Integrated Multitrophic Aquaculture: Filter Feeders Bivalves as Efficient Reducers of Wastes Derived from Coastal Aquaculture Assessed with Stable Isotope Analyses - Aquaculture and the Environment: A Shared Destiny - page 107

Particulate organic matter, was sampled by vacuum filtering 10 L of seawater, collected
using a 5 L Niskin bottle (General Oceanic, USA), through a pre-combusted (450ºC, 4 hours)
Whatman GF/F glass microfiber filter, 1 m above the floating cages while in control sites, it
was collected at the same depth as the filter feeders bivalves polyethylene bags.
Sedimentary organic matter was collected by SCUBA divers from a quadrant surface (400
cm 2 ) using hand cores.
2.2.2 Control sites
Similarly, in control sites, 6 specimens of filter feeder bivalves Mytilus galloprovincialis and
Chlamys varia were sampled. In control 1, Chlamys varia could not be sampled since the
polyethylene bags were lost after severe storm periods. Moreover, phytoplankton,
zooplankton, particulate organic matter (POM) and sedimentary organic matter (SOM) were
sampled seasonally and 5 samples of sedimentivorous echinoderms Holothuria forskalii were
sampled annually following the same procedure as in cage site (Table 1). All samples were
frozen immediately after sampling and kept at -20ºC till further processing.
2.3 Stable isotope analyses
Samples were dried in an oven at 60ºC for 24h and subsequently grounded to a fine powder
using a mortar and pestle. A minimum of two replicates for each sample was analyzed for
δ 13 C and δ 15 N isotopic signatures, except for commercial dry pellet feed which had a stable
and controlled composition (Trouw S.A). SOM samples for δ 13 C isotopic analysis were
acidified by adding 2N HCl (Carabel et al., 2006), while for δ 15 N analysis, non- acidified
replicates were used. From each sample 2 ± 0.1 mg of dry weight was placed in tin cups to
determine the stable isotope ratios of carbon and nitrogen. The analyses were run at the
SCTI (Scientific-Technical Services) from the Balearic Islands' University using a continuous
flow mass spectrometer (Thermo Finnegan Delta x-plus). Reference standards were Vienne
Pee Dee Belemnite (VPDB) for C and atmospheric nitrogen for N. One sample of an internal
reference, Bovine Liver Standard (1577b) (U.S. Department of Commerce, National Institute
of Standards and Technology, Gaithersburg, MD 20899), was analyzed every eight samples
in order to calibrate the system and compensate the drift over time. The analytical precision
of the stable isotope analyses was based on the standard deviation of the BSL samples; these
deviations were 0.08 ‰ for δ 13 C and 0.09 ‰ for δ 15 N. Isotope ratios were expressed in δ 13 C
and δ 15 N, with units of ‰, according to the following equation:
δ 13 C or δ 15 N= [(R sample / R reference ) - 1] x 1000
Where R is the corresponding 13 C/ 12 C or 15 N/ 14 N ratio.
2.3.1 Statistical analyses
A distance-based permutational analysis of variance (PERMANOVA) on 999 permutations
was employed to test the hypothesis that there were no differences in the isotopic signature
(δ 13 C and δ 15 N) of filter feeders bivalves between treatments and sampling periods. The factors
Treatment (with three levels: cage, control 1 and control 2), Year (with three levels: 2008,
2009 and 2010) and Season nested in Year (with three factors: spring, summer and
autumn) were treated as fixed. Additionally, a distance-based test for homogeneity of