Layout 1 INTRODUCTION Bottom trawling, along with dredging and dumping, represents one among the most severe physical distur- bances generated by human activities at sea (Thrush and Dayton, 2002). Typically, bottom trawling is carried out using heavy ground ropes and chains to drive fish and crustaceans from the seabed into nets (Johnson et al., 2015). Trawling is carried out on many types of grounds, from shallow waters down to the deep continental margins (Puig et al., 2012), by small and large vessels, and for a wide range of target species, including fish and crus- taceans (Hinz et al., 2009). These characteristics make bottom trawling one of the preferred methods of industrial fisheries worldwide but, at the same time, one among the human activities at sea most impacting highly vulnerable benthic marine ecosystems (e.g., cold-water corals or coralligenous bottoms; Fowler, 2003; Althaus et al., 2009; Bruckner, 2009; Heifetz et al., 2009; Bongiorni et al., 2010). Recently, not irrelevant damages determined by bottom trawling have been also documented on benthic communities and processes in deep-sea soft bottoms (Pusceddu et al., 2014). Previous investigations carried out in shallow marine ecosystems have revealed that bottom trawling can gen- erate a plethora of direct and indirect effects on pelagic and benthic environments and biota (Kaiser, 1998; Smith et al., 2003; Thrush and Dayton, 2002; Queiros et al., 2006; Hiddink et al., 2007; Smith et al., 2013). As well as having a direct impact on the stocks of the target species and the by-catch, bottom trawling can also alter the structure and physico-chemical characteristics of the trawled sediment and of the overlying water column (Jen- nings et al., 2001; Smith et al., 2003; Pusceddu et al., 2005b, 2005c; Puig et al., 2012). In the pelagic realm, bot- tom trawling can increase turbidity, internal nutrient loads, oxygen consumption, and possibly enhance phytoplank- ton primary production (Riemann and Hoffmann, 1991; Palanques et al., 2001; Durrieu de Madron et al., 2005). Advances in Oceanography and Limnology, 2015; 6(1/2): 21-32 ORIGINAL ARTICLE DOI: 10.4081/aiol.2015.5448 Quantity and biochemical composition of particulate organic matter in a highly trawled area (Thermaikos Gulf, Eastern Mediterranean Sea) Antonio Pusceddu,1* Silvia Bianchelli,2 Roberto Danovaro2 1Dipartimento di Scienze della Vita e dell’Ambiente, Università degli Studi di Cagliari, Via Fiorelli 1, 09126 Cagliari; 2Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy *Corresponding author: apusceddu@unica.it ABSTRACT Bottom trawling represents nowadays one of the most severe anthropogenic disturbances at sea, and determines large impacts on benthic communities and processes. Bottom trawling determines also local sediment resuspension and the effects of the injection of large amounts of surface sediments into the water column have been repeatedly investigated. Few studies have assessed the consequences of sediment resuspension caused by bottom trawling on the quantity, biochemical composition and bioavailability of suspended organic particles and how these eventually rival those exerted by natural storms. To provide insights on this poorly addressed issue, we inves- tigated concentrations and biochemical composition of total and enzymatically digestible pools of particulate organic matter (POM) in the Thermaikos Gulf (Mediterranean Sea) under calm sea conditions, during intensive trawling activities, and after a severe storm. We show here that sediment resuspension caused by trawling can cause large effects on POM quantity, biochemical composition and bioavailability. Both during trawling and after the storm, the relative importance of the carbohydrate pools increased (in the upper water column) and the total lipid concentrations decreased (in the intermediate and bottom layers) when compared to values measured during calm conditions. These results would suggest that bottom trawling could inject in the upper water column POM pools more refractory in nature (e.g., carbohydrates) than those present in calm or after-storm conditions. By contrast, we show also that the bioavailable frac- tion of biopolymeric C increased significantly during trawling in the upper water column of the shallowest stations and in the bottom water column layer of the deepest ones. These results provide evidence that bottom trawling can influence the overall trophic status of coastal waters, exerting effects similar or stronger than those caused by natural storms, though of variable amplitude depending on the water depth. Since bottom trawling is carried out worldwide and natural storms at sea can be frequent and intense, we claim for the need of assessing new adapting management strategies of bottom trawling in order to mitigate the synergistic impacts of anthropogenic and natural sediment resuspension on coastal biogeochemical cycles. Key words: Particulate organic matter; bottom trawling; eutrophication; Mediterranean Sea. Received: July 2015. Accepted: October 2015. No n- co mm er cia l u se on ly 22Bottom trawling impacts on particulate organic matter Bottom trawling can play also a key role in sustaining high productivity on some continental margins by accel- erating sedimentary C degradation (Polymenakou et al., 2005), nutrient turnover and, thus, enhancing phytoplank- ton blooms (Fanning et al., 1982; Christensen, 1989). One of the evident effects of bottom trawling consists in sedi- ment resuspension which generates visible and highly tur- bid plumes of suspended particles, with concentrations up to several hundred mg L–1 near the seabed (Schoellhamer, 1996; Durrieu de Madron et al., 2005). The injection of large amounts of surface sediments into the water column, particularly when trawling is carried out over soft bottoms (Black and Parry, 1994; Pilskaln et al., 1998), has been hypothesized to rival storms as the main agent for sedi- ment resuspension and transport on the middle and outer continental shelf of the Middle Atlantic Bight (Churchill, 1989). Whether indeed sediment resuspension induced by bottom trawling has more or less relevance than natural resuspension (as in the case of storms) in fuelling the water column with regenerated nutrients and suspended organic particles available as food for suspension feeders, remains to date a still largely unexplored issue. To provide insights on this issue, we investigated con- centrations and biochemical composition of both total and bioavailable (i.e., enzymatically hydrolizable) pools of suspended organic particles in the Thermaikos Gulf (Mediterranean Sea) along a putative decreasing gradient of anthropogenic influence, during three periods: Septem- ber 2001 (calm sea conditions and no trawling), October 2001 (trawling period), and February 2002 (no trawling, after a severe storm). More in details, we tested the null hypothesis by which the quantity, biochemical composi- tion and bioavailability of suspended particles along the whole water column are not affected by bottom trawling or severe storms at the sea surface. METHODS Study area and sampling The Thermaikos Gulf (Fig. 1) is a micro-tidal envi- ronment, located in the North Western Aegean Sea (East- ern Mediterranean), from 39°30’N to 40°38’N and 22°30’E to 23°19’E, with depths ranging from 30 to 200 m. The main circulation is characterized by more saline waters entering the outer shelf of the Gulf over the eastern part, then turning towards the northeast in the inner part; less saline waters flow southerly along the western coast- line (Poulos et al., 2000). The Thermaikos Gulf is char- acterized by an extended shelf (180 km long and 55 km wide) of very smooth relief and by meso-eutrophic con- ditions (Zervakis et al., 2005). The area under scrutiny, in particular, is characterized in its inner part by strong an- thropogenic influences, due to the Thessaloniki city’s har- bour and the adjacent industrial zone. Moreover, the Gulf receives important riverine inputs from three major rivers (Axios, Aliakmon and Pinios rivers; Karageorgis and Anagnostou, 2001). Five sampling stations (namely IP01, IP10, IP17, IP38, IP41 at 30, 41, 55, 51, and 54 m depth, respectively) were located along the coast and positioned along a north- to-south transect characterised by seasonally intensive bottom trawling activities (Fig. 1). At each station, water samples were collected at 2 m, 20 m and about 1 m above the bottom, in September, October 2001 and in February 2002. These periods were selected as putatively represen- tative of calm conditions and no trawling (September 2001), calm conditions with trawling (October 2001) and no trawling after a prolonged period of severe storms (February 2002). Water samples were collected by means of a rosette sampler equipped with 15 L Niskin bottles. After pre-filtration through a 200 µm mesh net to remove larger zooplankton, the water samples were filtered onto Whatman GF/F filters (pre-combusted at 450°C, 4 h), im- mediately after collection. Filters were stored at -20°C, until analyses. Data obtained from the bottom layer of the water col- Fig. 1. Study area and location of the sampling stations. No n- co mm er cia l u se on ly 23 A. Pusceddu et al. umn have been already partially discussed elsewhere (Pusceddu et al., 2005b). In this study we extended the analysis including also the data obtained from the interme- diate and surface layers of the water column, to document, if any, the effects of natural and anthropogenic sediment re- suspension on the quantity and food availability of (re)sus- pended organic particles along the entire water column. Biochemical composition of particulate organic matter Concentrations of total particulate proteins (TPRT), total particulate carbohydrates (TCHO) and total particu- late lipids (TLIP) were determined according to Hartree (1972) modified by Rice (1982) (proteins), Dubois et al. (1956) (carbohydrates), and Marsh and Weinstein (1966) and Bligh and Dyer (1959) (lipids), respectively. Concen- trations of the hydrolysable fractions of particulate proteins (HPRT) and carbohydrates (HCHO) were measured ac- cording to Dell’Anno et al. (2000), slightly modified for particulate samples (Pusceddu et al., 2005c). Filters were homogenised in 0.1 M Na-phosphate buffer (pH 7.5), son- icated three times for 1 min (with intervals of 30 s) before enzyme addition. Duplicate filters from each water depth (i.e., treated samples) were added to 100 µL of proteinase- K (1 mg mL–1) and 100 µL of protease (600 µg mL–1). An equal volume of Na-P buffer solution, without enzymes (i.e., control samples), was added to another set of filters. All the samples were incubated for 1 h at 37°C, under gen- tle agitation; they were filtered subsequently onto GF/F filters and rinsed twice with 5 mL of cold 0.1 M Na-P buffer (pH 7.5), in order to remove the digested protein fraction and the surplus of enzymes. Filters muffled at 450°C for 4 h and processed as described above were utilised as blanks. Hydrolysable protein analyses from these samples were carried out spectrophotometrically as described above. Difference in protein concentration be- tween the control and treated samples were assumed to represent the concentration of proteins actually hydrolysed by proteases (hydrolysed proteins, HPRT). For the enzy- matic digestion of particulate carbohydrates, the filters were homogenised with 0.1 M Na-phosphate, 0.1 M EDTA (pH 5.0) and sonicated three times for 1 min (with intervals of 30 s). Replicate filters (n=3, treated samples) were added to 100 µL of α-amylase, 50 µL of β-glucosi- dase, 100 µL of proteinase-K and 100 µL of lipase (stock solution of all enzymes was 1 mg mL–1). Another set of filters were treated by adding 0.1M Na phosphate, instead of enzyme solutions, and utilised as a control. Filters muf- fled at 450°C for 4 h and processed as described above, were utilised as blanks. After incubation, all the samples were centrifuged at 2000 g for 10 min and an aliquot of the supernatant was used to determine carbohydrates re- leased from POM hydrolysis. Soluble carbohydrates were determined from the supernatant of the control sample. Carbohydrates from all the supernatants were analysed spectrophotometrically, as described above. The actual fraction of enzymatically hydrolysed carbohydrates (HCHO) was obtained on the basis of the difference be- tween the carbohydrate concentrations determined in the supernatant of samples containing enzymes and the soluble fraction of the control. Total and hydrolysable carbohy- drate and protein and total lipid concentrations were con- verted into carbon equivalents, using 0.40, 0.49 and 0.75 mgC mg–1 conversion factors, respectively (Fabiano et al., 1995). The sum of the total protein, carbohydrate and lipid carbon equivalents was reported as particulate biopoly- meric organic carbon (BPC, Fabiano and Pusceddu, 1998). Particulate bioavailable organic carbon (BAOC), as a proxy of the organic carbon potentially available for con- sumers (Pusceddu et al., 2003, 2009), was defined as the sum of carbon equivalents of hydrolysable carbohydrates and proteins (Danovaro et al., 2001). Statistical analyses To test the null hypothesis that the quantity, biochem- ical composition and bioavailability of suspended parti- cles along the whole water column are not affected by bottom trawling nor by severe storms at the sea surface, three-way permutational analyses of variance (PER- MANOVA; Anderson, 2001; McArdle and Anderson, 2001) were carried out separately for each variable. The design included three orthogonal factors: period (P, 3 fixed levels: calm, trawling, storm), station (S, 5 fixed lev- els: IP01, IP10, IP17, IP38, IP41), and water depth (D, 3 fixed levels: surface, intermediate, bottom), with n=3 for the combination of factors. In the multivariate context, variations in the biochemical composition of suspended particles were assessed separately for the three water col- umn layers using a 2-way design with period (P, 3 fixed levels: calm, trawling, storm), and station (S, 5 fixed lev- els: IP01, IP10, IP17, IP38, IP41) as orthogonal sources of variance. The analyses were based on Euclidean dis- tances of previously normalized data, using 4999 random permutations of the appropriate units (Anderson and ter Braak, 2003). Because of the restricted number of unique permutations in the pairwise tests, p values were obtained from Monte Carlo samplings (Anderson and Robinson, 2003). Significant differences among water layers in each station and sampling period, as well as differences among sampling periods in each station and water column layer were then assessed using SNK post-hoc tests. Canonical analysis of principal coordinates (CAP) was used in the multivariate context to ascertain the allocation of experimental groups to those established a priori. Re- sults from the CAP were then used to visualize, using bi- plots, differences among experimental groups (i.e., among periods and stations). The PERMANOVA and CAP analy- ses were performed using the routines included in the PRIMER 6+ software (Clarke and Gorley, 2006). No n- co mm er cia l u se on ly 24Bottom trawling impacts on particulate organic matter RESULTS Total protein, carbohydrate, lipid, biopolymeric C, hy- drolysable protein and carbohydrate and bioavailable or- ganic carbon concentrations at each stations, water depth and sampling period are reported in Tab. 1. The results of the PERMANOVA tests revealed a significant interaction of the three tested factors for all of the investigated vari- ables (Tab. 2). Therefore, in order to identify the eventual significance of the major factor under scrutiny (i.e., trawl- ing vs storm effects), we used post-hoc SNK tests to dis- criminate differences in the concentration of suspended organic matter concentrations: i) among sampling depths during each period and at each station; ii) among sampling periods at each station and water column layer. The results of the post-hoc tests carried out to identify changes in the vertical distribution of suspended particles in the water column during the three different conditions (Tab. 3) reveal: i) the presence during calm conditions in September of a nepheloid layer (i.e., significantly higher concentrations in the bottom layer of the water column) in almost all stations; ii) a more homogeneous distribution of POM (i.e., values in intermediate and surface layers higher than or similar to those in the bottom layer) during trawling activities in October and, though to a lesser ex- tent, after-storm in February. These trends apply to almost all investigated variables and appear to be particularly ev- ident at the shallowest stations IP01 and IP10. The post-hoc tests carried out to identify variations in the concentration of suspended organic compounds among sampling periods in each water column layer and in all sampling stations (Tab. 4) reveal that: i) the stronger effects of bottom trawling - when compared to calm and after-storm conditions - are generally most evident in the intermediate and bottom layers of the water column, es- pecially for the carbohydrate pools; ii) in some stations (i.e., IP10, IP17, IP38) and pre-eminently for the protein pools, suspended particle concentrations during trawling in October are similar to those observed after-storm in February and consistently higher than those during calm conditions in September. The results of the 2-way PERMANOVA conducted to identify variations in the biochemical composition of par- ticulate organic matter among calm, trawling and after- storm conditions and among sampling stations in each of the three layers of the water column reveal the presence of a significant Period × Station interaction for each water column layer (Tab. 5). The bi-plots produced after the CAP analysis to better ascertain, separately for each layer of the water column, changes in the biochemical compo- sition of particulate organic matter during the three sam- pling periods reveal: i) in the surface layer of the water column a clear segregation of trawling conditions at all stations, with exception of IP17 and IP38, mostly driven by increasing concentrations of carbohydrate pools, whereas storm conditions mostly overlap with calm con- ditions (Fig. 2A); ii) in the intermediate and bottom layers of the water column a general segregation of trawling and storm (slightly overlapped one each other) from calm con- ditions, mostly explained by decreasing concentrations of total particulate lipids and increasing concentrations of total and hydrolisable carbohydrate and protein pools dur- ing trawling and after storm (Fig. 2B-C); iii) in the bottom layer of the water column, the best segregation among trawling, after-storm and calm conditions in the deepest stations (i.e., IP38 and IP41) (Fig. 2C). In the surface layer of the water column of all stations, with exception of IP17 and IP38, the bioavailable fraction of particulate biopolymeric C is much higher during trawl- ing activities than in calm and after-storm conditions (Fig. 3A). The positive effect of trawling activities on the bioavailability of particulate biopolymeric C observed in the surface layer of the water column is smoother in the in- termediate layer of the water column, where it is evident only in the deepest station IP41 (Fig. 3B). A higher bioavailability of particulate biopolymeric C during trawl- ing activities - when compared to calm and after-storm con- ditions - is again evident in the bottom layer of the water column, in the deepest stations IP38 and IP41 (Fig. 3C). DISCUSSION We show here that sediment resuspension caused by intensive bottom trawling can determine effects on par- ticulate organic matter (POM) quantity, biochemical com- position and bioavailability which are similar or even stronger than those eventually exerted by storms at the sea surface. On the one hand, this result is in accordance with previous findings from the Middle Atlantic Bight, which postulated that bottom trawling can rival natural resus- pension induced by storm conditions (Churchill, 1989). Previous studies, conducted in the Thermaikos Gulf and, comparatively, in the Gulf of Lions (NW Mediterranean Sea), reported changes in the quantitative characteristics of sinking POM, resulting in increased total suspended matter concentrations and gross sedimentation rates through alternate cycles of resuspension and sedimenta- tion associated with natural temporal variability (Grémare et al., 2003; Karageorgis and Anagnostou, 2001; Fernan- des et al., 2009; Zeri et al., 2009). Our results show also that the bottom layer of the water column in the Thermaikos Gulf during calm condi- tions is characterised generally by concentrations of POM (and almost all its biochemical constituents) higher than those in the intermediate and upper layers. The presence of such a nepheloid layer indicates that natural back- ground levels of sediment resuspension in the Thermaikos Gulf could be relatively high. In this sense, we must there- No n- co mm er cia l u se on ly 25 A. Pusceddu et al. Ta b. 1 .T ot al a nd h yd ro liz ab le p ro te in , t ot al a nd h yd ro liz ab le c ar bo hy dr at e, to ta l l ip id , b io po ly m er ic C (B PC ), an d bi oa va ila bl e C c on ce nt ra tio ns a t A ) s ur fa ce (2 m ), B ) i nt er m ed ia te (2 0 m ) a nd C ) b ot to m d ep th s in th e T he rm ai ko s G ul f u nd er c al m (S ep te m be r 2 00 1) , d ur in g tr aw lin g (O ct ob er 2 00 1) a nd a ft er s to rm (F eb ru ar y 20 02 ) c on di tio ns . P ro te in C ar bo hy dr at e To ta l l ip id B io po ly m er ic C B io av ai la bl e C S ta ti on C on di ti on To ta l H yd ro liz ab le To ta l H yd ro liz ab le µ g L –1 SD µg L –1 SD µ g L –1 SD µg L –1 SD µ g L –1 SD µ gC L –1 S D µ gC L –1 S D % o f B P C A ) I P0 1 C al m 8 3. 7 1 .3 2 0. 4 1 .5 6 7. 7 1 .9 1 4. 7 0 .1 2 3. 1 0 .0 8 5. 4 1 .4 3 3. 2 0 .8 3 8. 8 T r aw lin g 1 00 .2 0 .5 5 1. 3 4 .0 8 0. 0 5 .4 2 8. 2 0 .3 2 3. 2 3 .3 9 8. 5 4 .9 5 3. 8 4 .5 5 4. 6 St or m 1 17 .8 7 .5 8. 8 0 .1 10 6. 7 3. 2 8 .5 2. 1 2 3. 2 3 .3 11 7. 8 7. 5 2 5. 1 3 .4 2 1. 3 IP 10 C al m 3 8. 1 2 .1 2. 1 0 .3 3 2. 3 1 .4 0. 0 0 .0 1 0. 1 0 .0 3 9. 1 1 .6 8 .6 0. 2 2 1. 9 Tr aw lin g 99 .6 8 .4 6 6. 6 12 .2 1 26 .2 16 .3 3 8. 4 2 .0 9 .4 4. 7 10 3. 8 1 5. 7 52 .5 1 1. 8 50 .6 St or m 1 08 .5 6 .6 3. 8 0 .1 10 7. 1 4. 9 1 .0 0. 0 9 .3 4. 7 10 0. 5 1 0. 2 6 .8 5. 1 6 .7 IP 17 C al m 5 1. 0 0 .3 1 1. 2 1 .7 2 9. 1 1 .6 6. 5 1 .7 1 4. 3 2 .4 4 7. 3 2 .6 1 8. 8 3 .3 3 9. 7 Tr aw lin g 59 .5 2 .0 0. 6 0 .0 6 7. 0 10 .8 0 .0 0. 0 7 .2 1. 4 6 1. 3 6 .3 5 .7 1. 0 9 .3 St or m 80 .4 4 .4 1. 0 0 .0 9 5. 9 4 .5 6. 9 2 .4 7 .2 1. 4 8 3. 1 4 .9 8 .6 2. 0 1 0. 4 IP 38 C al m 5 5. 5 0 .3 9. 4 0 .4 4 6. 9 7 .4 3 3. 1 0 .3 1 1. 3 2 .4 5 4. 4 4 .9 2 6. 3 2 .1 4 8. 3 Tr aw lin g 65 .9 2 .3 1 0. 0 0 .6 8 0. 9 22 .4 1 1. 6 0 .0 7 .2 0. 0 7 0. 0 10 .1 14 .9 0 .3 2 1. 3 St or m 1 32 .1 8 .7 8 3. 3 4 .7 4 8. 4 2 .1 1. 0 0 .0 3 .1 1. 7 8 6. 4 6 .4 4 3. 5 3 .6 5 0. 4 IP 41 C al m 5 7. 6 6 .2 1 6. 0 0 .5 3 8. 6 2 .2 4. 9 0 .8 1 4. 1 2 .1 5 4. 3 5 .5 2 0. 3 2 .1 3 7. 5 Tr aw lin g 76 .1 1 6. 8 23 .2 4 .1 9 8. 8 23 .4 5 4. 8 0 .4 3 .1 1. 7 7 9. 1 18 .9 35 .6 3 .5 4 5. 0 St or m 84 .0 1 8. 2 26 .5 2 .2 7 7. 2 10 .4 1 2. 3 3 .3 5 .0 0. 0 7 5. 8 13 .1 21 .6 2 .4 2 8. 5 B ) I P0 1 C al m 12 8. 4 3 1. 9 46 .8 0 .8 7 1. 3 2 .4 5. 2 0 .1 3 0. 9 2 .9 11 4. 7 1 8. 7 48 .2 2 .6 4 2. 1 Tr aw lin g 1 32 .7 0 .5 4 5. 0 0 .2 9 1. 3 3 .1 0. 0 0 .0 2 1. 8 1 .4 11 7. 9 2. 5 3 8. 4 1 .2 3 2. 6 St or m 1 26 .1 2 .3 1 9. 8 3 .2 9 7. 4 1 .4 1. 0 0 .0 2 1. 8 1 .4 11 7. 1 2. 8 2 6. 5 2 .7 2 2. 6 IP 10 C al m 4 8. 6 0 .7 1. 9 0 .3 3 6. 5 2 .8 0. 6 0 .0 8 .9 1. 0 4 5. 0 2 .2 7 .8 0. 9 1 7. 4 Tr aw lin g 77 .7 1 .0 4 8. 5 3 .3 11 3. 6 1 8. 6 0 .0 0. 0 7 .3 1. 0 8 9. 0 8 .7 2 9. 3 2 .4 3 2. 9 St or m 84 .4 0 .9 1. 0 0 .0 7 4. 8 3 .2 1. 0 0 .0 7 .3 1. 0 7 6. 8 2 .5 6 .4 0. 7 8 .3 IP 17 C al m 3 7. 0 6 .4 0. 0 0 .0 2 7. 3 3 .6 5. 7 0 .6 1 3. 7 1 .0 3 9. 3 5 .3 1 2. 6 1 .0 3 2. 0 Tr aw lin g 72 .3 0 .7 1 8. 7 0 .6 8 4. 8 2 .1 0. 0 0 .0 3 .6 2. 4 7 2. 1 3 .0 1 1. 9 2 .1 1 6. 5 St or m 70 .3 3 .3 7. 8 0 .4 9 3. 5 1 .2 3 2. 5 3 .5 3 .6 2. 4 7 4. 6 3 .9 1 9. 5 3 .4 2 6. 2 IP 38 C al m 8 1. 0 2 .8 1 8. 9 6 .6 4 8. 3 4 .1 1 1. 9 0 .7 1 5. 8 2 .0 7 0. 9 4 .5 2 5. 9 5 .1 3 6. 6 Tr aw lin g 61 .4 5 .4 3. 2 0 .2 10 4. 1 8. 8 6 7. 8 1 .9 5 .0 1. 4 7 5. 5 7 .3 3 2. 4 1 .9 4 2. 9 St or m 1 05 .5 1 .3 4 7. 1 12 .7 65 .1 2 .2 2 1. 1 0 .1 3 .1 2. 4 8 0. 1 3 .3 3 3. 8 8 .0 4 2. 2 IP 41 C al m 11 3. 2 3. 7 0 .0 0. 0 4 9. 5 2 .9 0. 0 0 .0 1 6. 6 1 .0 8 7. 7 3 .7 1 2. 4 0 .8 1 4. 2 Tr aw lin g 63 .8 4 .7 2 2. 2 5 .5 11 5. 0 9. 7 5 0. 6 14 .0 3 .1 2. 4 7 9. 6 8 .0 3 3. 4 10 .1 4 2. 0 St or m 70 .9 9 .1 2 3. 8 0 .3 9 3. 1 1 .3 1. 0 0 .0 2 .1 1. 2 7 3. 5 5 .9 1 3. 6 1 .0 1 8. 5 C ) I P0 1 C al m 17 3. 3 6. 1 7 6. 7 3 .7 15 0. 6 5. 8 7 3. 5 5 .3 4 0. 4 1 .0 17 5. 4 6. 0 9 7. 3 4 .7 5 5. 4 Tr aw lin g 1 35 .1 10 .3 1 8. 8 1 .1 12 3. 4 1 7. 6 42 .0 0 .9 2 2. 2 1 .9 13 2. 2 1 3. 5 42 .6 2 .3 3 2. 2 St or m 1 85 .9 5 .2 4 6. 0 1 .9 15 1. 5 1 1. 0 67 .6 4 .1 2 2. 2 1 .9 16 8. 3 8. 4 6 6. 2 4 .0 3 9. 3 IP 10 C al m 8 4. 9 8 .0 1 2. 9 0 .7 8 5. 6 2 .2 3 9. 3 1 .9 2 9. 9 0 .5 9 8. 3 5 .2 4 4. 5 1 .4 4 5. 3 Tr aw lin g 1 08 .2 1 .4 2 3. 8 5 .7 15 3. 0 3 6. 1 40 .2 9 .3 1 0. 0 2 .9 12 1. 7 1 7. 3 35 .3 8 .7 2 9. 0 St or m 1 05 .2 7 .5 2 8. 4 4 .9 10 5. 1 2. 5 1 .0 0. 0 1 0. 0 2 .9 10 1. 1 6. 8 2 1. 8 4 .5 2 1. 6 IP 17 C al m 7 6. 0 0 .9 2 1. 2 0 .7 8 4. 1 2 .5 4 8. 9 8 .2 2 7. 6 0 .0 9 1. 6 1 .5 5 0. 7 3 .7 5 5. 3 Tr aw lin g 1 05 .9 6 .6 2 1. 1 2 .8 9 8. 1 14 .1 2 7. 3 6 .1 1 .3 0. 8 9 1. 7 9 .8 2 1. 8 4 .7 2 3. 8 St or m 1 35 .1 13 .1 5 0. 0 0 .8 14 9. 0 4. 2 1 .0 0. 0 4 .0 0. 4 12 8. 7 8. 4 2 7. 9 0 .7 2 1. 6 IP 38 C al m 10 3. 5 0. 5 2 6. 2 9 .6 7 4. 3 8 .0 4 4. 0 1 .0 1 7. 5 2 .9 9 3. 5 5 .6 4 3. 6 7 .2 4 6. 6 Tr aw lin g 11 2. 8 4. 2 6 4. 0 6 .2 17 4. 5 4. 6 10 9. 8 1 9. 3 4 .0 0. 4 12 8. 1 4. 2 7 8. 2 11 .0 6 1. 0 St or m 1 28 .4 6 .6 5 3. 0 1 .3 11 4. 6 5. 6 7 9. 6 5 .4 6 .0 0. 0 11 3. 3 5. 5 6 2. 3 2 .8 5 5. 0 IP 41 C al m 8 8. 9 14 .6 1 .0 0. 1 6 5. 9 0 .2 2 5. 7 5 .5 3 4. 3 3 .8 9 5. 7 10 .1 36 .5 5 .1 3 8. 1 Tr aw lin g 77 .3 4 .7 1 6. 1 1 .4 13 9. 0 7. 5 9 7. 0 7 .5 6 .0 0. 0 9 8. 0 5 .3 5 1. 2 3 .6 5 2. 2 St or m 1 00 .5 5 .6 3 9. 7 8 .0 5 7. 4 3 .4 7. 5 0 .6 6 .7 1. 9 7 7. 2 5 .5 2 7. 5 5 .6 3 5. 6 SD , S ta nd ar d de vi at io n (n =3 ). No n- co mm er cia l u se on ly 26Bottom trawling impacts on particulate organic matter Tab. 2. Results of 3-way PERMANOVA testing for differences in the investigated variables among sampling periods, stations and sampling depths. Source DF MS F P Total protein Period (P) 2 8.2 134.9 *** Station (S) 4 12.1 199.4 *** Depth (D) 2 14.1 232.4 *** P x s 8 1.5 24.2 *** P x D 4 1.3 21.7 *** S x D 8 1.2 19.1 *** P x S x D 16 0.6 9.9 *** Residual 90 0.1 Hydrolysable protein Period (P) 2 4.0 117.6 *** Station (S) 4 5.7 165.0 *** Depth (D) 2 4.5 131.1 *** P x s 8 5.1 148.0 *** P x D 4 1.3 38.4 *** S x D 8 1.1 31.8 *** P x S x D 16 2.3 66.9 *** Residual 90 0.0 Total carbohydrate Period (P) 2 21.4 292.7 *** Station (S) 4 2.0 26.7 *** Depth (D) 2 17.3 236.3 *** P x s 8 2.9 39.8 *** P x D 4 0.4 5.8 ** S x D 8 1.1 14.6 *** P x S x D 16 0.5 7.4 *** Residual 90 0.1 Hydrolysable carbohydrate Period (P) 2 7.1 270.2 *** Station (S) 4 4.5 173.7 *** Depth (D) 2 19.7 755.9 *** P x s 8 2.9 109.4 *** P x D 4 0.7 25.2 *** S x D 8 1.7 66.0 *** P x S x D 16 1.3 49.6 *** Residual 90 0.0 Total lipid Period (P) 2 21.2 397.8 *** Station (S) 4 14.1 264.8 *** Depth (D) 2 4.0 75.2 *** P x s 8 0.4 8.5 *** P x D 4 3.2 60.8 *** S x D 8 0.4 7.7 *** P x S x D 16 0.2 3.3 ** Residual 90 0.1 Biopolymeric C Period (P) 2 4.9 66.8 *** Station (S) 4 11.6 156.8 *** Depth (D) 2 20.9 282.8 *** P x s 8 1.5 19.7 *** P x D 4 1.2 15.7 *** S x D 8 0.8 11.4 *** P x S x D 16 0.4 5.6 *** Residual 90 0.1 Bioavailable organic C Period (P) 2 2.0 39.3 *** Station (S) 4 9.3 183.6 *** Depth (D) 2 19.6 387.0 *** P x s 8 2.4 47.8 *** P x D 4 1.1 21.5 *** S x D 8 0.6 12.8 *** P x S x D 16 1.3 25.2 *** Residual 90 0.1 DF, degrees of freedom; MS, mean square; F, F value; ***P<0.001; **P<0.01. Fig. 2. Bi-plots after CAP analysis illustrating spatial (among sta- tions) and temporal (among calm, trawling and after-storm con- ditions) variations in the biochemical composition of particulate organic matter in the Thermaikos Gulf, in the superficial (A), in- termediate (B) and bottom (C) water layers. Vectors are propor- tional to the correlation of variables with the two major axes. tPRT, total proteins; tCHO, total carbohydrates; tLIP, total lipids; hPRT, hydrolysable proteins; hCHO, hydrolysable carbohydrates. No n- co mm er cia l u se on ly 27 A. Pusceddu et al. Tab. 4. Visual representation of post-hoc tests carried out to ascertain variations in the concentration of particulate organic compounds in the Thermaikos Gulf during calm (September, C), trawling (October 2001, T) and after storm (February 2002, S) conditions in the three water column layers. Green/white, yellow, light blue and red cells indicate missing effects of both trawling and storm, stronger effects of storm, similar effects of storm and trawling, stronger effects of trawling, respectively. Water layer Variable IP1 IP10 IP17 IP38 IP41 Surface Total protein S>T>C S,T>C S,T>C S,T>C ns Hydrolysable protein T>C>S T>S>C C>S>T S>C,T S,T>C Total carbohydrate S>T>C S,T>C S>T>C ns S,T>C Hydrolysable carbohydrate T>C>S T>S>C S>C>T C>T>S T>S>C Total lipid ns ns C>T,S C>T>S C>T,S Biopolymeric C S>T>C S,T>C S>T>C S>C ns Bioavailable organic C T>C>S T>C>S C>S>T S>C>T T>C Intermediate Total protein T>S S>T>C S,T>C S>C>T C>T,S Hydrolysable protein C>T>S T>C>S T>S>C S>C>T S,T>C Total carbohydrate S>T>C T>S>C T>S>C T>S>C T>S>C Hydrolysable carbohydrate C>S>T C>S>T S>C>T T>S>C T>S>C Total lipid C>T,S ns C>T,S C>T,S C>T,S Biopolymeric C ns S,T>C S,T>C S>C C>S Bioavailable organic C C,T>S T>C>S S>C>T ns T>S>C Bottom Total protein C,S>T S,T>C T>S>C S>T>C S>T Hydrolysable protein C>S>T S,T>C S>T,C S,T>C S>T>C Total carbohydrate ns T>S>C S>T,C T>S>C T>S>C Hydrolysable carbohydrate C,S>T C,T>S C>T>S S,T>C T>C>S Total lipid C>T,S C>T,S C>S>T C>S>T C>S,T Biopolymeric C C,S>T ns S>T,C T>S>C C,T>S Bioavailable organic C C>S>T C>S C>T,S S,T>C T>C,S ns, not significant. Tab. 3. Visual representation of post-hoc tests carried out to ascertain variations in the distribution of particulate organic compounds in the water column of the Thermaikos Gulf during calm (September 2001), trawling (October 2001) and after storm (February 2002) conditions. Green, yellow and red cells indicate missing, weak and strong signatures, respectively, of sediment resuspension in the upper water column layer. Condition (period) Variable IP01 IP10 IP17 IP38 IP41 Calm (Sept 2001) Total protein B>S B>I>S B>S>I B>I>S B,I>S Hydrolysable protein B>I>S B>I,S B>S>I B>S S>B>I Total carbohydrate B>I,S B>I,S B>I,S B>I,S B>I>S Hydrolysable carbohydrate B>S>I B>I>S B>I,S B>S>I B>S>I Total lipid B>I>S B>I,S B>I,S B>S B>I,S Biopolymeric C B>I,S B>I>S B>I,S B>I>S B,I>S Bioavailable organic C B>I>S B>I,S B>S>I B>I,S B>S>I Trawling (Oct 2001) Total protein B,I>S B,S>I B>I>S B>I,S B>I Hydrolysable protein S>I>B S,I>B B,I>S B>S>I ns Total carbohydrate B>I>S ns B>S B>I,S B>I Hydrolysable carbohydrate B>S>I B,S>I B>S,I B>I>S B>I,S Total lipid ns ns S>B S>B B>I,S Biopolymeric C B,I>S B>I B>I,S B>I,S B>I Bioavailable organic C S>B,I S>I B>I>S B>I>S B>S After storm (Feb 2002) Total protein B>S,I B,S>I B>S>I B,S>I B>I Hydrolysable protein B>I>S B>S>I B>I>S S>I,B I>B Total carbohydrate B>S>I B,S>I B>I,S B>I>S I>S>B Hydrolysable carbohydrate B>S>I ns I>S>B B>I>S B,S>I Total lipid ns ns S>B B>S B,S>I Biopolymeric C B>S,I B,S>I B>S,I B>S,I ns Bioavailable organic C B>S,I B>S,I B>I>S B>S,I B,S>I B, bottom; I, intermediate; S, surface. No n- co mm er cia l u se on ly 28Bottom trawling impacts on particulate organic matter fore acknowledge that a certain (unknown) proportion of variance among calm, trawling, and after-storm condi- tions is most likely associated to natural temporal vari- ability in the POM quantity and biochemical composition in the Thermaikos Gulf. Indeed, variations in the concen- tration of POM along the water column, as well as in its biochemical composition, can be the result of biological processes, including among the others primary productiv- ity and particle consumption (Fabiano and Pusceddu, 1998; Fabiano et al., 2001). Moreover, concentration and composition of POM can be affected, in particular along the continental shelf, by rivers discharge (Goñi et al., 2013). This latter, however, was most likely not the case as the analysis of meteorological data in the region during the study period revealed a very dry season in autumn 2001, accompanied by very low levels of river discharge (Tragou et al., 2005) Differences in the environmental characteristics among the sampling periods appeared to be particularly relevant between September-October 2001 and February 2002. In fact, the hydrological characteristics of the Ther- maikos Gulf did not change markedly from September (calm conditions) to October 2001 (during trawling), when pronounced thermoclines and haloclines were recorded at ca. 35-40 m depth in the whole study area (Tragou et al., 2005; Zervakis et al., 2005). On the other hand, a series of cold fronts that passed over the region in late January 2002 completely homogenised the thermo- haline structure of the water column in February 2002 (Tragou et al., 2005; Zervakis et al., 2005). Moreover, during this period, a low salinity, low temperature water front occurred along the western boundary of the Gulf, characterised by strong, vertically homogeneous south- ward velocities of up to 20 cm s–1 (Zervakis et al., 2005). These large differences would make in principle difficult interpreting the differences in POM characteristics among the September-October 2001 and February 2002 periods. However, the lack of significant variations in the physico- chemical characteristics from calm to trawling conditions, at least, allows us to corroborate the hypothesis by which bottom trawling is a major factor stimulating sediment re- suspension and, as a consequence, increasing POM con- centrations in the water column as well as significantly modifying its biochemical composition and bioavailabil- ity for consumers. This result is indeed consistent with previous investigations carried out by means of both cor- relative and manipulative approaches, which generally demonstrated that bottom trawling can significantly inject large amounts of sediments and associated POM into the overlying water column (Durrieu de Madron et al., 2005; Pusceddu et al., 2005a, 2005c; Martín et al., 2014b). Our results paradigmatically demonstrate that along with the well-known mechanisms of pelagic-benthic coupling as- sociated with sedimentation processes of POM (Graf, 1992), the reverse exchange of material (i.e., benthic- pelagic coupling, sensu Marcus and Boero, 1998) stimu- lated by sediment resuspension caused by anthropogenic and, to a lesser extent, natural processes can have impor- tant consequences on POM stocks in the water column. We report here also that the transition from calm to trawling and to after-storm conditions is characterised by clear changes in the biochemical composition of POM. In particular, we show that both during trawling in October and after-storm in February, the relative importance of total and hydrolysable carbohydrates increases signifi- cantly when compared to values measured during calm conditions. At the same time, suspended POM during trawling and after the storm is also characterised by lipid Tab. 5. Results of PERMANOVA analysis testing for differences in the biochemical composition of particulate organic matter consid- ering all investigated variables in the three layers of the water column. Source DF MS F P Surface Period 2 34.302 85.383 *** Station 4 15.271 38.012 *** Period x Station 8 9.7823 24.349 *** Residual 30 0.40175 Total 44 Intermediate Period 2 27.632 102.07 *** Station 4 21.758 80.37 *** Period x Station 8 8.6978 32.128 *** Residual 30 0.27072 Total 44 Bottom Period 2 28.789 106.5 *** Station 4 21.519 79.606 *** Period x Station 8 8.5294 31.553 *** Residual 30 0.27032 Total 44 DF, degrees of freedom; MS, means square; F, F value; ***P<0.001. No n- co mm er cia l u se on ly 29 A. Pusceddu et al. concentrations significantly lower than those in calm con- ditions. The gross biochemical composition of POM is the result of a complex multiple-source array of biotic and abiotic factors (Danovaro et al., 2000) and variations in the relative importance of protein, carbohydrate and lipid contents can be highly indicative of changes in the labile vs refractory nature of particles (Fabiano et al., 2001). Carbohydrates are usually associated with organic matter pre-eminently refractory in nature (Grémare et al., 2003; Pusceddu et al., 2009), so that our results strongly sug- gests that sediment resuspension caused by trawling and, though to a lesser extent, by storms can lower the food availability of suspended organic particles. This hypoth- esis is also corroborated by the significant decrease, dur- ing trawling, in the concentration of particulate lipids, whose major fraction is generally highly labile (Carreira et al., 2010). Altogether these results would suggest that OM particles resuspended after bottom trawling could more refractory in nature than those present in the water column in calm or after-storm conditions. Nevertheless, we report here that, in contrast with what hypothesized from the gross biochemical composition only, the bioavailable fraction of particulate biopolymeric C in- creases significantly during trawling, though not consis- tently at all stations and water layer. This apparent incongruence is due to the fact that while total protein, lipid and carbohydrate pools include generally a very het- erogeneous complex of organic compounds which are not Fig. 3. Variations in the bioavailable fraction of particulate organic matter (in terms of percentage fraction of biopolymeric C enzymatically digestible) in the Thermaikos Gulf during calm (September 2001), trawling (October 2001) and after-storm (February 2002) conditions. No n- co mm er cia l u se on ly 30Bottom trawling impacts on particulate organic matter equally reactive to degradation (Pusceddu et al., 2009), the enzymatically digestible fractions of protein and car- bohydrate pools (i.e., the bioavailable fraction of organic C; Dell’Anno et al., 2000; Pusceddu et al., 2003) are more reliable descriptors of organic matter food availability to consumers than the gross biochemical composition alone (Fabiano and Pusceddu, 1998; Pusceddu et al., 1999; Danovaro et al., 2001). The apparent positive effect of bottom trawling on the bioavailability of suspended POM is however, most clearly confined to the bottom layer of the deepest stations investigated in this study. This result does not allow us concluding that sediment resuspension induced by trawl- ing can cause the injection of labile organic compounds in the whole water column, nor that the faster mobilisation rates of organic C buried in the sediment after trawling in coastal sediments is a major factor contributing to coastal eutrophication (Polymenakou et al,. 2005; Pusceddu et al., 2005c). Previous studies carried out in the field and under lab- oratory conditions have demonstrated that sediment resus- pension caused by storms or trawling activities can enhance either suspended or sedimentary POM quantity and bioavailability (Pusceddu et al., 2005a, 2005b, 2005c). In particular, benthic microbes exposed to O2-rich waters caused by sediment disturbance induced by trawling can stimulate a faster mobilisation of organic C buried in the sediment, injecting more labile molecules into the system (Polymenakou et al., 2005). Other studies have also demon- strated that bottom trawling in coastal waters creates plumes of resuspended sediments which last just a few hours in the water column (Schoellhamer, 1996; Palanques et al., 2001; Durrieu de Madron et al., 2005). Nevertheless, volumes moved from the sediment to the water column can be huge if trawling activities are chronic as observed in sev- eral regions of the Mediterranean Sea (Martín et al., 2014a). In such conditions we can hypothesize that the overall en- hancement of C cycling in the water column and in the upper layers of the sediment exposed to chronic bottom trawling might stimulate an increase of available nutrients able, in turn, to sustain increased levels of primary produc- tivity, ultimately leading to a potential internal eutrophica- tion process (Polymenoakou et al., 2005). Overall, the results of this study are very different from what, instead, observed in deep-sea sediments ex- posed to chronic trawling activities, where the lack of a conspicuous re-deposition from the water column deter- mines a dramatic lowering of organic C sedimentary con- tents and turnover rates (Pusceddu et al., 2014). CONCLUSIONS Although limited to a very short-term analysis, likely biased by the uncontrolled seasonal variations in the quan- tity and biochemical composition of POM, our results cor- roborate the most recent literature which demonstrates that bottom trawling represents a major threat not only for commercially exploited target species and the associated by-catch, but can have also important consequences on the biogeochemical cycling of organic C. Our results have also shown that the effects of intensive trawling activities can rival those (temporarily) exerted by natural events (e.g., storms). Bottom trawling represents the most com- mon fishing practice worldwide, it is being carried out at progressively deeper depths (Puig et al., 2012) and is ex- erting severe consequences on the submersed seascape (Martín et al., 2014a) as well on the biodiversity and func- tioning of marine ecosystems (Pusceddu et al., 2014). Since bottom trawling is carried out worldwide and natu- ral storms at sea can be frequent and intense, we claim for the need of assessing new adapting management strategies of bottom trawling in order to mitigate the synergistic im- pacts of anthropogenic and natural sediment resuspension on coastal biogeochemical cycles.. ACKNOWLEDGMENTS This work has been supported by the project INTER- POL (EVK3-2000–0023) of the European Commission. REFERENCES Althaus F, Williams A, Schlacher TA, Kloser RJ, Green MA, Barker BA, Bax NJ, Brodie P, Schlacher-Hoenlinger MA, 2009. Im- pacts of bottom trawling on deep-coral ecosystems of seamounts are long-lasting. Mar. Ecol. Prog. 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