Applicative implications of Carcinus maenas and ...

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Applicative implications of Carcinus maenas and Ruditapes philippinarum in biomonitoring studies after oil spills ab

b

a

G.V. Aguirre-Martínez , C. Morales-Caselles , T.A. Del Valls & ab

M.L. Martín-Díaz a

Department of Physical Chemistry, Faculty of Marine and Environmental Sciences, Universidad de Cádiz-Rio San Pedro Campus, 11510 Puerto Real, Cádiz, Spain b

Andalusian Center of Marine Science and Technology (CACYTMAR), Campus Universitario de Puerto Real, 11510 Puerto Real, Cádiz, Spain Published online: 02 Jul 2014.

To cite this article: G.V. Aguirre-Martínez, C. Morales-Caselles, T.A. Del Valls & M.L. Martín-Díaz (2014): Applicative implications of Carcinus maenas and Ruditapes philippinarum in biomonitoring studies after oil spills, Chemistry and Ecology, DOI: 10.1080/02757540.2014.932780 To link to this article: http://dx.doi.org/10.1080/02757540.2014.932780

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Chemistry and Ecology, 2014 http://dx.doi.org/10.1080/02757540.2014.932780

Applicative implications of Carcinus maenas and Ruditapes philippinarum in biomonitoring studies after oil spills


G.V. Aguirre-Martíneza,b∗ , C. Morales-Casellesb , T.A. Del Vallsa and M.L. Martín-Díaza,b a Department of Physical Chemistry, Faculty of Marine and Environmental Sciences, Universidad de Cádiz-Rio San Pedro Campus, 11510 Puerto Real, Cádiz, Spain; b Andalusian Center of Marine Science and Technology (CACYTMAR), Campus Universitario de Puerto Real, 11510 Puerto Real, Cádiz, Spain

(Received 23 October 2013; final version received 17 April 2014) Biomarkers have been tested in order to address the most suitable battery for determining adverse effects of crude oil spills on marine invertebrates. An oil spill with increasing degrees of severity was simulated by mixing crude oil (0%, 0.5%, 2%, 8%, 16%, 32%) with sediment. Carcinus maenas and Ruditapes philippinarum were exposed to this sediment for seven days with the aim of comparing their applicability in biomonitoring studies. Four biomarkers including ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), glutathione peroxidase (GPx) and lipid peroxidation (LPO) were analysed in gill and digestive gland tissues of clams; and in gill and hepato-pancreas tissues of crabs. EROD, GST and GPx enzymatic activities were significantly induced in gill and digestive gland tissues of clams when increasing oil concentrations (p < .01). In crabs all the biomarkers were significantly activated in gill tissues, whereas EROD and LPO activities were induced only in hepato-pancreas tissues (p < .01). Gill and digestive gland in clams and gill in crabs were found to be the most reliable tissues for analysis of biomarkers. The biomarkers selected are thus considered suitable for assessing toxicity of sediments after a marine crude oil spill accident. Both species were found to be sensitive and suitable for biomonitoring purposes. Keywords: crab; clam; biomarkers; bioassay; marine invertebrates; sediment

1.

Introduction

In recent decades, industrial development and anthropogenic activities including maritime transport and port activities, particularly those associated with the petroleum industry, have raised the levels of petrochemical products and derivatives discharged into aquatic environments.[1] However, in the context of these activities, the single most serious source of oil pollution at sea are spill accidents which cause severe damage to the marine biota.[2–5] The best-known examples of major marine spills of crude oil include the Bohai Bay oil spill in China (2011), the Deep Water Horizon rig in the Gulf of Mexico (2010), the sinking of the oil tanker Prestige off the Spanish Atlantic Coast (2002) and Erika disaster in France (1999). Damage in the aquatic environment is caused most evidently by the physical properties and movement of the oil spilled, which impedes normal life. Nevertheless, the severity of the adverse biological effect is difficult to predict as it also depends on the toxicity resulting from the chemical composition of hydrocarbons and the diversity, variability and sensitivity of the particular biological systems affected.[6,7] Some hydrocarbons are considered persistent organic pollutants ∗ Corresponding

author. Email: [email protected]

© 2014 Taylor & Francis


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capable of persisting unchanged in the environment for a long time without altering their toxic properties.[8] The semi-volatile properties of some hydrocarbons make them highly mobile [9] so contamination in one area is readily extended to other areas, thus affecting marine bottoms as well as rocky shores, often at considerable distance from the site of the accident. It has been documented that the impact of a crude oil (petroleum) spill in the marine environment becomes more prolonged when the displacement of the oil includes transport to the sediment where it can persist for years,[10] with long-term effects on the survival and well-being of many organisms.[4,11,12] Petrochemical products can be taken up easily by marine organisms, due to the ability of these compounds to interact with cellular molecules following binding to lipophilic sites.[13] After the xenobiotic uptake, the organism is forced to intensify the production of reactive oxygen species (ROS) by several mechanisms that can lead to cellular damage through protein oxidation, lipid peroxidation (LPO) and DNA damage. As the organism’s response to this potential damage, reactions that include enzymatic and non-enzymatic activities are stimulated to eliminate contaminant-generated ROS, allowing the organism to overcome or minimise oxidative stress in polluted environment.[14,15] To evaluate these sublethal responses in organisms, several authors recommend the use of biomarkers [16–21] because they can demonstrate whether or not an organism has been significantly affected after being exposed to a certain contaminant.[19,22] The biomarker approach is considered to be a sensitive response for determining the bioavailable fraction of pollutants and their possible toxic effects in an aquatic ecosystem, thus providing early warnings of possible environmental risks.[21–25] The aim of this study is to identify a suitable battery of biomarkers capable of detecting the bioavailability of compounds bound to the oil and to detect sublethal effects provoked by crude oil (petroleum) present in sediment, using two benthic bioindicator species, and to compare among the species and tissues and their implication in the risk assessment of accidents involving petrochemical contamination in the marine environment. To reach these aims two benthic invertebrates with distinctive feeding habits and different physiological characteristics were selected in this research as bioindicator species. The shore crab Carcinus maenas is a deposit feeder and the clam Ruditapes philippinarum is a filter feeder. Given the considerable knowledge existing about these species, including their biology and behaviour, they have previously been applied as indicator species in the assessment of contaminated sites [24,26–28] and experiments have been reported of their application as biomarkers to evaluate exposure and effects.[25,29–32]

2. 2.1.

Material and methods Selection of organisms

All individuals used in this research were of similar size and length: males of C. maenas (carapace width of 51 ± 5 mm) and R. philippinarum (42 ± 0.9 mm) and were purchased from an aquaculture farm located at a clean site on the Atlantic coast of southern Spain (Cadiz). In the laboratory, organisms were acclimatised separately over a 48 h period, in tanks of 300 L capacity with filtered seawater, supplied with constant aeration. Conventional parameters including pH (7.8–8.2), T (19◦ C ± 1◦ C), salinity (33.8 ± 0.3) and dissolved oxygen (>5 mg L−1 , 60% sat) were periodically controlled and maintained under a 12 h light: 12 h dark regime. 2.2. Laboratory bioassay The study was carried out applying crude oil (petroleum) extracted from that discharged by the wrecked oil tanker Prestige,[33,34] mixed with clean sediment from the Bay of Cádiz,

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Spain,[29,30] in different proportions taking into consideration dry weight of sediment and crude oil (0.5%, 2%, 8%, 16% and 32%). A control treatment with clean sediment from the Bay of Cadiz was included (0% of crude oil). Each mixture of oil and sediment was placed in a 20 L aerated glass aquarium and filtered seawater was added in a proportion of 1:4, sediment to water. Individual clams and crabs (n = 8) in duplicate were exposed during seven days to the contaminated sediment. Abiotic parameters including pH (7.8–8.2), T (19◦ C ± 1◦ C), salinity (33.8 ± 0.3) and dissolved oxygen (>5 mg L−1 , 60% sat) were routinely monitored, under a 12 h light: 12 h dark regime.


2.3. Biochemical analysis Eight crabs and eight clams were collected per replicate (n = 8) after seven days of exposure, then the whole tissue from clams (gill and digestive gland) and from crabs (gill and hepatopancreas) were extracted and stored at −80◦ C. Later, samples were homogenised and centrifuged at 15, 000 × g for 20 min at 2◦ C.[35] The supernatant fraction was carefully extracted and stored at −80◦ C. 2.3.1. Ethoxyresorufin O-deethylase activity Mixed function oxidised activity was measured using the adapted ethoxyresorufin O-deethylase (EROD) assay.[36] In dark microplates (96 flat bottom wells) duplicates of 50 μL of the supernatant were added to 160 μM 7-ethoxyresorufin and 10 μM reduced nicotinamide adenine dinucleotide phosphate (NADPH), in 100 mM KH2 PO4 buffer (pH 7.4). Determination of 7ethoxyresorufin in the samples was carried out using a standard calibration curve of resorufin (5 μM) concentration. Hydroxyresorufin was determined fluorometrically using 516 nm (excitation) and 600 nm (emission) filters. Results were expressed as pmol/min mg wet weight (ww). 2.3.2. Glutathione S-transferase activity The procedure utilised to determine glutathione S-transferase (GST) activity was adapted from Boryslawskyj et al.[37] A sample of 50 μL of supernatant (S15) was added to 200 L of 1 mM glutathione and 1 mM 1-chloro-2.4-dinitrobenzene in a buffer of 10 mM Hepes-NaOH, pH 6.5, containing 125 mM NaCl. Absorbance was measured at 340 nm every 5 min for 30 min. Results were expressed as nmol/min mgww. 2.3.3. Glutathione peroxidase activity The procedure applied for glutathione peroxidase (GPx) activity was adapted from Mcfarland et al.[38] Each sample was run in duplicate; GPx activity was measured spectrophotometrically at 340 nm at 3 s intervals for 3 min. The decrease in NADPH absorbance measured at 340 nm during the oxidation of NADPH to NADP was indicative of GPx activity. Results were expressed as nmol/min mgww. 2.3.4.

Lipid peroxidation

The thiobarbituric acid method [39] was used to determine LPO on homogenate samples of tissues by measuring the production of malonaldehyde. In a 1.5 mL Eppendorf tube, 150 L homogenate was mixed with 300 L of 10% trichloroacetic acid containing 1 mM FeSO4 and

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150 L of 0.67% thiobarbituric acid. Fluorescence was measured at 540 nm. Blanks and standards of tetramethoxypropane (stabilised form of malonaldehyde) were prepared in homogenisation buffer. Results of LPO were expressed as nmol/mgww. 2.4. Statistical analysis


Biomarker responses were analysed using a SPSS/PC+ statistical package®. Significant differences between tissues of individuals exposed to the uncontaminated sediment (control) and to the contaminated sediment (sediment containing 0.5, 2, 8, 16 and 32% of crude oil) were determined using a one-way ANOVA followed by a multiple comparison of Dunett’s tests, and correlation was estimated by Pearson analysis. The significance level was set at p < .01 and p < .05.

3. 3.1.

Results Biochemical responses in R. philippinarum

EROD activity measured in gill tissues of clams varied from 18.26 to 141.15 pmol/min mgww (Figure 1). EROD activity was significantly induced after exposure to the crude oil mixture of 16%, compared to control, and at this proportion reached 7.3 times the activity measured in control clams (p < .01). However, a decrease of 43% in EROD activity, compared with controls, was observed when clams were exposed to sediments containing the highest percentage of crude (32%). GST enzymatic activity evaluated in gill tissues ranged from 20.5 to 365.6 nmol/min mgww. Significant induction, compared with controls, was observed at a crude oil mixture of 2% (p < .01), reaching 1.4 times the activity measured in clams exposed to control treatment; in contrast, a significant decrease in GST activity, compared to control clams, was measured in gill of clams exposed to the other crude oil mixtures tested: 0.5%, 8%, 15% and 32% (p < .01) (Figure 1). Values of GPx activity measured in gill tissues ranged from 3.10 to 87.35 nmol/min mgww. This enzymatic activity increased following an exponential tendency when exposed to the different mixtures of crude oil and sediment (Figure 1), and this activity was significantly higher, compared to control, in clams exposed to the 16% of crude oil mixture (p < .01) (28 times control). However, it was observed that the highest mixture of crude oil tested in this study (32%) decreased GPx activity by 21% compared with control. LPO did not show significant differences with respect to control organisms. There was no evidence of increasing LPO in gill tissues of the exposed organisms (values ranged from 4.01 to 6.0 nmol/mgww) (Figure 1). Regarding digestive gland tissues of R. phillipinarum, EROD activity ranged from 15.7 to 119.7 pmol/min mgww, reaching 7.6 times the activity measured in control organisms. Significant induction, compared with control organisms, was observed after exposure to crude oil mixtures of 0.5% and 16% (p < .01) (Figure 2). GST activity measured ranged from 3.9 to 74.4 (nmol/min mgww) in exposed organisms. This activity was significantly induced by exposure to a 0.5% crude mixture (p < .05) but there was a significant decrease in enzymatic activity, below basal levels, at crude oil mixtures of 16% and 32% (p < .01) (Figure 2). The measurements of GPx in digestive gland tissues show that this activity varied from 26.6 to 116.3 nmol/min mgww. Two significant peaks were observed at crude oil mixtures of 2% and 8% (p < .01) (Figure 2) followed by a decrease in GPx at the 16% of the crude oil mixture. Measurements of LPO activity varied between 2.3 and 3.8 nmol/mgww; however, this activity was not significantly different from that detected in control organisms.

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Figure 1. Representation of EROD, GST, GPx and LPO activities measured in gill tissues of R. philippinarum exposed to the simulated crude oil spill (mean ± SE, n = 8). Asterisks indicate significant differences between means compared with control organisms (p < .05).

The Pearson correlation between biomarkers and crude oil mixtures tested in gill tissues showed a significant positive relationship between induction of both EROD and GPx activities and an increasing proportion of crude oil in the mixtures tested (p < .01; r = 0.53 and 0.43, respectively). A negative correlation was found between both GST and LPO activities and crude oil mixture proportion (p < .01; r = 5.3 and r = 52, respectively) (Table 1). In digestive gland tissues, a


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Figure 2. Representation of EROD, GST, GPx and LPO activities measured in digestive gland tissues of R. philippinarum exposed to the simulated crude oil spill (mean ± SE, n = 8). Asterisks indicate significant differences between means compared with control organisms (p < .05).

positive correlation was observed between both EROD activity and LPO and crude oil proportion, although the correlation was not significant. Significant negative correlation was found between GST and the crude oil mixture (p < .01; r = 0.58) (Table 2). 3.2.

Biochemical responses in C. maenas

EROD activity measured in gill tissues of crabs ranged from 26.5 to 60.4 pmol/min mgww. A significant increase in this activity, compared to control, was observed in organisms exposed


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Table 1. Pearson’s correlation between biomarkers and mixture of crude oil and sediment analysed in gill tissues from R. philippinarum (n = 8). % Crude oil EROD GST GPx LPO ∗∗

0.508∗∗ −0.528∗∗ 0.543∗∗ −0.515∗∗

EROD −0.507∗∗ 0.820∗∗ −0.370

GST −0.366 0.256

GPx −0.600∗∗

p < .01.


Table 2. Pearson’s correlation between biomarkers and mixture of crude oil and sediment analysed in digestive gland tissues from R. philippinarum (n = 8). % Crude oil EROD GST GPx LPO ∗∗

0.059 −0.581∗∗ −0.041 0.235

EROD 0.027 0.053 0.037

GST −0.581∗∗ −0.365

GPx −0.344

p < .01.

for seven days to a 16% crude oil mixture (p < .01) (2 times control) (Figure 3). GST activity varied from 16.1 to 69.5 nmol/min mgww following exposure to the crude oil mixtures. Significant induction of GST activity, compared to control, was observed in crabs exposed to a 32% of crude oil mixture (p < .01) (3.5 times control). A minimal induction of GST activity below basal levels was found in gill tissues of crabs exposed to crude oil mixtures of 8% and 16%, although this was not significant (Figure 3). The highest GPx activity in gill tissues (125 nmol/min mgww) was measured for exposure to the 32% of sediment and crude oil mixture; this was significantly different from that in control crabs (p < .05) (3.2 times control) (Figure 3). LPO was measured in the range of 1.14 and 3.0 nmol/mgww with significant induction produced by 0.5% fuel dilution (p < .01) (Figure 3). In hepato-pancreas tissues of crabs, induction of EROD activity ranged from 6.8 to 145.7 pmol/min mgww. There was a significant increase in this activity in organisms exposed to the crude oil mixtures of 0.5% (p < .01) and 16% (p < .05) (Figure 4). GST activity ranged from 7.1 to 63.8 nmol/min mgww in the seven-day bioassay. A significant decrease in GST activity was found in organisms exposed to sediment with 2%, 8% and 32% of crude oil (p < .01), but an increase in GST activity was observed in crabs exposed to a crude oil mixture of 16%, although this was not significant. Similarly, GPx activity measured in hepato-pancreas tissues was found to be below the values recorded in control organisms (374.6 nmol/min mgww). The lowest activity significantly different from controls was observed in organisms exposed to crude oil mixtures of 0.5% and 2% (108.7 and 148 nmol/min mgww, respectively) (p < .01) (Figure 4). Significant induction of LPO activity compared to control organisms was found in organisms exposed to crude oil mixtures of 0.5% (23.4 nmol/mgww) and 32% (42.1 nmol/mgww) (p < .01); this activity reached 4.3 and 7.7 times, respectively, the activity values recorded in control organisms (Figure 4). Analysis of the Pearson correlation between exposed C. maenas tissues and sublethal effects demonstrated a positive correlation between EROD, GST (p < .05; r = 0.4) and GPx activities (p < .01; r = 0.59) in crabs’ gill tissues and increasing proportions of crude oil in the sediment tested. However, a negative correlation was observed between the LPO level in gill tissues and the crude oil mixtures tested, although it was not significant (Table 3). In hepato-pancreas tissues analysed, a significant positive correlation was observed between the LPO level (p < 0.01;

G.V. Aguirre-Martínez et al. 250



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Figure 3. Representation of EROD, GST, GPx and LPO activities measured in gill tissues of C. maenas exposed to the simulated crude oil spill (mean ± SE, n = 8). Asterisks indicate significant differences between means compared with control organisms (p < .05).

r = 0.66) and the crude oil mixtures, and a positive relationship between EROD activity and the crude oil mixtures, although this correlation was not significant (Table 4). 4.

Discussion

Results demonstrate that certain tissues in R. philippinarum and C. maenas are measurably sensitive to compounds present in crude oil and give consistent responses after the organisms’ exposure

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Figure 4. Representation of EROD, GST, GPx and LPO activities measured in hepato-pancreas tissues of C. maenas exposed to the simulated crude oil spill (mean ± SE, n = 8). Asterisks indicate significant differences between means compared with control organisms (p < .05).

to sediment mixed with increasing concentrations of crude oil. These results are in agreement with previous research indicating adverse effects measured in the mollusk Austrocochlea porcata [40] and observed in R. philippinarum and C. maenas after being exposed to crude oil.[31,32] Among the biomarkers tested, in R. philippinarum, EROD and GPx activities determined in both gill and digestive gland tissues were significantly induced (p < .01) in organisms exposed to sediments contaminated with crude oil. In C. maenas EROD activity measured in gill and hepato-pancreas tissues showed significant induction (p < .01), compared to controls. These results agree with

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G.V. Aguirre-Martínez et al. Table 3. Pearson’s correlation between biomarkers and mixture of crude oil and sediment analysed in gill tissues from C. maenas (n = 8). % Crude oil EROD GST GPx LPO ∗

0.196 0.382∗ 0.590∗∗ −0.181

EROD −0.292 0.687 −0.544∗∗

GST 0.511∗∗ 0.249

GPx 0.067

p < .05. p < .01.

∗∗


Table 4. Pearson’s correlation between biomarkers and mixture of crude oil and sediment analysed in hepato-pancreas tissues from C. maenas (n = 8). % Crude oil EROD GST GPx LPO ∗

0.023 −0.380∗ −0.197 0.659∗∗

EROD 0.304 −0.292 0.143

GST 0.078 −0.416∗∗

GPx −0.084

p < .05. p < .01.

∗∗

those indicating induction of EROD activity in hepato-pancreas tissues of C. maenas and R. philippinarum after in situ exposure to sediments contaminated with crude oil spilled from the Prestige.[33] The substantial induction of EROD activity, as observed in R. philippinarum and C. maenas, has been widely reported in many other species of invertebrates, including crab and clam species.[35] In addition, it has been stated that exposure to polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenils (PCBs) can induce EROD activity in C. maenas and R. philippinarum.[29] In the present research work, EROD activity can be considered the most reliable sublethal response. EROD enzymatic induction was observed in all tissues tested in clams and crabs after exposure to sediments contaminated with crude oil. As a biomarker EROD is responsive to a wide range of chemicals, but it is usually used as indicator of exposure to hydrocarbons, specifically to PAHs, although not all PAHs induce EROD equally.[41] EROD induction was found in this research to be significantly activated to a greater degree than the other biomarkers tested. This could mean that some of the known inducers of EROD activity, such as PAHs, could have been present in the crude oil–sediment mixtures tested. GST activity, on the other hand, was significantly induced in gill tissues of C. maenas exposed only to sediment containing the highest percentage of crude oil tested (32%) (p < .01), while in gill tissues a significant induction in GST activity was observed in both clams (p < .01) and crabs (p < .05) exposed to the 2% sediment–crude oil mixture.A less clear picture was found in digestive gland and hepato-pancreas tissues in the two species; a decrease in GST activity was observed, compared to control organisms. For this enzyme it has been reported higher induction of activity in gill tissues than in digestive gland, mantle, foot or gonad tissues of Mytilus galloprovincialis after exposure to organic xenobiotic.[42] Similarly researchers have indicated low values for GST induction in digestive gland tissues of R. philippinarum after exposure to crude oil.[31,32] As an explanation of these findings, Cossu et al. [43] reported that GST activity responses of aquatic species to pollution are conflicting because a relationship between the pollutant present and enzyme induction has not always been observed. GST is a Phase II type enzyme and catalyses the synthetic conjugation reaction of the xenobiotic parent compounds and their metabolites in order to facilitate the excretion of chemicals. Levels of GST in tissues can be modified by a large range



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of xenobiotics and also by abiotic factors. Given its involvement in the detoxification process of xenobiotics, GST activity has been proposed as a biomarker for several aquatic invertebrate species including crustaceans and mollusks.[42] In the present study, the GST activity measured might be the result of exposure not only to hydrocarbons such as PAHs but also to other chemicals such as metals, which have been reported to be present in the crude oil carried by the Prestige.[34,44,45] It is known that xenobiotics can produce toxic effects related to oxidative stress. Oxygen toxicity is defined as injurious effects due to cytotoxic ROS, which are also called free radicals.[46] These reduction products of molecular oxygen (O2 ) are the superoxide anion radical (O− 2 ), hydrogen peroxide (H2 O2 ) and the hydroxyl radical (OH), the latter being an extremely potent oxidant capable of reacting with critical cellular macromolecules, possibly leading to enzyme inactivation, LPO, DNA damage and ultimately cell death.[47] It has been indicated that antioxidant systems act to prevent oxidative damage by eliminating the ROS, and they may be induced as an adaptive response after an exposure to PAHs, allowing the organism to overcome oxidative stress, partially or totally, in a polluted environment.[46] In the present research, strong GPx enzyme induction (p < 0.01), compared to the control, was observed in tissues of both species exposed to sediment contaminated with high proportions of crude oil, except in crabs hepato-pancreas tissues where GPx was barely induced. This is in agreement with studies describing high values of GPx induction in digestive gland tissues of M. galloprovincialis exposed to organic pollutants.[48] Results from this work agree with previous research that reported a strong correlation between GPx and EROD activities in clam tissues (p < .05).[29] However in our research, GPx activity in crab tissues presented a strong relationship with GST activity (p < .01). These results agree with those of Reid and Macfarlane [40] who studied potential biomarkers in A. porcata indicating that GPx activity was the only biochemical parameter to show a significant positive relationship to exposure to crude oil reflecting a compensatory adaptation of the specie to a contaminated environment. When pro-oxidant forces overwhelm antioxidant defences, a cellular oxidative stress is established.[49] However in some cases, prolonged exposure to petroleum contaminants may also result in depletion of components of the antioxidant response to toxic effect.[40] Depletion of enzymatic GPx activity was detected in this work in digestive gland tissues of clams and in hepato-pancreas tissues of crabs exposed to the two highest oil–sediment mixtures tested (16% and 32%). GPx is considered an efficient protective enzyme against LPO [47] and its reduced activity has been linked to LPO in the fish Limanda limanda exposed to sediments contaminated with PAHs and PCBs.[43] These findings explain the significant GPx activity induction observed in gill and digestive gland tissues of clams, and in gill tissues of crabs, while the LPO level decreased. It could be expected that induction of LPO measured in this research occurred when the organism’s antioxidant system was overwhelmed, as observed in hepato-pancreas tissues of C. maenas. LPO is a very important consequence of oxidative stress.[15] The process of LPO proceeds by a chain reaction and, as in the case of redox cycling, demonstrates the ability of a single radical species to propagate a number of deleterious biochemical reactions.[50,51] The LPO level found in this work were similar to those measured in gill and digestive gland tissues of the mussel Perna viridis.[52] However, the present research did not reveal evidence of a significant increase in LPO in tissues of the species tested compared to control organisms. Low LPO levels from this study match those measured in the visceral mass of A. Porcata exposed to crude oil [40] and those reported by Cossu et al. [43] who did not detect any substantial LPO in digestive gland tissues of the fresh water bivalve Unio tumidus after exposure to polluted sites. The responses of many biomarkers are not directly associated with real harmful effects in the target organism; some biomarkers are designed to indicate exposure and other biomarkers measure effects such as compensation or repair responses. Therefore, a lack of response could reflect the absence of any inducers or, on the other hand, it could mean that an efficient repair has taken place in the organism.


12


Whether a biomarker response is observable or not depends not only on the level of exposure, but also on the timing of the measurement made after the exposure.[19] In this regard, probably the enzymatic activity of the biomarkers tested in this research would vary if a longer term experiment were performed. It has been stated that, if biomarkers follow a linear aetiology over time, it could be expected that initial EROD induction would protect the cell from immediate injury; this would be followed by induction of antioxidant enzymes GST and GPx to prevent oxidative stress, and LPO if or when EROD were overwhelmed.[53] However, in this study this linear aetiology was not observed. In addition to the time of exposure, routes of exposure also contribute to the magnitude of the promoted toxic effect.[54] In our research work, it was demonstrated that EROD activity, GST activity and the LPO level were significantly induced in gill tissues of both species which is the major route of entrance of the contaminants. However, high sensitivity of gill tissues to oxidative stress was also indicated by the induction of antioxidant parameters, often more pronounced in this tissue than in digestive gland and hepato-pancreas tissues. Furthermore, GPx activity was significantly activated in digestive gland tissues R. philippinarum (p < .01) when exposed to the sediment contaminated with 2% and 8% of crude oil; these results are in agreement with a previous research indicating that GPx induction is higher in digestive gland than in gill tissues of the fresh water bivalve U. tumidus.[43] In this regard, it has been stated that the digestive gland is the organ most responsible for antioxidant responses, through dietary accumulation.[55] In crabs, probably the increase in EROD activity, GST activity and the LPO level, together with inhibition of GPx activity in hepato-pancreas tissues is related to its feeding habits; C. maenas is a deposit feeder therefore its food was probably in contact with the crude oil bound to the sediment.

5.

Conclusions

Both R. philippinarum and C. maenas are species measurably susceptible to environmental contaminants; therefore, they are proposed for use in environmental risk assessment. Moreover, an assay involving two invertebrate species with different feeding habits should allow a better understanding of the biological effects of contaminants bound to sediments. The present work has demonstrated enzymatic induction of a range of biomarkers associated with crude oil present in the sediment. Consistent responses were observed for EROD and GPx activities measured in tissues of R. philippinarum and C. maenas, but GST activity and LPO showed contradictory trends. EROD and GPx activities can provide sensitive biomarkers for monitoring the exposure of these species to pollutants of this type. Furthermore, these biomarkers also showed some potential in the prediction of toxicity. More studies are needed to increase knowledge of relationships between the degree of deficiency of antioxidant system and cell injury in order to predict harmful effects in the organism. It can be assumed, for further studies of sublethal effects in responses to organic pollutants, that EROD activity is consistently and markedly induced in all the tissues analysed. The most suitable tissues for EROD, GST and GPx enzymatic analysis are gill tissues of both species; LPO activity was induced more measurably in digestive gland tissues of R. philippinarum and in hepato-pancreas tissues of C. maenas. It is recognised that sublethal responses depend not only on fuel concentration and toxicity, but also on the period of exposure, the route of uptake and the species studied. Therefore, differences in responses arising from differences in species, tissues, biomarkers or time of exposure have to be considered in future toxicology studies. This type of study provides useful tools for environmental biomonitoring and ensuring environmental safety; in particular, the biomarkers analysed in this study are considered useful for the determination of toxicity from exposure to sediments contaminated with crude oil following marine spills.


13

Acknowledgements This research was supported as part of the Erasmus mundus UNESCO/UNITWIN/WiCoP activities at Cádiz, Spain. The work was part of the project ‘Desarrollo y mejora del análisis integrado para la evaluación de la calidad de sedimentos litorales, incluidos los materiales de dragado portuario (INTEGRAL)’ financed by Spanish Ministry of Science and Education (CTM2005-07282-C03-C01/TECNO) and the project ‘Caracterización de la calidad ambiental de ecosistemas costeros afectados por vertidos de petróleo: comparación entre casos de vertidos accidentales (impacto agudo) frente a derrames continuos (impacto crónico) (TRIADA)’, financed by the Spanish Ministry of Science and Education (VEM200320563/INTER). The authors would like to thank Lesley Lusty for the English language review.


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