5.1 ANÁLISIS DE VARIABLES DEMOGRÁFICAS Y PREOPERATORIAS.

At each site five sediment cores were collected using a hand held 150 mm diameter PVC pipe corer that was pushed into the sediment to a depth of 100 mm. Benthic sampling was restricted to shallow subtidal assemblages or just below the low water mark to prevent the inclusion of information relating to across-estuary vertical gradients in faunal assemblage structure that are commonly associated with tidal inundation (Edgar and Barrett, 2002, Ysebaert et al., 2003). Sediment cores were taken at 0.0, 0.1, 0.2, 0.3, and 0.5 m depths (where applicable) along a transect line running perpendicular from the

Montagu River Black River Detention River Duck River Port Sorell Pipers River

low tide water mark. Cores were collected at these depths in order to maximise the range of animals collected at a site. To reduce the effects of sampling at different tide heights at each location the estuaries were sampled from lower to upper (where practical) between the hours of one and half hours before and one and half hours after the predicted low tide. The five samples collected at each site were pooled and sieved (1 mm mesh) in the field prior to fixing in 10% buffered formalin. Pooling of replicate cores within a site did not allow estimates of variability at this scale, but this was deemed less important than variability at the scales of location and estuary and allowed greater allocation of effort to sample at larger spatial scales (Carey and Keough, 2002, Ross et al., 2007). In the laboratory, macroinvertebrates were rinsed in freshwater and washed through a sieve stack (8, 4, 2 and 1 mm). Material retained in the 1 mm sieve was divided into two or four (using a Jones riffle splitter) depending on the amount of material contained. Fauna in each of the sieve size classes were sorted, identified to the lowest practical taxon and counted. Counts from the 1 mm fraction were multiplied by either two or four to obtain an estimate of total abundance for that size class.

3.2.2.2 Environmental variables

To measure pore water salinity, three cores (diameter = 23 mm, depth = 20 mm) of sediment were collected and amalgamated in the field. Sediment pore water was extracted in the field using a syringe and a wadded cotton wool filter. Salinity was measured in the field using a hand-held refractometer. Dissolved oxygen concentrations were recorded for the bottom waters

overlying the area of benthos sampled at each site using a dissolved oxygen probe (HACH HQ40d multimeter) with values presented as percentage saturation.

Sediment samples were collected for estimates of total nitrogen and total phosphorus, percentage organic carbon and microphytobenthos (MPB) biomass (using sediment chlorophyll a as a proxy). Sediments for the analysis of total nitrogen and total phosphorus were collected by scraping sediments into clean 300 ml glass sample jars provided by the NATA-

accredited Analytical Services Tasmania (AST). These samples were

immediately frozen. Analysis of total nitrogen (TN) and total phosphorus (TP) were measured in the laboratory using internal methods based on standard procedures: TN: Total Kjeldahl Nitrogen (TKN) present in the sample was determined by converting the nitrogen to ammonium sulphate in a sulphuric acid potassium sulphate digestion procedure. Ammonium–nitrogen was subsequently determined by colorimetric auto-analyser method based on APHA Standard method (2005) 4500-Norg D. Analysis was performed using a Lachat Flow Injection analyser. TP: Phosphorus compounds were

converted to orthophosphate in the Kjeldahl digest, and the resulting solution was analysed by Flow Injection analysis. This method was developed by Lachat Instruments based on APHA Standard methods 4500-P G. Results for TN and TP are reported as mg/kg DMB (dry mass basis). Sediments retained for the analysis of percentage organic carbon content were collected using a 45mm diameter core to a depth of 50 mm. Percent organic carbon in

sediments was calculated after drying by loss of organic carbon on ignition at 500 °C.

Sediment chlorophyll a was determined from three replicate sediment cores (23 mm diameter) inserted to a depth of 20 mm following the method of Light and Beardall (1998). Sediments were frozen and returned to the laboratory for analysis. Chlorophyll a was extracted from the sediments with acetone (90% v/v) over a 24 hour period in darkness at 4 °C and then centrifuged (4500 rpm for 15 min) to remove sediments. The concentration of chlorophyll a and the breakdown product (pheophytin) where determined

spectrophotometrically. Concentration (mg/m2) of chlorophyll a (corrected for pheopigments) were obtained using the equations of Lorenzen (1967).

Sediment retained after the extraction of MPB was used to determine the mean particle size and the percentage silt/clay content of the sediments. The sediment was dried in an oven at 60 °C and the organics in the sample were removed by adding 10 % hydrogen peroxide. The mean particle size and percentage silt/clay content was determined using a Saturn Digisizer 5200 laser diffractometer. The mean particle size was provided in the output of the

Digisizer and converted to the phi (Ф) scale for further analysis. The

percentage of the sample comprising the silt/clay component in the samples was calculated by pooling the percentage volume frequencies of particle sizes <0.063 mm calculated by the Digisizer.

Sediments for stable isotope analysis were collected using five 23 mm diameter cores inserted to a depth of 20 mm and were subsequently

amalgamated for analysis. In the laboratory, sediment samples were rinsed through a 315 µm sieve with deionised water (Milli – Q ≥ 18 MΩ.cm) and dried in an oven at 60 °C for at least 48 h. Dried samples were pulverised in a mortar with a pestle until the sample had been reduced to a fine powder. Sediments were transferred to a clean 5 ml glass vial and treated with 10% hydrochloric acid to remove carbonates to provide a more accurate

assessment of δ13C.

Stable isotope samples were analysed using a continuous flow-isotope ratio mass spectrometer (GV Isoprime Eurovector EA 3000, Manchester UK) at Griffith University. Isotopic ratios of samples are expressed in conventional notation as δ values (δ13C or δ15N) and were calculated using the equation:

δ13

C or δ15N = [(Rsample/Rstandard) – 1] x 1000

where R is the corresponding isotopic ratio 13C: 12C or δ14N: δ15N. Reference material was PeeDee Belemnite (PDB) for carbon, and atmospheric air for nitrogen.