**3. "Classic" biomarkers versus "***omics***" methodologies**

### **3.1 Assessing pollution effects in fish**

After the initial studies of the UCO group, the use of "classic" biomarkers became increasingly popular to assess the effects of pollutants on fish. One example was the ''Prestige'' oil spill in November 2002 off the Galician coast (NW Spain). Due to the heavy nature of the crude oil and its low solubility in sea water, its dispersion was low and it remained *in situ* in oil patches adhered to rocks and sediments. The biological effects of these oil patches were tested in *S. aurata* (Morales-Caselles et al., 2006), using MT levels, EROD activity and histology as biomarkers, without significant results. Biomarkers including CAT, GSHPx, GSSGrase and DT-diaphorase, AcChE, CbE, GST and MDA were also analysed in *S. senegalensis*, with modest alterations (Solé et al., 2008).

Antioxidative and phase II and III biotransforming enzymes were used to follow the effects of linear alkylbenzene sulphonates in sole via an *in vivo* continuous-flow assay (Alvarez-Muñoz et al., 2007) that was also used in *S. aurata* and *S. senegalensis* to assess the toxicity of sediments from littoral areas in northern and southern Spain (Jiménez-Tenorio et al., 2007), using MTs, EROD and histopathology. Jimenez-Tenorio et al. (2008) assessed the sediment toxicity caused in *S. senegalensis* by acute or chronic spills using histopathology, EROD and GST as biomarkers. Oxidative stress biomarkers and PAH contents were studied in Huelva soles near a petrochemical plant (Oliva et al.*,* 2010). Significant correlations were found among the levels of GST, GPx and CAT, hepatic levels of PAH metabolites and PAH contents in sediments.

The effect of waterborne copper (Cu) was studied in *S. senegalensis* in static conditions (Fonseca et al., 2009) by assessing the following characteristics: biomarkers such as MTs or MDA, mass indices, and biochemical condition indices such as the RNA/DNA ratio and the lipid and protein content. Cu triggers a biomarker response and lowers growth and condition, without changes in morphometric indices. Decreased condition shows that lipid reserves enable fish to respond to toxicity and to maintain growth and protein synthesis, although with lower rates than control fish.

The effects of Mexel®432 and NaClO antifoulings were studied in *S. senegalensis* by assessing osmolality, Na+/K+-ATPase, stress, histology, oxidative damage, antioxidant defences and detoxification (López-Galindo et al*.*, 2010a,b). NaClO increases plasma cortisol, glucose and lactate after an acute stress, with a later recovery. Gill GST and AChE are sensitive to NaClO. Hepatic markers initially respond to NaClO but longer exposures are toxic. Mexel®432 initially increases cortisol, which later returns to basal values, but glucose, lactate and triglycerides decresase. Gills have a lowered Na+,K+-ATPase activity, causing an imbalance in osmoregulation. Moderate changes are found in KAT, GSSGrase and GPX but not in MDA, GST or CbE. A multi-biomarker approach was used in *Dicentrarchus labrax*, *S. senegalensis* and *Pomatoschistus microps*, from the Aveiro and Tejo (Portugal) estuaries (Fonseca et al., 2011), which were affected by anthropic activities, without highly significant results.

Detection of DNA damage by biomarkers for genotoxicity was used in *S. senegalensis* (Costa et al., 2008a) exposed to sediments from three Sado Estuary (W Portugal) sites. The two blood parameters used were erythrocyte nuclear abnormalities (ENA) and DNA strandbreaks (DNA-SB). The levels of metals, PAHs, PCBs and DDTs were determined in the

After the initial studies of the UCO group, the use of "classic" biomarkers became increasingly popular to assess the effects of pollutants on fish. One example was the ''Prestige'' oil spill in November 2002 off the Galician coast (NW Spain). Due to the heavy nature of the crude oil and its low solubility in sea water, its dispersion was low and it remained *in situ* in oil patches adhered to rocks and sediments. The biological effects of these oil patches were tested in *S. aurata* (Morales-Caselles et al., 2006), using MT levels, EROD activity and histology as biomarkers, without significant results. Biomarkers including CAT, GSHPx, GSSGrase and DT-diaphorase, AcChE, CbE, GST and MDA were

Antioxidative and phase II and III biotransforming enzymes were used to follow the effects of linear alkylbenzene sulphonates in sole via an *in vivo* continuous-flow assay (Alvarez-Muñoz et al., 2007) that was also used in *S. aurata* and *S. senegalensis* to assess the toxicity of sediments from littoral areas in northern and southern Spain (Jiménez-Tenorio et al., 2007), using MTs, EROD and histopathology. Jimenez-Tenorio et al. (2008) assessed the sediment toxicity caused in *S. senegalensis* by acute or chronic spills using histopathology, EROD and GST as biomarkers. Oxidative stress biomarkers and PAH contents were studied in Huelva soles near a petrochemical plant (Oliva et al.*,* 2010). Significant correlations were found among the levels of GST, GPx and CAT, hepatic levels of PAH metabolites and PAH

The effect of waterborne copper (Cu) was studied in *S. senegalensis* in static conditions (Fonseca et al., 2009) by assessing the following characteristics: biomarkers such as MTs or MDA, mass indices, and biochemical condition indices such as the RNA/DNA ratio and the lipid and protein content. Cu triggers a biomarker response and lowers growth and condition, without changes in morphometric indices. Decreased condition shows that lipid reserves enable fish to respond to toxicity and to maintain growth and protein synthesis,

The effects of Mexel®432 and NaClO antifoulings were studied in *S. senegalensis* by assessing osmolality, Na+/K+-ATPase, stress, histology, oxidative damage, antioxidant defences and detoxification (López-Galindo et al*.*, 2010a,b). NaClO increases plasma cortisol, glucose and lactate after an acute stress, with a later recovery. Gill GST and AChE are sensitive to NaClO. Hepatic markers initially respond to NaClO but longer exposures are toxic. Mexel®432 initially increases cortisol, which later returns to basal values, but glucose, lactate and triglycerides decresase. Gills have a lowered Na+,K+-ATPase activity, causing an imbalance in osmoregulation. Moderate changes are found in KAT, GSSGrase and GPX but not in MDA, GST or CbE. A multi-biomarker approach was used in *Dicentrarchus labrax*, *S. senegalensis* and *Pomatoschistus microps*, from the Aveiro and Tejo (Portugal) estuaries (Fonseca et al., 2011), which were affected by anthropic activities, without highly significant

Detection of DNA damage by biomarkers for genotoxicity was used in *S. senegalensis* (Costa et al., 2008a) exposed to sediments from three Sado Estuary (W Portugal) sites. The two blood parameters used were erythrocyte nuclear abnormalities (ENA) and DNA strandbreaks (DNA-SB). The levels of metals, PAHs, PCBs and DDTs were determined in the

**3. "Classic" biomarkers versus "***omics***" methodologies** 

also analysed in *S. senegalensis*, with modest alterations (Solé et al., 2008).

**3.1 Assessing pollution effects in fish** 

contents in sediments.

results.

although with lower rates than control fish.

sediments. Scarcely polluted sediments are weaker inducers of genotoxic damage, whereas those under urban, industrial or agricultural influences significantly increase ENA and DNA-SB. A strong correlation exists between PAH and PCB content and genotoxicity, while metals have a weaker correlation. In a parallel study, *S. senegalensis* were exposed to Sado Estuary sediments (Costa et al., 2008b). Livers had more histological lesions than gills, and sediments contaminated by organics caused more damage to both organs than those contaminated by metals. Two "classic" biomarkers, MT and CYP1A, were also assessed. Lethality and biomarker responses do not linearly depend on the cumulative levels of contaminants but rather of their bioavailability and synergistic effects (Costa et al., 2009). In a parallel study, exposure to contaminated sediments induced DNA fragmentation and clastogenesis (Costa et al., 2011). Still, the most contaminated sediment revealed an antagonistic effect between metals and organics, enhanced by higher bioavailability. The laboratory assay caused a more pronounced increase in ENA, whereas a significant increase in DNA-SB exists in field-tested fish exposed to reference sediment.
