Environmental quality in sediments of Cadiz and Algeciras Bays based on a weight of evidence approach (southern Spanish coast)

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by Elsevier in Marine Pollution Bulletin, Vol. 110, Issue 1 on September 2016, available at: https://doi.org/10.1016/j.marpolbul.2016.06.078 © 2016 Elsevier. En idUS Licencia Creative Commons CC BY-NC-ND Environmental quality in sediments of Cadiz and Algeciras Bays based on a weight of evidence approach (southern Spanish coast) José Antonio Useroa, Daniel Rosado*a,b , José Useroa, José Morilloa a Department of Chemical and Environmental Engineering, Universidad de Sevilla, 41092 Sevilla, Spain b Department of Chemistry, Universidad Técnica Particular de Loja, 11 01 608 Loja, Ecuador HIGHLIGHTS:  A WOE approach has been applied to sediments of Cadiz and Algeciras Bays.  In Cadiz Bay, the Inner Bay showed highest degradation.  In Algeciras Bay, industrialised areas were the most degraded.  Some areas exceed guidelines for aqua regia extractable metals and toxicity. CORRESPONDING AUTHOR: * Department of Chemical and Environmental Engineering, Universidad de Sevilla, Camino de los Descubrimientos, s/n, 41092 Sevilla, Spain. Tel.: 0034 954 487 274; E-mail address: d[email protected]s, [email protected] (Daniel Jesús Rosado Alcarria). 1 Abstract This research applies an integrated sediment quality assessment method using a weight of evidence approach to Cadiz and Algeciras Bays (southern Spain). The method is composed of several analysis (particle size profile, aqua regia extractable metals, acid labile metals, total organic carbon, toxicity bioassay with Photobacterium phosphoreum and macrobenthic community alteration). The proposed method provides a single result, the environmental degradation index (EDI). EDI defined samples as low degraded (outer areas of both bays) and moderately degraded (Inner Bay of Cadiz Bay, surroundings of Algeciras port and the north of Algeciras Bay). These samples showed the highest concentration of aqua regia extractable metals, which exceeded effects range-low (ERL) for Zn (51-176 mg/l), Cu (11-54 mg/l), As (4.3-9.5 mg/l), Hg (0.17-0.28 mg/l), Ni (23-82 mg/l), and. Cr (37-134 mg/l). They also exceeded some quality criteria for total organic carbon (4.0-6.5%) and toxicity (120-240 TU/g) and showed poor results for macrobenthic community. Keywords Sediment quality; ecological risk assessment; trace metals; sediment toxicity; benthic macroinvertebrates; Cadiz Algeciras Bays. 2 1 Introduction Waste has been dumped into the aquatic ecosystems for decades (Ahuja, 2013; Earnhart, 2013), and pollution is reaching worrying levels in some areas (Baldassin et al., 2016; Vandermeersch et al., 2015; Zhou et al., 2016). In the aquatic environment, many pollutants bind onto sediments and can reach concentrations higher than those in the water column. Changes in environmental conditions, such as pH or redox potential, can release a fraction of the contaminants retained in the sediments and re-solubilize them in water (Wang et al., 2015) . Thus, sediments may act as a sink or a source of contamination and, in the latter case, may affect aquatic life, or even humans, through the food chain (Mackay et al., 2016). This was the case of Minamata Bay and the Jinzu River basin (Horiguchi, 2014; Mason et al., 2006; Tomiyasu et al., 2006). The population of Minamata Bay was exposed to methylmercury through the consumption of seafood during the 50s and 60s of the last century. Methylmercury was discharged into the sea by a nearby factory and caused mental disorders to between 700 and 4,000 people and death to a percentage of them (Harrison, 2001). Regarding the Jinzu River, mining companies released cadmium into the river during the last century. The river was used mainly for irrigation of rice fields. This led to high cadmium levels in the people fed with this rice and itai itai desease (Horiguchi, 2014). As of July 2009, 195 persons were officially designated as Itai-Itai disease patients in the region (Nogawa and Suwazono, 2011). Because of this, contaminants in the sediments of aquatic ecosystems have become one of the most important environmental problem at present (Chapman and Wang, 2001; Ruiz et al., 2008; Sainz and Ruiz, 2006) and many researches are carried out in this topic (Chapman and Smith, 2012; Chapman et al., 2013; Foster et al., 2015; Nowell et al., 2016; Rosado et al., 2016, 2015a; Testa et al., 2013; Yunker et al., 2015). 3 Trace elements are bioavailable and persistent in the environment causing bioaccumulation and toxicity effects in the biota. Typically, benthic invertebrates start bioaccumulation by both the absorption from interstitial water and ingested sediment and transfer them to higher trophic levels, extending the hazard (Conti et al., 2016). The uptake of trace metals by benthic organisms depends largely on their chemical forms (Morillo et al., 2008). Integrated sediment quality assessment methods using a weight of evidence (WOE) approach measure and integrate metrics from different lines of evidence, e.g., chemical characterization, toxicity testing, and biological surveys (Anderson et al., 2003; Chapman, 2002, 1992, 1990; Chapman et al., 1997; Cherry, 2001; Crane, 2003; Ghirardini et al., 1999; Khosrovyan et al., 2015; Long and Chapman, 1985; Qi et al., 2015). This allows to conduct a comprehensive assessment of the quality of sediment, as they provide more reliable information than the use of single techniques (Buruaem et al., 2013; Chapman and Hollert, 2006; Chapman, 2007; Riba et al., 2004). This research has two main objectives. First, to test a modified version of an integrated sediment quality assessment method previously proposed by Rosado et al. (2015b) using a WOE approach which is simple, low-cost and provides an easily understandable and comparable result. Second, to implement the modified version of the integrated sediment quality assessment method in the Algeciras and Cadiz Bays, where anthropogenic sources of pollutants and areas of high ecological and economic value coexist in a relatively small zone. 2 Study area The areas studied are the bays of Cadiz and Algeciras, located in southern Spain (Figure 1), whose sediments are affected by discharges from major urban centers, large and small industries, intense port activity and, to a lesser extent, farming. 4 2.1 Cadiz Bay Cadiz Bay is located in the southwest of the Spanish Atlantic coast, between the town of Rota and the city of Cadiz, and covers an area of 110 km2. It can be divided into two main maritime regions. The Inner Bay, located south of the bridge José Leon de Carranza, is characterized by a maximum depth of five meters and a low rate of water renewal, which favors accumulation of pollutants discharged. The Outer Bay, that is extended northward from the bridge up to Rota, is deeper, open to the Atlantic Ocean and thus, the renewal of its waters is faster (Araújo et al., 2009; Ligero et al., 2002, 2004). The prevailing currents in the coasts of the Gulf of Cadiz come from west-northwest generating an anticlockwise circular current in the Outer Bay. The Inner Bay is protected from the surge and, partially, the wind, so water dynamics are tidally controlled causing a less defined current with several gyres (Ligero et al., 2002; Periáñez, 2009; Periáñez et al., 2013). In the Outer Bay empties the Guadalete River, the main river in the area. Cadiz Bay hosts several towns (Rota, El Puerto de Santa María, Puerto Real, San Fernando, Chiclana de la Frontera and Cadiz) whose population exceeds 400,000 inhabitants (Carrasco et al., 2003). In this bay also takes place a remarkable industrial activity, including shipyards and car and aircraft components manufacturers. Furthermore, there is a high maritime traffic due to merchant ships and, increasingly, to cruises and ferries between the Iberian Peninsula and the north of Africa and the Canary Islands. Cadiz Bay includes some areas cataloged as Special Protection Area under European Union's Birds Directive: the Cadiz Bay Natural Park and the Trocadero Island and Sancti Petri Marshes nature reserves. 5 2.2 Algeciras Bay Algeciras Bay is located on the southern end of the Iberian Peninsula, next to the Strait of Gibraltar, between two important water bodies, the Atlantic Ocean and the Mediterranean Sea, and two continents, Europe and Africa. This bay is a semi-enclosed water body in a horseshoe shape, with a maximum width of 9 km and a north-south length of about 10 km. The endpoints that define the mouth of the bay are Punta Carnero in the west and Punta Europa in the east, defining a water surface of about 75 km2 with a depth of up to 400 m (Sammartino et al., 2014). The main rivers which drain into the bay are the Guadarranque and Palmones rivers. Westerly winds are predominant in the area and they lead to a clockwise water circulation, i.e., runs along the coast starting at the west end (Punta Carnero) to the east end (Punta Europa). This current can be switched to anticlockwise in the case of easterly winds (Periáñez, 2012; Sánchez-Garrido et al., 2014). The maximum tidal stands at 0.8 m along the entire shore (Periáñez, 2012). Five important cities located around the bay (Algeciras, Los Barrios, San Roque, La Línea de la Concepción and Gibraltar) are home to more than 250,000 inhabitants (Kosore et al., 2015). The major industries in the area are mainly located in the northern part of the bay and include petrochemical and petroleum refineries, a stainless steel manufacturing plant, four power plants and a biofuel production plant (Tarazona et al., 1991). Two ports are also located in this bay: Gibraltar and Algeciras. The first is one of the Europe's top port for refueling. The latter is ranked among the most important ports of the world (Kosore et al., 2015). Thus, bay waters are subject to one of the most intense maritime traffic all over the Mediterranean Sea (Díaz-de Alba et al., 2011) and large amounts of pollutants are discharged into the bay (Carballo and Naranjo, 2002; Morillo et al., 2007; Tarazona et al., 1991). 6 Within the Algeciras Bay is located the Palmones River Marshes Nature Reserve, cataloged as a Special Protection Area under European Union's Birds Directive, where birds stop for their migrations. Next to the southwestern end of the Algeciras Bay there is another protected area, the Strait of Gibraltar Natural Park. Figure 1. Location of the sampling points in Cadiz and Algeciras Bays. 3 Materials and methods 3.1 Sampling and sample pre-treatment 5 sampling points were located in Algeciras Bay (A1-A5), 5 in Cadiz Bay (C1-C5) and 1 in a pollution free zone (R) of Cadiz Littoral to serve as a reference (see Table 1 and Figure 1). 7 Table 1. Coordinates of the sampling points. Station North West R 36o17'34'' 6o09'09'' A1 36o05'42'' 5o25'27'' A2 36o08'31'' 5o25'53'' A3 36o09'35'' 5o25'46'' A4 36o10'42'' 5o24'06'' A5 36o09'39'' 5o22'22'' C1 36o36'14'' 6o17'04'' C2 36o34'32'' 6o13'52'' C3 36o32'33'' 6o14'24'' C4 36o30'16'' 6o14'42'' C5 36o29'26'' 6o13'20'' Sediment samples were taken in February 2015 with a Van Veen grab and in duplicate at each station. Only grabs that achieved adequate penetration (2/3 of total volume) to collect the first 5 cm of the sediment and that showed no evidence of leakage or surface disturbance were retained, transferred and stored in a dark cooler at 4 °C. In the laboratory, samples were divided into two fractions as shown in Table 2: fraction 1, without any further treatment, to determine particle size profiles, and fraction 2, dried (60 oC) and sieved (<63 µm), to perform metal analysis (aqua regia extractable and acid labile fractions), total organic carbon and the toxicity bioassay. All conventional sediment parameters were expressed as percentages of total dry sample weight (105 ºC). Organism samples were taken in duplicate in the same sampling points and dates as the sediment samples. Every sample was composed of five subsamples collected with a 0.05 m2 surface Van Veen grab. Subsamples were sieved in situ through a 0.5-mm mesh and were preserved in a solution made of ethanol and bengal rose stain (0.4 g of bengal rose in 1 L of ethanol) for one week. 14 Table 4. Results of the tests conducted in the laboratory (mean ± SD). R C1 C2 C3 C4 C5 A1 A2 A3 A4 A5 Particle size profiles Sediment type 1 FS FS VFS MS VFS S FS FS MS FS VCS Aqua regia extractable metals (mg/kg) Zn 38 ± 0.63% 46 ± 1.04 % 85 ± 2.38 % 78 ± 3.34 % 124 ± 0.23 % 176 ± 0.46 % 39 ± 2.93 % 154 ± 1.6 4% 43 ± 0.45% 51 ± 4.43% 46 ± 4.79% Cu 5.6 ± 5.68% 6.2 ± 5.98 % 13 ± 0.32% 12 ± 5.24% 35 ± 7.55 % 41 ± 3.63 % 8.5 ± 2.43 % 54 ± 0.65 % 8.9 ± 8.65% 11 ± 3.21% 9.4 ± 8.41% Cd 0.3 ± 1.79 % 0.3 ± 8.23 % 0.4 ± 10.61 % 0.3 ± 7.25% 0.3 ± 14.59 % 0.4 ± 13.48 % 0.4 ± 13.37 % 0.5 ± 9.70 % 0.4 ± 12.44% 0.7 ± 8.04% 0.4 ± 14.81% Pb 6.1 ± 8.70% 7.5 ± 4.07 % 11 ± 6.43 % 10 ± 4.68% 28 ± 0.16 % 30 ± 8.29 % 7.2 ± 2.15 % 23 ± 9.67 % 7.8 ± 8.64% 16 ± 6.87% 7.9 ± 2.20% Fe 2 22 ± 0.43% 22.5 ± 0.76 % 23.4 ± 0.82 % 24.1 ± 3.75% 29 ± 4.06 % 28.5 ± 3.20 % 21.4 ± 4.52 % 24.2 ± 3.51% 21.7 ± 2.54% 26.2 ± 0.04% 22.5 ± 1.40% As 3.7 ± 8.74% 3.9 ± 4.98 % 7.3 ± 6.61 % 4.2 ± 9.62 % 7.9 ± 0.23 % 9.5 ± 6.13 % 3.9 ± 9.59 % 7.8 ± 4.62 % 4.2 ± 6.29% 4.3 ± 4.08% 4.2 ± 7.07% Hg 0.10 ± 7.23% 0.11 ± 5.78% 0.14 ± 7.24% 0.12 ± 9.91% 0.28 ± 4.34 % 0.24 ± 10.51 % 0.12 ± 11 . 00 % 0.17 ± 1.69 % 0.13 ± 5.97% 0.27 ± 14.44% 0.14 ± 8.40% Mn 410 ± 0.65% 404 ± 4.22% 430 ± 0.72% 420 ± 2.59% 416 ± 3.43 % 395 ± 1.91 % 410 ± 1.59 % 460 ± 4.81% 324 ± 0.48% 368 ± 3.11% 350 ± 2.11% Ni 4.0 ± 7.41% 4.0 ± 2.35% 12 ± 6.81% 15 ± 1.85% 23 ± 5.8 5 % 25 ± 9.19 % 19 ± 6.44 % 50 ± 1.68% 20 ± 7.64% 82 ± 6.32% 50 ± 8.50% Cr 14 ± 4.53% 16 ± 1.65% 18 ± 0.26% 25 ± 0.36 % 37 ± 1.69 % 52 ± 1.70 % 25 ± 0.8 3 % 95 ± 4.31% 28 ± 3.68% 134 ± 4.50% 80 ± 2.31% Acid labile metals (mg/kg) Zn 1.7 ±6.14% 2.9 ±0.71% 7.3 ±5.08% 3.6 ±6.32% 6.9 ±8.29% 13 ±1.40% 2.5 ±2.00% 13 ±6.49% 3.1 ±3.97% 6.1 ±7.05% 3.6 ±9.94% Cu 0.6 ±2.85% 0.7 ±6.37% 1.3 ±8.39% 1.4 ±5.19% 4.6 ±9.17% 6.6 ±9.61% 1.2 ±10.73% 6.5 ±9.20% 1.2 ±8.73% 2.0 ±2.00% 1.4 ±9.55% Cd 0.03 ±3.82% 0.03 ±0.24% 0.06 ±9.54% 0.03 ± 11.24 % 0.04 ± 10.05 % 0.06 ±11.28% 0.04 ±1.07% 0.06 ±1.24% 0.04 ±5.59% 0.11 ±9.99% 0.06 ± 12.21 % Pb 1.0 ±6.26% 1.1 ±11.28% 2.1 ±6.94% 1.9 ±9.98% 3.9 ±4.98% 5.7 ±6.04% 1.4 ±13.73% 6.9 ±3.38% 0.7 ±2.24% 2.7 ±11.59% 1.0 ±3.30% Fe 264 ±3.49% 360 ±4.32% 234 ±1.05% 193 ±0.38% 232 ±4.23% 399 ±2.44% 257 ±2.47% 436 ±4.51% 412 ±1.00% 550 ±0.52% 360 ±2.40% Mn 119 ±2.73% 117 ±2.65% 155 ± 3.20% 139 ±1.15% 125 ±4.25% 134 ±2.07% 119 ±1.85% 161 ±2.84% 94 ±0.66% 121 ±0.71% 109 ±0.52% Ni 0.44 ±2.39% 0.48 ±4.66% 1.3 ±7.68% 1.5 ±8.72% 1.8 ±0.96% 3.5 ±9.15% 2.7 ± 7.73% 9.0 ±1.09% 3.6 ±1.31% 17 ±3.60% 6.5 ±9.07% Cr 1.1 ±7.85% 2.1 ±5.81% 2.5 ±1.97% 2.0 ±9.84% 4.4 ±13.18% 7.8 ±3.33% 2.5 ±6.73% 15 ±13.62% 2.7 ±0.04% 27 ±14.37% 10 ±10.53% Total organic carbon (%) 1.2 ± 4.70% 1.3 ± 5.21% 3.9 ± 9.48% 1.8 ± 1.45% 5.3 ± 8.95% 6.5 ± 5.39% 1.5 ± 8.48% 5.0 ± 2.78% 1.8 ± 5.87% 4.0 ± 8.67% 2.4 ± 0.86% Toxicity bioassay (TU/g) 39 ± 6.95% 42 ± 13.17% 90 ± 8.36% 48 ± 5.06% 120 ± 6.97% 240 ± 3.43% 43 ± 1.44% 210 ± 4.57% 62 ± 11.31% 170 ± 4.66% 51 ± 0.21% Macrobenthic community Number of families 48 ±8.20% 43 ± 6.20% 33 ± 1.37% 40 ± 1.21% 30 ± 7.54% 24 ± 6.02% 43 ± 3.83% 28 ± 9.05% 50 ± 3.34% 31 ± 6.75% 56 ± 4.91% M - AMBI index 0.89 ± 5.29% 0.86 ± 5.16% 0.76 ± 0.31% 0.76 ± 0.44% 0.65 ± 5.18% 0.62 ± 4.38% 0.81 ± 0.56% 0.70 ± 1.92% 0.93 ± 2.36% 0.75 ± 4.77% 0.99 ± 2.99% 1 VCS: very coarse sand; CS: coarse sand; MS: medium sand; FS: fine sand; VFS: very fine sand; S: silt; 2 Fe total content is expressed in g/kg. 15 4.1.2 Aqua regia extractable metals In Cadiz Bay, the highest concentrations of most metals are found in the Inner Bay (C4 and C5), as shown in Table 4. Pb, As, Ni and Hg differences are significant (p<0.05) between the Inner Bay and the concentrations in other sampling points of Cadiz Bay. The Inner Bay is an area of shallow draft (with depths below 5 meters), fine sediment grain size (fine sand or silt) and low water renewal, which favors the accumulation of pollutants from sewage, factories, maritime traffic, etc. Maximum concentrations of most metals (Zn, Cu, Pb, As, Ni, and Cr) in Cadiz Bay are located at the station C5 since it is more isolated from the open sea than the sampling point C4. Differences between stations C5 and C4 are significant (p<0.05) for Zn, Cu and Cr. However, highest concentrations of Hg and Fe are found in the station C4. Aqua regia extractable metals in the Outer Bay (C1 to C3) are similar or slightly higher than those in the reference station (R), far from sources of pollution. These results are explained by higher depth, coarser particle size and short flushing time (Periáñez et al., 2013), which favors the dispersion of the pollutants discharged. The lowest concentrations of most metals in the Outer Bay are found in station C1. However, only Zn shows a significant difference (p<0.05) between C1 and both C2 and C3. By contrast, in station C2 occur the highest concentrations of Zn, Cu, Pb, Cd, As and Mn by the influence of the Guadalete river. Nevertheless, only As shows a significant difference between C2 and both C1 and C3. Mn in station C2 reach the highest concentration in Cadiz Bay, including the Inner Bay. In Algeciras Bay, the highest concentrations of metals (p<0.05) are located at sampling points A2 (Cu, Zn, Pb, As and Mn), in the vicinity of Algeciras municipality and its port, and A4 (Ni, Cr, Cd and Hg), which concentrates the largest industrial activity in the bay. 16 Highest concentration of Fe is also found at sampling point A4. However, difference is not significant between A4 and A2 (p>0.05). Lowest concentrations of metals occur in stations A1 (Zn, Cu, Pb, As, Fe, Cr, Ni and Hg) and A3. Nonetheless, only Mn shows significant difference between A1 and A3 (p<0.05). Reference station (R) shows the lowest concentrations of Zn, Cu, Pb, As, Cr, Hg and Ni in all areas studied. The reference point is also most likely to be the lowest value for Cd given the lower standard deviation. The reference point is above the minimum value recorded in some metals (Fe and Mn) whose natural concentrations are higher and the influence of pollution is less relevant. Table 5 shows the quality criteria for sediments proposed by both Long et al. (1995) and the Centro de Estudios y Experimentación de Obras Públicas (CEDEX) (DelValls et al., 2004). Long et al. (1995) criteria are based on the potential to induce toxic effects on marine organisms. The criteria are divided into two values: effects range-low (ERL) and range-mediated effects (ERM). The ERL refers to the concentration at which toxic effects were identified in the lower 10th percentile for each chemical. The ERM denotes the same concept but applied to the median, or 50th percentile. CEDEX’s criteria are widely used by Spanish scientists to decide the proper management for dredged material in Spanish ports (DelValls et al., 2004). When concentrations of all metals are below AL1, sediments are considered not contaminated and can be discharged freely into the sea. If the concentration of any metal is between AL1 and AL2, the sediment is defined as moderately contaminated. In this case, it is required to select a disposal area justifying its suitability. It is also necessary to study the possible transport of the fine fraction, estimate the impact on the ecosystem and design an environmental monitoring program. If the concentration of any metal exceeds AL2, the sediment is classified as highly contaminated. Thus, justification about treatment prior to disposal and confinement is necessary. 17 Table 5. Quality criteria for total metal concentrations in sediments (mg/kg, dry weight). Metal Long et al. (1995) DelValls et al. (2004) ERL1 ERM2 AL13 AL24 Zn 150 410 500 3000 Cu 34 270 100 400 Cd 1.2 9.6 1.0 5.0 Pb 46.7 218 120 600 Fe - - - - As 8.2 70 80 200 Hg 0.15 0.71 0.6 3.0 Mn - - - - Ni 20.9 51.6 100 400 Cr 81 370 200 1000 1 ERL: effects range-low. 2 ERM: effects range-median. 3 AL1: action level 1. 4 AL2: action level 2. In Cadiz Bay, aqua regia extractable metals in the sediments of the Outer Bay are below the quality criteria proposed by Long et al. (1995), whereas both stations in the Inner Bay (C4 and C5) exceed the ERL's for Ni, Cu and Hg. Furthermore, C5 station exceeds ERL for Zn and As. In Algeciras Bay, station A2 exceeds ERLs for Zn, Ni, Cr, Cu and Hg and station A4 exceeds ERL for Cr and ERM for Ni. In the reference station (R) none of the quality criteria proposed by Long et al. (1995) are exceeded. None of the analyzed samples exceed CEDEX recommended action level 1, indicating that these sediments could be dumped into the sea without taking special precautions. 4.1.3 Acid labile metals Around 30% of Mn is part of the acid soluble fraction, being the metal with the highest percentage (Table 4). Thus, Mn is bound more weakly to sediments, has the greatest mobility and is, probably, the most bioavailable. These high percentages of Mn in acid soluble fraction are probably related to a large amount of manganese carbonates in sediments (Dassenakis et al., 2003). Other studies of marine sediments also found high 18 percentages of Mn in the acid soluble fraction (Kiratli and Ergin, 1996; Marin and Giresse, 2001; Morillo et al., 2002). Fe is the metal with the lowest percentage in the acid soluble fraction (slightly above 1%) and thus, the strongest attached to sediment and the least mobile. It is probably due to its fundamentally natural origin and its abundance in the Earth's crust. These results agree with those published by Morillo et al. (2007) who found that most of Fe in sediments forms crystalline oxides (goethite, limonite, magnetite, etc.) strongly attached to the sediment. Zn shows the second lower percentage in the acid soluble fraction (less than 9%). These results agree with those obtained in other studies (Dawson and Macklin, 1998; Hudson- Edwards et al., 1996; Moalla et al., 1997) in which most of the Zn was in the reducible fraction (second step of the BCR protocol). In the reference station (R), the lowest percentages of extraction is obtained for most elements. This fact is related to the natural origin of metals in this station, which tend to be more firmly attached to the sediment than those from an anthropogenic source (Rosado et al., 2015b). By contrast, in Algeciras Bay are located higher percentages in the acid soluble fraction for Ni, Cr, Cu and Zn. 4.1.4 Total organic carbon The total organic carbon shows no significant difference between the Cadiz and Algeciras Bays (p>0.05), although large differences within each area are appreciated. The highest concentrations (p<0.05) of total organic carbon in the Cadiz Bay (Table 4) are located in the Inner Bay (C4, 5.3%; C5, 6.5%), followed by the surroundings of the Guadalete river mouth (C2, 3.9%). The Inner Bay accumulates pollutants present in sewage from urban areas near the Bay (San Fernando, Puerto Real, Puerto de Santa Maria and Cadiz) by low 19 water renewal and shallow draft. The Guadalete River is affected by sewage from large populations, such as Jerez and Arcos, discharges from winemaking and sugar industries, effluents from agricultural activities, etc. In Algeciras Bay, the largest average concentrations of total organic carbon found in the vicinity of Algeciras municipality and its port (A2, 5.0%), followed by the surroundings of the oil refinery (A4, 4.0%), which concentrates the largest industrial activity in the bay. The values reached in these sampling points are significantly higher (p<0.05) than the rest of the stations in the area and the median total organic carbon content in the sediments of coastlines around the world, set at 1.5% (Seiter et al., 2004). Lowest levels of total organic carbon in the sediments are located at the reference station (R, 1.2%), in the outer areas of the bays (C1, 1.3%; A1, 1.5%) and the outermost stations of discharges (C3, 1.8%; A3, 1.8%). The total organic carbon values of these stations show no significant difference with the reference point (p>0.05) and are similar to the median of total organic carbon content in sediment coastlines around the world, set at 1.5% (Seiter et al., 2004). 4.1.5 Toxicity bioassay with P. phosphoreum The results of the toxicity bioassay (Table 4) are similar to those of total organic carbon and metals in terms of areas and sampling points with highest and lowest values. Thus, major toxicities of Cadiz Bay (p<0.05) occur in the Inner Bay (C5, 240 TU/g; C4, 120 TU/g). These are followed by the surroundings of the Guadalete river mouth (C2, 90 TU/g). In Algeciras Bay, the area with greater toxicity (p<0.05) corresponds to the surroundings of Algeciras municipality and its port (A2, 210 TU/g) and the vicinity of the oil refinery (A4, 170 TU/g). These values are similar to those found by Martínez-Lladó et al. (2007) in the sediments of the Port of Barcelona (average 240 TU/g). 20 The lowest toxicity is located in the reference station (R, 39 TU/g) and in the outer areas of both bays (C1, 42 TU/g; A1, 43 TU/g). These values are similar to those obtained in a nearby coast, the Huelva Littoral, with an average of 35 TU/g (Usero et al., 2008). Considering the sediment toxicity quality criteria established in 100 TU/g (Environment Canada, 2002) and 133 TU/g (Casado-Martínez et al., 2006; DelValls et al., 2004; Morales-Caselles et al., 2008), C5, A2 and A4 samples are considered toxic by both thresholds, while C4 is only considered toxic according to the threshold set by Environment Canada (2002). None of the other stations are considered toxic. 4.2 Organism sample analysis The analyses of the macrobenthic community in sediments have identified a total of 11 taxonomic groups, three of which are well represented: annelids (25 families), crustaceans (32 families) and molluscs (28 families). To a lesser extent are also present echinoderms (3 families) and cnidarians (2 families) and 6 other groups with only one family each (nemertea, phoronida, cephalochordate, sipuncula, tunicate and turbellaria). The predominant group corresponds to the annelids, which has the largest number of families in all sampling points, except for the L2, O1 and T1. The group of annelids is only represented by polychaete class, mainly organisms of families Spionidae, Capitellidae and Syllidae. These families are composed of opportunistic species or primary colonizers who take advantage of any alteration of the environment to occupy a large ecological niche. Crustaceans are the next largest group, accounting for 20-45% of families in all sampling points. Among them, amphipods class is the most widely represented (15 families) including families Apseudidae, Corophiidae and Haustoridae. Molluscs are the third largest group, ranging from 8-35% of families in all sampling points. They represent 35% of all 21 families in station A1, becoming the most abundant taxonomic group. The other groups, composed of 8 taxa, only represent between 5-17% of families in all sampling points. Regarding the number of families and the M-AMBI index, results show a wider diversity in macrobenthic community where pollution is lower. As shown in Table 4, number of families and M-AMBI index are lower in stations A2 and A4 in Algeciras Bay and C4 and C5, the Inner Bay of Cadiz Bay. The latter (C5) reaches the lowest value of the entire study area. This fact is undoubtedly related to two peculiarities. First, C5 hosts the highest levels of Zn, Pb, As and toxicity of the study area. Second, the particle size is very fine (silt), which complicate organisms to inhabit the sediment due to the small gaps between particles. Overall, in similar sediments the number and diversity of macrobenthic community is higher in coarser particle size (Rosado et al., 2015b). 4.3 Integrated sediment quality assessment method 4.3.1 Pollution indices As shown in the Figure 2, the results of four indices (ICI, IB, ICO and IT) follow a common pattern. In Cadiz Bay, the maximum values of these indices are located in the Inner Bay (C4 and C5), followed by the surroundings of the Guadalete river mouth (C2). In Algeciras Bay, these indices reach their highest in the vicinity of Algeciras municipality and its port (A2). The lowest values of these indices are located in the outer areas of the bays (C1 and A1) and the reference station (R). The latter (R) shows the lowest values for the four indices, indicating that fulfills its function correctly as a reference sample. 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