The exchange processes between marine sediment and the overlying water on coasts play a large role in the ecosystem function and productivity of both habitats. The most notable exchange is the provision of nutrients in coastal waters from the sediment on the continental shelf below [1-3]. It is estimated that approximately a third to one half of nutrients for the primary production in these waters is sourced from the sediment [1]. This large exchange of nutrients is heavily influenced by the activity of infauna [1-5]. The manipulation of sediment by organisms is known as bioturbation and predominantly occurs in the upper centimetres of sediment [2, 6]. Bioturbation increases the sediment-water interface through irrigation, creating some of the biochemical and physical properties of sediment that allow for efflux of nutrients into the water column [1-5].

The sediment-water interface firstly allows for large nutrient production through aiding mineralisation. Mineralisation in marine sediment occurs as decaying organic matter from above and within sediment is broken down through oxidation by bacteria and fungi into inorganic nutrients. These mineralised nutrients are then able to be used by photosynthesising organisms [3, 7].  Increased irrigation promotes this through diffusion of oxygen into oxygen limited water pores in the sediment [5, 7-9]. This oxygen is used by decomposers for mineralisation and the irrigation creates channels the mineralised nutrients can move into the water column through [3, 7].

The degree of sediment irrigation through bioturbation is dependent on size, abundance and feeding method of the organisms [3, 5]. Larger bioturbators have also been found to work more sediment, increasing the water-sediment interface [5]. Amphinomids are abundant in sandy reefs around the world, with several species known to burrow beneath the sediment to scavenge, hunt or hide during the day [10]. Despite their global distribution, abundance in coastal regions and relatively large size, the impact of Amphinomid bioturbation has not been studied [10]. This study investigates the impact of burrowing Amphinomids on water flow into sediment to access if they play a role in aiding the sediment-water nutrient cycle. Due to their abundance and typically mobile lifestyle it is predicted that Amphinomids will be able to irrigate sediment effectively, allowing more pore water to form deeper in the sediment for sediment processes than in their absence.

A tracer experiment was conducted by measuring how far water containing dye would travel through sediment containing Amphinomid worms.

Six grams of sediment was left aside to dry for one week. Macrobenthos and large particles were then removed from the dry sediment and from approximately 42g of wet sediment using tweezers. Roughly 13 drops of red food colouring was added to the dried sediment and mixed until sediment was completely coated in red. Amphinomid specimens were collected from the aquarium and placed into petri dishes filled with filtered sea water shortly before commencement of the trials.

Analysis
One hour after being covered with nescofilm, microcosms were photographed and printed to scale. A 0.5cmx0.5cm grid was drawn over the image to create four horizontal layers, each 0.5cm thick. The area below these layers was omitted as the cuvette begins to curve inwards, reducing sediment space compared to layers above. The percentage cover of red in each of the four layers was then recorded with the aid of the grid. A linear regression was performed on control and worm microcosm data and an additional log transformed regression was performed on the control data.

General Observations
Most Amphinomids burrowed within the one hour of being placed into microcosms however several remained on the sediment surface as seen in Figure 1. The first sediment layer was found to have 100% dyed surface water for all microcosms which is expected as it contained the original 1g of dyed sediment. The dyed water was observed to easily move into the sediment in all microcosms. This movement was seen to occur around worms, through gaps within the sediment and particularly along the edges of the cuvette where channels of red supernatant water lead to the base of the cuvette as in Figure 2.

Statistical Analysis
The red dye observed at different sediment depth shows a general negative trend in both control and treatment data as seen in Figure 3. The regression analysis found this to be a significant linear relationship for the treatment data, with the modelled line shown in Figure 3 as well (F=15.81, df=1,14, p=0.001, α=0.05). This significant linear relationship was not found for the control data (F=5.686, df=1,6, p=0.054, α=0.05), with a log transformed model not explaining the relationship between variables either (F=3.54, df=16, p=0.1089, α=0.05).  A formal statistical comparison between the treatment and controls could not performed as the controls could not be modelled, and the sample size of the controls was too small (n=2) for a comparison of results for each layer

The average percentage red at different depths are very similar between control and treatments as seen in Table 1 until the deepest layer where the control trials have a higher average of percentage cover of red (46%±25) than the treatment trials (37.75%±21.22). The variance in percentage cover red observed in each layer is very large as the standard error in Table 1 and the spread of data points in Figure 2 shows. For example, in the last sediment layer, the results of percentage red in control trials were 21% and 71%, and treatment trials ranged from 5%-100%.

Table 1: The average percentage red observed in each of four sediment layers in control (no worms) and treatment (with worms) microcosms.

The results show that the burrowing Amphinomids used in the study may create a negative, linear relationship between sediment depth and water flow into sediment that is not found without them. Without worms, deeper sediment (from 10 to 20mm), had on average more surface-sourced water than with worms however both averages had large standard error. The results showing a general decrease reflect the literature of sediment profiles. This result is however contradictory to the hypothesis that Amphinomids would irrigate efficiently and could be explained by the range in results, sample size and the burrowing behaviour and environment of the worms.

Variation and Sample Size
The large range and small sample size lessen the reliability of the results as estimation and comparison of true averages becomes difficult. Outliers and noise are not compensated for and small effects of variables are likely not to be detected.

The large range in percentage red found in the lower sediment layers may be attributable to confounding variables such as water movement eased by the glass edges of the microcosms. This created a vertical channel for water to travel through in some of the cuvette which in at least one sample from control and treatments created a large pool at the bottom of the cuvette, increasing the value for % red in bottom layers. These results did not represent influence from Amphinomids yet due to extremely small sample size in the control trials, they held greater weight in the control than treatment averages.

This noise and not the presence of Amphinomids may be responsible for creating the greater average %red found in the control trials than the treatment trials however a larger sample size is needed to confirm this as it would reduce the standard error, potentially help narrow in on a true average and confirm if the two averages are significantly different through analysis. Using deeper microcosms with curved edges may also help eliminate the noise created by the confounding variable as it will reduce water flowing artificially by glass and pooling.

Amphinomid Environment and Behaviour
As some worms did not burrow in trials yet hastily burrowed when collected in wet sediment, the red food dye may have influenced burrowing behaviour. This could have altered the path of burrowing worms and/or their activity, possibly compromising their bioturbation capability which has been shown to occur in several bioturbators with exposure to various substances.

The Heron Island sediment may also not provide the best environment for testing effects of bioturbation. Mermillod-Blondin found that bioturbation has a greater influence on water transport into sediment when the environment is diffusion dominated and not advection dominated [2]. Diffusion dominated systems have their water transport dominated by movement through diffusion, advection systems move water through pores [2, 11].  Heron island sediment has been found to be advection dominated due to its permeable sands [11, 12]. This transport into sediment through random pores of water was observed in all trials and was greater than anticipated, possibly due to sediment only loosely being added to cuvettes created many random pores. This may be responsible for some of the variation in the % red results. This may also mean that Amphinomids potential impact on water transport could have been masked by the influence of advection transport in this environment, consequently making it difficult to detect with the small sample and large variation in this study.

Further investigation should be conducted that reduces the impact of advection transport to determine if there is an impact of Amphinomids, albeit small. This was attempted through shaking and poking of sediment in cuvettes with tweezers before trials to reduce random water pores. This was found to possibly be too successful in compacting sediment as little to no water transport occurred and Amphinomids did not burrow at all. This may indicate that burrowing behaviour is deterred with sediment compaction in Amphinomids. Investigation into threshold of sediment compaction or amount of pores in sediment necessary for burrowing should be conducted as this could have implications for bioturbation rates in different Heron Island environments. Assessing the impact of this family in diffusion dominated sediment should also be conducted as the worms may have greater importance in water transport.

Conclusions
The results of this study are generally inconclusive as no comparison between control and treatment could be done, and sample size and method limitations make comparisons difficult to draw. The general distribution of average percentage red appears to be similar in both treatment and control trials when limitations are considered. If an effect of bioturbation by the worms on water transport is present it is likely to be small due to the properties of Heron Island reef sediment and may therefore have little impact on the nutrient provisioning to the water column. The implications for further research from these are great as a potential sediment specific effect of burrowing behaviour may be indicated and the need for many, deep microcosms when investigating Amphinomid bioturbation as their influence is likely to be hard to detect.

I’d like to thank Bernard and Sandie Degnan and all tutors for their advice and guidance in forming this investigation, identifying the family of worm and use of their aquarium. I would not have been able to create such a finicky project without them.

1.           Middelburg, J.J., K. Soetaert, and P.M.J. Herman, Empirical relationships for use in global diagenetic. Deep Sea Research part 1: Oceanographic Research Papers, 1997. 44(2): p. 327-344.

2.           Mermillod-Blondin, F., The functional significance of bioturbation and biodeposition on biogeochemical processes at the water–sediment interface in freshwater and marine ecosystems. Journal of the North American Benthological Society, 2011. 30(3): p. 770-778.

3.           Welsh, D.T., It's a dirty job but someone has to do it: The role of marine benthic macrofauna in organic matter turnover and nutrient recycling to the water column. Chemistry and Ecology, 2003. 19(5): p. 321-342.

4.           Aller, R.C., Experimental Studies of Changes Produced by Deposit Feeders on Pore Water Sediment, and Overlying Water Chemistry. American Journal of Science, 1978. 278: p. 1185-1234.

5.           Thrush, S.F. and P.K. Dayton, Disturbance to Marine Benthic Habitats by Trawling and Dredging: Implications for Marine Biodiversity. Annual Review of Ecology and Systematics, 2002. 33(1): p. 449-473.

6.           Meysman, F.J., J.J. Middelburg, and C.H. Heip, Bioturbation: a fresh look at Darwin's last idea. Trends Ecol Evol, 2006. 21(12): p. 688-95.

7.           Lohrer, A.M., S.F. Thrush, and M.M. Gibbs, Bioturbators enhance encosystem function through complex biogeochemical interactions. Nature, 2004. 431: p. 1092-1095.

8.           Thrush, S.F., et al., Functional Role of Large Organisms in Intertidal Communities: Community Effects and Ecosystem Function. Ecosystems, 2006. 9(6): p. 1029-1040.

9.           Ziebis, W., M. Huettel, and S. Forster, Impact of biogenic sediment topography on oxygen fluxes in permeable seabeds. Marine Ecology Progress Series, 1996. 140: p. 227-237.

10.         Borda, E., et al., Revamping Amphinomidae (Annelida: Amphinomida), with the inclusion ofNotopygos. Zoologica Scripta, 2015. 44(3): p. 324-333.

11.         Santos, I.R., et al., Diel coral reef acidification driven by porewater advection in permeable carbonate sands, Heron Island, Great Barrier Reef. Geophysical Research Letters, 2011. 38(3): p. n/a-n/a.

12.         Huettel, M., et al., Hydrodynamical impact on biogeochemical processes in aquatic sediments. Hydrobiologia, 2003. 494: p. 231-236.