Marine Pollution Bulletin 94 (2015) 185–198
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Histopathological indices in sole (Solea solea) and hake (Merluccius merluccius) for implementation of the European Marine Strategy Framework Directive along the Basque continental shelf (SE Bay of Biscay) Nagore Cuevas a,⇑, Izaskun Zorita b, Pedro M. Costa c, Iñaki Quincoces a, Joana Larreta b, Javier Franco b a
AZTI-Tecnalia, Txatxarramendi ugartea z/g, 48395 Sukarrieta, Spain AZTI-Tecnalia, Herrera Kaia, Portualdea z/g, 20110 Pasaia, Spain c MARE-Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia, Faculdade de ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b
a r t i c l e
i n f o
Article history: Available online 12 March 2015 Keywords: Merluccius merluccius Solea solea Histopathology Contaminants Basque continental shelf
a b s t r a c t Sole and hake, together with sediments, were collected during two campaigns along the Basque continental shelf to study the utility of two existing histopathological indices for assessing the biological effects of contaminants to implement the European Marine Strategy Framework Directive. Hepatic and gonadal histopathology were measured, and metal and/or organic contaminants were determined in both liver and sediments. Sediments from the Basque continental shelf were found to be moderately impacted by metals but non-impacted by organic compounds. Metal bioaccumulation and histopathological lesions in liver were higher in sole than in hake, although non-specific and early non-neoplastic toxicopathic lesions were observed in both species. No gross alterations were recorded in gonad. The two histopathological indices applied were highly correlated in both organs but the lack of correlation between sediment contamination levels, bioaccumulation and histopathological indices suggests that other factors, rather than pollution alone, are responsible for the biological effects observed. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The European Marine Strategy Framework Directive (MSFD; Directive 2008/56/EC) aims to achieve or maintain ‘‘Good Environmental Status’’ (GEnS) in European marine waters by 2020. This directive contains the first conceptual and practical guidelines for the development of efficient monitoring programmes in order to determine the status of marine environments (Borja et al., 2010; Lyons et al., 2010). Thus, the MSFD bases GEnS on eleven qualitative descriptors, with Descriptor 8 being formulated as ‘‘Concentrations of contaminants are at levels not giving rise to pollution effects’’, thereby implying that contamination is to be monitored and assessed in order to determine whether the levels of toxicants in the environment reach levels that are able to induce adverse effects in biota, thus becoming pollution. These contaminants that may cause harmful biological effects have been selected as priority chemical pollutants of EU-wide concern (Borja et al., 2010). The majority of these chemical substances are organic ⇑ Corresponding author. Tel.: +34 94 657 40 00; fax: +34 94 657 25 55. E-mail address: [email protected] (N. Cuevas). http://dx.doi.org/10.1016/j.marpolbul.2015.02.030 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.
compounds (e.g. polychlorinated biphenyls), although four metals (i.e. Cd, Ni, Pb and Hg) are also considered as priority substances (Directive 2008/105/EC). The achievement of GEnS for Descriptor 8 relies on monitoring programmes covering the concentrations of chemical contaminants determined in different matrices, such as water, sediment or biota, and measuring the biological effects of pollutants on marine organisms (Law et al., 2010). According to the MSFD, such an assessment must be carried out for each marine region or subregion, for example the Bay of Biscay and Iberian coasts, within the North East Atlantic Ocean (Borja, 2006). Consequently, some authors have already proposed standardised sampling strategies using diverse sentinel organisms for biomonitoring in European marine waters within the scope of the MSFD (e.g. Benedetti et al., 2014; Costa et al., 2013). However, a detailed understanding of the responsiveness of potential target species towards toxicants and environmental parameters is required in order to achieve an adequate level of quality assurance (Lyons et al., 2010). Flatfishes (Teleostei: Pleuronectiformes) have been widely employed as sentinel species, partly due to their benthic behaviour, which makes them appropriate organisms for biomonitoring marine
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sediments (Reynolds et al., 2003; Stentiford et al., 2003). Species such as dab (Limanda limanda), European flounder (Platichthys flesus) and English sole (Pleuronectes vetulus) are the main target species for monitoring purposes in Northern Europe and North-Western America (see for instance Köhler et al., 1992; Lang et al., 2006; Myers et al., 1998; Vethaak and Wester, 1996). However, as these species are less abundant around the Iberian Peninsula, other alternative species are likely to be more appropriate sentinels. One of the most abundant flatfish found on the Basque continental shelf (South-Eastern Bay of Biscay) is the sole (Solea solea), although the presence of Senegalese sole (Solea senegalensis) is also remarkable in offshore waters and estuaries (Franco et al., 2012; Quincoces et al., 2011). In this respect, adult soles are characterised by a high habitat fidelity, thus making them a site-specific species (Pawson, 1995), although they do undergo short seasonal migrations from deeper offshore areas to shallower spawning grounds. The European hake (Merluccius merluccius) has been also targeted as a sentinel organism for the Basque continental shelf, although to a lesser extent than flatfishes (Marigómez et al., 2006; Raingeard et al., 2009). The largest European hake nurseries are located in the Bay of Biscay (Casey and Pereiro, 1995), thus making this species an ideal candidate for biomonitoring approaches along the Basque continental shelf. Hake performs nictemeral migrations due to food availability; thus, it is a demersal species by day and pelagic at night (Sánchez and Gil, 2000). In addition, adults concentrate in canyons and on the rocky bottoms of the shelf break areas (Sánchez, 1993). As such, commercially exploited species, such as sole and hake, are of particular value for determining a plausible link between ecological and human risk. Fish diseases and histopathological alterations are increasingly used as indicators of pollution effects since they provide a relevant biological end-point of historical exposure (Stentiford et al., 2003). In this regard, essentially qualitative histopathological approaches have provided vital information regarding the description of histological alterations in field-collected or tested aquatic organisms (e.g. Costa et al., 2009; Stehr et al., 1998). Nonetheless, the absence of some sort of quantification makes it difficult to establish statistically significant cause-effect relationships between pathology and contaminants and to assess the significance of the differences between areas or campaigns (Costa et al., 2009; Lang et al., 2006). Therefore, semi-quantitative histopathological approaches, especially those that consider the biological significance in addition to the dissemination of histopathological changes, may effectively circumvent this issue while conferring a wider degree of biological significance (Costa et al., 2009; Vethaak and Wester, 1996). Although different semi-quantitative histopathological indices are employed for the assessment of health status in target species (Bernet et al., 1999; Triebskorn et al., 2008; van Dyk et al., 2007), the present research is focused on the adaptation, application and comparison of two existing indices, one developed by Costa et al. (2009) for sole and another one by Lang et al. (2006) for flounder. Fish livers and gonads are considered to be ideal target organs for the assessment of biological effects caused by pollution due to their role in xenobiotic transformation, storage and elimination (Hinton et al., 2001) and their relationship with the reproductive cycle and liability for upcoming generations (Blazer, 2002), respectively. Moreover, several emerging pollutants, known as endocrine disrupting chemicals, may affect the reproductive potential of gonads, thereby provoking alterations, such as intersex, that can be detected histologically (Bateman et al., 2004). In this work, the concentrations of some priority substances (according to the Directive 2008/ 105/EC) with high potential to cause adverse biological effects have been assessed; metals have been measured both in the sediments and the fish tissues; and polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) and some organochloride pesticides were analysed in the sediments.
The present work aims to: (1) test potential sentinel fishes (sole and hake) for biomonitoring approaches along the North Atlantic Iberian shelf, namely in the Basque continental shelf; (2) adapt, apply and compare two existing histopathological indices: that developed by Costa et al. (2009) and that proposed by Lang et al. (2006) in the liver and gonads of sole and hake; and (3) evaluate fish histopathology as an indicator of environmental pollution along the Basque continental shelf with regard to implementation of the European Marine Strategy Framework Directive. 2. Materials and methods 2.1. Study area and sample collection The Basque continental shelf is located in the South-Eastern Bay of Biscay and its total length is approximately 150 km (Fig. 1), with a width, which is characterised by its narrowness, in the range 7– 20 km (Uriarte et al., 1998). Coastal waters in this region are influenced by both terrestrial climate and open ocean water masses (Valencia et al., 2004). Within the Basque continental shelf, soft bottom substrates below 100 m depth are dominated by mud and sand and occupy an area of 419 km2 (Galparsoro et al., 2010). Human pressures (such as treated waste effluents) within the studied area are located inland, especially in the vicinity of estuaries (Borja et al., 2006). Nevertheless, there are additional pressures for offshore waters, such as a gas storage platform, four dredged sediment disposal sites and fisheries (Borja et al., 2011). Sediment was collected annually during five years (from 2009 to 2013) with the aid of a Smith–McIntyre grab at two sampling sites, termed West and East Basque continental shelf (Fig. 1). Hake and sole were collected during two years by bottom trawling in July 2012 and July 2013. A total of 30 individuals from each species were collected at each sampling site. Owing to the high mobility of hake and the reduced width of the studied area, the Basque continental shelf was considered as a single overall sampling area. In the case of sole, as a more restricted mobility was expected (Pawson, 1995), two sampling areas located in accordance with sediments in the West and East of the Basque continental shelf were considered independently. Fish were transferred to aerated water tanks where they were anesthetized and then their biometric parameters (length and weight), together with externally visible diseases, were registered on-board. Liver and gonad samples were subsequently excised. Two liver pieces were collected; one piece was frozen for subsequent bioaccumulation analyses, while the other one was processed immediately for histological assessment. Gonads were collected and fixed for histology. Sagittae otoliths were only removed from sole for age determination. In the case of hake, as there is no agreed protocol for otolith reading, the age was estimated according to the well-established lengthage regressions (Piñeiro and Saínza, 2003). 2.2. Sediment characterisation In order to determine the sediment granulometry, dried sediments were run through a column of sieves (gravel >2 mm; sand 2 mm – 63 lm; mud
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Histopathological indices in sole (Solea solea) and hake (Merluccius merluccius) for implementation of the European Marine Strategy Framework Directive along the Basque continental shelf (SE Bay of Biscay) Nagore Cuevas a,⇑, Izaskun Zorita b, Pedro M. Costa c, Iñaki Quincoces a, Joana Larreta b, Javier Franco b a
AZTI-Tecnalia, Txatxarramendi ugartea z/g, 48395 Sukarrieta, Spain AZTI-Tecnalia, Herrera Kaia, Portualdea z/g, 20110 Pasaia, Spain c MARE-Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia, Faculdade de ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b
a r t i c l e
i n f o
Article history: Available online 12 March 2015 Keywords: Merluccius merluccius Solea solea Histopathology Contaminants Basque continental shelf
a b s t r a c t Sole and hake, together with sediments, were collected during two campaigns along the Basque continental shelf to study the utility of two existing histopathological indices for assessing the biological effects of contaminants to implement the European Marine Strategy Framework Directive. Hepatic and gonadal histopathology were measured, and metal and/or organic contaminants were determined in both liver and sediments. Sediments from the Basque continental shelf were found to be moderately impacted by metals but non-impacted by organic compounds. Metal bioaccumulation and histopathological lesions in liver were higher in sole than in hake, although non-specific and early non-neoplastic toxicopathic lesions were observed in both species. No gross alterations were recorded in gonad. The two histopathological indices applied were highly correlated in both organs but the lack of correlation between sediment contamination levels, bioaccumulation and histopathological indices suggests that other factors, rather than pollution alone, are responsible for the biological effects observed. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The European Marine Strategy Framework Directive (MSFD; Directive 2008/56/EC) aims to achieve or maintain ‘‘Good Environmental Status’’ (GEnS) in European marine waters by 2020. This directive contains the first conceptual and practical guidelines for the development of efficient monitoring programmes in order to determine the status of marine environments (Borja et al., 2010; Lyons et al., 2010). Thus, the MSFD bases GEnS on eleven qualitative descriptors, with Descriptor 8 being formulated as ‘‘Concentrations of contaminants are at levels not giving rise to pollution effects’’, thereby implying that contamination is to be monitored and assessed in order to determine whether the levels of toxicants in the environment reach levels that are able to induce adverse effects in biota, thus becoming pollution. These contaminants that may cause harmful biological effects have been selected as priority chemical pollutants of EU-wide concern (Borja et al., 2010). The majority of these chemical substances are organic ⇑ Corresponding author. Tel.: +34 94 657 40 00; fax: +34 94 657 25 55. E-mail address: [email protected] (N. Cuevas). http://dx.doi.org/10.1016/j.marpolbul.2015.02.030 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.
compounds (e.g. polychlorinated biphenyls), although four metals (i.e. Cd, Ni, Pb and Hg) are also considered as priority substances (Directive 2008/105/EC). The achievement of GEnS for Descriptor 8 relies on monitoring programmes covering the concentrations of chemical contaminants determined in different matrices, such as water, sediment or biota, and measuring the biological effects of pollutants on marine organisms (Law et al., 2010). According to the MSFD, such an assessment must be carried out for each marine region or subregion, for example the Bay of Biscay and Iberian coasts, within the North East Atlantic Ocean (Borja, 2006). Consequently, some authors have already proposed standardised sampling strategies using diverse sentinel organisms for biomonitoring in European marine waters within the scope of the MSFD (e.g. Benedetti et al., 2014; Costa et al., 2013). However, a detailed understanding of the responsiveness of potential target species towards toxicants and environmental parameters is required in order to achieve an adequate level of quality assurance (Lyons et al., 2010). Flatfishes (Teleostei: Pleuronectiformes) have been widely employed as sentinel species, partly due to their benthic behaviour, which makes them appropriate organisms for biomonitoring marine
186
N. Cuevas et al. / Marine Pollution Bulletin 94 (2015) 185–198
sediments (Reynolds et al., 2003; Stentiford et al., 2003). Species such as dab (Limanda limanda), European flounder (Platichthys flesus) and English sole (Pleuronectes vetulus) are the main target species for monitoring purposes in Northern Europe and North-Western America (see for instance Köhler et al., 1992; Lang et al., 2006; Myers et al., 1998; Vethaak and Wester, 1996). However, as these species are less abundant around the Iberian Peninsula, other alternative species are likely to be more appropriate sentinels. One of the most abundant flatfish found on the Basque continental shelf (South-Eastern Bay of Biscay) is the sole (Solea solea), although the presence of Senegalese sole (Solea senegalensis) is also remarkable in offshore waters and estuaries (Franco et al., 2012; Quincoces et al., 2011). In this respect, adult soles are characterised by a high habitat fidelity, thus making them a site-specific species (Pawson, 1995), although they do undergo short seasonal migrations from deeper offshore areas to shallower spawning grounds. The European hake (Merluccius merluccius) has been also targeted as a sentinel organism for the Basque continental shelf, although to a lesser extent than flatfishes (Marigómez et al., 2006; Raingeard et al., 2009). The largest European hake nurseries are located in the Bay of Biscay (Casey and Pereiro, 1995), thus making this species an ideal candidate for biomonitoring approaches along the Basque continental shelf. Hake performs nictemeral migrations due to food availability; thus, it is a demersal species by day and pelagic at night (Sánchez and Gil, 2000). In addition, adults concentrate in canyons and on the rocky bottoms of the shelf break areas (Sánchez, 1993). As such, commercially exploited species, such as sole and hake, are of particular value for determining a plausible link between ecological and human risk. Fish diseases and histopathological alterations are increasingly used as indicators of pollution effects since they provide a relevant biological end-point of historical exposure (Stentiford et al., 2003). In this regard, essentially qualitative histopathological approaches have provided vital information regarding the description of histological alterations in field-collected or tested aquatic organisms (e.g. Costa et al., 2009; Stehr et al., 1998). Nonetheless, the absence of some sort of quantification makes it difficult to establish statistically significant cause-effect relationships between pathology and contaminants and to assess the significance of the differences between areas or campaigns (Costa et al., 2009; Lang et al., 2006). Therefore, semi-quantitative histopathological approaches, especially those that consider the biological significance in addition to the dissemination of histopathological changes, may effectively circumvent this issue while conferring a wider degree of biological significance (Costa et al., 2009; Vethaak and Wester, 1996). Although different semi-quantitative histopathological indices are employed for the assessment of health status in target species (Bernet et al., 1999; Triebskorn et al., 2008; van Dyk et al., 2007), the present research is focused on the adaptation, application and comparison of two existing indices, one developed by Costa et al. (2009) for sole and another one by Lang et al. (2006) for flounder. Fish livers and gonads are considered to be ideal target organs for the assessment of biological effects caused by pollution due to their role in xenobiotic transformation, storage and elimination (Hinton et al., 2001) and their relationship with the reproductive cycle and liability for upcoming generations (Blazer, 2002), respectively. Moreover, several emerging pollutants, known as endocrine disrupting chemicals, may affect the reproductive potential of gonads, thereby provoking alterations, such as intersex, that can be detected histologically (Bateman et al., 2004). In this work, the concentrations of some priority substances (according to the Directive 2008/ 105/EC) with high potential to cause adverse biological effects have been assessed; metals have been measured both in the sediments and the fish tissues; and polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) and some organochloride pesticides were analysed in the sediments.
The present work aims to: (1) test potential sentinel fishes (sole and hake) for biomonitoring approaches along the North Atlantic Iberian shelf, namely in the Basque continental shelf; (2) adapt, apply and compare two existing histopathological indices: that developed by Costa et al. (2009) and that proposed by Lang et al. (2006) in the liver and gonads of sole and hake; and (3) evaluate fish histopathology as an indicator of environmental pollution along the Basque continental shelf with regard to implementation of the European Marine Strategy Framework Directive. 2. Materials and methods 2.1. Study area and sample collection The Basque continental shelf is located in the South-Eastern Bay of Biscay and its total length is approximately 150 km (Fig. 1), with a width, which is characterised by its narrowness, in the range 7– 20 km (Uriarte et al., 1998). Coastal waters in this region are influenced by both terrestrial climate and open ocean water masses (Valencia et al., 2004). Within the Basque continental shelf, soft bottom substrates below 100 m depth are dominated by mud and sand and occupy an area of 419 km2 (Galparsoro et al., 2010). Human pressures (such as treated waste effluents) within the studied area are located inland, especially in the vicinity of estuaries (Borja et al., 2006). Nevertheless, there are additional pressures for offshore waters, such as a gas storage platform, four dredged sediment disposal sites and fisheries (Borja et al., 2011). Sediment was collected annually during five years (from 2009 to 2013) with the aid of a Smith–McIntyre grab at two sampling sites, termed West and East Basque continental shelf (Fig. 1). Hake and sole were collected during two years by bottom trawling in July 2012 and July 2013. A total of 30 individuals from each species were collected at each sampling site. Owing to the high mobility of hake and the reduced width of the studied area, the Basque continental shelf was considered as a single overall sampling area. In the case of sole, as a more restricted mobility was expected (Pawson, 1995), two sampling areas located in accordance with sediments in the West and East of the Basque continental shelf were considered independently. Fish were transferred to aerated water tanks where they were anesthetized and then their biometric parameters (length and weight), together with externally visible diseases, were registered on-board. Liver and gonad samples were subsequently excised. Two liver pieces were collected; one piece was frozen for subsequent bioaccumulation analyses, while the other one was processed immediately for histological assessment. Gonads were collected and fixed for histology. Sagittae otoliths were only removed from sole for age determination. In the case of hake, as there is no agreed protocol for otolith reading, the age was estimated according to the well-established lengthage regressions (Piñeiro and Saínza, 2003). 2.2. Sediment characterisation In order to determine the sediment granulometry, dried sediments were run through a column of sieves (gravel >2 mm; sand 2 mm – 63 lm; mud
