Factores Claves del Clima

2-4-1 Chlorophyll a by fluorescence

Chlorophyll (chl) concentrations in water samples were measured by fluorescence using the method of Welschmeyer (1994). Water samples (25ml) were filtered under light positive pressure through 25 mm GF/F (Whatman) filters using a syringe attached to an in line filter holder.

Alternatively, chlorophyll was size fractionated (Fchl) by passing 25 or 50ml water samples through a sequence of 25mm polycarbonate filters with pore sizes of 20, 5, 2 and 0.2µm. Filtration through the 20 and 5µm filters was done under gravity, but through the 2 and 0.2 µm filters under gentle vacuum (<20cmHg) using a hand vacuum pump (NALGENE Brand products), following the method of Iriarte and Purdie (1994). All filters were stored at –20 oC prior to extraction.

To measure the chlorophyll a, each filter was placed in a 15-ml centrifuge tube and 5ml of 90% acetone was added and shaken thoroughly to extract the pigments. The samples were than immediately sonicated for 30 seconds, or they were allowed to stand in the dark (4oC) overnight. The samples were then centrifuged at 3000 rpm for 10 minutes at room temperature, and the supernatant fluorescence measured in a 10- Av fluorometer (Turner Designs). The fluorometer was calibrated regularly using

concentration of the chlorophyll standard was determined by spectrophotometry according to the equation given by Jeffery and Humphrey (1975):

chla = 11.85 E664 – 1.54 E647 – 0.08 E630

Where E = the absorbance readings of the spectrophotometer at 664, 647 and 630nm, using a 1cm cell, after correction for turbidity by subtracting the reading at 750nm.

The sample chlorophyll a concentrations were determined from the following

equation:

chl (mg/m3_{) = R * v/V}

Where R = Calibrated fluorescence value, v = Volume of acetone used for extraction (ml) and V = Volume of sample filtered (ml) (Parsons et al., 1984).

In this study, all samples of chl were measured in triplicate and mean values calculated; the standard deviation was <5%, although maximum deviations up to 25% were found when chlorophyll was lower than 1mg m-3.

In addition to total chlorophyll a (Tchl) measurements with different water sample volumes, an experiment was carried out for size fractionated chlorophyll a. Three (a- c) 25 ml water samples were size fractionated through four different polycarbonate filters, with pore sizes 20, 5, 2, 0.2 µm, as described above. The results are shown in Table 2 -1.

The total chlorophyll a (Tchl) obtained from the different volumes (25 and 50 ml) of sample water gave similar values (2.2, 2.3 mg m-3, respectively) to the average of summation of size fractionated chlorophyll a (Fchl) (see Table 2-1). The highest variability was found for the 5-2 and < 2µm fractions.

Table 2-1: Results of the size fractionated chlorophyll a experiment

size fract. A B C range mean %

> 20µm 1.07 1.04 1.06 1.04-1.07 1.06 46

5-20 µm 0.43 0.41 0.47 0.41-0.47 0.44 19

2-5 µm 0.36 0.28 0.50 0.28-0.50 0.38 17

< 2 µm 0.38 0.41 0.47 0.38-0.47 0.42 18

2-4-2 High Performance Liquid Chromatography (HPLC) of phytoplankton pigments High Performance Liquid Chromatography (HPLC) was used to analyse the pigment content of samples, using the method of Mantoura and Llewellyn (1983), as

described by Barlow et al (1993). A Thermoquest HPLC system (gradient pump,

vacuum degasser, autosampler, UV/V photodiode array and Fluorescence Detector) incorporating a 3µm C-18 HPLC column, was used. The solvents were A: 80% methanol and 20% 1M ammonium acetate and B: 60% methanol and 40% acetone, with a decreasing gradient from 100% A to 100% B for 10 minutes, followed by an isocratic stop at 100% B for 7.5 minutes. A second gradient over 2.5 minutes was then used to return to the initial condition of 100% A. The total run time per sample was approximately 17.5 minutes.

Chlorophyll and carotenoids were measured by absorbance at 440 nm, and detected phaeopigments were measured by fluorescence using an excitation wavelength of 410nm and emission at 670nm. Data collection and integration utilised the Chromquest software on a Dell 1100 computer.

Pigment identities were established by co-elution with authentic pigment standards (Sigma Chemical Co, DHI). Peak identity was further confirmed by on-line photodiode array spectroscopy. The consistency of the HPLC values are within an error of ±5%, and the correlation (R2) for chl standards was 0.99, as shown in Figure 2-2. Figure 2-3 shows a chromatogram for a pigment mixture standard, with different concentrations of each pigment, used as a standard before sample analysis.

Figure 2-2: HPLC chla calibration. P e a k a re a 0 50 1 00 15 0 20 0 25 0 Weig ht inj ec ted (ng) 0 2 4 6 8 10 12 Y = 0 .0 4X - 0 .01 R2 _{= 0.9 9} n = 9

Figure 2-3: HPLC chromatogram for a standard pigment mixture.

(Pigment identification after solvent peak: 1= chlorophyll c3, 2=chlorophyll c1+c2, 3=peridinin,

4=19-butanoyloxyfucoxanthin, 5=fucoxanthin, 6=19-hexanoyloxyfucoxanthin, 7=brasinoxanthin,

8=violaxnthin, 9=diadinoxanthin, 10=alloxanthin, 11=zeaxanthin, 12= chlorophyll b, 13= chlorophyll

a, 14=ß-carotene).

Four 250ml water samples were filtered onto four 25mm GF/F filters using a Millipore glass filtration system, giving duplicate pairs of samples. The filters were stored at -70 to -85oC for up to three months. One pair of filters (the other pair is stored as back-up) was placed in 3ml HPLC grade 90% acetone, sonicated in a 15ml centrifuge tube for 30 seconds, centrifuged at 3000 rpm for 10 minutes to remove cellular debris, and then filtered through a 0.2µm nylon filter. 1ml of the extract was transferred to a small glass vial and placed in the HPLC auto-sampler. Then an aliquot of 500µl of clarified extract was automatically mixed with 500µl of 1M ammonium acetate. A mixture of 100µl of that was injected into the HPLC.

Pigment concentrations (Pc) were calculated according to the following equation (Barlow et al., 1993): Pc (µg l-1_{or mg m}-3_{) = (Pa . v . 1000 / Pr . Vi . V 0.5)/1000} Minutes 0 2 4 6 8 10 12 14 16 m AU 0 20 40 mAU 0 20 40 11 1 ₄ 12 13 14 9 8 10 3 7 2 6 5

Where Pa = Peak area at 440nm, v = Volume of extracted acetone (ml), Pr = Pigment response factor, Vi = Volume injected in the column (100 µl), V = Volume of filtered sample (l) and 0.5 = the buffer dilution factor