Experimental plots were established at three sites within mature stands of Rhizophora stylosa (FLNS1, FLNX8, FLNY9), in an area approved for reclamation by the Gladstone Port Authority. The sites were matched visually for tidal elevation, mangrove tree composition and condition, sediment composition and benthic invertebrate populations. Five plots ranging in area from 26 m² to 53 m² were constructed at each site, so that each plot enclosed at least 10 mangrove trees. Around each plot, prop roots were cut in a path about 0.5 m wide to allow installation of plastic retaining walls. The retaining walls were dug into the mud to a depth of 20 cm and supported to a height of 1 m. A gate was installed to allow tidal waters to move in and out while retaining the oil. Plots at three sites, located outside the reclamation area (FLNN, FLSS, CISS), which were established earlier for dispersant experiments were also used as undisturbed control plots for the mangrove bioremediation experiments.
Five replicate plots were established at each of four sites (ABS-1, ABS-2, ABS-3 and ABS-4) in a Halosarcia sp. dominated salt marsh area, approved for reclamation by the Gladstone Port Authority. Enclosures consisted of circular walls of light weight plastic sheeting (Corflute), approximately 1.25 m in diameter, buried 7 cm into the sediment, and standing 33 cm above it. Regular tidal flushing in each plot, occurred through two plastic U tubes, each ~12 mm in diameter, and buried with their outlets on either side of the sheeting wall, within and outside the plot. The salt marsh plots did not have an undisturbed control plot.
Before oiling, each mangrove plot was sampled to determine sediment grain size, hydrocarbon content and total organic carbon content. Replicate 7 cm diameter cores were collected from each plot, sliced and the 0-2, 10-12 and 20-22 cm sections from each replicate were pooled.
One of five treatments was applied to each plot: Gippsland crude oil only; Gippsland crude oil and bioremediation; Bunker C fuel oil only; Bunker C fuel oil and bioremediation; and controls with no oil. The oils were preweathered prior to application and both oil types were applied at the rate of 5L/m² for for mangrove plots and 2L/m² for salt marsh plots. The average rate of dosing in each plot was calculated from four pooled surface sediment (0-2 cm) samples collected from each plot 40 hours after oiling.
Bioremediation treatments were applied 40 hours after oiling. The bioremediation strategy used for the mangrove plots involved forced aeration of mangrove sediments and the addition of nutrients. For the salt marsh plots, only nutrients were applied.
At 1, 2, 5 or 6 and 12 or 13 months, 4 replicate 7 cm diameter cores were collected from each mangrove plot, sliced and the 0-2, 10-12 and 20-22 cm sections from each replicate were pooled. For the salt marsh plots, cores were collected with 2 cm diameter plastic tubes after 1, 3 and 9 months. Four replicate 0-1 cm and the 9-10 cm core sections from each plot were combined for analysis. Observations, including the presence of roots, animals, burrows and oil were made before the samples were frozen for later analysis.
The following analyses were conducted on the sediment samples: Water content of sediments; Total hydrocarbons determined by gas chromatography with flame ionisation detection (GC-FID); The percentage of unresolved hydrocarbons; Concentrations of individual isoprenoid and n-alkanes (µg/g dry weight); Ratios of isoprenoid to n-alkenes as biodegradation indices.
At the end of the experiment (August, 1998), mangrove sediments were sampled for a range of nutrients, including total nitrogen, phosphorus and carbon. Sediment samples were taken from 0-5 cm deep cores using a plastic open mouth syringe, approximately 2 cm in diameter, placed in plastic screw top containers and kept on ice until returned to the AIMS laboratory at Townsville for analysis.
