University of Bahrain College of Science Department of Biology 2nd Semester 2012/2013

Chlorophyll Content Determination of 4 stations in sea water off the East Coast of Asker

Name: Bisma Bashir Mohammad ID # 20094704 Date: May 5, 2013 Instructors name: Dr. Mohammad Al-Rumaidh

2 Chlorophyll Determination of 4 stations in sea water off the East Coast of Asker

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To determine the chlorophyll content of 4 different stations 1km apart in sea water collected on April 28, 2013 off the East Coast of Askar.

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Chlorophyll is the photosynthetic pigment found in plants and plantlike organisms which uses sunlight and CO2 to produce their own food by the help of chlorophyll. Structure of chlorophyll shows that it has Mg chelated by porphyrin ring which consists of Mg, H and other elements such as N and O [1]. In sea water, the plant-like organisms are the phytoplanktons ranging from microscopic to macroscopic which carry out photosynthesis in water. Cyanobacteria, diatoms, dinoflagellates, green algae are some examples of phytoplanktons [2]. The structure of typical chlorophyll is shown in figure 1. Chlorophyll measurements are required for checking the biological activity of water [1]. Different methods are present which measures the chlorophyll content of the water such as:
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Trichromatic method which determines chlorophyll a, b and c spectrophotometrically. Fluorometric technique which determines chlorophyll and pheophytin. High Performance Liquid Chromatography (HPLC).

For any of the above method, basic principle involves the mechanical Figure 1: Structure of typical rupture of cells containing chlorophyll and then extracting them in Chlorophyll molecule [1] organic solvent. This extract is then analyzed either spectrophotometrically, fluorometrically or by HPLC [3]. From these 3 methods, the spectrophotometric method is easy to handle as it has less analytical sensitivity and higher detection limit but from this method, chlorophyll a is overestimated when pheophytin a is present. Pheophytin a is a degradation product of chlorophyll a along with pheophorbide a. Both of these degradation products absorb UV in the same spectral region as of chlorophyll a and thus gives an overestimation of chlorophyll a pigment [4]. The absorbance properties of the chlorophyll pigments help in their quantitative and qualitative analysis [5]. The objective of this lab is to determine the chlorophyll content off the East Coast of Askar at 4 different stations separated 1km from each other. The method adopted is Trichromatic method which measures chlorophyll content spectrophotometrically.

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Sampling was done on April 28, 2013 off the East Coast of Askar from 1:00-3:00pm. Water samples were collected in 1L bottle from 4 stations located at a distance of approximately 1km vertical from each other. Temperature, PSU, DO and pH of those 4 stations were also determined and given in table 1. The samples were mixed with 5% Formalin immediately after collection.

3 Chlorophyll Determination of 4 stations in sea water off the East Coast of Asker

Figure 2: Study Site for chlorophyll determination

These water samples were then brought to the laboratory and filtered, using a filter paper of 45m pore size, as shown in Figure3. After filtration, the filter papers were placed in labeled plastic bags and stored in the freezer until analysis. For chlorophyll extraction, each filter paper was placed in a homogenizer tube along with small amount of MgCO3 and 10mL of 90% acetone and homogenized thoroughly (Figure4). The contents of the homogenizer tube were then transferred to plastic centrifuge tubes and centrifuged for 10 minutes. After 10min., the supernatant was transferred in a 25mL volumetric flask and filled with acetone to make a total volume of 25mL. To determine chlorophyll concentration, trichromatic method was followed. In this method, the absorbance of the extracted solution was measured at 630, 647, 664 and 750nm for each sample using a spectrophotometer.

Figure 3: Trichromatic Method for Chlorophyll determination in sea water

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The Enviornmental parameters recorded during sampling from the 4 stations are as follows:
Table 1: Enviornmental Parameters off the East Coast of Askar collected on April 28, 2013 at 01:00pm

Station # PSU DO (mg/m3) pH Temperature (C) 1 25.7 49.30 0.64 9.02 2 25.6 49.44 0.76 9.48 3 25.7 49.59 0.76 9.35 4 25.1 49.79 0.79 9.46 Mean Standard deviation 25.50.30 49.50.21 0.740.07 9.30.21 The absorbance of each sample measured at the wavelengths of 630, 647, 664 and 750nm, according to the trichromatic method, was recorded in Table 2. The absorbance at 750nm was measured as turbidity blank. This was subtracted from other absorbance values and recorded as corrected absorbance in same table 2.

4 Chlorophyll Determination of 4 stations in sea water off the East Coast of Asker Table 2: Absorbance values of chlorophyll extracts from sea water samples collected off the East Coast of Askar Station # 630nm 0.363 0.298 0.342 0.29 0.417 0.422 0.057 0.053 0.09 0.371 0.39 0.316 Absorbance 645nm 663nm 0.264 0.004 0.117 0.057 0.125 0.013 0.106 0.049 0.207 0.065 0.291 0.035 0.059 0.06 0.054 0.054 0.088 0.087 0.265 0.01 0.161 0.028 0.201 0.019

750nm 0.088 0.134 0.1 0.127 0.164 0.127 0.062 0.057 0.086 0.102 0.131 0.097

Absorbance taken at 750nm was used to subtract the turbidity from the solution and the corrected absorbances are given in table 2.
Table 3: Corrected Absorbance values and their average of chlorophyll extracts from sea water samples collected off the East Coast of Askar Station # Corrected Absorbance E630 E645 E663 0.275 0.176 -0.084 0.164 -0.017 -0.077 0.242 0.025 -0.087 0.163 -0.021 -0.078 0.253 0.043 -0.099 0.295 0.164 -0.092 -0.005 -0.003 -0.002 -0.004 -0.003 -0.003 0.004 0.002 0.001 0.269 0.163 -0.092 0.259 0.03 -0.103 0.219 0.104 -0.078

Average Corrected Absorbance

E630 0.2270
0.2370 0.2493

E645 0.0613
0.0620 0.0743

E663 -0.0827
-0.0897 -0.0773

-0.0017 0.2490

-0.0013 0.0990

-0.0013 -0.0910

The average corrected values, from Table 3, were then used to calculate the chlorophyll content according to the trichromatic equations: Chlorophyll a: Ca = 11.64(E663) 2.16(E645) 0.10(E630) Chlorophyll b: Cb = 20.97(E645) 3.94(E663) 3.66(E630) Chlorophyll c: Cc = 54.22(E630) 14.81(E645) 5.53(E663) The total chlorophyll content of each water sample was determined according to the following equation:

Where, C = sum of chlorophyll a, b and c i.e. Ca + Cb + Cc (g/L) v = volume of acetone added (mL) V = volume of seawater (L) l = pathlength of cuvette = 1cm

5 Chlorophyll Determination of 4 stations in sea water off the East Coast of Asker

Volume of acetone added was 25ml, whereas the volume of sea water samples was 500mL for Station 1 and 2 whereas it was 250mL for Station 3 and 4. The values of chlorophyll a, b and c and the total chlorophyll concentration of all samples, calculated according to the above equations, are recorded in table4.
Table 4: Chlorophyll Content of sea water samples collected from 4 stations off the East Coast of Asker

Station # 1

Chlorophyll type Ch a Ch b Ch c Ch a Ch b Ch c Ch a Ch b Ch c Ch a Ch b Ch c

Chlorophyll Content (mg/m3) -53.601 39.05233 592.837 -57.697 -51.7893 39.30033 47.54517 621.3888 642.2815 -1.28067 -1.66067 -6.32467 -124.818 152.323 1253.782

Total Chlorophyll (mg/m3) 578.2883

638.0373 602.9921667

-9.266

1281.287

Figure 4: Chlorophyll Content Comparison of 4 stations off the East Coast of Asker

Figure 4 shows that the chlorophyll content of the 4 stations off the East Coast of Asker. Here, station 4 showed highest chlorophyll content whereas station 3 showed the least chlorophyll content.

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Chlorophyll content determination is one of the ways to check the productivity of sea water. Productivity gives an indication of the amount of fishes and other marine organisms in the sea which can then be consumed by the

6 Chlorophyll Determination of 4 stations in sea water off the East Coast of Asker

people. The primary productivity of sea water off the East Coast of Asker was estimated by measuring its chlorophyll content. The chlorophyll content was measured using the Trichromatic method. After immediate collection, the water samples were fixed with 5% formalin with an intention to kill the zooplanktons and thus prevent them from eating up the phytoplankton. These phytoplanktons are important as they account for the chlorophyll in water. In lab, the water samples were then filtered using a filter paper of 45m pore size because of the larger size of phytoplanktons. During extraction procedure, a small amount of MgCO3 was used with acetone because it prevents the degradation of chlorophyll. Acetone which is an organic solvent was used to dissolve chlorophyll in the solution. Table 1 shows the environmental parameters of the 4 stations. No much difference was seen in any of the 4 parameters i.e. the temperature, pH, D.O and PSU were similar in the 4 stations. This could be due to the reason of less distance from each other and the shore. To observe a visible change in these parameters, the distance between the 4 stations should be as large as 10km or above because the difference in these parameters between station 1 and 4 are also not much even though the distance is 4km and this is also not enough. Chlorophyll concentration was estimated by UV spectroscopy because chlorophyll a, b and c absorb radiations in the UV region, however, there absorption maxima are slightly different. Chlorophyll a absorbs the most at 664nm while chlorophyll b absorbs maximum at 647nm and chlorophyll c absorbs maximum at 630nm. Hence, the absorption of the solution was taken at the three wavelengths as shown in table 2. Moreover, the absorption was taken at 750nm as a blank to remove any absorption due to turbidity. Hence, the absorption at 750nm was subtracted from the absorption at the other three wavelengths as shown in table 3. Table 4 shows the final chlorophyll content of each station. Also for each station, the different chlorophyll types were calculated. Chlorophyll a in each station showed ve results which were much unexpected as chlorophyll a indicates the presence of green algae and green algae are very abundant in seas but our results showed none so this could be due to any kind of error during the extraction or centrifugation step. The centrifuge used was not accurate i.e. all the particles didnt settle down leaving the solutions turbid. This turbid solution didnt give proper measurement with UV spectroscopy. This could also be explained form the 750nm reading which should not exceed 0.05 but for all the stations this value was higher so mostly likely explanation to this error is poor centrifugation step. Chlorophyll b and c were abundant in station 1, 2 and 4 but again an unexpected result was seen in station 3 where both the chlorophylls were again in ve. Chlorophyll b is abundant in brown algae whereas chlorophyll c is in diatoms. These two are also expected to be in large amount in sea water specially diatoms. The number of diatoms found in other 3 stations was the most among the other chlorophylls followed by chlorophyll b and then a. Summing up the 3 chlorophylls for each station, figure 4 shows which station had the highest chlorophyll content. According to figure 4, station 4 showed the highest concentration of total chlorophyll followed by 2 and 1. The difference between the total chlorophyll content of station 1 and 2 is very small which could be less distance between the 2. Station 3 showed the least which is supposed to be more than station 4 and less than station 1 and 2 because as we move away from shore, the productivity of water decreases but our results are opposite i.e. station 4 which is supposed to be least showed highest and station 1 which is supposed to show highest, showed lower than 2 and 4. No proper assumption can be made on these results as they are not accurate and so should be repeated with care.

7 Chlorophyll Determination of 4 stations in sea water off the East Coast of Asker

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We can conclude based on our results that highest concentration of total chlorophyll is seen in station 4 whereas least was found in station 3. But literature suggests as we go away from the coast, the productivity decreases i.e. chlorophyll concentration will also decrease. Therefore, no proper assumption can be made on these results as they are not accurate and so should be repeated with care.

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1. The Basis of Chlorophyll Measurements (2005). Accessed date: May 10, 2013. Available: http://www.ysi.com/media/pdfs/T606-The-Basics-of-Chlorophyll-Measurement.pdf 2. Phytoplankton (no date). Accessed date: May 10, 2013. Available: http://nerrs.noaa.gov/doc/siteprofile/acebasin/html/biores/phyto/pytext.htm 3. Dos Santos A. C. A. et al. (2003): Comparison of three methods for chlorophyll determination: Spectrophotometry and Fluorometry in samples containing pigment mixtures and Spectrophotometry in samples with separate pigments through High Performance Liquid Chromatography. Acta Limnologica Brasiliensia. 15(3): 7-18. 4. Applicability of Chlorophyll a methods (2011). Accessed date: May 10, 2013. Available: http://www.dep.state.fl.us/water/sas/qa/docs/application-chlorophyll-a-methods.pdf 5. Dere, S., Gunes, T. and Sivaci, R. (1998). Spectrophotometric Determination of Chlorophyll- A, B and Total Carotenoid Contents of Some Algae Species Using Different Solvents. Tr. J. of Botany, 22:13-17.