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Microbial and Meiofaunal Response to Intensive Mussel-Farm Biodeposition in Coastal Sediments of the Western Mediterranean
PII: S0025-326X(99)00209-X
Marine Pollution Bulletin Vol. 40, No. 3, pp. 244±252, 2000 Ó 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0025-326X/00 $ - see front matter
Microbial and Meiofaunal Response to Intensive Mussel-Farm Biodeposition in Coastal Sediments of the Western Mediterranean S. MIRTO à*, T. LA ROSAà, R. DANOVARO § and A. MAZZOLAà Instituto Scienze del Mare, Facolta di Scienze, Universit a di Ancona, Via Brecce Bianche, Monte D' Ago, 60131 Ancona, Italy àDip. Biologia Animale, Universit a di Palermo, Via Archira®, 18, 90123 Palermo, Italy §Dip. Zoologia, Universit a di Bari, Via Orabona 4, 70125 Bari, Italy We studied the impact of organic loads due to the biodeposition of a mussel farm in a coastal area of the Tyrrhenian Sea (Western Mediterranean). Sediment chemistry, microbial and meiofaunal assemblages were investigated from March 1997 to February 1998 on monthly basis at two stations: the ®rst was located under the mussel farm, while the second was at about 1-km distance and served as control. Benthic response to changes in the biodepositional regime was investigated in terms of biochemical composition of the sedimentary organic matter, phytopigment content, bacterial abundance and composition and meiofaunal community structure. A large accumulation of chloroplastic pigments, proteins and lipids was observed under the mussel farm. Such changes in the sedimentary conditions re¯ected the accumulation of faeces and pseudo-faeces and led to the creation of reducing conditions. Microbial assemblages beneath the mussel cultures increased their densities and displayed, when compared to the control, a larger cyanobacterial importance associated to a strong decrease of the picoeukaryotic cell density. Farm sediments displayed signi®cant changes in meiofaunal density: turbellarian, ostracod and kinorhynch densities decreased signi®cantly, while copepods remained constant or increased possibly pro®ting of the enrichment in microphytobenthic biomass associated to mussel biodeposits. The comparative analysis of the mussel biodeposition and ®sh-farm impact on sediments beneath the cultures revealed that mussel farms induced a considerably lower disturbance on benthic community structure. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: mussel farm; bacteria; cyanobacteria; picoeukaryotes; meiofauna; Mediterranean Sea. *Corresponding author. Tel.: +39-71-220-4654; fax: +39-71-2204650. E-mail address: [email protected]
244
In the Mediterranean Sea, aquaculture activities (particularly ®sh and mussel farms) have expanded signi®cantly during recent years (Rana, 1998), inducing an increasing interest and concern for their potential impact on coastal marine environments, with important management implications (Barg and Phillips, 1998). Mussel farming is known to be responsible for intensive biodeposition of faeces and pseudo-faeces that might cause strong changes in the physical and chemical characteristics of the sediments beneath the long lines (Dahlback and Gunnarsson, 1981; Gilbert et al., 1997; Kaspar et al., 1985; Mirto et al., 1999b). The eects of mussel farms on benthic environments are likely to be of limited spatial extension and appear less relevant than those due to intensive ®sh farm activities (that employ external sources of organic matter; Mazzola et al. 1999a,b). Nonetheless, in certain cases, mussel biodeposition can aect benthic community structure (Castel et al., 1989; Dinet et al., 1990; Kr oncke, 1996) and might induce reducing conditions over a wide bottom area (Dahlback and Gunnarsson, 1981), thus aecting organic matter composition and cycling (Kaspar et al., 1985). Despite the growing interest in the critical eects of coastal aquaculture on the environment, studies on the benthic response to intensive mussel farms are still scarce, and no clear biological indicators of intensive mussel biodeposition have been identi®ed yet (Dinet et al., 1990; Mattson and Linden, 1983; Radziejewska, 1986; Tenore et al., 1982). Microbial assemblages, for their role in biogeochemical cycles, and meiofauna, for their high sensitivity to environmental changes, have been recently proposed as tools for monitoring the impact of organic enrichment following intensive aquaculture activities (Mazzola et al., 1999a,b; Mirto et al., 1999a).
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This study was designed to investigate the eects of a mussel farm on the coastal benthos of a temperate area of the Western Mediterranean. Sediment characteristics, microbial assemblages and meiofaunal parameters were investigated in order to evaluate: (a) changes in sediment characteristics, organic matter composition and pigment content due to the mussel-farm biodeposition; (b) changes in bacterial and meiofaunal density and community structure. Finally the results reported here will be compared to the impact due to ®sh-farm in an adjacent coastal area (Mazzola et al., 1999a,b) to investigate which parameters best describe the dierences between the two aquaculture activities.
Materials and Methods Study site and sampling This study was conducted from March 1997 to February 1998 in the Gaeta Gulf (Tyrrhenian Sea, NWMediterranean Sea, Fig. 1). The area is characterized by microtidal regime (about 30 cm), temperature ranging from 14.0°C in January to 25.3°C in August and the presence of two river estuaries. Dominant currents ¯ow in SE±NW direction following the cyclonic circulation of the Tyrrhenian Sea. The study area is sheltered and characterized by the presence of sandy-muddy sediments. Posidonia oceanica meadows are present in the northern part of the study area, although of limited extent (Ferretti et al., 1989). Visibility was highly reduced in the entire study area independently from the presence of the farms. The mussel farm under investigation is located in the inner part of the Gulf and produce a biomass equivalent to about 400 ton yearÿ1 . The culture technique used by local mussel farmers is the longline technique, which means that mussel larvae
Fig. 1 The sampling stations in the Gaeta Gulf, Tyrrhenian Sea (Western Mediterranean). The shadow box represents the mussel-farm area.
settle on ropes hanging down from long horizontal anchored lines, suspended by buoys. Sediment samples were collected on a monthly basis manually by SCUBA divers. A preliminary survey was carried out on a wide area and a grid of six stations was selected to identify the control station. To better follow the temporal evolution of the mussel-farm impact, two stations were selected: the Mussel station (at 10 m depth) and the Control station (about 800 m far from the long lines, in a southern area not aected by the aquaculture plants at a depth of 10 m). In the Gulf of Gaeta is also present a ®sh farm installation composed of 20 ¯oating cages, which, however, did not aect the hydrology or trophic conditions of the mussel area. Meiofaunal samples were collected in three replicate cores (inner diam. 4.0 cm, 12.6 cm2 surface area) down to a depth of 10 cm (which appeared to be adequate for quantitative analysis, after meiofaunal counting in the top 17-cm of the sediment core). The sediment of two additional cores was sectioned into dierent sediment layers: 0±1, 1±5 and 5±10 cm and frozen at ÿ20°C for the analysis of photosynthetic pigments and organic matter parameters. Environmental parameters Redox potential discontinuity (RPD) layer was visually estimated at the depth at which sediment colour turns from brown to black. Lipids were extracted from sediment samples by direct elution with chloroform and methanol according to Bligh and Dyer (1959) and Marsh and Weinstein (1966). Protein analyses were carried out according to Hartree (1972). Concentrations are presented as albumin equivalents. Carbohydrates were analysed according to Gerchacov and Hatcher (1972), speci®cally adapted for carbohydrate determination in sediments. For each analysis about 0.5 g of sediment was used. Carbohydrate, protein and lipid concentrations were converted to carbon equivalent assuming a conversion factor of 0.40, 0.49 and 0.75, respectively (Fichez, 1991). The sum of protein, lipid and carbohydrate carbon was referred to as biopolymeric fraction (BPF, Fabiano and Danovaro, 1994). For each biochemical analysis, blanks were made using the same previously calcinated sediments (450°C, 2 h). All analyses were carried out in three replicates. Only the results of the top 1-cm layer are reported in this study. Chlorophyll-a analysis n 3 was carried out according to Lorenzen and Jerey (1980). Chloroplastic pigment equivalents (CPE) were referred as the sum of chlorophyll-a and phaeopigment concentrations. Sediment organic matter concentrations were normalized to dry weight after desiccation at 60°C until constant weight. Microbial analyses Microbial analyses were carried out according to the procedure described by Maugeri et al. (1990), Porter and Feig (1980), Montagna, (1982). Brie¯y, each sediment replicate (n 3, about 1 cm3 , 0.2 lm ®ltered seawater 245
246 TABLE 1
a
1.4 2.1 1.9 3.2 2.9 1.4 1.6 1.7 1.5 1.4 1.8 2.0 1.9
0.2 0.2 0.4 1.0 0.5 0.2 0.4 0.3 0.4 1.0 0.8 0.5 0.6
SD
SD Standard Deviation.
March April May June July August September October November December January February Annual
cm
Control
1.1 1.1 1.2 1.6 1.3 1.3 1.3 0.6 1.2 1.1 1.9 2.0 1.3
0.2 0.2 0.3 0.2 0.3 0.8 0.4 0.5 0.3 0.2 0.4 0.5 0.4
SD
Mussel
cm
RPD
1778.3 2522.8 3897.6 4052.8 2442.9 2976.3 2567.8 1741.8 2597.8 2148.4 1617.9 3304.9 2637.4
324.0 352.2 1036.4 527.0 119.5 290.1 272.6 178.9 355.3 236.5 68.9 889.3 799.3
SD 3052.4 4222.7 5552.2 5175.6 4085.8 3841.2 3410.7 3758.7 4362.0 3954.5 2619.4 7718.9 4312.8
lg g 280.9 680.9 19.4 356.1 808.2 626.5 283.7 1165.4 985.2 607.4 283.4 649.0 1344.1
SD
ÿ1
ÿ1
lg g
Mussel
Control
PRT
5257.1 1240.9 1125.7 1656.2 2814.6 1811.4 1582.5 820.8 643.4 1105.9 503.8 1108.4 1639.2
lg g
ÿ1
1065.2 487.7 245.1 769.9 1470.4 903.7 408.6 181.8 107.1 1037.3 15.0 246.5 1294.9
SD
Control
3471.3 1296.7 1406.1 4180.4 4656.2 1715.2 1762.9 2554.8 1541.9 930.1 1099.7 1979.1 2216.2
ÿ1
693.0 112.2 302.5 1322.4 1173.7 168.0 508.1 449.1 168.3 385.4 91.5 1195.5 1238.4
SD
Mussel lg g
CHO
489.1 1219.8 367.7 1037.3 1435.7 1321.0 1009.7 2096.0 894.6 331.3 603.6 830.8 969.7
lg g
ÿ1
111.7 409.5 150.7 111.3 250.3 292.3 243.5 332.0 215.3 58.3 108.6 347.2 507.9
SD
Control
LIP
932.3 1971.9 871.5 2474.1 2899.7 2690.4 2490.4 2504.2 1942.5 1962.4 1376.1 2282.0 2033.1
lg g
ÿ1
85.0 594.7 104.8 211.1 605.3 174.9 159.4 1.5 506.1 271.3 364.6 1487.5 666.1
SD
Mussel
2315.2 1279.6 1220.5 2869.6 2293.5 1925.2 2018.7 1626.6 2463.1 1877.6 876.8 2399.3 1930.5
lg g
ÿ1
367.4 109.2 232.8 488.0 568.3 780.1 319.0 831.7 1502.6 330.1 267.1 270.7 588.2
SD
Control
BPC
1941.5 2185.0 1391.0 3590.7 4432.9 2995.8 4798.9 1337.0 2150.4 4714.5 2545.6 5190.5 3106.2
lg gÿ1
402.2 432.7 328.1 1376.3 881.4 474.1 905.1 438.8 591.9 833.8 445.3 997.5 1391.8
SD
Mussel
Environmental parameters: RPD: Redox Potential Discontinuity layer depth (cm), PRT: Proteins, CHO: Carbohydrates, LIP: Lipids and BPC: Biopolymeric carbon concentrations (as the sum of lipid, protein and carbohydrate carbon) in the top 1-cm sediment layer.a
Marine Pollution Bulletin
Volume 40/Number 3/March 2000
containing neutralized 2% formalin) was sonicated three times (Soni®er Transonic Labor 2000, 50 W for 1 min.). Total bacterial counts were obtained by staining cells with DAPI (40 6-diamidino-2-phenylindole). Bacterial cells were counted using epi¯uorescence microscopy on at least 10 ®elds randomly selected for a total count of more than 400 cells. Pico-sized prokaryotic and eukaryotic cells were classi®ed according to their auto¯uorescent spectrum: phycoerythrin±phycocyanin rich cyanobacteria (yellow±orange ¯uorescence) and chlorophyll-dominant (red±green ¯uorescence), respectively (Waterbury et al., 1986) counted as auto¯uorescent cells. Data were normalized to dry weight after desiccation (60°C, 24 h). Meiofaunal analysis Samples were ®xed with 4% buered formaldehyde in 0.4 lm pre®ltered seawater solution. Sediments were sieved through 1000 and 37 lm mesh sieves. The fraction remaining on the 37 lm mesh sieve was centrifuged three times with Ludox HS (density 1.18 g cmÿ3 ) as described by Heip et al. (1985). All meiobenthic animals were counted and classi®ed per taxon under a stereo microscope after staining with Rose Bengal (0.5 g lÿ1 ).
Results Environmental parameters The results of the environmental parameters investigated (RPD, proteins, carbohydrates, lipids and biopolymeric carbon) are reported in Table 1. The RPD depth was signi®cantly higher (t-test, p < 0:01) at the Control than beneath the mussel farm and ranged from 1.4 to 2.9 cm (in March and July, respectively) at the Control station and from 0.6 to 2.0 cm (in October and February, respectively) at the Mussel station. Chlorophyll-a concentrations were signi®cantly higher at the Mussel station (t-test, p < 0:01), and ranged from 2.3 1.0 to 21.9 5.6 lg gÿ1 (in February and May, respectively) at the Control station and from 9.0 2.2 to 54.0 23.2 lg gÿ1 (in August and May, respectively) at the Mussel station (Fig. 2a). Similar temporal patterns were displayed by chloroplastic pigment equivalent concentrations that, at all sampling periods, maintained signi®cantly higher values at the Mussel station than at the Control (Fig. 2b). Proteins were the dominant biochemical class of organic compounds ranging from 1617.9 68.9 to 4052.8 527.0 lg gÿ1 at the Control station (in January and June, respectively), and from 2619.4 283.4 to 7718.9 649.0 lg gÿ1 at the Mussel station, (in January and February, respectively). Total carbohydrates ranged from 503.8 15.0 to 5257.1 1065.2 lg gÿ1 at the Control station (in July and March, respectively) and from 930.1 385.4 to 4656.2 1173.7 lg gÿ1 at the Mussel station (in December and July, respectively). Finally, lipids ranged from 331.3 58.3 to 2096.0 332.0 lg gÿ1 at the Control station (in December and October, respectively)
and from 871.5 104.8 to 2899.7 605.3 lg gÿ1 at the Mussel station (in May and July, respectively). Biopolymeric carbon concentrations (BPC) displayed a similar temporal pattern and data reported here for the top 1-cm of the sediment core are not signi®cantly different than those integrated down to 10-cm depth (Mirto, 1998). Microbial density and composition Total bacterial numbers (TBN) ranged from 0.30 0.10 to 1:80 0:70 108 cells gÿ1 (in May and March, respectively) at the Control station and from 0.60 0.07 to 2:90 0:90 108 cells gÿ1 (in May and March, respectively) at the Mussel station (Fig. 3a). Auto¯uorescent microbial cells density (de®ned here as `pico-phytobenthos' PPB) ranged from 0.16 0.05 to 0.73 0.40106 cells gÿ1 (in December and July, respectively) at the Control station, whilst at the Mussel station ranged from 0.50 0.06 to 6:00 2:80 106 cells gÿ1 (in March and October, respectively; Fig. 3b). PPB density at the Mussel station was signi®cant higher than at the Control (t-test, p 0:005) and dierences between stations were particularly evident from June to October. The contribution of the eukaryotic cells to the total density of auto¯uorescent cells is illustrated in Fig. 4. At the Control station, eukaryotic cells accounted from 4% to 88% (in July and March, respectively) of the total PPB density. Eukaryotic cell contribution to PPB
Fig. 2 Temporal changes in chlorophyll-a and Chloroplastic Pigment Equivalents (CPE) concentrations in the top 1-cm of the sediment. Reported are the results of the student's t-test: p < 0:05; p < 0:01, no indication is reported when dierence is not signi®cant.
247
Marine Pollution Bulletin
density decreased markedly at the Mussel station where it accounted from 1% to 46% (October and March, respectively).
Fig. 3 Temporal variations of total bacteria and auto¯uorescent microbial density at the Control station and at the Mussel station. Data are expressed as cell. gÿ1 SD. Reported are the result of the student t-test: p < 0:05; p < 0:01, no indication is reported when dierence is not signi®cant.
Meiofaunal abundance and community structure Meiofaunal density in the top 10-cm of the sediments ranged from 1137 1060 to 2950 254 ind. 10 cmÿ2 (in December and September, respectively) in the Control, whilst in the Mussel station meiofaunal density ranged from 870 263 to 2238 178 ind. 10 cmÿ2 (in October and June, respectively, Fig. 5). Meiofaunal densities beneath the mussel farm during the 12 months of sampling were signi®cantly lower than at the Control station (2071 181 and 1551 121 ind. 10 cmÿ2 ) at Control and Mussel station respectively; t-test, p 0:014), and biodeposition impact upon meiofaunal density was particularly evident in July, September and October (when dierences were statistically signi®cant). Temporal changes of the main meiofaunal taxa are illustrated in Fig. 6, while the comparison of meiofaunal community structure at Control and Mussel stations is illustrated in Fig. 7. A similar meiofaunal composition was observed in the two stations: nematodes accounted on average for 75% of the total density at both stations, followed by copepods (10% and 15% at the Control and Mussel respectively), polychaetes (4% in both stations). Only kinorhynch, ostracod and turbellarian densities strongly decreased at the mussel station.
Discussion Mussel-farm impact on sediment characteristics The mussel farm induced evident changes in the sediment characteristics. Oxygen penetration into the bottom sediments (measured as RPD depth) was signi®cantly reduced at the Mussel station, except in January and February, when water column mixing also induced an increase of oxygen availability in the upper layers of the sediments (La Rosa, 1998). The biodepo-
Fig. 4 Bacterial community structure in the Control site and under the mussel farm.
248
Fig. 5 Temporal variations of total meiofaunal density at the Control station and at the Mussel station. Data are expressed as ind. 10 cmÿ2 SD. Reported are the result of the student t-test: p < 0:05; p < 0:01, no indication is reported when dierence is not signi®cant.
Volume 40/Number 3/March 2000
sition due to mussel farm activities led to a signi®cant increase in phytopigment concentrations. In agreement with Navarro and Thomposon (1997), such accumulation was apparently related to spring phytoplankton blooms that were ®ltered and compacted by mussels and discharged to the sediments, reaching concentrations of 50 lg chlorophyll-a gÿ1 . The signi®cant dierence observed between Mussel and Control station (t-test, p < 0:01) suggests that total phytopigments (as chlorophyll-a or CPE) might be used as tracers of mussel biodeposition. Phytopigment concentrations reported here were much higher than those reported by Grenz et al. (1990) from mussel farms of the Gulf of Fos (NW Mediterranean) or in other New Zealand and French sites (Barranguet et al., 1993; Kaspar et al., 1985). Previous studies carried out on mussel farms suggested that high values of chlorophyll-a are due to deposition of pseudo-faeces, while high phaeopigment concentrations are related to the deposition of faeces (Dahlback and Gunnarsson, 1981; Navarro and Thomposon, 1997). In
this study, we observed a phaeopigment content 2±4 times higher than chlorophyll-a and, on average, CPE content in the sediments beneath the mussels was about 6 times higher than in the Control (13.52 1.59 vs 86.84 14.25 lg gÿ1 ). Sedimentary organic carbon enrichment has been reported in most studies dealing with aquaculture impacts (Castel et al., 1989; Gilbert et al., 1997; Grenz et al., 1990), but total organic content appears to be more conservative than biopolymeric organic carbon (BPC, Mazzola et al. 1999). Mussel biodeposition eects were evident in terms of biopolymeric carbon accumulation that at all sampling periods at the Mussel stations showed values signi®cantly higher than in the Control (t-test, p < 0:01). BPC enrichment of the mussel-farm sediments was less evident in spring, during the phytoplankton bloom, due to the high organic matter input to the sediments (reported also in the sediments of the Control stations). The analysis of the dierent biochemical classes of organic compounds contributing to
Fig. 6 Temporal variations in meiofaunal density in the Control site and under the mussel farm. Reported are: (a) nematodes; (b) copepods; (c) polychaetes; (d) kinorhynchs; (e) ostracods; (f) turbellarians. Data are expressed as ind. 10 cmÿ2 SD. Reported are the result of the student t-test: p < 0:05; p < 0:01, no indication is reported when dierence is not signi®cant.
249
Marine Pollution Bulletin
contribution to the total auto¯uorescent microbial density. Therefore, we might conclude that the microbial parameters that best highlight the microbiological effects of mussel-farm biodeposition are the increase of the autotrophic component of the total bacteria community and the decrease in importance of eukaryotic cells.
Fig. 7 Meiofaunal community structure in the Control site and under the mussel farm.
the biopolymeric pool revealed that protein and lipids accumulated preferentially in mussel-farm sediments, being signi®cantly higher than in the Control (t-test, p < 0:02), whereas carbohydrates appeared to be more conservative (Table 1). Mussel-farm impact on bacterial assemblages Bacterial densities recorded in this study at Control station (on average 1:11 0:16 108 cells gÿ1 ) are comparable with those reported in most coastal sites of the Mediterranean Sea (Danovaro et al., 1998; La Rosa, 1998). Sediment organic enrichment and the modi®cation of the characteristics of the benthic environment led to an increase of total bacterial density of about 64%, but the increase was signi®cant in April (t-test, p < 0:01), May and June (t-test, p < 0:05) and October (t-test, p 0:01). Additional evidence of the impact of the mussel activities on the benthic bacterial communities is provided by the signi®cant increase of auto¯uorescent cells (as PPB density) that were three times as numerous at the mussel station compared to the Control. Changes in the community structure of the bacteria beneath the mussel farm were also evident in terms of reduced contribution of the eukaryotic density to the total microbial density. Such changes are likely to be the result of the combined eect of a larger organic matter input and a reduction of oxygen penetration into the sediments. Changes occurring in microbial assemblages from mussel-farm sediments can be summarized as follows: (1) increase of the total bacterial density; (2) increase of the cyanobacteria contribution to the total microbial density and (3) strong decrease of the picoeukaryotic 250
Mussel-farm impact on meiofaunal assemblages Mussel biodeposition had a signi®cant impact on meiofaunal assemblages. Meiofaunal density at the Mussel station was signi®cantly lower than at the Control station (on average 2071 625 and 1550 420 ind. 10 cmÿ2 at Control and Mussel station respectively; t-test, p < 0:01). Biodeposition by bivalves generally provides a strong input of organic matter of high quality and availability to benthic assemblages and previous studies reported a positive response (i.e., an increase) of meiofaunal density to this organic enrichment (Castel et al., 1989; Dinet et al., 1990; Guelorget et al., 1994). By contrast, we reported a clear decrease of the total meiofaunal density apparently due to changes in the sedimentary characteristics (i.e., the layer of faecal material reduced oxygen ¯uxes at water-sediments interface). The eects of mussel biodeposition on meiofaunal assemblages were more dicult to point out in terms of assemblage composition. Nematodes accounted on average for 75% of total meiofaunal density at both stations. Copepods displayed good tolerance to mussel biodeposition and their density remained rather constant with time or even increased. Such copepod response, characterized by harpacticoid dominance, is likely due to their capability of exploiting the enrichment in microphytobenthic biomass (expressed as chlorophyll-a) associated to mussel biodeposits. Polychaete densities, on average, were similar at the two stations but decreased signi®cantly at the Mussel station in April and August (t-test, p < 0:05), September and November (t-test, p < 0:01). Other taxa, such as turbellarians, ostracods and kinorhynchs, displayed a signi®cant decrease in the mussel-farm sediments, and such dierences were particularly evident for kinorhynchs that disappeared almost completely from July to February. Nematodes are assumed to be quite resistant to sediment organic enrichment and the resulting reducing conditions and some species are permanently found in suboxic sediments (Danovaro, 1999; Danovaro et al., 1999; Giere, 1993; Powell, 1989). However, the results of this study pointed out, on annual average, a signi®cant impact of the organic enrichment on nematode abundance (t-test, p 0:01). Similar results were reported by Dinet et al. (1990) that demonstrated a strong impact of bivalve biodeposition on meiofauna density, which decreased drastically (by a factor of four) when compared to the Control site. Conversely, other authors reported an increase of the total meiofaunal density induced by mussel biodeposition (Castel et al., 1989; Guelorget et al., 1994; Radziejewska, 1986). However, a more
Volume 40/Number 3/March 2000
detailed analysis revealed that such a positive response was observed in high energy environments (such as coastal lagoons), where resuspension events reduced OM accumulation and enhanced oxygen penetration into the sediments. The presence of ®sh farm installations in the Gulf of Gaeta enabled us to compare the impact of two dierent aquaculture activities on this coastal system. Previous studies have indeed demonstrated that organic enrichment due to ®sh farm resulted in a strong and statistically signi®cant reduction of the total meiofaunal density (which decreased about 50%), mostly due unexpected nematode sensitivity to this kind of disturbance (Mazzola et al. 1999a,b). In this regard, mussel farm biodeposition had a clearly lower impact, as meiofaunal density decreased only 25%. Changes induced by biodeposition were also observed in terms of meiofaunal community structure, and it is worth noting that both mussel and ®sh farm impact determined an increase of the copepod percentage at the expense of nematodes, which together with kinorhynchs, turbellarians and ostracods might represent useful indicators of biodeposition disturbance. The authors are particularly indebted with the sta of the Med®sh s.r.l. (Gaeta) for precious collaboration during sampling. Thank are due to Dr C. Gambi and Dr M. Cecchi (University of Ancona) for help in meiofaunal sorting. Our thanks are due also to Dr E. Favaloro and Dr B. Savona (University of Palermo) for kind support during sampling. A special thanks is due to Prof. T. Maugeri (University of Messina) for suggestions and for providing laboratory facilities. This work is part of the doctoral thesis of S.M. and T.L.R. and was supported by a grant of the Ministero dell'Universit a e Ricerca Scienti®ca e Tecnologica and Ministero per le Politiche Agricole, Italy. Barg, U. and Phillips, M. J. (1998) Environment and Sustainability. FAO Fisheries Circular No. 886, Rev.1, Rome, FAO. 163 pp. Barranguet, C., Alliot, E. and Plante-Cuny, M. R. (1993) Benthic microphytic activity at two Mediterranean shell®sh cultivation sites with reference to benthic ¯uxes. Oceanologica Acta 17 (2), 211±221. Bligh, E. G. and Dyer, W. (1959) A rapid method for total lipid extraction and puri®cation. Canadian Journal of Biochemistry and Physiology 37, 911±917. Castel, J., Labourg, P. J., Escaravage, V., Auby, I. and Garcia M. E. (1989) In¯uence of Seagrass Beds and Oyster Parks on the Abundance and Biomass Patterns of Meio-and Macrobenthos in Tidal Flats. Estuarine, Coastal and Shelf Science 28, 71±85. H. (1981) Sedimentation and Dahlback, B. and Gunnarsson, L. A. Sulfate Reduction Under a Mussel Culture. Marine Biology 63, 269±275. Danovaro, R. (1999) Benthic microbial loop and meiofaunal response to oil induced disturbance in coastal sediments: a review. International Journal of Environmental Pollution, in press. Danovaro, R., Della Croce, N. and Fabiano, M. (1998) Biochemical composition of particulate organic matter and bacterial dynamics at the sediment±water interface in a Mediterranean seagrass system. Hydrobiologia 363, 241±252. Danovaro, R., Della Croce, N., Martorano, D., Parodi, P., Pusceddu, A. and Fabiano, M. (1999) Biological indicators of oil induced disturbance in coastal sediments of the Ligurian Sea. Pertanika, in press. Dinet, A., Sornin, J. M., Sabliere, A., Delmas, D. and Feuillet-Girard, M. (1990) In¯uence de la biodeposition de bivalves ®ltreurs sur les peuplements meiobenthiques dÕun marais maritime. Cahiers de Biologie Marine 31, 307±322. Fabiano, M. and Danovaro, R. (1994) Composition of organic matter in sediment facing a river estuary (Tyrrhenian Sea): relationships with bacteria and microphytobenthic biomass. Hydrobiologia 277, 71±84.
Ferretti, O., Niccolai, I., Bianchi, C. N., Tucci, S., Morri, C. and Veniale, F. (1989) An environmental investigation of a marine coastal area: Gulf of Gaeta (Tyrrhenian Sea) Hydrobiologia 176/ 177, 171±187. Fichez, R. (1991) Composition of fate of organic matter in submarine cave sediments: implications for the biogeochemical cycle of organic carbon. Oceanologica Acta 14, 369±377. Gerchacov, S. M. and Hatcher, P. G. (1972) Improved technique for analysis of carbohydrates in sediments. Limnology and Oceanography 17, 938±943. Giere, O. (1993) Meiobenthonology. Springer, Berlin, pp. 1±328. Gilbert, F., Souchu, P., Bianchi, M. and Bonin, P. (1997) In¯uence of shell®sh farming activities on nitri®cation, nitrate reduction to ammonium and denitri®cation at the water-sediment interface of the Thau lagoon, France. Marine Ecology Progress Series 151, 143± 153. Grenz, C., Hermin, M. N., Baudinet, D. and Daumas, R. (1990) In situ biochemical and bacterial variation of sediments enriched with mussel biodeposits. Hydrobiologia 207, 153±160. Guelorget, O., Perthuisot, J. P., Lamy, N. and Lefebre, A. (1994) Structure et organisation de lÕetang de Thau dÕapres la fauna benthique (macrofaune, meiofaune) Relations avec le con®nement. Oceanologia Acta 17, 105±114. Hartree, E. F. (1972) Determination of proteins: a modi®cation of the Lowry method that give a linear photometric response. Analytical Biochemistry 48, 422±427. Heip, C., Vincx, M. and Vranken, G. (1985) The ecology of marine nematodes. Oceanography and Marine Biology Annual Review 23, 399±489. Kaspar, H. F., Gillespie, P. A., Boyer, I. C. and MacKenzie, A. L. (1985) Eects of mussel aquaculture on the nitrogen cycle and benthic communities in Kenepuru Sound, Malborough Sounds, New Zealand. Marine Biology 85, 127±136. Kr oncke, I. (1996) Impact of biodeposition on macrofaunal communities in intertidal sand¯ats. Marine Ecology 17 (1±3), 159±174. La Rosa, T. (1998) Comunit a microbiche in aree interessate da impianti di maricoltura. Ph.D. Thesis, University of Messina, 94 pp. Lorenzen, C. and Jerey, J. (1980) Determination of chlorophyll in sea water. Unesco Technical Papers in Marine Science 35, 1±20. Marsh, J. B. and Weinstein, W. J. (1966) A simple charring method for determination of lipids. Journal of Lipid Research 7, 574±576. Mattson, J. and Linden, O. (1983) Benthic macrofauna succession under mussel, Mytilus edulis L. (Bivalva), cultured on hanging longlines. Sarsia 68, 97±102. Maugeri, T. L., Acosta Pomar, M. L. C. and Bruni, V. (1990) Picoplancton. Nova Thalassia 11, 199±205. Mazzola, A., Mirto, S. and Danovaro, R. (1999a) Initial ®sh-farm impact on meiofaunal assemblages in coastal sediments of the western Mediterranean. Marine Pollution Bulletin, 38, 1126±1133. Mazzola, A., Mirto, S., Danovaro, R. and Fabiano, M. (1999b) Fish farming eects on benthic community structure in coastal sediments: analysis of the meiofaunal resilience. ICES Journal Marine Science, in press. Mirto, S. (1998) Risposta delle comunit a meiobentoniche a biodepositi di impianti di maricoltura nel Golfo di Gaeta. Ph.D. Thesis, University of Messina, 185 pp. Mirto, S., Fabiano, M., Danovaro, R., Manganaro, A. and Mazzola, A. (1999a) Use of meiofauna for detecting ®sh farming disturbance in coastal sediments: preliminary results. Biologia Marina Mediterranea 6 (1), 331±334. Mirto, S., Gambi, C., Cecchi, M. and Manganaro, A. (1999b) In¯uences of biodeposition due to intensive mussel culture on a coastal meiofaunal assemblages: preliminary results. Atti 13° Congresso AIOL, in press. Montagna, P. A. (1982) Sampling design and enumeration statistics for bacteria from marine sediments. Applied and Environmental Microbiology 43, 1376±1372. Navarro, J. M. and Thomposon, R. J. (1997) Biodeposition by the horse mussel Modiolus modiolus (Dillwyn) during the spring diatom bloom. Journal of Experimental Marine Biology and Ecology 209, 1±13. Porter, K. G. and Feig, Y. S. (1980) The use of DAPI for identifying and counting of aquatic micro¯ora. Limnology Oceanography 25, 943±948. Powell, E. N. (1989) Oxygen, sulphide and diusion: why thiobiotic meiofauna must be sulphide-insensitive ®rst-order respirers. Journal of Marine Research 47, 887±932.
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Marine Pollution Bulletin Radziejewska, T. (1986) On the role of Mytilus edulis aggregations in enhancing meiofauna communities o the southern Baltic coast. Ophelia supp. 4, 211±218. Rana, K. J. (1998) Global overview of production and production trends. FAO Fisheries Circular No. 886, Rev.1, Rome, FAO. 163 pp. Tenore, K. R., Boyer, L. F., Cal, R. M., Corral, J., Garcia-Fernandez, C., Gonzalez, N., Gonzalez-Gurriaran, E., Hanson, R. B., Iglesias, J., Krom, M., Lopez-Jamar, E., McClain, J., Pamatman, M., Perez,
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A., Rhoads, D. C., de Santiago, G., Tietjen, J., Westrich, J. and Windom, H. L. (1982) Coastal upwelling in the Rõas Bajas, NW Spain: contrasting the benthic regimes of the Rõas de Arosa and the Muros. Journal Marine Research 40, 701±772. Waterbury, J. B., Watson, S. W., Valois, F. W. and Franks, D. G. (1986) Biological and ecological characterization of marine unicellular cyanobacterium Synechococcus. Canadian Bulletin of Fisheries and Aquatic Science 214, 71±120.
Marine Pollution Bulletin Vol. 40, No. 3, pp. 244±252, 2000 Ó 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0025-326X/00 $ - see front matter
Microbial and Meiofaunal Response to Intensive Mussel-Farm Biodeposition in Coastal Sediments of the Western Mediterranean S. MIRTO à*, T. LA ROSAà, R. DANOVARO § and A. MAZZOLAà Instituto Scienze del Mare, Facolta di Scienze, Universit a di Ancona, Via Brecce Bianche, Monte D' Ago, 60131 Ancona, Italy àDip. Biologia Animale, Universit a di Palermo, Via Archira®, 18, 90123 Palermo, Italy §Dip. Zoologia, Universit a di Bari, Via Orabona 4, 70125 Bari, Italy We studied the impact of organic loads due to the biodeposition of a mussel farm in a coastal area of the Tyrrhenian Sea (Western Mediterranean). Sediment chemistry, microbial and meiofaunal assemblages were investigated from March 1997 to February 1998 on monthly basis at two stations: the ®rst was located under the mussel farm, while the second was at about 1-km distance and served as control. Benthic response to changes in the biodepositional regime was investigated in terms of biochemical composition of the sedimentary organic matter, phytopigment content, bacterial abundance and composition and meiofaunal community structure. A large accumulation of chloroplastic pigments, proteins and lipids was observed under the mussel farm. Such changes in the sedimentary conditions re¯ected the accumulation of faeces and pseudo-faeces and led to the creation of reducing conditions. Microbial assemblages beneath the mussel cultures increased their densities and displayed, when compared to the control, a larger cyanobacterial importance associated to a strong decrease of the picoeukaryotic cell density. Farm sediments displayed signi®cant changes in meiofaunal density: turbellarian, ostracod and kinorhynch densities decreased signi®cantly, while copepods remained constant or increased possibly pro®ting of the enrichment in microphytobenthic biomass associated to mussel biodeposits. The comparative analysis of the mussel biodeposition and ®sh-farm impact on sediments beneath the cultures revealed that mussel farms induced a considerably lower disturbance on benthic community structure. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: mussel farm; bacteria; cyanobacteria; picoeukaryotes; meiofauna; Mediterranean Sea. *Corresponding author. Tel.: +39-71-220-4654; fax: +39-71-2204650. E-mail address: [email protected]
244
In the Mediterranean Sea, aquaculture activities (particularly ®sh and mussel farms) have expanded signi®cantly during recent years (Rana, 1998), inducing an increasing interest and concern for their potential impact on coastal marine environments, with important management implications (Barg and Phillips, 1998). Mussel farming is known to be responsible for intensive biodeposition of faeces and pseudo-faeces that might cause strong changes in the physical and chemical characteristics of the sediments beneath the long lines (Dahlback and Gunnarsson, 1981; Gilbert et al., 1997; Kaspar et al., 1985; Mirto et al., 1999b). The eects of mussel farms on benthic environments are likely to be of limited spatial extension and appear less relevant than those due to intensive ®sh farm activities (that employ external sources of organic matter; Mazzola et al. 1999a,b). Nonetheless, in certain cases, mussel biodeposition can aect benthic community structure (Castel et al., 1989; Dinet et al., 1990; Kr oncke, 1996) and might induce reducing conditions over a wide bottom area (Dahlback and Gunnarsson, 1981), thus aecting organic matter composition and cycling (Kaspar et al., 1985). Despite the growing interest in the critical eects of coastal aquaculture on the environment, studies on the benthic response to intensive mussel farms are still scarce, and no clear biological indicators of intensive mussel biodeposition have been identi®ed yet (Dinet et al., 1990; Mattson and Linden, 1983; Radziejewska, 1986; Tenore et al., 1982). Microbial assemblages, for their role in biogeochemical cycles, and meiofauna, for their high sensitivity to environmental changes, have been recently proposed as tools for monitoring the impact of organic enrichment following intensive aquaculture activities (Mazzola et al., 1999a,b; Mirto et al., 1999a).
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This study was designed to investigate the eects of a mussel farm on the coastal benthos of a temperate area of the Western Mediterranean. Sediment characteristics, microbial assemblages and meiofaunal parameters were investigated in order to evaluate: (a) changes in sediment characteristics, organic matter composition and pigment content due to the mussel-farm biodeposition; (b) changes in bacterial and meiofaunal density and community structure. Finally the results reported here will be compared to the impact due to ®sh-farm in an adjacent coastal area (Mazzola et al., 1999a,b) to investigate which parameters best describe the dierences between the two aquaculture activities.
Materials and Methods Study site and sampling This study was conducted from March 1997 to February 1998 in the Gaeta Gulf (Tyrrhenian Sea, NWMediterranean Sea, Fig. 1). The area is characterized by microtidal regime (about 30 cm), temperature ranging from 14.0°C in January to 25.3°C in August and the presence of two river estuaries. Dominant currents ¯ow in SE±NW direction following the cyclonic circulation of the Tyrrhenian Sea. The study area is sheltered and characterized by the presence of sandy-muddy sediments. Posidonia oceanica meadows are present in the northern part of the study area, although of limited extent (Ferretti et al., 1989). Visibility was highly reduced in the entire study area independently from the presence of the farms. The mussel farm under investigation is located in the inner part of the Gulf and produce a biomass equivalent to about 400 ton yearÿ1 . The culture technique used by local mussel farmers is the longline technique, which means that mussel larvae
Fig. 1 The sampling stations in the Gaeta Gulf, Tyrrhenian Sea (Western Mediterranean). The shadow box represents the mussel-farm area.
settle on ropes hanging down from long horizontal anchored lines, suspended by buoys. Sediment samples were collected on a monthly basis manually by SCUBA divers. A preliminary survey was carried out on a wide area and a grid of six stations was selected to identify the control station. To better follow the temporal evolution of the mussel-farm impact, two stations were selected: the Mussel station (at 10 m depth) and the Control station (about 800 m far from the long lines, in a southern area not aected by the aquaculture plants at a depth of 10 m). In the Gulf of Gaeta is also present a ®sh farm installation composed of 20 ¯oating cages, which, however, did not aect the hydrology or trophic conditions of the mussel area. Meiofaunal samples were collected in three replicate cores (inner diam. 4.0 cm, 12.6 cm2 surface area) down to a depth of 10 cm (which appeared to be adequate for quantitative analysis, after meiofaunal counting in the top 17-cm of the sediment core). The sediment of two additional cores was sectioned into dierent sediment layers: 0±1, 1±5 and 5±10 cm and frozen at ÿ20°C for the analysis of photosynthetic pigments and organic matter parameters. Environmental parameters Redox potential discontinuity (RPD) layer was visually estimated at the depth at which sediment colour turns from brown to black. Lipids were extracted from sediment samples by direct elution with chloroform and methanol according to Bligh and Dyer (1959) and Marsh and Weinstein (1966). Protein analyses were carried out according to Hartree (1972). Concentrations are presented as albumin equivalents. Carbohydrates were analysed according to Gerchacov and Hatcher (1972), speci®cally adapted for carbohydrate determination in sediments. For each analysis about 0.5 g of sediment was used. Carbohydrate, protein and lipid concentrations were converted to carbon equivalent assuming a conversion factor of 0.40, 0.49 and 0.75, respectively (Fichez, 1991). The sum of protein, lipid and carbohydrate carbon was referred to as biopolymeric fraction (BPF, Fabiano and Danovaro, 1994). For each biochemical analysis, blanks were made using the same previously calcinated sediments (450°C, 2 h). All analyses were carried out in three replicates. Only the results of the top 1-cm layer are reported in this study. Chlorophyll-a analysis n 3 was carried out according to Lorenzen and Jerey (1980). Chloroplastic pigment equivalents (CPE) were referred as the sum of chlorophyll-a and phaeopigment concentrations. Sediment organic matter concentrations were normalized to dry weight after desiccation at 60°C until constant weight. Microbial analyses Microbial analyses were carried out according to the procedure described by Maugeri et al. (1990), Porter and Feig (1980), Montagna, (1982). Brie¯y, each sediment replicate (n 3, about 1 cm3 , 0.2 lm ®ltered seawater 245
246 TABLE 1
a
1.4 2.1 1.9 3.2 2.9 1.4 1.6 1.7 1.5 1.4 1.8 2.0 1.9
0.2 0.2 0.4 1.0 0.5 0.2 0.4 0.3 0.4 1.0 0.8 0.5 0.6
SD
SD Standard Deviation.
March April May June July August September October November December January February Annual
cm
Control
1.1 1.1 1.2 1.6 1.3 1.3 1.3 0.6 1.2 1.1 1.9 2.0 1.3
0.2 0.2 0.3 0.2 0.3 0.8 0.4 0.5 0.3 0.2 0.4 0.5 0.4
SD
Mussel
cm
RPD
1778.3 2522.8 3897.6 4052.8 2442.9 2976.3 2567.8 1741.8 2597.8 2148.4 1617.9 3304.9 2637.4
324.0 352.2 1036.4 527.0 119.5 290.1 272.6 178.9 355.3 236.5 68.9 889.3 799.3
SD 3052.4 4222.7 5552.2 5175.6 4085.8 3841.2 3410.7 3758.7 4362.0 3954.5 2619.4 7718.9 4312.8
lg g 280.9 680.9 19.4 356.1 808.2 626.5 283.7 1165.4 985.2 607.4 283.4 649.0 1344.1
SD
ÿ1
ÿ1
lg g
Mussel
Control
PRT
5257.1 1240.9 1125.7 1656.2 2814.6 1811.4 1582.5 820.8 643.4 1105.9 503.8 1108.4 1639.2
lg g
ÿ1
1065.2 487.7 245.1 769.9 1470.4 903.7 408.6 181.8 107.1 1037.3 15.0 246.5 1294.9
SD
Control
3471.3 1296.7 1406.1 4180.4 4656.2 1715.2 1762.9 2554.8 1541.9 930.1 1099.7 1979.1 2216.2
ÿ1
693.0 112.2 302.5 1322.4 1173.7 168.0 508.1 449.1 168.3 385.4 91.5 1195.5 1238.4
SD
Mussel lg g
CHO
489.1 1219.8 367.7 1037.3 1435.7 1321.0 1009.7 2096.0 894.6 331.3 603.6 830.8 969.7
lg g
ÿ1
111.7 409.5 150.7 111.3 250.3 292.3 243.5 332.0 215.3 58.3 108.6 347.2 507.9
SD
Control
LIP
932.3 1971.9 871.5 2474.1 2899.7 2690.4 2490.4 2504.2 1942.5 1962.4 1376.1 2282.0 2033.1
lg g
ÿ1
85.0 594.7 104.8 211.1 605.3 174.9 159.4 1.5 506.1 271.3 364.6 1487.5 666.1
SD
Mussel
2315.2 1279.6 1220.5 2869.6 2293.5 1925.2 2018.7 1626.6 2463.1 1877.6 876.8 2399.3 1930.5
lg g
ÿ1
367.4 109.2 232.8 488.0 568.3 780.1 319.0 831.7 1502.6 330.1 267.1 270.7 588.2
SD
Control
BPC
1941.5 2185.0 1391.0 3590.7 4432.9 2995.8 4798.9 1337.0 2150.4 4714.5 2545.6 5190.5 3106.2
lg gÿ1
402.2 432.7 328.1 1376.3 881.4 474.1 905.1 438.8 591.9 833.8 445.3 997.5 1391.8
SD
Mussel
Environmental parameters: RPD: Redox Potential Discontinuity layer depth (cm), PRT: Proteins, CHO: Carbohydrates, LIP: Lipids and BPC: Biopolymeric carbon concentrations (as the sum of lipid, protein and carbohydrate carbon) in the top 1-cm sediment layer.a
Marine Pollution Bulletin
Volume 40/Number 3/March 2000
containing neutralized 2% formalin) was sonicated three times (Soni®er Transonic Labor 2000, 50 W for 1 min.). Total bacterial counts were obtained by staining cells with DAPI (40 6-diamidino-2-phenylindole). Bacterial cells were counted using epi¯uorescence microscopy on at least 10 ®elds randomly selected for a total count of more than 400 cells. Pico-sized prokaryotic and eukaryotic cells were classi®ed according to their auto¯uorescent spectrum: phycoerythrin±phycocyanin rich cyanobacteria (yellow±orange ¯uorescence) and chlorophyll-dominant (red±green ¯uorescence), respectively (Waterbury et al., 1986) counted as auto¯uorescent cells. Data were normalized to dry weight after desiccation (60°C, 24 h). Meiofaunal analysis Samples were ®xed with 4% buered formaldehyde in 0.4 lm pre®ltered seawater solution. Sediments were sieved through 1000 and 37 lm mesh sieves. The fraction remaining on the 37 lm mesh sieve was centrifuged three times with Ludox HS (density 1.18 g cmÿ3 ) as described by Heip et al. (1985). All meiobenthic animals were counted and classi®ed per taxon under a stereo microscope after staining with Rose Bengal (0.5 g lÿ1 ).
Results Environmental parameters The results of the environmental parameters investigated (RPD, proteins, carbohydrates, lipids and biopolymeric carbon) are reported in Table 1. The RPD depth was signi®cantly higher (t-test, p < 0:01) at the Control than beneath the mussel farm and ranged from 1.4 to 2.9 cm (in March and July, respectively) at the Control station and from 0.6 to 2.0 cm (in October and February, respectively) at the Mussel station. Chlorophyll-a concentrations were signi®cantly higher at the Mussel station (t-test, p < 0:01), and ranged from 2.3 1.0 to 21.9 5.6 lg gÿ1 (in February and May, respectively) at the Control station and from 9.0 2.2 to 54.0 23.2 lg gÿ1 (in August and May, respectively) at the Mussel station (Fig. 2a). Similar temporal patterns were displayed by chloroplastic pigment equivalent concentrations that, at all sampling periods, maintained signi®cantly higher values at the Mussel station than at the Control (Fig. 2b). Proteins were the dominant biochemical class of organic compounds ranging from 1617.9 68.9 to 4052.8 527.0 lg gÿ1 at the Control station (in January and June, respectively), and from 2619.4 283.4 to 7718.9 649.0 lg gÿ1 at the Mussel station, (in January and February, respectively). Total carbohydrates ranged from 503.8 15.0 to 5257.1 1065.2 lg gÿ1 at the Control station (in July and March, respectively) and from 930.1 385.4 to 4656.2 1173.7 lg gÿ1 at the Mussel station (in December and July, respectively). Finally, lipids ranged from 331.3 58.3 to 2096.0 332.0 lg gÿ1 at the Control station (in December and October, respectively)
and from 871.5 104.8 to 2899.7 605.3 lg gÿ1 at the Mussel station (in May and July, respectively). Biopolymeric carbon concentrations (BPC) displayed a similar temporal pattern and data reported here for the top 1-cm of the sediment core are not signi®cantly different than those integrated down to 10-cm depth (Mirto, 1998). Microbial density and composition Total bacterial numbers (TBN) ranged from 0.30 0.10 to 1:80 0:70 108 cells gÿ1 (in May and March, respectively) at the Control station and from 0.60 0.07 to 2:90 0:90 108 cells gÿ1 (in May and March, respectively) at the Mussel station (Fig. 3a). Auto¯uorescent microbial cells density (de®ned here as `pico-phytobenthos' PPB) ranged from 0.16 0.05 to 0.73 0.40106 cells gÿ1 (in December and July, respectively) at the Control station, whilst at the Mussel station ranged from 0.50 0.06 to 6:00 2:80 106 cells gÿ1 (in March and October, respectively; Fig. 3b). PPB density at the Mussel station was signi®cant higher than at the Control (t-test, p 0:005) and dierences between stations were particularly evident from June to October. The contribution of the eukaryotic cells to the total density of auto¯uorescent cells is illustrated in Fig. 4. At the Control station, eukaryotic cells accounted from 4% to 88% (in July and March, respectively) of the total PPB density. Eukaryotic cell contribution to PPB
Fig. 2 Temporal changes in chlorophyll-a and Chloroplastic Pigment Equivalents (CPE) concentrations in the top 1-cm of the sediment. Reported are the results of the student's t-test: p < 0:05; p < 0:01, no indication is reported when dierence is not signi®cant.
247
Marine Pollution Bulletin
density decreased markedly at the Mussel station where it accounted from 1% to 46% (October and March, respectively).
Fig. 3 Temporal variations of total bacteria and auto¯uorescent microbial density at the Control station and at the Mussel station. Data are expressed as cell. gÿ1 SD. Reported are the result of the student t-test: p < 0:05; p < 0:01, no indication is reported when dierence is not signi®cant.
Meiofaunal abundance and community structure Meiofaunal density in the top 10-cm of the sediments ranged from 1137 1060 to 2950 254 ind. 10 cmÿ2 (in December and September, respectively) in the Control, whilst in the Mussel station meiofaunal density ranged from 870 263 to 2238 178 ind. 10 cmÿ2 (in October and June, respectively, Fig. 5). Meiofaunal densities beneath the mussel farm during the 12 months of sampling were signi®cantly lower than at the Control station (2071 181 and 1551 121 ind. 10 cmÿ2 ) at Control and Mussel station respectively; t-test, p 0:014), and biodeposition impact upon meiofaunal density was particularly evident in July, September and October (when dierences were statistically signi®cant). Temporal changes of the main meiofaunal taxa are illustrated in Fig. 6, while the comparison of meiofaunal community structure at Control and Mussel stations is illustrated in Fig. 7. A similar meiofaunal composition was observed in the two stations: nematodes accounted on average for 75% of the total density at both stations, followed by copepods (10% and 15% at the Control and Mussel respectively), polychaetes (4% in both stations). Only kinorhynch, ostracod and turbellarian densities strongly decreased at the mussel station.
Discussion Mussel-farm impact on sediment characteristics The mussel farm induced evident changes in the sediment characteristics. Oxygen penetration into the bottom sediments (measured as RPD depth) was signi®cantly reduced at the Mussel station, except in January and February, when water column mixing also induced an increase of oxygen availability in the upper layers of the sediments (La Rosa, 1998). The biodepo-
Fig. 4 Bacterial community structure in the Control site and under the mussel farm.
248
Fig. 5 Temporal variations of total meiofaunal density at the Control station and at the Mussel station. Data are expressed as ind. 10 cmÿ2 SD. Reported are the result of the student t-test: p < 0:05; p < 0:01, no indication is reported when dierence is not signi®cant.
Volume 40/Number 3/March 2000
sition due to mussel farm activities led to a signi®cant increase in phytopigment concentrations. In agreement with Navarro and Thomposon (1997), such accumulation was apparently related to spring phytoplankton blooms that were ®ltered and compacted by mussels and discharged to the sediments, reaching concentrations of 50 lg chlorophyll-a gÿ1 . The signi®cant dierence observed between Mussel and Control station (t-test, p < 0:01) suggests that total phytopigments (as chlorophyll-a or CPE) might be used as tracers of mussel biodeposition. Phytopigment concentrations reported here were much higher than those reported by Grenz et al. (1990) from mussel farms of the Gulf of Fos (NW Mediterranean) or in other New Zealand and French sites (Barranguet et al., 1993; Kaspar et al., 1985). Previous studies carried out on mussel farms suggested that high values of chlorophyll-a are due to deposition of pseudo-faeces, while high phaeopigment concentrations are related to the deposition of faeces (Dahlback and Gunnarsson, 1981; Navarro and Thomposon, 1997). In
this study, we observed a phaeopigment content 2±4 times higher than chlorophyll-a and, on average, CPE content in the sediments beneath the mussels was about 6 times higher than in the Control (13.52 1.59 vs 86.84 14.25 lg gÿ1 ). Sedimentary organic carbon enrichment has been reported in most studies dealing with aquaculture impacts (Castel et al., 1989; Gilbert et al., 1997; Grenz et al., 1990), but total organic content appears to be more conservative than biopolymeric organic carbon (BPC, Mazzola et al. 1999). Mussel biodeposition eects were evident in terms of biopolymeric carbon accumulation that at all sampling periods at the Mussel stations showed values signi®cantly higher than in the Control (t-test, p < 0:01). BPC enrichment of the mussel-farm sediments was less evident in spring, during the phytoplankton bloom, due to the high organic matter input to the sediments (reported also in the sediments of the Control stations). The analysis of the dierent biochemical classes of organic compounds contributing to
Fig. 6 Temporal variations in meiofaunal density in the Control site and under the mussel farm. Reported are: (a) nematodes; (b) copepods; (c) polychaetes; (d) kinorhynchs; (e) ostracods; (f) turbellarians. Data are expressed as ind. 10 cmÿ2 SD. Reported are the result of the student t-test: p < 0:05; p < 0:01, no indication is reported when dierence is not signi®cant.
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contribution to the total auto¯uorescent microbial density. Therefore, we might conclude that the microbial parameters that best highlight the microbiological effects of mussel-farm biodeposition are the increase of the autotrophic component of the total bacteria community and the decrease in importance of eukaryotic cells.
Fig. 7 Meiofaunal community structure in the Control site and under the mussel farm.
the biopolymeric pool revealed that protein and lipids accumulated preferentially in mussel-farm sediments, being signi®cantly higher than in the Control (t-test, p < 0:02), whereas carbohydrates appeared to be more conservative (Table 1). Mussel-farm impact on bacterial assemblages Bacterial densities recorded in this study at Control station (on average 1:11 0:16 108 cells gÿ1 ) are comparable with those reported in most coastal sites of the Mediterranean Sea (Danovaro et al., 1998; La Rosa, 1998). Sediment organic enrichment and the modi®cation of the characteristics of the benthic environment led to an increase of total bacterial density of about 64%, but the increase was signi®cant in April (t-test, p < 0:01), May and June (t-test, p < 0:05) and October (t-test, p 0:01). Additional evidence of the impact of the mussel activities on the benthic bacterial communities is provided by the signi®cant increase of auto¯uorescent cells (as PPB density) that were three times as numerous at the mussel station compared to the Control. Changes in the community structure of the bacteria beneath the mussel farm were also evident in terms of reduced contribution of the eukaryotic density to the total microbial density. Such changes are likely to be the result of the combined eect of a larger organic matter input and a reduction of oxygen penetration into the sediments. Changes occurring in microbial assemblages from mussel-farm sediments can be summarized as follows: (1) increase of the total bacterial density; (2) increase of the cyanobacteria contribution to the total microbial density and (3) strong decrease of the picoeukaryotic 250
Mussel-farm impact on meiofaunal assemblages Mussel biodeposition had a signi®cant impact on meiofaunal assemblages. Meiofaunal density at the Mussel station was signi®cantly lower than at the Control station (on average 2071 625 and 1550 420 ind. 10 cmÿ2 at Control and Mussel station respectively; t-test, p < 0:01). Biodeposition by bivalves generally provides a strong input of organic matter of high quality and availability to benthic assemblages and previous studies reported a positive response (i.e., an increase) of meiofaunal density to this organic enrichment (Castel et al., 1989; Dinet et al., 1990; Guelorget et al., 1994). By contrast, we reported a clear decrease of the total meiofaunal density apparently due to changes in the sedimentary characteristics (i.e., the layer of faecal material reduced oxygen ¯uxes at water-sediments interface). The eects of mussel biodeposition on meiofaunal assemblages were more dicult to point out in terms of assemblage composition. Nematodes accounted on average for 75% of total meiofaunal density at both stations. Copepods displayed good tolerance to mussel biodeposition and their density remained rather constant with time or even increased. Such copepod response, characterized by harpacticoid dominance, is likely due to their capability of exploiting the enrichment in microphytobenthic biomass (expressed as chlorophyll-a) associated to mussel biodeposits. Polychaete densities, on average, were similar at the two stations but decreased signi®cantly at the Mussel station in April and August (t-test, p < 0:05), September and November (t-test, p < 0:01). Other taxa, such as turbellarians, ostracods and kinorhynchs, displayed a signi®cant decrease in the mussel-farm sediments, and such dierences were particularly evident for kinorhynchs that disappeared almost completely from July to February. Nematodes are assumed to be quite resistant to sediment organic enrichment and the resulting reducing conditions and some species are permanently found in suboxic sediments (Danovaro, 1999; Danovaro et al., 1999; Giere, 1993; Powell, 1989). However, the results of this study pointed out, on annual average, a signi®cant impact of the organic enrichment on nematode abundance (t-test, p 0:01). Similar results were reported by Dinet et al. (1990) that demonstrated a strong impact of bivalve biodeposition on meiofauna density, which decreased drastically (by a factor of four) when compared to the Control site. Conversely, other authors reported an increase of the total meiofaunal density induced by mussel biodeposition (Castel et al., 1989; Guelorget et al., 1994; Radziejewska, 1986). However, a more
Volume 40/Number 3/March 2000
detailed analysis revealed that such a positive response was observed in high energy environments (such as coastal lagoons), where resuspension events reduced OM accumulation and enhanced oxygen penetration into the sediments. The presence of ®sh farm installations in the Gulf of Gaeta enabled us to compare the impact of two dierent aquaculture activities on this coastal system. Previous studies have indeed demonstrated that organic enrichment due to ®sh farm resulted in a strong and statistically signi®cant reduction of the total meiofaunal density (which decreased about 50%), mostly due unexpected nematode sensitivity to this kind of disturbance (Mazzola et al. 1999a,b). In this regard, mussel farm biodeposition had a clearly lower impact, as meiofaunal density decreased only 25%. Changes induced by biodeposition were also observed in terms of meiofaunal community structure, and it is worth noting that both mussel and ®sh farm impact determined an increase of the copepod percentage at the expense of nematodes, which together with kinorhynchs, turbellarians and ostracods might represent useful indicators of biodeposition disturbance. The authors are particularly indebted with the sta of the Med®sh s.r.l. (Gaeta) for precious collaboration during sampling. Thank are due to Dr C. Gambi and Dr M. Cecchi (University of Ancona) for help in meiofaunal sorting. Our thanks are due also to Dr E. Favaloro and Dr B. Savona (University of Palermo) for kind support during sampling. A special thanks is due to Prof. T. Maugeri (University of Messina) for suggestions and for providing laboratory facilities. This work is part of the doctoral thesis of S.M. and T.L.R. and was supported by a grant of the Ministero dell'Universit a e Ricerca Scienti®ca e Tecnologica and Ministero per le Politiche Agricole, Italy. Barg, U. and Phillips, M. J. (1998) Environment and Sustainability. FAO Fisheries Circular No. 886, Rev.1, Rome, FAO. 163 pp. Barranguet, C., Alliot, E. and Plante-Cuny, M. R. (1993) Benthic microphytic activity at two Mediterranean shell®sh cultivation sites with reference to benthic ¯uxes. Oceanologica Acta 17 (2), 211±221. Bligh, E. G. and Dyer, W. (1959) A rapid method for total lipid extraction and puri®cation. Canadian Journal of Biochemistry and Physiology 37, 911±917. Castel, J., Labourg, P. J., Escaravage, V., Auby, I. and Garcia M. E. (1989) In¯uence of Seagrass Beds and Oyster Parks on the Abundance and Biomass Patterns of Meio-and Macrobenthos in Tidal Flats. Estuarine, Coastal and Shelf Science 28, 71±85. H. (1981) Sedimentation and Dahlback, B. and Gunnarsson, L. A. Sulfate Reduction Under a Mussel Culture. Marine Biology 63, 269±275. Danovaro, R. (1999) Benthic microbial loop and meiofaunal response to oil induced disturbance in coastal sediments: a review. International Journal of Environmental Pollution, in press. Danovaro, R., Della Croce, N. and Fabiano, M. (1998) Biochemical composition of particulate organic matter and bacterial dynamics at the sediment±water interface in a Mediterranean seagrass system. Hydrobiologia 363, 241±252. Danovaro, R., Della Croce, N., Martorano, D., Parodi, P., Pusceddu, A. and Fabiano, M. (1999) Biological indicators of oil induced disturbance in coastal sediments of the Ligurian Sea. Pertanika, in press. Dinet, A., Sornin, J. M., Sabliere, A., Delmas, D. and Feuillet-Girard, M. (1990) In¯uence de la biodeposition de bivalves ®ltreurs sur les peuplements meiobenthiques dÕun marais maritime. Cahiers de Biologie Marine 31, 307±322. Fabiano, M. and Danovaro, R. (1994) Composition of organic matter in sediment facing a river estuary (Tyrrhenian Sea): relationships with bacteria and microphytobenthic biomass. Hydrobiologia 277, 71±84.
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