Bivalves provide a wealth of ecological services as they provide natural filtration to improve water quality as well as reduce the outbreaks of algal blooms. In recent years there has been additional strains on our marine environment with more metal pollutants entering the waterways and contaminating marine species. In small quantities copper is non-lethal however in large quantities it can be detrimental to the function of bivalve organisms and even lethal at prolonged exposure. In this in vitro experiment I examine the effects of varying copper concentrations on mussels (Mytilus Galloprovincialis). Individuals were placed in a mixture of seawater and copper sulphate and were provided with an algal feeding solution. Filtration rate was monitored over 4 hours by using a spectrophotometer to measure optical density of the water and calculating the number of algae cells that had been consumed. In higher concentrations, individuals consumed less algae cells (1.93E+08 cells per hour) than the control group (3.57E+08 cells per hour). This would suggest that either the copper was having a detrimental effect on the functionality of the organism or that the organism has the ability to sense the presence of the copper in the water and regulate filtration.

Bivalves have a significant role in marine ecosystems in controlling and maintaining water quality. With the rise of industrialization and the increase in agriculture there has been an increase in nutrients, specifically nitrate and phosphorus, into the water column which has given rise to the number of algal and cyanobacterial blooms (Mclaughlan & Aldridge 2013). In large quantities cyanobacterial blooms can have detrimental effects on other marine species as it lowers the amount of absorbed oxygen in the water as well as blocks available sunlight essential for the photosynthetic plants that provide food resources for other species (Mclaughlan & Aldridge 2013). As seen in the example of the Hudson River the presence of bivalves has had significant effect on reducing the outbreak of algae blooms (Mclaughlan & Aldridge 2013). As well as feeding on algae in the water, bivalves can also sequester nitrate and phosphorus from the waterway which would otherwise be available to phytoplankton to thrive on (Mclaughlan & Aldridge 2013).

With the increase in industrial areas, especially in close vicinity to river systems there has also been an increase in copper released into the waterways. In small concentrations, copper can be excreted or detoxified by bivalves however large concentrations can be lethal to organisms as the metabolic rate can not keep up with the metal intake (Fukunaga & Anderson 2011). While bivalves provide ecological benefits to marine systems, they are also a nuisance to fishing and marine aquaculture. In aquaculture, bivalves cause damage to nets and mechanics as well as providing additional competition for food resources (Fitridge et al 2012). It is conservatively estimated that approximately 1.5 - 3 billion dollars of aquaculture production costs is contributed towards biofouling control in the US. While copper based antifouling paints protect ship hulls from biofouling, it has detrimental effects on the bivalve populations by leeching into the waterways. In a study of copper contamination from antifouling paints, Schiff et al (2004) found that a 9.1m boat using antifouling paint had a dissolved copper emission of 26.7g per month. Another source of copper pollution influx comes from stormwater runoff where dissolved copper concentration levels are elevated during and shortly after storm events (Fukunaga & Anderson 2011). Metals in shellfish and molluscs are not just detrimental to the organism but also to humans through consumption. In Japan, heavy dumping of mercury into the Minamata Bay between 1932 and 1968 resulted in bioaccumulation in the marine species which in turn resulted in a large number of mercury poisoning cases (over 10000 people have received financial compensation) through seafood consumption (Gosling 2015).

In this in vitro study that was performed at the University of Queensland, I examine the effects of varying copper concentration levels on the filtration rates of mussels (Mytilus Galloprovincialis). Individuals were placed in separate containers with different concentrations of copper sulphate and sea water and provided with algae feeding solution. It was expected that specimens in the higher copper concentration treatments would have a lower filtration rate than those in the control group with no copper sulphate added.

Study Setup

Mytilus Galloprovincialis were purchased from a local seafood vendor and stored in sea water at room temperature for 24 hours prior to the experiment.

An algal feeding solution was prepared by combining 10mL Isochrysis 1800, 10mL Nannochloropsis 3600 and 10mL Pavlova 1800 in a tube. The solution was stirred to ensure mixing of the various algae. 2 litres of copper sulphate and sea water solutions were prepared with 3 different copper sulphate concentrations; 0.25, 0.5 and 0.75 mg/L. Each concentration was divided equally into 5 containers, 1 for each replicate, with 400mL in each container. For the control group, 5 containers were each filled with 400mL of sea water. A pipette was used to transfer 500µL of the feeding solution into each container and each container was stirred to ensure the algae feeding solution was diluted evenly. Mytilus Galloprovincialis were then placed in a container with one specimen per container, as shown in figure 1 below, and monitored for 4 hours. At the end of the monitoring period each specimen was measured, and length was recorded.

Measuring Filtration Rates

At the beginning of the experiment 400mL of seawater was poured into a container and 500µL of algae feeding solution was added and stirred thoroughly. 10µL was transferred into a haemocytometer and the number of algae cells were counted under a microscope to calculate the total algae cells in the 400ml solution. Figure 2 below shows the dilution of algae in the base solution at 100x magnification. 2mL of the solution was then transferred into a cuvette and the colour density of the solution was measured using a spectrophotometer. This provided a base measurement for the number of cells per optical density of the solution. At time 0, 2, 3 and 4 hours, 2 cuvettes were filled each with 2mL of water from each container and optical density was measured using the spectrophotometer. The average of the 2 readings was calculated for each sample, and the number of algae cells filtered was calculated.

Statistical Analysis

Data was recorded and entered into an excel spreadsheet and the average algae cell filtration rates for each treatment concentration was plotted on a graph along with the control group. RStudio was then used to perform an ANCOVA analysis.

As shown in figure 3 below, individuals exposed to the higher concentrations of copper had a much lower filtration rate than those in the control group. Results from the ANCOVA analysis also confirmed that there was a fairly significant decrease in filtration (N=20, df=16, p=0.077) for specimens subjected to 0.75mg/L copper concentrations. From the ANCOVA analysis it was also determined that there was no significant interaction (p=0.15) between the size of the mussel and the filtration rate. In terms of filtration rate per hour, individuals in the control group averaged 3.57E+08 cells per hour, whereas in the 0.75mg/L copper treatment group the average filtration rate was 1.93E+08 cells per hour. Also note that the number of algae consumed did not constantly go up over the 4-hour period in the 0.75mg/L copper concentration group, with a decline in algae consumed between the 2nd and 3rd hour. While the higher copper treatment group had a lower filtration rate, the same did not hold true for the 0.5mg/L copper treatment group. Individuals in the 0.25mg/L copper group and the 0.5mg/L copper treatment group had similar filtration rates of 2.81E+08 and 2.90E+08 cells per hour respectively, although the filtration for 0.5mg/L copper treatment group was slightly higher than the lower concentration.

From our results we can see that there was a lower filtration rate in the individuals that were subjected to the higher copper treatments, suggesting that copper may be having detrimental effects. To understand the effects that copper has on the organism it is important to first examine feeding mechanisms of bivalves.

In feeding, water is taken across the gills where particles are separated from the water column and passed to the stomach for digestion and absorption (Gosling 2015). Cilia on the gills create a flow of water through the mantle cavity and gills. Water is filtered by the cirri, which acts as a strainer, removing particles from the water. Particles are trapped in a fine mucus layer which is then transported to the mouth and oesophagus which is lined with cilia necessary for passing the food to the stomach. In the stomach particles are processed by the digestive gland through intracellular digestion and waste is passed through to the intestine where waste nutrients are either excreted or processed and passed to other functions of the body.

In previous studies of the biological effects of copper on mussels, Al-Subiai et al. (2011), found that Mytilus Edulis exposed to copper concentrations for 5 days experienced a loss of cilia around the gills and a loss of tubule definition in the digestive tract. While it is unclear whether these effects could happen in a shorter time period with much higher copper concentrations, the damages they discovered would have detrimental effects on filtration. With the process of filtration, a loss of cilia around the gills would limit the food particles that are being sifted out of the water and therefore lead to lower filtration rates. Digestive tubules are responsible for two functions; transferring of food to the digestive gland and transporting waste back out of the digestive gland to the intestine. Within the tubes themselves there are cylindrical digestive cells which are responsible for intracellular digestion. Any damage to these cells as suggested by Al-Subiai el al (2011) would also slow the process of filtration. With the 0.25mg/L and 0.5mg/L copper treatment groups, damage may not have started to happen yet at 4 hours, explaining why we see similarities in the filtration rates. 

Lower filtration rates in higher copper concentration levels might also suggest the presence of chemoreceptors in the organism sensing the presence of high copper concentrations and regulating filtration. Through the study of bivalves two opposing views have arisen regarding filtration rate regulation. While the less popular belief is that filtration in bivalves is an automatic process determined by the capacity of the pump and the retention efficiency of the gills (Gosling 2015), the more popular belief is that the process is physiological, and that mussels control filtration based on the availability of food particles. Throughout the filtration process there is particle selection processes that occur, both pre and post- ingestive. Pre digestive particle selection occurs at the gills and is facilitated by the latero‐frontal cilia which restricts size of particles that are passed onto the mouth. As well as size selection particles are also selectively processed based on particle stickiness, electrostatic charge or molecular excretions (Gosling 2015). Another form of pre-ingestive particle processing is the production of pseudofaeces which is facilitated through the production of different types of mucus. High viscous mucus that flow counter current to the gills and remove particles directly from the inhalant flows whereas low viscous mucus is responsible for capturing particles destined for the ingestive tract. The mucus is then processed by the labial palps and excreted as pseudofaeces before reaching the intestines or digestive tracts. The final process of particle sorting in post-ingestive, where larger particles or small dense particles that have made it past previous particle sorting processes are filtered by ridges and grooves in the stomach and transferred to the midgut where excreted through faecal pellets. Through this process there is several opportunities for copper to be filtered out and the reduced rate of filtration may instead be caused by extra efforts to strain out unwanted particles. This post-ingestion selection may explain why there was a decline in the number of algae consumed in the higher copper concentrations after the 2nd hour as contaminated particles may have been returned to the water. While these particle sorting functions maybe adequate for short term exposure to copper, as discovered by Al-Subiai et al (2011) long term exposure would start to damage the cilia around the gills and disable the pre-digestive sorting mechanisms.

While this study has shown that the presence of copper has detrimental effects on the filtration rates of Mytilus Galloprovincialis more study is needed to explore the internal workings of the organism to explain why. The lower filtration rates could be the result of copper causing direct damage on internal organs or it may be caused by delays in filtration due to selective particle sorting. Regardless, long exposure to copper concentrations even at small doses (+100 µg/L) can have lethal effects (Martin 1979) and limiting copper pollution in waterways should be of utmost importance. While currently there are non-copper based antifouling paints available on the market today, these are more expensive and less effective than the traditional copper based antifouling solutions that have been used for years (Carson et al 2009). Introducing vegetated filter strips between pollutant sources, including farms and industrial estates, and waterways would also minimise the concentration of metals entering the waterway as the provide a natural filter (Larson, R.A. & Safferman 2012).

A big thanks to Sandie and Bernie Degan for helping to plan and prepare for this study as well as Davade and Matt for sharing their expertise

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