, MCV, MCH and MCHC in both sampling times. However, the total numbers of RBC decreased in seabass exposed to MPs compared to fish from Cd and MPs-Cd mixture
30 groups at day 5. Contrarily, an increase of total WBC numbers was observed in fish exposed to Cd compared to the values found in control and exposed fish to MPs at day 5. At the end of trial (10 days), fish exposed to Cd showed an increase and a decrease of total RBC and WBC numbers, respectively, compared to the unexposed group (control) (Table 3).
Table 3. Haematocrit (Ht), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cells (RBC) and white blood cells (WBC) in European sea specimens unexposed (control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1 and 0.25 mg L-1,
respectively). Values are expressed as means ± SEM (n=8). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks denote significant differences within each experimental group regarding time (two-way ANOVA; P≤0.05). ns: not significant.
Parameters Control Cd MPs MPs-Cd mixture
Ht (%) Day 5 34.63 ± 1.64 37.38 ± 1.25 34.13 ± 2.08 38.88 ± 1.23 Day 10 29.75 ± 1.00* 33.57 ± 1.13* 30.88 ± 2.26 30.71 ± 1.89* Hb (g dl-1) Day 5 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 Day 10 0.02 ± 0.00 0.03 ± 0.00 0.03 ± 0.01 0.03 ± 0.01 MCVa (µm3) Day 5 12.12 ± 0,85 11.05 ± 0.40 13.68 ± 1.39 11.73 ± 0.44 Day 10 11.27 ± 0.68 10.61 ± 0.32 10.84 ± 0.90 10.47 ± 0.55 MCHb (pg cell-1) Day 5 0.10 ± 0.01 0.10 ± 0.01 0.11 ± 0.00 0.09 ± 0.01 Day 10 0.09 ± 0.01 0.09 ±0.01 0.12 ± 0.02 0.12 ± 0.03 MCHCc (g 100 ml-1) Day 5 0.09 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 0.08 ± 0.01 Day 10 0.08 ± 0.01 0.08 ± 0.01 0.11 ± 0.02 0.10 ± 0.02 RBC (×106 µl)
Day 5 2.92 ± 0.16 (ab) 3.40 ± 0.13 (a) 2.64 ± 0.25 (b) 3.33 ± 0.11 (a)
Day 10 2.69 ± 0.14 (a) 2.94 ± 0.24 (b) 2.88 ± 0.09 (ab) 2.90 ± 0.12 (ab)*
WBC (×104 µl)
Day 5 5.08 ± 0.86 (a) 6.28 ± 0.41 (b) 4.55 ± 0.32 (a) 5.28 ± 0.34 (ab)
Day 10 8.35 ± 0.67 (a)* 5.55 ± 0.47 (b) 6.29 ± 0.48 (ab)* 6.79 ± 0.47 (ab)*
Comparing sampling times within each experimental group, significant variations were recorded in Ht values, RBC and WBC numbers. More specifically, a decrease was
31 observed in the Ht values in unexposed fish, exposed to Cd and MPs-Cd mixture at 10 days compared to values obtained at 5 days. Similarly, RBC numbers dropped at the end of trial in fish exposed to MPs-Cd mixture respect to results obtained at day 5. In the case of total WBC numbers, a significant increase was observed in fish unexposed and exposed to MPs and MPs-Cd mixture after 10 days of exposure compared to values recorded at day 5.
5.1. Immune parameters measured in plasma
Lysozyme activity in plasma of fish exposed to Cd increased in respect to values found in fish exposed to MPs-Cd mixture (Figure 5A) being the increment also significant comparing the results obtained in the fish from the same experimental group at the day 5. The peroxidase activity (Figure 5B) was not modified in the plasma of experimental fish after 5 days of exposure whilst this activity increased in the fish from Cd group compared to control and MPs groups at day 10. Interestingly, a significant variation was observed in peroxidase activity in plasma of all experimental fish sampling at the day 10 in comparing to the fish exposed for 5 days whilst the opposite pattern was observed in the protease activity (Figure 5D). In contrast, no significant variations were recorded in antiprotease activity (Figure 5C) between experimental groups. At 10 days, protease activity increased in plasma of fish exposed to MPs-Cd mixture compared to control and MPs groups (Figure 5D).
5.1. Immune parameters measured in skin mucus
The lysozyme activity measured in skin mucus showed variations in both sampling
times (
Figure 6A
). This activity decreased in skin mucus of fish exposed to MPs respectto values found in control and MPs-Cd mixture groups at day 5. After 10 days of exposure, the lysozyme activity increased in skin mucus of fish exposed to Cd respect to control and MPs-Cd mixture groups. Over time, lysozyme activity increased in fish exposed to Cd and MPs (individually administered) whilst the values observed in unexposed group were reduced.
The values of peroxidase activity increased in skin mucus of fish exposed to MPs
during 10 days respect to fish unexposed (
Figure 6B
). Comparing sampling times withineach experimental group, peroxidase activity augmented in fish exposed to Cd and MPs-Cd mixture at day 10 compared to day 5. Regarding the protease activity (Figure 7), no variations were observed in the experimental fish throughout the trial.
32
Figure 5. Lysozyme (A), peroxidase (B), antiprotease (C) and protease (D) activities measured in plasma of European seabass specimens unexposed (control) or exposed to Cd (0.1 mg Cd L-1),
MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1 and 0.25 mg L-1, respectively). Bars
represent the mean ± SEM (n=8). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks represent the differences between experimental groups.
Figure 6. Lysozyme (A) and peroxidase (B) activities on skin mucus of European seabass
specimens unexposed (control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd
mixture (0.1 mg Cd L-1 and 0.25 mg L-1, respectively). Bars represent the mean ± SEM (n=8).
Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks represent the differences between experimental groups.
33
Figure 7.Protease activities on skin mucus of European seabass specimens unexposed (control)
or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1 and 0.25
mg L-1, respectively). Bars represent the mean ± SEM (n=8).
5.1. Bactericidal activity in plasma and skin mucus
The bactericidal activity of the plasma against V. harveyi and P. damselae subsp.
piscicida is shown in Figure 8A and Figure 8B, respectively. In both cases, the bactericidal
activity of the plasma showed variations at the end of the trial (after 10 days of exposure). In the case of V. harveyi, the bactericidal activity of plasma against this bacterium decreased in fish exposed to Cd and MPs respect to values observed in the control group. In addition, the reduction of this activity of fish from these groups (Cd and MPs) was significant compared to the values obtained at the day 5.
Figure 8. Vibrio harveyi bacteria (A) and Photobacterium damsalae subsp. piscicida (B) bacteria
on bactericidal activity on plasma of European seabass specimens unexposed (control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1 and 0.25 mg L-1,
respectively). Bars represent the mean ± SEM (n=8). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks represent the differences between experimental groups.
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Figure 9. Bactericidal activity of Vibrio harveyi bacteria (A) and Photobacterium damsalae subsp. piscicida bacteria (B) on skin mucus of European seabass specimens unexposed (control) or
exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1 and 0.25 mg
L-1, respectively). Bars represent the mean ± SEM (n=8). Different letters indicate statistically
significant differences (P<0.05, Tuckey test). Different letters denote significant differences between experimental groups (two-way ANOVA; P≤0.05). Asterisks represent the differences between experimental groups.
Regarding bactericidal activity of the plasma against P. damselae subsp. piscicida, the values declined in fish exposed to MPs respect to those found in the fish unexposed or exposed to MPs-Cd mixture. Over time, the bactericidal activity against P. damselae in the MPs group decreased at the day with respect to values found at the first sampling point whilst an opposite pattern was observed in the control group.
In the case of skin mucus, the bactericidal activity increased in fish from Cd group respect to unexposed group at the day 5 whilst this activity decreased over time (Figure 9A). Similarly, the bactericidal activity of fish skin mucus from Cd group incubated with V.
harveyi was reduced over time. In contrast, the skin mucus of fish exposed to Cd showed
an increase when the samples were incubated with P. damselae subsp. piscicida compared both sampling times (Figure 9B).
5.1. Hepatic Oxidative Stress
The LPO and CAT activity measured in the liver of experimental fish were not altered in any group at any sampling point (Figure 10A and Figure 10B). In the same way, no variations were observed in any experimental group versus time.
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Figure 10. Lipid peroxidation (A) and catalase (B) on European seabass specimens unexposed
(control) or exposed to Cd (0.1 mg Cd L-1), MPs (0.25 mg L-1) and MPs-Cd mixture (0.1 mg Cd L-1
and 0.25 mg L-1, respectively). Bars represent the mean ± SEM (n=8). Different letters denote