Materials and Methods

The study site is located in a drain along Upper Changi Road East, Singapore at latitude 1° 20' 2.48" S and longitude 103° 57' 23.63" E.  The drain transports ground water from Upper Changi Road and spans a total of about 400 metres before depositing the water into a concretised natural canal, Sungei Ketapang.  Near the junction where the drain meets the canal, it is flanked by two narrow strips of sediment with little grass with a total area of about 4.32 square metres.
Sungei Ketapang then transports to water to the sea at East Coast Park some three kilometres away.  Water level in Sungei Ketapang is consequently affected by the diurnal tides experienced by the Singapore coastlines.  During low tides, the canal carry water from the land to the sea and the converse happens during high tides.  Along with the incoming seawater from the high tide, aquatic organisms are transported inland.  During rising tides beyond 1.8 metres in height, water from the concretised canal starts to overflow into the drain (Plate 1) and at a height of two metres, the water floods the sediment strips behind the concrete slabs of the drain and completely quickly submerges the sediment strips.

Fig. 1. Location of study site and other sites (Pasir Ris Park and East Coast Park) with similar Uca annulipes communities and layout of the study site showing connection between drain and concretised Sungei Ketapang (inset).

On these sediment strips, Uca annulipes and U. rosea can be seen emerging and feeding when the sediment strips are exposed.  In the cracks of the drain other species of crustaceans, Episesarma spp., Scylla olivacea, Varuna yui, Perisesarma eumolpe, can be seen as well.  Along the perimeter of the drain, mud mounds belonging to mud lobsters Thalassina anomala were also observed.  As the most visible and common crustacean seen in the drain is the Uca annulipes, this species has been selected for the purpose of this study.

Plate 1. (a) concretised Sungei Ketapang overflowing into the drain (circled) during a rising tide, (b) water from concretised Sungei Ketapang (background) overflowing into the drain during a rising tide, (c) sediment strips exposed during receding tide and
(d) dominant crab species Uca annulipes seen foraging on sediment strips.

The water flowing in the drain was collected at the junction of the drain and concretised Sungei Ketapang, mid and beginning of the drain (Fig.1 inset), at one hour intervals over a six hours period twice a month for a period of 12 months.  Salinity of the water collected was measured using a refractometer.  The corresponding tidal height was also recorded.

            Sediment samples were collected from (i) behind the concrete slabs of the drain at the junction where the drain meets the concretised canal, (ii) mid-point and
(iii) beginning of the drain and weighed.  Samples were then dried in an oven at 80 °C for 24 hours and then weighed again to determine the amount of water in the samples.  Fixed amounts of water were then added to the samples and re-suspended in a shaker.  After 24 hours, salinity of the suspensions was determined using a refractometer.  These replicates were processed for each sediment sample. Sediment samples were also collected from East Coast Park and Pasir Ris Park (Fig. 1) where
Uca annulipes were also found for comparisons.
Sediment from the junction between the drain and the canal were collected and placed in airtight Ziploc bags and transported back to the laboratory for analysis of particle size using the method outlined by Buchanan (1984).  The analysis repeated for East Coast Park and Pasir Ris and the results compared to determine if there were any differences in the samples using GRADISTAT v.4.0.
            The presence of transient fauna were observed and documented with a digital camera and identified.  The transient fauna was compared with those typically found in Singapore’s waterways and mangroves.  Data collection for drain water salinity, population density and fauna comparison were carried out twice a month, over a period of 12 months.
            Average population density of the most visible species, Uca annulipes, was estimated by enumeration of occupied fiddler crab burrows twice a month over a period of 12 months.  Burrows with chimneys were typically ignored because they were not typically made by U. annulipes.  The resulting density was then compared densities of similar populations found naturally in Singapore.
Uca annulipes were collected individually from the study site, East Coast Park and Pasir Ris Park.  They were then placed in buckets along with about 1.5cm depth of sediment from the same location.  In order to ensure the sustainability of the population, only male crabs were captured.  They were then acclimatised at room temperature in their sediment and buckets in which they were collected for at least 24 hour period before subjected to 72-hour static acute toxicity tests.  The tests were carried out according to the guidelines on acute toxicity testing as described by Parrish (1985) and Rand & Petrocelli (1985).  The aim of the acute toxicity tests was to determine the mean lethal salinity limits (LC50).
Ten male crabs (7mm ≤ carapace width ≤ 15mm) were placed in each of the five 15-litre tanks.  Solutions in each tanks were made up to the required salinities of 0 , 10 , 20 , 30 and 40 using Instant Ocean® salt and aerated, de-chlorinated tap water.  Evaporation was minimised by covering the lids to reduce water loss and variations in salinity.  The crabs in the tanks were checked for mortality after 6, 12, 24, 36, 48, 60 and 72 hours.  Crabs which have an inactive appearance were repeatedly prodded with a glass rod and then subsequently turned upside down. After five minutes, if no movement was detected, the crabs were declared dead.  The crabs’ behaviour were also recorded as ‘slightly torpid’ if the crabs became active after gentle prodding and ‘very torpid’ if only little or slight sluggish movement was detected after repeated prodding as recommended by Owen (2002). Replicates tests were carried out and experiment repeated on crabs from East Coast Park and Pasir Ris Park.
The percentage mortality from the duplicates of each site were analysed and determined if they were significantly different from each other.  If there is no significant difference (p > 0.05), they were combined to increase the confidence of the estimate for LC50 for each salinity (Owen, 2002).  Time-toxicity graphs were plotted using the 24, 48, 72 hour median mortality data.  These will serve as an indication whether and when acute lethality stopped in each tests.  The point of the graph where the curves are asymptotic with the time axis will be the LC50.  This is also an indication for the salinity at which 50% of the test population can live for an indefinite period (Rand & Petrocelli, 1985).  The analysis were carried out using MINITAB (MINITAB, Inc., Release 16.1.0, 2010)

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