Environ Sci Pollut Res DOI 10.1007/s11356-013-2440-0
RESEARCH ARTICLE
Genotoxicity and histological alterations in grey mullet Mugil liza exposed to petroleum water-soluble fraction (PWSF) Cauê Bonucci Moreira & Ricardo Vieira Rodrigues & Luis Alberto Romano & Emeline Pereira Gusmão & Bianca Hartwig Seyffert & Luís André Sampaio & Kleber Campos Miranda-Filho
Received: 31 March 2013 / Accepted: 9 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Responsible editor: Philippe Garrigues
gills for histopathological study. For these procedures, seven fish were sampled per concentration tested. The LC50-96 h was estimated at 37.5 % of the PWSF. The time required for MN induction was 96 h of exposure. The time of clearance was sufficient to achieve a MN frequency similar to that of the control group. Histopathological studies showed severe changes in the gill and liver tissues. The most relevant histopathology in the gills was telangiectasia. Hepatic histopathology such as cholestasis, dilated sinusoids and inflammatory infiltrates were commonly described. The MN test and histological study effectively detected damages caused by medium-term exposition to the PWSF, and despite the toxicity, a few days without exposure can minimize PWSF genotoxicity in juveniles of M. liza.
Electronic supplementary material The online version of this article (doi:10.1007/s11356-013-2440-0) contains supplementary material, which is available to authorized users.
Keywords Petroleum hydrocarbon . Micronucleus . Histopathology . Toxicity . Aquaculture
Abstract Petroleum hydrocarbons are considered one of the main organic chemicals found in water bodies. In the present study, the median lethal concentration (LC50) was estimated for mullet Mugil liza after acute exposure to petroleum watersoluble fraction (PWSF). Furthermore, histopathological studies and micronuclei frequency were also performed in order to observe deleterious effects of medium-term exposition to PWSF. Mullets (25±2.3 g) were exposed to chronic concentrations (1.7, 3.5 and 7 % of PWSF), plus the control group, for 14 and 7 days of clearance time. Throughout the experimental period (1, 4, 14 and 21 days), blood samples were collected for analysis of micronucleus (MN) and liver and
C. B. Moreira : R. V. Rodrigues : E. P. Gusmão Programa de Pós-Graduação em Aquicultura, Laboratório de Piscicultura Estuarina e Marinha, Universidade Federal do Rio Grande, CEP 96201-900, Rio Grande, RS, Brazil L. A. Romano : L. A. Sampaio Laboratório de Piscicultura Estuarina e Marinha, Instituto de Oceanografia, Universidade Federal do Rio Grande, CEP 96201-900, Rio Grande, RS, Brazil B. H. Seyffert Laboratório de Ecotoxicologia e Microcontaminantes Orgânicos, Instituto de Oceanografia, Universidade Federal do Rio Grande, CEP 96201-900, Rio Grande, RS, Brazil K. C. Miranda-Filho (*) Laboratório de Aquacultura, Departamento de Zootecnia, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CEP 31270-901 Belo Horizonte, MG, Brazil e-mail:
[email protected] K. C. Miranda-Filho e-mail:
[email protected]
Introduction In recent years, concentrations of pollutants in aquatic ecosystems have increased alarmingly as a result of human activity (Cajaraville et al. 2000). Among the organic pollutants, the petroleum hydrocarbons require greater concern due to their environmental impact (Varanasi 1989). The toxicity of oil is a matter of continuing investigation, and the knowledge of its toxicity to different ontogenetic levels of aquatic biota is required (Rodrigues et al. 2010). The bioavailability and toxicity of petroleum are different depending on the type and degree of biological, chemical and physical degradation, which must be considered in toxicological assessment studies (Rice 1985). Toxicity tests are an important analytical tool for assessing the effects of chemical agents on living organisms under standardized conditions, enabling the comparisons to other chemical agents. They
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can be designed to detect acute or chronic effects, generated by lethal or sublethal concentrations, respectively (Rand and Petrocelli 1985). The petroleum water-soluble fraction (PWSF) is the portion which is soluble in water, containing mainly monoaromatic hydrocarbons of the BTEX group (benzene, toluene, ethyl-benzene and xylene). PWSF is highly toxic and soluble and can be rapidly absorbed by organisms (Stephens et al. 1997; Dórea et al. 2007). The concentration of polycyclic aromatic hydrocarbons (PAHs)—non-polar chemicals comprising two to ten aromatic rings (Barra et al. 2006; Hylland 2006)—is small in comparison to monoaromatic hydrocarbons found in PWSF. These pollutants reported are known to be important in environmental studies, due to their high carcinogenic potential (Newman 2009). The biotransformation of petroleum hydrocarbons in fish is facilitated primarily by the group of enzymes known as cytochrome P450 1A, which belongs to the first phase of biotransformation by performing reductive and oxidative reactions and transforming the xenobiotics into more hydrosoluble compounds, facilitating their excretion (Goksoyr and Förlin 1992). However, this transformation can generate intermediate compounds that are more toxic, mutagenic and carcinogenic than the original (Nebert and Gonzales 1987; Buhler and Williams 1988). The micronucleus test is considered a cytogenetic evaluation and has been used with great success to evaluate the mutagenicity of environmental pollutants through the appearance of micronuclei in erythrocytes (Klobucar et al. 2003). This test evaluates the effects of irreversible genetic damages of clastogenic substances, which cause chromosome breakage, and aneugenic agents, which affect the cell division through loss or gain of whole chromosomes (Ribeiro et al. 2003). According to the literature, there is an association between the frequency of micronuclei exposure to toxic agents (Ayllon and Garcia-Vazquez 2002; Gustavino et al. 2001; Pacheco and Santos 2002; Ferraro et al. 2004: Vanzella et al. 2007). Histopatology is usually used as a tool to show a relation between tissues and their exposure to environmental pollutants (Haensly et al. 1982; Solangi and Overstreet 1996; Khan 2003). In accordance to Heath (1995), gills are important in respiration, osmoregulation, ionic balance and excretion of nitrogenous compounds, constituting the larger contact area between the internal and external environment. For these reasons, this organ has been successfully used in biomonitoring activities (Schwaiger et al. 1997). Petroleum WSF induces microscopical changes in the gill and liver tissues under chronic exposures (Khan 2003; Simonato et al. 2008). In a study conducted by Brand et al. (2001), salmon (Oncorhynchus gorbuscha) were exposed to sub-chronic concentrations of PWSF and several liver histopathologies were found and associated with PAHs, including proliferation of
epithelial cells in the bile duct, hepatocellular necrosis and pleomorphism. The mullet belongs to the family Mugilidae and is widely distributed in coastal waters and estuaries throughout the world. Many species are important to the fishing and aquaculture activities. Mugil platanus (now Mugil liza, Menezes et al. 2010) occurs from Brazil to Argentina and juveniles are easily captured in the environment (Menezes and Figueiredo 1985). These characteristics can be useful to compare the environmental integrity in relation to pollution in different ecosystems along the coast. Despite the importance of studying the harmful effects caused by petroleum hydrocarbons, information about the toxicity of PAHs and mainly monoaromatic compounds (BTEX) in marine organisms is still needed. Therefore, the aim of this study was to investigate the lethal and sublethal effects (histopathological studies and analysis of micronuclei frequency) caused by the exposure of grey mullets (M. liza) juveniles to the soluble fraction of light crude oil.
Material and methods Preparation of petroleum water-soluble fractions (PWSFs) The light crude oil used in the present study was donated by the Brazilian Agency for Petroleum and Gas (ANP). The hydrosoluble fraction was prepared in a fume hood, free of luminous radiation, in a ratio of nine parts water for one part oil. The temperature, pH and salinity of the water were 23 °C, 7.98 and 29‰, respectively. The PWSF was slowly mixed with a magnetic stirrer (Quimis, Q241, Brazil) in a 10-L “mariotti” flask during approximately 22 h, according to the methodology proposed by Anderson et al. (1974) and modified by Rodrigues et al. (2010). After 1 h of rest, PWSF was removed and taken to the toxicity tests. Physicochemical parameters The mean values and standard error of temperature, dissolved oxygen and pH for the different PWSF concentrations are presented in tables as supplementary material. These parameters were not statistically different in medium-term and acute toxicity tests. Hydrocarbons analysis The PAHs were analyzed with a gas cromatograph (PerkinElmer Clarus® 500) with mass spectrometer (GC-MS) (Method 8270D–EPA). The BTEX analysis that followed employed a gas cromatograph (Perkin-Elmer Clarus® 500) with flame ionization detector (GC-FID) and a headspace Turbomatrix HS-40 sampler (Perkin-Elmer) (Method
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8015B–EPA). All the analyses were performed at ISATEC laboratories (Research, Development and Chemical Analysis Ltd.) in Rio Grande, RS, Brazil. Acute toxicity tests A preliminary acute toxicity test (96 h) was performed in order to define the PWSF lethal range to juvenile mullet. PWSFs nominal concentrations in the definitive 96-h test were 20, 25, 30, 35, 40 and 45 %, and there was a control group without addition of PWSF. The mean weight of the juvenile mullet was 0.9±0.15 g. All treatments were conducted in triplicate in 1-L beakers (n per treatment=21; seven mullets per beaker), with gentle aeration and complete exchange of solutions every 24 h. During the experiment, photoperiod was maintained at 12 h and light intensity varied 750–1,000 lx (Chauvin Arnoux, CA 810, France). Food was ceased during the test. Salinity was measured daily with hand refractometer (Atago, S/Mill, Japan), dissolved oxygen with digital oxygen meter (Yellow Spring International, 55/12 FT, USA) and pH using a digital pH meter (Hanna, 221, Romania). The fish mortality was observed every 24 h.
Brazil) and paraplast embedded. The tissues were sectioned in a microtome (MRP03 LUPE, Brazil) with 5-μm thickness and stained with hematoxylin and eosin. The slides were observed in an optical microscope (Olympus B201, EUA) and photomicrographs were taken with a digital camera. The histopathologies were described according to Romano and Cueva (1988).
Micronuclei (MN) analysis The blood of the previously anesthetized juvenile mullet was sampled from the caudal vein after 1, 4, 14 and 21 days of experiment. The blood was spread on glass slides, previously cleaned in 5 % hydrochloric acid. The smears were then fixed in absolute methanol for 10 min and stained with Giemsa diluted in distilled water at a concentration of 10 % (Barsiené et al. 2004) to observe peripheral erythrocytes. The frequency of micronuclei was evaluated every 1,000 cells using an optical microscope (Nikon E-200, Japan). A total of 3,000 cells with intact membranes and nuclei were analyzed per fish (seven per treatment). Only micronuclei that had 1/3– 1/20 of nuclei size were counted.
Medium-term toxicity test The medium-term toxicity test was carried out during 21 days (14 days of exposure to the PWSF and 7 days of depuration in water free of PWSF), in a semi-static system. Four polypropylene circular tanks (200 L) were used to test the control group and three nominal concentrations of PWSF, without replicates. Juveniles of M. liza (25 g of mean weight) were used in four treatments (n per treatment=40; 40 mullets per tank). Dry food (Guabi®, 35 % of protein) and gentle aeration were provided. According to Cohen and Nugegoda (2000), aeration does not cause significant losses of petroleum hydrocarbons in tests performed in experimental tanks. Chronic concentrations were based in the LC50-96 h, multiplied by the application factor (0.1) proposed by Sprague (1971), resulting in the safety level (SL). Therefore, the concentrations tested were C1=1.7 % of PWSF (below SL); C2= 3.5 % of PWSF (equivalent to SL); and C3=7 % of PWSF (twice the SL). The physicochemical parameters (oxygen, temperature and salinity) were daily monitored as described in “Hydrocarbons analysis.” The experimental solutions were 90 % renewed every 48 h.
Statistical analysis The median lethal concentrations (LC50) of the PWSF and their confidence intervals (95 %) were estimated for juvenile mullet at 24, 48, 72 and 96 h using the software Trimmed Spearman–Karber method (Hamilton et al. 1977) and the SL was estimated according to Sprague (1971). The water quality parameters were compared with one-way analysis of vriancea. To analyze the differences between the frequencies of micronuclei, non-parametric Kruskal–Wallis test was used. All analyses were performed with the software Statistica 7.0.
Results PWSFs analysis The total concentration of PAHs and BTEX was estimated at 52.87 and 15,431.92 μg/L, respectively, and the sum of total hydrocarbons of petroleum was calculated in 15,484.79 μg/L for non-diluted PWSF samples (Table 1).
Histological analysis The histological study was performed only in the mediumterm experiment. Gills and liver were sampled of seven specimens (euthanized with 100 ppm of benzocaine) at 1, 4, 14 and 21 days and fixed in 10 % buffered formalin. All samples were processed in automated tissue processor (LUPE-PT05,
Acute toxicity test The LC50 results estimated for 24, 48, 72 and 96 h and the safe level (SL) for juvenile mullet M. liza exposed to PWSF are shown in Table 2.
Environ Sci Pollut Res Table 1 Total concentrations (μg/L) of monocyclic aromatic hydrocarbons (BTEX) and polycylic aromatic hydrocarbons (PAHs) in samples of the PWSF Hydrocarbons
Petroleum
Benzene
6,622.29
Toluene
5,672.53
Ethyl-benzene
98.74
Xylene
3,038.36
∑ BTEX
15,431.92
Naphtalene
51.085
Acenaphtalene
Nd
Acenaphtene
Nd
Fluorene
0.766
Phenantrene
1.024
Anthracene
Nd
Fluorantene
Nd
Pyrene
Nd
Benzo[a]Anthracene
Nd
Crysene
Nd
Benzo[b]Fluoranthene
Nd
Benzo[k]Fluoranthene
Nd
Benzo[a]Pyrene
Nd
Indene[1.2.3-C.D]Pyrene
Nd
Dibenzo[a,i,h]Anthracene
Nd
Benzo[g,h,i]Perylene
Nd
∑ PAHs
52.87
∑ Petroleum hydrocarbons
15,484.79
Nd, not detected
Micronuclei analysis Relative frequencies of micronuclei in erythrocytes of M. liza exposed to PWSF in the medium-term test can be observed in Fig. 1a. A high incidence of micronuclei after 4 and 14 days of exposure to PWSF is observed in Fig. 1b, highlighting the action of aneugenic and/or clastogenic agents present in the tested PWSF. Statistical differences between exposed animals and the control group were not observed at 24 h. After 96 h, the average micronuclei in different PWSF concentrations were
Table 2 Median lethal concentrations (LC50 for different periods), confidence interval and safe level for juvenile grey mullet Mugil liza exposed to PWSF Time (h)
LC50 (%)
Safe level (%)
24
51.27 (53.72–48.93)
5.1
48
47.18 (51.22–43.46)
4.7
72
39.05 (43.31–35.20)
3.9
96
37.15 (41.21–37.15)
3.7
superior to the control group; however, there was no statistically significant difference observed for the lowest concentration (1.7 % of PWSF). The mean values of the micronuclei were 0.4 % in the control group and 1.6, 2.5 and 3.8 % in the concentrations 1, 2 and 3, respectively. After 14 days of exposure, all the concentrations differed significantly from the control group and the micronuclei means were 0.3 % control group and 2.3, 1.6 and 1.7 % for concentrations 1, 2 and 3, respectively. In general, the time required for the formation of the micronuclei decreased while the PWSF concentrations increased. After 7 days of depuration period, the micronuclei frequency decreased and no statistical difference was observed among treatments. The highest frequency of micronuclei was registered after 96 h of exposure.
Histological analysis In the concentration of 3.5 % of PWSF, there was an increase in the chloride cells in the gills after 4 days of medium-term exposure. The histopathologies were mainly observed in the gills and liver of juvenile mullet (Table 3). The alterations were more severe with the increase of PWSF concentrations. The most significant lesions found in the gills were elevation of the respiratory epithelium (Fig. 2b) and telangiectasia (Fig. 2c). The distal and diffuse hyperplasia (Fig. 2d) were commonly observed, mainly the diffuse hyperplasia, which was not observed in the control group. After the depuration period, the occurrence of histopathologies decreased, though telangiectasia and distal hyperplasia were still observed in the highest concentration of PWSF. No histopathology was observed in the hepatic tissues of the control group (Fig. 3a). The lesions were more severe with the increase of PWSF concentrations. Focal inflammatory infiltrates (Fig. 3b) and around the bile duct (Fig. 3c) were found with higher relevance after 96 h of exposure in the concentrations of 3.5 and 7 % of PWSF and not observed in the period of depuration. However, cholestasis (Fig. 3d) and eosinophyl granulocytes (Fig. 3e) were only observed in the lowest concentration (1.7 %). In higher concentrations (3.5 and 7 %), an increased concentration of lipids was observed in all the periods of PWSF exposure. Dilatation of sinusoids with accumulation of erythrocytes was observed only in 7 % of PWSF and in the periods of 96 h and 14 days of exposure (Fig. 3d). The histopathologies decreased during the depuration period, but the presence of inflammatory infiltrates around the bile duct and the presence of eosinophilic cells, both in the highest concentration, suggest an inflammatory process.
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a 6
b
Micronucleus Frequency /3000 cells
Fig. 1 a Micronucleus frequency induced in erythrocytes of grey mullet of Mugil liza exposed to different concentrations of PWSF. Different letters represent statistical differences (p
