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Monitoring the biological effects of pollution on the Algerian west coast using mussels Mytilus galloprovincialis
Algerian west coast
Zohei'r M. Taleb* Sofiane Benghali Amina Kaddour Zitouni Boutiba
Reseau de Surveillance Environnementale (RSE), Department ofBiology, University ofOran Es Senia, 31000 Oran, Algeria;
* corresponding author
Received 13 April 2007, revised 19 September 2007, accepted 6 November 2007.
The Algerian west coast is the prime recipient ofseveral forms of pollution; hence, the necessity for an impact assessment of this coastal pollution using a suite ofrecommended marine biomarkers, including lysosomal membrane stability in living cells by the Neutral Red Retention Time (NRRT) method, the evaluation ofmicronucleus (MN) frequency, and the determination ofacetylcholinesterase (AChE) activity in mussels Mytilus galloprovincialis, sampled from the large, pollutedOranHarbour (OH) andthe Maarouf(Mrf) marine mussel farm between July 2005 and April 2006. The difference in the variations ofthe annual physical parameters between OH and Mrfcorresponds to the influence ofthe domestic and industrial sewage discharged by the city ofOran. The biological data ofthe mussels (condition index, protein content) recorded at both sites were related to their natural reproductive cycle. This indicated that intrinsic variation between the sites due to different mussel development phases was minimal. The variation in the AChE activity ofsome organs ofOH and Mrfmussels, with minimal inhibition
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in July and a higher NRRT recorded in the granular haemocytes in the Mrfthan in the OH mussels during the autumn and spring, depends on the quality ofthe biotope and on generic stress factors. Moreover, the variation in MN frequency, in general reflecting a non-significant seasonal and spatial genotoxic effect ofthe contamination at the two sampling sites, requires further investigations regarding biotic and abiotic variations.
Biomonitoring has become one ofthe ways ofpredicting changes in the global environment. Many scientific programmes in different Mediterranean countries are taking this approach to the biological effects ofcontaminants with the aim ofpromoting a common and integrated strategy ofusing marine biomarkers in recommended sentinel species (UNEP/FAO/IOC 1993, UNEP 1997, Viarengo et al. 1997, UNEP/RAMOGE 1999, Cajaraville et al. 2000, Viarengo et al. 2000a,b, ICES 2004). Biomarkers, for example, mussels Mytilus spp., are early warning biological tools able to detect pre-pathological changes or disturbances as responses to environmental pollutants at the cellular and organism levels (Moore 1985, Amiard et al. 1986, Viarengo et al. 1990, Lionetto et al. 2003, Regoli et al. 2004, Gravato
et al. 2005).
The increase in the human population (more than 40% ofAlgeria's population inhabits the littoral zone), industrial development in western Algeria and the absence ofurban and/or industrial sewage treatment plants have turned the coastal marine environment into a prime recipient ofseveral forms ofpollution. Our research laboratory's assessment ofmarine pollution by contaminants bioaccumulated in marine species (e.g. mussels, sea urchins, crustaceans, fish and cetaceans) from several sites on the Algerian west coast during the last ten years has revealed high concentrations ofheavy metals (Hg, Cd, Pb, Zn, Cu, Mn, Ni, Mg), organochlorine compounds (PCB and chlorinated pesticides) and polyaromatic hydrocarbons (chrysene, phenanthrene) (Taleb 1997, Taleb et al. 1997, Taleb & Boutiba 1999, 2007, Boutiba et al. 2003). This merely underlines the crucial importance of evaluating the impacts ofthe pollution gradients in this coastal area of Algeria.
In accordance with the current national priority environmental policy (National Action Plan for the Environment and Sustainable Development, Algerian Ministry ofthe Environment (PNAE-DD 2002)), the first regional marine biomonitoring project 'Use ofbiomarkers for the assessment ofmarine pollution impacts in the western Algerian coastal area' was recently developed by our laboratory. This project introduced certain
recommended marine biomarkers (UNEP 1997, UNEP/RAMOGE 1999,
Bocquene & Galgani 2004): determination oflysosomal membrane stability
in living cells by the Neutral Red Retention Time (NRRT) method (general stress), evaluation ofmicronucleus (MN) frequency (genotoxic effects), and determination ofacetylcholinesterase (AChE) activity (presence of organophosphorus compounds, carbamates and some heavy metals) in mussels Mytilus galloprovincialis, the most frequently used sentinel organism in Mediterranean marine environmental biomonitoring programmes. As filter feeders, these animals have the capacity to accumulate organic and inorganic xenobiotics present in their environment (Jernelov 1996, Boutiba
et al. 2003, Taleb & Boutiba 2007).
Lysosomes are subcellular organelles containing hydrolytic enzymes capable ofprocessing damaged or redundant cellular components. They are also able to accumulate and detoxify a wide range of toxic metals and organic pollutants, capable ofdamaging cells (Moore 1985, Viarengo et al. 1987). However, the uptake oftoxic compounds can affect lysosomal membrane integrity, which may cause lysosomal contents to leak into the cytoplasm. Changes to the permeability ofthe lysosomal membrane caused by several environmental pollutants can be monitored in vitro by using the NRRT assay (Lowe & Pipe 1994, Lowe et al. 1995b, Ringwood et al. 1998, Dailianis et al. 2003, Harding et al. 2004, Koukouzika & Dimitriadis 2005). In an unstressed state, lysosomes will accumulate and retain the cationic neutral red dye for an extended period of time. However, following a stressor, the destabilized lysosomes will coalesce to form larger lysosomal structures and the neutral red dye will leak into the cytosol ofthe cell across damaged membranes (Moore 1980, Lowe et al. 1995a). The NRR in mussel haemocytes is one ofthe most widely recommended biomarkers in marine
biomonitoring programmes (UNEP 1997, UNEP/RAMOGE 1999).
An MN is formed during the metaphase/anaphase transition of mitosis. It may arise from an intact lagging chromosome (a eugenic event leading to chromosome loss) or from an acentric chromosome fragment detaching from a chromosome after breakage (a clastogenic event) that does not integrate into the daughter nuclei. The MN test has been used in different aquatic organisms (Hose 1985, Burgeot et al. 1995, Hagger et al. 2002, Banni et al. 2003), widely so in the gills and haemocytes ofbivalve molluscs (Brunetti et al. 1988, Scarpato et al. 1990, Wrisberg et al. 1992, Burgeot et al. 1996, Bolognesi et al. 1996, Venier et al. 1997, Bolognesi et al. 1999, Dailianis et al. 2003, Koukouzika & Dimitriadis 2005). Some studies on micronuclei in mussels have focused on evaluating other nuclear abnormalities, like bi-nucleated cells, and eight-shaped, fragmented nuclei or nuclear buds (Venier et al. 1997, Barsiene et al. 2003, Dailianis et al. 2003).
The role ofAChE (EC 126.96.36.199) in cholinergic transmission is to control the nerve impulse by reducing the concentration ofacetylcholine (ACh) at
the synaptic junctions by a catalytic reaction ofACh hydrolysis; muscular tetany and death are thus avoided. Nevertheless, AChE inhibition leads to severe physiological weakening in marine organisms (McHenery et al. 1997, Ozmen et al. 1998). In this field, several studies have emphasized AChE inhibition in bivalves such as M. galloprovincialis as a biomarker in species exposed to organophosphates, carbamates and some heavy metals (Bocquene et al. 1990, Galgani et al. 1992, Bocquene et al. 1997, Najimi et al. 1997, Narbonne et al. 1999, Dellali et al. 2001, Dailianis et al. 2003).
2. Material and methods
2.1. Sampling sites
Mussels (M. galloprovincialis) were collected from the large Oran Harbour (OH; Oran Bay, Algerian west coast), into which 90 million m3 ofuntreated wastewaters are discharged annually by the Oran metropolis and many industrial units. High levels ofheavy metals, polyaromatic hydrocarbons and bacterial density were recorded in the tissues of M. galloprovincialis at this site (Boutiba et al. 2003). The second sampling site was a marine mussel farm located in the rural area of Maarouf(Mrf) in the
0 100 km
Figure 1. Sampling sites located along the Algerian west coast
extreme north-west ofAlgeria, approximately 200 km distant from the first site (Figure 1).
To assess the seasonal pattern ofnaturally (physical and biological) induced stress responses, seawater temperature, salinity, pH and dissolved oxygen were monitored in situ. At the laboratory the food supply (chlorophyll a) (Lorenzen 1967), the protein content (Bradford 1976) in some organs and the mussel condition index were calculated as follows: CI = tissue wt (g)/shell length (mm) x 100 (Kagley et al. 2003). The sampling sites were geo-referenced with Garmin GPS 12 (Table 1).
Table 1. Data from the Oran Harbour (OH) and Maarouf(Mrf) sampling sites
Sampling Sampling Position Temp. Salinity Turbidity Dissolved Chl a
3] [mg m 3
October 2005 35°42'663"N
January 2006 00° 39'320"W
November 2005 35° 04'249"N
March 2006 02° 03'790"W
2.2. Mussel collection and handling
In each season, both male and female mussels were sampled at random from mussel stocks and shipped in coolers with ice packs to the laboratory, where they were maintained in flow-through raceway systems ofseawater at ambient temperature and salinity for at least 2 days prior to experimental use. Depuration ofmussels facilitates the removal ofany residual sediment in the soft tissues or body cavity.
2.3. Evaluation of NRRT in haemolymph
The analytical method was performed according to Lowe et al. (1992) and Lowe et al. (1995b), and the procedure proposed by UNEP/RAMOGE (1999), with slight modifications.
Haemolymph was withdrawn from the posterior adductor muscle of ten mussels in physiological saline so as to obtain a 1:1 v/v suspension ofcell/physiological saline. The suspension obtained from each mussel was spread on Poly-L-Lysine (1/10) prepared slides and transferred to a lightproof humidity chamber for 15 min to allow the cells to attach. After incubation, 40 /Л ofNR working solution were dropped onto each slide. At the end of15 min, the slides were quickly examined at 400x magnification and the images digitalized using computer-enhanced automatic image
analysis. The system included a charged couple device (CCD) Sony colour camera mounted on a Zeiss light microscope. Image software (Pinnacle Studio, v. 8) electronically captured the microscopic images displayed on a television screen (Sony Trinitron) and stored them on a personal computer. Where there was evidence ofdye loss from the lysosomes to the cytosol in at least 50% ofthe cells examined (granular haemocytes), the time following the NR probe application represented the NRRT for the mussel.
2.4. MN in the haemolymph and gill tissue
The micronuclei frequency was determined according to the procedure proposed by UNEP/RAMOGE (1999). Haemolymph was withdrawn from the posterior adductor muscle often mussels in physiological saline so as to obtain a 1:1 v/v suspension ofcell/physiological saline. Suspensions were spread on slides, transferred to a lightproof humidity chamber, and allowed to attach. Cells were then fixed in methanol:acetic acid (3:1), stained with 3% Giemsa and mounted in Eukitt. Gill cells were isolated by enzymatic digestion with a solution ofDispase I (Neutral protease, Boehringer Mannheim, Germany). The cellular suspension obtained by filtration was centrifuged, and aliquots of the resuspended pellet were fixed in methanol:acetic acid (3:1) overnight, spread on slides, stained with 3% Giemsa and mounted in Eukitt. The stained slides were analyzed under the same Zeiss light microscope at a final magnification of 1000x under oil immersion. The scoring ofslides involved examining more than 1000 agranular haemocytes and epithelial-like gill cells. The criteria used for identifying micronuclei are given in UNEP/RAMOGE (1999).
2.5. Determination of AChE activity
Haemolymph was collected from the posterior adductor muscles of five mussels with a sterilized syringe and placed in Eppendorftubes. Digestive gland, haemolymph, gills and mantle/gonad complex were ground in Tris buffer (0.1M, pH 7.5). The haemolymph samples and tissue homogenates obtained were centrifuged at 9000 g for 20 min at 4°C. Aliquots of the supernatant (S9 fraction) were frozen at — 80°Cuntil analysis. The S9 containing the cytosolic proteins was removed and used to determine AChE activity. Protein concentrations were determined according to the Bradford (1976) method using bovine serum albumin (BSA) as standard. AChE activity was determined using the Ellman et al. (1961) method. Acetylthiocholine was hydrolysed by AChE, producing thiocholine and acetic acid. The released thiocholine reacts with 5,5'-dithio-bis-2-nitrobenzoate (DTNB) to produce 5-thio-2-nitrobenzoate (TNB), a yellow compound which absorbs at 412 nm. For this propose, 50 jj\ ofthe stock
solution containing AChE fractions (S9) was added to a reaction mixture containing 850 Tris 100 mM pH 7.5 and 50 of 1.875 mM DTNB (Sigma-Aldrich). After pre-incubation, the reaction was started by the addition of50 //l of8.25 mM acetylthiocholine (Sigma-Aldrich). AChE activity was determined by kinetic measurement for 30 min at 20°Cusingan Anthelie Advanced Junior n°285 spectrophotometer. Results were expressed as nmoles thiocholine produced per min and per mg protein.
2.6. Statistical analysis
Statistical analysis ofthe data - condition index; protein content; haemocyte NRR; MN frequency of haemocytes and gill cells; AChE activity ofhaemolymph, digestive gland, gills and mantle/gonad complex - were based on Duncan's test for multiple comparison and Student's t-test between pairs ofmean values using Microsoft STATISTICA (v. 6.0) statistical software. The significance level for all statistical tests was set at p < 0.05.
3. Results and discussion
3.1. Physical parameters and chlorophyll a
The annual physical parameters in Oran Harbour (OH) summarized in Table 1 reflect the influence ofthe untreated domestic and industrial wastewater released by the city ofOran, mainly in July (when the salinity and dissolved oxygen were lower and the temperature and turbidity were higher). However, at the Maarouf(Mrf) station, all the physical parameters remained relatively constant during the two months ofsampling, except Chl a, whose concentrations were appropriate to the seasonal phytoplankton bloom.
3.2. Biological parameters
The lengths ofthe mussels collected did not vary markedly during the sampling period, ranging from 50 to 82 mm in the natural mussel population (OH) and from 55 to 84 mm in the mussel farm population (Mrf) (Table 2). However, there was an increase in the somatic weight ofthe OH mussels during July and April compared to the other months (October and January) and ofthose from Mrf between November and March (Table 2). The condition index reflects this seasonal somatic weight change
The protein level in the digestive glands, haemolymph, gills and mantle/gonad complex ofthe OH mussels varied significantly during the sampling periods, except in the digestive gland between April and October and in the haemolymph between April and January.
Table 2. Shell length [mm], tissue weight complex [g] and condition index (CI) of mussels, Mytilus galloprovincialis (values are means ± SD, n = 10 per month, per site) sampled from Oran Harbour (OH) and Maarouf(Mrf)
66.70 ± 6.00
7.16 ± 2.17
26.38 ± 7.06cb
64.90 ± 9.80
6.33 ± 3.20
24.20 ± 10.88b
69.80 ± 5.40
6.24 ± 1.76
13.76 ± 6.79a
68.20 ± 5.50