Abstract
Human activities have a significant influence on marine life. Pollutants from landfills, dumps, mines, farms, etc. can pollute the ocean and later through the food chain cause harm to all land animals and humans as well. Protein is a very important structural component of an organism. Using protein for energy is an inefficient form of metabolism and in the long run detrimental to the organism. A significant low level of protein content was noticed in Mytilus californianus or (mussels) obtained from the polluted environment when compared to the tissue of Mytilus californianus obtained from pristine environment. Organism that are actively growing and reproducing tend to use more energy than those that are not. Thus estimating the energy content in their tissue will give an idea about their health. Mussels from polluted environment have higher energy content stored in its tissue when compared to mussels from pristine environment. The availability of food, the amount of work spent in finding food, can influence the size and weight of the M. californianus obtained from both habitats. However, we could not observe any such difference from the data collected in the lab. The effect of shell color on the ability to dissipate heat in M. californianus were also looked into, in the ecology lab, and no difference was observed.
Introduction: Pollutants from landfills, dumps, mines and farms can pollute the oceans. Later, through the food chain, these pollutants can cause harm to living organisms on land. These pollutants and thrash that are dumped into the oceans can be directly toxic or deprive the oxygen in the water, thereby harming the plants and animals in the ecosystem. The sewage that is disposed into oceans, can lead to a condition called nutrient loading or eutrophisim. These pollutants, cause a decrease in the ocean’s oxygen content and increase in its carbon dioxide content, leading to acidification of ocean water. As the content of dissolved carbon dioxide in water increases, the pH of the water decreases. This acidification can have an adverse effect on shell fishes like M. californianus. Carbon dioxide reacts with water to form carbonic acid that dissolves the shell. Californian mussel or Mytilus californianus is an example of a marine living organism with a shell and can be used to study the effect of pollution on marine life (Sarkar et al., 2006). The NACP coastal survey cruise in 2007 reported ocean acidification of North American Continental Shelf and these waters travel all the way to reach the water along the inland shores of the coast. Many researchers have reported that shell fishes from various polluted coasts are unsafe for human consumption as they were loaded with toxin (Wu and Wang, 2008).
Protein is a very important structural component of an organism. It is required for body building and in situations where there is carbohydrate and lipid shortage, it also serves as a source of nutrient. Comparing the protein content in the M.californianus obtained from two different sources, will help predict energy utilization status in different habitats. In this lab experiment, M. californianus was collected from two different lab environments: polluted and pristine sites, and their protein content was estimated. The M.californianus collected from Santa Monica Pier is considered as the one from the polluted sites. The Santa Monica pier is a popular tourist attraction and the environment of this place is polluted by human activities. On the other hand, Ventura Pier, does not have many human visitors, and is isolated and away from human activity. The environment in this place is not polluted by human activities.
The hypothesis of the study was that organism that are dependent on protein metabolism, break down tissue protein and this would affect fitness. We expect to notice a difference in the tissue protein content of M.californianus obtained from the polluted and pristine environment. Citrate synthase is a key Krebs cycle enzyme and a regulator of flux through the energy pathway. The citric acid cycle can oxidize end products of carbohydrate, fat and protein metabolism to CO2, and generate 10 to 12 ATP.
The energy content in the tissue can be estimated by the heat of combustion released, when burning it in a bomb calorimeter. In a bomb calorimeter, very high electric current is used to completely burn the tissue placed in the chamber. The heat that is given off in the exothermic reaction that follows, is used to calculate the energy content in the tissue. The technique is accurate and efficient. In the lab experiments the tissue energy content in the M. californianus obtained from the polluted and the pristine environment was estimated.
Weight tends to increase faster than size. Nevertheless, the size can place a restriction on the amount of weight an organism can gain (Downing et al., 1993). According to the rules of allometric scaling, the change is the size of the entire organism need not happen in proportion to changes in size of individual traits like area of the gills, volume of the mantle cavity, tissue mass, mass of muscular foot, etc. However, according to isometric scaling the changes in size of the entire organism, happen at a rate that is in proportion to that of the individual traits. The scaling of M. californianus body part experiment, was done to determine whether, the changes in organism size followed an allometric or isometric change.
Pollution in the water can adversely affect respiratory rate of marine life (Scott and Major, 1972). The respiration rate of M. californianus were measured using an oxygen electrode. A fiber optic oxygen electrode (Ocean optici) was used to measure the oxygen flux. The respiration rate was measured at ambient temperature for the M. californianus obtained from polluted environment, obtained from pristine environment and for the M. californianus that was incubated in a controlled environment. For measuring the respiration rate in a controlled environment, the M. californianus were incubated in a closed chamber for 30-60 minutes.
Temperature extreme can have adverse effects on delicate sea animals like M. californianus. In time of the day, when the tide is low, there is less chance for the organism to dissipate its heat through contact with water. During summer, the temperature may soar up to 40oC and this can cause severe burn injuries to the organism. During winter, the mussel is enclosed in the ice, and this can injure the tissues. The effect of temperature on the M. californianus physiology was done in another ecology lab experiment and is discussed towards the end of the report.
Methods:
i. Estimation of protein using the Lowry’s method: Lowry’s reagent (diluted copper tartrate) is used to estimate protein in the tissue sample. It was added to the diluted tissue homogenate of the organism. Later Folin’s reagent was added. Based on the concentration of protein, there is a visible color change in the reaction mixture. The intensity of color change was measured by taking the absorbance at 750 nm. The absorbance of the test sample was used to calculate the concentration of the protein in the sample using the regression line equation. The regression line that fits the calibration curve was drawn, by plotting the standards of known protein concentration against their absorbance at 750 nm.
ii. Estimating the energy content of the tissue using Bomb calorimeter: In physiological ecology lab, a scaled down version of the bomb calorimeter called the micro bomb calorimeter was used to measure energy stored in the tissue. The micro-bomb calorimeter has a stainless steel chamber with a frying pan, in which the dried tissue pellets to be burned are kept. It is then filled completely with oxygen. The bomb calorimeter is kept immersed in an insulated water bath during the experiment. The electric current passes through the chamber and this burns the tissue completely. The heat that is released when the tissue burns, increases the temperature of the water surrounding the calorimeter. The rise in temperature is used to measure the energy released in the unit joules/g. The last calculation is done automatically by a computer program that is linked to the instrument.
iii. Scaling the M. californianus experiment: For this experiment, M. californianus of different sizes were chosen. First, their whole body size was measured using a measuring scale, following which they were dissected and the size of each individual body part was measured. The wet weight of each body part was determined using a weighing balance. After this, the body parts were dried for 3 to 4 days in a hot air oven set at 60oC. The dry weight of the body parts was then determined using a weighing balance.
The gills of M. californianus, were carefully dissected out without damaging it and placed in a Petri dish containing sea water. A picture of the isolated gill was taken using a digital camera. Then the size of the gill in the picture was measured using a scale. This experimental step should be done before the step of drying the body parts in the hot air oven.
The volume of the M. californianus shell was determined by filling the shell with water up-to its lips and then measuring the volume of the filled water by pouring it into a measuring cylinder. At the end of the labor spend doing this experiment, one would have values for the following variables: length, foot mass, tissue mass, gill area, shell volume and shell weight.
iv. Recording the respiration rate: To record the respiration rate, the oxygen electrode was first calibrated. The M. californianus whose respiration rate is to be determined is cleaned and then placed in a respiration chamber that is connected to the oxygen electrode. Following this, the chamber is sealed with a cork. The chamber is then immersed in a water bath that contain sea water maintained at an ambient temperature. After assuring that the water flows through the oxygen electrode, respiration rate was recorded using a computer that was connected to the instrument. The respiration rate was recorded for 60minutes and the data was reported at every 1minute interval. At the end of 60 minutes, the recorded data were transferred to an excel file for further evaluation. A calibration curve of time vs oxygen (mg/l) was drawn and the linear regression equation was calculated. On substituting the values for the different constants in the regression equation, the oxygen required per mg of M. californianus tissue was known. This is multiplied with the body weight to get the amount of oxygen utilized/mussel/min.
v. Effect of various condition on temperature characteristic of M. californianus: For this experiment, IR camera was used to measure the temperature of the M. californianus at the area of various shell parts. Mussel mimics are used for this purpose. The mimic was created by filling a clay model of M. californianus in-between the shell. Shells of different size and color were used in this study. This will help determine the thermal characteristic of the shell at various conditions like: color, presence of tissue, aggregation, breeze, moisture, etc. The image was taken with an IR camera at 0, 5 and 10th minute, after exposure to test temperatures. The FLIR software was used to determine the temperature from the IR images using Flying Spot. The temperature at the beginning of the warming (or Cooling) and the final temperature at 5th and 10th minute can be determined using the technique. The data thus obtained is analyzed to understand the significance of thermal exchange on the organism physiology.
Results:
i. Estimation of protein using the Lowry’s method: * the client has directed that he will incorporate the result of the first lab experiment himself.
ii. Estimating the energy content of the tissue using Bomb calorimeter: The average of energy released from burning M. californianus obtained from pristine environment was 19.34±0.45 KJ/g and for the M. californianus obtained from polluted environment was 15.08 ± 3.96 KJ/g. The M. californianus from pristine environment had higher energy content than those from polluted environment. The difference was statistically significant at 95% CI (p-value=0.02 as estimated by students t-test)
iii. Scaling the mussel body parts and shell: The comparative size and weight of the whole body and the individual body parts of M. californianus, obtained from the pristine and polluted environment are shown in table 1. The significance of the Difference in Mean between the two, was tested using students t-test and the p value obtained in shown in Table 1. Though M. californianus obtained from pristine environment have slightly lower figures for all variables, the difference in the scale between pristine and polluted environment is not statistically significant.
iv. Effect of various condition on temperature characteristic of M. californianus: The difference in temperature variation that was measured in the colored and white shell M. californianus is represented in Figure 2. The difference observed is not statistically significant. The temperature increase in colored shell mussel was 1.41 ± 0.48 o C and in white mussel was 1.82 ± 0.46 o C . Nevertheless, the difference is not statistically significant when determined by student’s t-test.
Figure 2: Temperature of Colored and White shell M. californianus.
Discussion:
Human activities have a significant influence on marine life. Marine life, including coral reefs has an important impact on human and animal life on this planet. Any adverse changes happening in the sea, not only harms the marine organisms, but can also have extended effects on life on land.
The level of protein in the organism can be an indication of M. californianus’s, energy metabolism. Using protein for energy is an inefficient form of metabolism, and in the long run it is detrimental to the organism’s wellbeing. Carbohydrates and fat metabolism are an efficient and favorable form of metabolism. Comparing the protein content in the tissue using the Lowry’s method, and the energy content using bomb calorimeter will help determine the quality of tissue in M. californianus obtained from the two sources: polluted and pristine environment.
Organisms that are actively growing and reproducing, tend to use more energy than those that are not (Smolders et al., 2004). Thus estimating the energy content in the tissue will give an idea about their health. By determining the energy content stored in the tissues of the organism, it is possible to predict the energy budget or the proportion of energy that is allotted for different activities.
Spatial heterogeneity will help the organisms adapt to variation in their habitat and eventually these organisms may evolve to give rise to new species (Downing et al., 1993). According to one theory, organisms with a phenotype that helps it to survive in a given environment will linger, while the others will disperse to an environment that is suitable for them. Size and shape are important phenotypic determinants, and its variation can be easily assessed in M. californianus from the polluted and pristine environment. In this lab study, it is assumed that the size of the organism, is directly proportional to its weight. This is just an assumption and may not hold true in all situations.
The respiratory rate of M. californianus can be adversely affected by the polluted waters in the ocean. There is no literature support in this area with respect to M. californianus. Nevertheless, defective respiration has been reported for other marine organisms that live in polluted water. (Scott and Major, 1972)
In addition to all the physiological parameters, the effect of the organism’s phenotype that helps it to adapt to its environment was also assessed in the lab. The color of the shell can influence the M. californianus’s ability to absorb and dissipate heat.
In the ecology lab, different experiments were conducted to determine the difference in protein content, the total energy, size of body parts of M. californianus that were collected from the two different environments. The pristine environment is untouched by human activity and the M. californianus that are found in this environment are not exposed to toxic pollutants unlike the ones collected from polluted environment. The result from the experiments indicate that there is a significant difference in protein and energy content between M. californianus obtained from the pristine and polluted environment. The organisms from pristine environment have a higher mean protein and energy content. This indicates better metabolism and the health status of these mussels over those obtained from polluted environment. There was no significant difference in the size of the mussels and their individual body parts, between those obtained from the pristine and polluted environment.
In another experiment, the temperature rise in colored and white shelled M. californianus, exposed to high temperature was determined. There was no statistically significant difference in the temperature increase between the two, though, the white shelled mussel had a slightly higher rise in mean temperature than the colored shelled ones. This experiment was done to understand the role of shell color in heat dissipation and retention.
Introduction of a pollutant can affect the stability of the marine ecosystem and cause harm to the organisms living in it. The Mytilus californianus, is a native of the North American coastline, and can be spotted in abundance on the open rocky ground along the coast. They prefer areas of high salinity and low sedimentation. The effect of marine pollution on living organism was investigated using this species as a model. While the impact of pollution is evident in the protein content and energy content of this organism, its impact on other parameters like body size and traits is less appreciable.
References
Downing, J., Rochon, Y., Pérusse, M. and Harvey, H. (1993). Spatial Aggregation, Body Size, and Reproductive Success in the Freshwater Mussel Elliptio complanata. Journal of the North American Benthological Society, 12(2), pp.148-156.
Sarkar, A., Ray, D., Shrivastava, A. and Sarker, S. (2006). Molecular Biomarkers: Their significance and application in marine pollution monitoring. Ecotoxicology, 15(4), pp.333-340.
Scott, D. and Major, C. (1972). The Effect of Copper (II) on Survival, Respiration, and Heart Rate in the Common Blue Mussel, Mytilus edulis. Biological Bulletin, 143(3), p.679.
Smolders, R., Bervoets, L., De Coen, W. and Blust, R. (2004). Cellular energy allocation in zebra mussels exposed along a pollution gradient: linking cellular effects to higher levels of biological organization. Environmental Pollution, 129(1), pp.99-112.
Wu, J. and Wang, J. (2008). Impacts of Pollution from Different Sources on Ecological Quality of a Multiple-use Coast. Water Air Soil Pollut, 193(1-4), pp.25-35.
