You are here: Research Projects > Student Project 2015 | Nils Jonsson
Student Project
---> Methods
---> Estimating the Number of Cells
---> Sponge Identification
---> Locating the Microplastics in the Sponge
---> Analyzing the Data
---> Algal Clearance Rate
---> Clearance Rate of Microplastics
---> Conclusion
A Comparison in Clearance Rates Between Microplastics(10µm) and Different Algal Cell Sizes (Nano <2µm, 4-7µm and 7-20µm) by theDemospongiae Amphimedon sp.
Nils Jonsson 2015
Sponges (Porifera) are very efficient filterers, being able to absorb a large interval of different sized particles. This study aimed to investigate the clearance rate of different algal sizes (Nano <2µm, 4-7µm and 7-20µm) by Amphimedon sp. The study found that there was little difference in the clearance rate, however algae of the size 4-7µm was cleared the fastest, and the Nano (<2µm) sized cells were cleared the slowest. This agrees with previous research (Turon, 1997) and is here discussed to be a result of the different algal sizes being absorbed by the sponge on different locations. The bigger particles being absorbed by the pinacocytes and the smaller ones being absorbed by the choanocytes. The study also sought to investigate the clearance rate of microplastics (10µm) in comparison with equally sized algal cells. The results showed that the microplastics were being cleared by the sponge at a rate slower than the different algal cell sizes. Furthermore, the results indicate that the Amphimedon sp. was able to exclude the plastic, once it had entered the sponge, thus being able to recognize food from nonfood particles. This study raises further questions about the effects of microplastics on sponges, and concludes that further research is needed in this area.
Sponges have, during the last couple of years been discovered to be very important organisms in the oceanic ecosystems around the world's oceans. Not only are they a vital food source for some animals (Rogers & Paul, 1991). But recent research have shown that the sponges ability to filtrate a vast amount of water each day play a critical role in recycling nutrients to the habitat (de Goeij et al., 2013). With a changing climate, and increasing runoff from land, it is likely that the role of sponges will be increasingly important. Sponges are able to filtrate out particles smaller than 0.7 µm (Yahel et al., 2001). However their main food source consists of micron sized cells (Reiswig, 1971). The different sized particles tend to be taken up by the sponge on different locations. Particles between 5 - 50µm are generally taken up by pinacocytes that line the channel walls of the sponge. Smaller particles <5µm are taken up by the choanocyte chambers (Reiswig, 1971). This could potentially affect the clearance rate of different sized particles.
There is currently a worldwide increase in demand for plastics (Andrady, 2011). How much of the plastics produced each year, that enters the ocean has not been reliably estimated, however with an increasing use of the ocean, mainly in terms of industrial fishing, coupled with an increasing population along coast lines, the amount of plastics entering the ocean will most likely increase (Ribic et al., 2010). Plastics are becoming an increasingly difficult problem for marine animals, where some species now face extinction due to plastics in the ocean (Mrosovsky et al., 2009). Recently, the discovery of microplastics in the ocean have spawned new research on its effect on filter feeding animals. Although the potential bio-magnification of microplastics in the food web has yet to be studied in detail (Andrady, 2011). There are evidence that animals can ingest microplastics (Browne et al., 2008) should the microplastics enter the food chain through a filter feeder, for instance, some of the plastics could be excreted. The feacal pellets could then be digested by other suspension feeders and detrivores (Wright et al., 2013). The potential ingestion of microplastics seem to be highly species specific, should the plastics be ingested it can have toxic effects (Andrady, 2011), (Endo et al., 2005). Microplastics have been seen to be ingested by several planktivores and filter feeders, should the size be similar to that of normal food particles (Browne et al., 2008), (Christaki et al., 1998). However very little research have been done about the effect of microplastics on sponges. The sponges have been shown to be highly selective in their feeding, being able to differentiate between symbiotic bacteria and food bacteria, even of the same size (Wehrl et al., 2007). The selective feeding of sponges could mean that they are more resilient to the increasing concentration of microplastics in the ocean.
This study aimed to investigate the effect of different sized algae on the clearance rate by the Amphimedon sp. This data would later be the foundation for a follow up experiment, where the clearance rate of microplastics were compared to the clearance rate of the different sized food particles. It was hypothesized that the sponge would be able to distinguish plastic from food, hence the clearance rate would be lower than that of algae fed sponges.
The sponge used for this experiment was the demospongiae Amphimedon sp. of the family niphatidae (fig 1). The individuals were sampled off the East Coast of Australia, and put in an aquaria. A small amount of tissue was removed from two sponges and checked under a compound microscope, to see if the individuals were alive before the experiment started. Nine live individuals of similar volume (≈1000mm3) were picked out. The volume was measured using a caliper (±0.01mm). For the study four different particles were filtrated by the sponges: Microplastics of the size 10µm and algae of the sizes Nano (<2µm), 4-7µm and 7-20µm. The experiments took place during two different lab sessions two weeks apart. The first week no plastics were filtrated. The sponges were separated into individual cups with 100ml of seawater (this was enough to cover all the sponges in water). They were then separated into three different groups, i.e. 3×3 sponges. The first group were exposed to algae of the Nano size, the second group 4-7µm sized algae and the third group 7-20µm. 2 µl of each algal concentration was added to the cups (fig 2). The algae was stirred, then 1ml of the solution was removed and put into cuvettes, for absorbance testing. This was the 0 hour mark. Three cuvettes were used per cup, to account for the variance of the spectrophotometer. This procedure was repeated at the 1 hour and 2 hour mark. The algae was kept suspended in the liquid by stirring the cup every 10 min. The clearance rate was measured using a spectrophotometer, using OD 600nm. Once the data was recorded, the contents of the cuvette were poured back into the cup.
After two weeks a similar experiment was carried out. Here the Nano sized algae was replaced with microplastics of the size 10µm. This size was chosen due to the results of the first experiment. The absorbance was tested every half hour in this experiment, which ran for two hours. Again, there was in total 3×3 sponges. One group was fed microplastics 10µm, one was fed 4-7µm algae and the third group was fed 7-20µm algae. The cups were stirred every 10min. For the spectrophotometer 1ml of the solution was put in a cuvette, three cuvettes per cup was used, to account for the variance of the spectrophotometer. Before the absorbance was read, the cuvettes were manually shaken in order to suspend any particles that had settled at the bottom of the cuvette. Once the data was recorded, the contents of the cuvette were poured back into the cup.
Estimating the Number of Cells
For the experiment, 2µl of highly concentrated algae was added to 100ml of sea water. In order to find roughly how many algal cells were in each cup, a hemocytometer was used. The hemocytometer was loaded with 10µl of the particle mixed sea water. The hemocytometer was then placed on a compound microscope, where the number of cells were counted, using the hemocytometer's grid lines. From this, the number of cells per 1ml of sea water could be calculated. Plastic: 33000cells/ml, 4-7µm: 22000cells/ml, 7-20µm: 30000cells/ml.
Sponge Identification
In order to identify the sponge, the spicules had to be extracted. This was done by covering a small piece of the sponge with bleach, in an Eppendorf tube. The solution was left for approximately 40min, until the tissue was dissolved. The solution was then extracted and put on a slide, and checked using a Compound microscope.
Locating the Microplastics in the Sponge
In order to tell what happened with the plastics in the aforementioned experiment, one sponge was pressed through a filter, in order to try and isolate the choanocyte chambers. The sponge contents were mixed in salt water, the solution was then put on a slide and checked using a compound microscope. Photographs were taken, as a record of the results.
Analyzing the Data
The data was analyzed using Microsoft Excel 2013, where the data was plotted in scatterplot graphs. Exponential trendlines were calculated along with r2 values.
In figure 4 we see that the absorbance went down the fastest in the sponges fed with algae of the size range 4-7 microns. Due to the slope being steeper (y = 0,11e-1,768x, where -1,768= the slope). The second fastest reduction in absorbance was with the algae of size 7-20 (slope = -1,586) with the Nano sized algae, being filtrated the slowest (slope = -1,413). For all three algal sizes the majority of the algae was filtered out during the first hour, resulting in exponential trendlines.
In figure 5 we find that there is a very strong correlation between decreasing absorbance, i.e. the number of cells, and time in sponges that have been fed algae, with R2 values very close to 1. The sponges that were fed microplastics, however, show a lesser correlation between the absorbance and time, in fact we find on two different occasions, at 1 hour and 2 hour mark, that the number of free floating plastics had increased compared to the previous data point. Something that never happened in the algal fed sponges.
In figure 6 the results of the plastics experiment (fig 2), was paired with the results of the first experiment (fig 1). This was mainly for illustrative purposes, as the absorbances at 0h were much closer to each other. Since the method was standardized, the curves are comparable, with the only difference being the increased number of data point for the plastic curve. The figure illustrates a clear difference between the algal sizes and microplastics, where the microplastics had a much lower slope (-0,745), and R2 value.
After the experiment, the sponge tissue of a plastic fed sponge was removed to further investigate, what happened with the plastics. The results are illustrated by photographs in figure 7 & 8. The photos indicate that, indeed, the plastics were taken up by the sponge.
Algal Clearance Rate
This study have found that there generally is a very small difference in the clearance rate between different sized algae, using the same sized sponges (fig 4). However the trend that could be noticed, was that the medium sized cells 4-7µm were cleared faster than the other algal sizes, with the Nano sized cells (<2µm) being filtered the slowest. The results could suggest that food particles are taken up slower by the choanocytes compared to the pinacocytes lining the channel walls of the sponge. Previous studies have shown that the particle sizes less than 0.2 microns are generally filtered out slower compared to larger micron sized particles (Turon, 1997). Turon (1997) found, however, that particles of the size 1µm was retained the most efficient, which disagrees with the results in this study. However, it is possible that the Nano sized algae contained a large percentage of cells smaller than 1µm. This should be investigated for future research. Worth noting, is that the sponges used in the Turon (1997) experiment were different from the species used here. Now, the data in this study was very limited in terms of replicates, and so to further investigate the relationship between particle size and clearance rate, I suggest having more replicates. This will grant more statistical power. Interestingly, if the theory of different sized particles being picked up at different locations in the sponge, either pinacocysts or choanocytes, is applicable for the Amphimedon sp. then the results (fig 4) suggests that the efficiency of retaining particles is not very different between the two systems. For further research I suggest looking at the pinacocysts and choanocytes to see if different sized particles are found at these locations.
The study found that regardless of the size of the algae, the clearance rate of the sponges followed an exponential curve (fig 4, 5, 6). This was expected, since the sponges are filter feeders and not actively seeking the food particles. So when the algal concentration is high in the beginning, a higher percentage of the food particles will be sucked in by the flagellar movement of the choanocyte chamber. As the concentration of algae decreases, less and less algae will be close enough to the sponge to be caught by the ingoing current. This agrees with (Turon, 1997). If the contents of the cup would have been continuously stirred, then it could have potentially impacted the slope of the clearance rate. Since the sponge would be more regularly exposed to the algae. In order to get a better picture of how many cells was retained by the sponges, I suggest counting the actual number of cells in the cups. This is most likely a more reliable way of measuring the clearance rate. Another way of measuring the amount of cells could be to measure the dry weight of the sponge, however this implies that the algal cells way the same, and that the sponges are of equal size.
Clearance Rate of Microplastics
This study also found that microplastics are soaked up by the sponge (fig 7 & 8). An attempt at locating intact choanocyte chambers was made, unfortunately none were found. Thus the only conclusion that can be draw from figure 5, 6 and 7 is that the plastics seem to be taken up by the sponge. However, where exactly they go in the sponge and how it affects the sponge is still unknown. For further research I suggest to try and find intact choanocyte chambers, and see if the plastics are able to enter the choanocytes, or potentially clog them There is evidence that a high concentration of food particles can clog the pores of the sponge (Maldonado et al., 2010). Whether or not microplastics enables clogging or not needs to be researched. Another follow up experiment would be to test how plastic exposed sponges compare in clearance rate against control sponges. This would enable us to further understand how microplastics may affect the clearance rate of sponges. Furthermore, allowing the plastic covered sponge to recover in a healthy environment, could tell us more about the sponges ability to rid itself of plastics. It is important to note that the microplastic concentration used in this experiment was significantly higher than what we would expect to find in the ocean. However the results do show that the plastics can take up microplastics, and to some extent figure 5 and 6 show that the sponges can exclude some amount of the plastics, once they've entered the sponge. However, the results suggest that far from all plastic particles are excluded by the sponge. This could potentially lead to a scenario where sponges have to spend more and more energy, as the microplastic concentration rises, to get rid of the unwanted particles. This could result in less food uptake by the sponge.
The choice of measuring the optical density for 600nm was made as we would expect the turbidity of the solution to decrease as algae and plastics were consumed by the sponge. However, the light will scatter differently depending of the size of the cell. Furthermore, for the second experiment, although the same amount of algae was used as in the first experiment (2µl), the spectrophotometer recorded very different absorbances for the 0h mark. This was very surprising, and could indicate a malfunction of the spectrophotometer, or some sort of human error. In order to get more reliable data I suggest counting the number of particles in the 100ml cup over time, and set up the experiment to have the same amount of particles in each replicate.
Another issue that was encountered during the experiment was that the microplastics sank very fast once they were added to the seawater. This may have had two implications. Firstly, it is possible, that the plastic beads spent most of the time at the bottom of the cup, not being able to be soaked up by the sponge. Continuous stirring, or shortening the interval between stirs, instead of the 10min interval that was used this study, may have allowed the sponges to have a better chance of filtrating the microplastics. Secondly, the rapid sinking rates may explain the generally larger standard deviation found in the microplastic data (fig 5 & 6). As the water was added to the cuvette, the plastics might have sunk down to the bottom of the cuvette, before the absorbance could be measured. This was, however, accounted for by manually shaking the cuvettes before the OD 600nm was measured. For future research automatically stirring of the cups, using a magnetic stirrer for instance, along with more replicates and a rapid read of the OD 600nm should be implemented.
This study found that there is a very small difference in clearance rate between particle sizes Nano (<2µm), 4-7µm and 7-20µm for the sponge Amphimedon sp. the Different particle sizes are normally picked up by the sponge on different locations, pinacocytes and choanocytes. This study concludes that there is a very small difference in retention rates between these two sites, however that the Nano (<2µm) are retained the slowest, which agrees with previous studies (Turon, 1997), and that the 4-7µm sized algae are picked up the fastest. The study also found that microplastics of the size 10µm do not behave exactly the same way as algal cells of similar size. The data suggests that the sponges can recognize plastic as a nonfood particle and, to some extent, dispose of it. Sponges being highly specific in their food uptake have been shown before (Wehrl et al., 2007). The results showed that the majority of the microplastics were taken up by the sponge, however. Further research is needed on what effect this will have on sponges in the long term.
The author wishes to thank Dr. John N.A. Hooper for his assistance in identifying the sponge genus.
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