The primary productivity of phytoplankton, macroalgae, and seagrasses forms the base of marine ecosystem structuring in aquatic environments. Primary productivity is affected by various environmental factors and ecological processes that usually interact in a complex manner. The rate of primary production usually governs the overall ecosystem health and ecological productivity of a water body, and any observed trends may reveal the occurrence of potential stresses on existing ecosystems. Along the Saudi Gulf coast, primary productivity monitoring may help provide the basis for identifying the potential stressors to the coastal marine environments. Foremost among the considerations is the potential adverse effect of excessive anthropogenic nutrient loadings, which may lead to eutrophication events that can adversely impact on ecosystem health. In addition, high nutrient loads from man-made activities may trigger the excessive growth of some toxic phytoplankton species, potentially resulting in harmful algal blooms (HABs) with serious human health risks and negative economic impacts. This study is geared towards monitoring the primary productivity levels in selected areas of the Saudi Gulf waters to identify areas of concern as regards hyper-nutrification, ecological disturbance, and potential hot spots for HAB events. Nutrient loadings and the identification of potential HAB organisms will form a special focus of the investigations.

1. 1
The 14C-radiotracer method to measure the primary
productivity of phytoplankton
SECTION 1 INTRODUCTION:
The primary productivity of phytoplankton, macroalgae, and seagrasses
forms the base of marine ecosystem structuring in aquatic environments.
Primary productivity is affected by various environmental factors and
ecological processes that usually interact in a complex manner. The rate of
primary production usually governs the overall ecosystem health and
ecological productivity of a water body, and any observed trends may reveal
the occurrence of potential stresses on existing ecosystems. Along the
Saudi Gulf coast, primary productivity monitoring may help provide the
basis for identifying the potential stressors to the coastal marine
environments. Foremost among the considerations is the potential adverse
effect of excessive anthropogenic nutrient loadings, which may lead to
eutrophication events that can adversely impact on ecosystem health. In
addition, high nutrient loads from man-made activities may trigger the
excessive growth of some toxic phytoplankton species, potentially resulting
in harmful algal blooms (HABs) with serious human health risks and
negative economic impacts.
This study is geared towards monitoring the primary productivity levels
in selected areas of the Saudi Gulf waters to identify areas of concern as
regards hyper-nutrification, ecological disturbance, and potential hot spots
for HAB events. Nutrient loadings and the identification of potential HAB
organisms will form a special focus of the investigations.

2. 2
SECTION 2 OBJECTIVES
The specific objectives of this study are as follows:
1. Conduct measurements of primary productivity at a selected area
along the Saudi Gulf coast;
2. Collect samples of phytoplankton at different sampling locations and
seasons, and determine the spatiotemporal changes in composition,
distribution, and abundance of the major phytoplankton taxa in the
study area;
3. Conduct simultaneous measurements of the known environmental
correlates of primary productivity, including dissolved nutrients, and
elucidate any significant patterns of variations among the observed
variables;
4. Conduct field measurements to determine the respective
contributions of the macroalgae and seagrasses to the primary
production in the area;
5. Identify and characterize any observed patterns of the phytoplankton
species that have been associated with harmful algal blooms; and
6. Perform a focused analysis of the potential role of nutrient loadings
on the observed patterns of primary productivity in the study area.
SECTION 3 MATERIALS AND METHODS
The 14C-radiotracer method will be applied to measure the primary
productivity of phytoplankton

3. 3
Appendix
EQUIPMENT:
• Go-Flo bottles
• kevlar hydroline
• teflon messengers
• stainless steel weight
• temperature- and light-controlled deck incubation system
(NORDA/USM incubation system)
• free-drifting productivity array (including polypro line, spreader bars,
surface floats, buoy, radio
• transmitter and strobe le light)
• 500 ml wide-mouth polycarbonate bottles
• vacuum filtration system
• liquid scintillation counting (LSC) vials
• pipettes
• glassware
• vortex mixer
• liquid scintillation counter (Packard model 4640; United
Technologies Inc.)
REAGENTS
• distilled deionized water (DDW)
• HCl for trace metal analysis (Baker Instra-Analyzed)
• Na2CO3 (99.999%)
• NaH-14
CO3 solution (cat #CMM-50, Research Products Inc.)
• β-phenethylamine
• Aquasol-II (Dupont)
• 2 M HCl

4. 4
CLEANING
Due to the potentially toxic effects of trace metals on phytoplankton
metabolism in waters, the following procedure will be used to minimize
the contact between water samples and possible sources of
contamination.
3.1. HCl solution (1M) is prepared with high purity hydrochloric acid and
freshly-prepared glass distilled deionized water (DDW).
3.2. 300 ml poly Vinyl transparent bottles are rinsed twice with 1M HCl and
left overnight filled with the same acid solution. The acid is removed
by rinsing the bottles three times with DDW before air drying.
3.3. Go-Flo bottles, fitted with teflon-coated springs, are rinsed three times
with 1M HCl and DDW before use.
3.4. Pipette tips used in the preparation of the isotope stock and in the
inoculation of samples are rinsed three times with concentrated HCl,
three times with DDW and once with the sodium carbonate solution
and stored in a clean polyethylene glove until used.
4. ISOTOPE STOCK PREPARATION
4.1. The preparation of the isotope stock is performed wearing
polyethylene gloves. A 25 ml acid-washed teflon bottle and a 50 ml
acid-washed polypropylene centifuge tube are rinsed three times with
DDW.
4.2. 0.032 g of anhydrous Na2CO3 (ALDRICH 20,442-0, 99.999% purity) are
dissolved in 50 ml DDW in the centrifuge tube to provide a solution of
6 mmol Na2CO3 per liter.

5. 5
4.3. 1 ampoule of NaH-14
CO3 (4µCi per ml) is mixed with 16.5 ml of the
above prepared Na2CO3 solution in the teflon bottle.
4.4. The new stock activity is checked by counting triplicate 10 µl samples
with 1 ml β-phenethylamine in 10 ml Aquasol-II.
4.5. Triplicate 10 µl stock samples are also acidified with 1 ml of 2 M HCl,
mixed intermittently for 1-2 hours and counted in 10 ml Aquasol-II to
confirm that there is no 14
C-organic carbon contamination. The
acidification is done under the hood. The acidified dpm should be
<0.001% of the total dpm of the 14
C preparation.
5. INCUBATION SYSTEMS
Typically, we will measure primary production using in situ incubation
techniques.
5.1. A free-floating array equipped with buoy was used for the in-situ
incubations. Incubation bottles were attached to a 10 m, 1/2"
polypropylene in situ line at the depths corresponding to the sample
collections.
5.2. Generally, two incubation depths will be selected (surface and bottom).

6. 6
6. SAMPLING
6.1. Approximately at 8 O’clock, seawater samples were collected with
acid- washed, Niskin bottles using Kevlar line, metal-free sheave,
teflon messengers and a stainless-steel bottom weight. A dedicated
hydro winch was used for the primary productivity sampling
procedures in a further effort to reduce/eliminate all sources of trace
metal contamination.
6.2. Under low light conditions, water samples (250 ml) were transferred to
the incubation bottles (300 ml bottles) and were stored in the dark.
Polyethylene gloves were worn during sample collection and
inoculation procedures.
7. ISOTOPE ADDITION AND SAMPLE INCUBATION
7.1. Two light bottles, two dark bottles (bottles covered with double layered
black cloth) and 1 time-zero control were collected at each depth
(surface and bottom) for in situ incubation.
7.2. After all water samples were drawn from the appropriate Niskin, 400 µl
of the 14
C-sodium carbonate stock solution were added to each sample
using a specially-cleaned pipette tip. The incubation period was 2
hours.
7.3. After 2 hours, the free-floating array was recovered and all in situ
bottles were immediately placed in the dark and processed as soon as
possible. The time of recovery was recorded.

7. 7
8. FILTRATION
8.1. Filtration of the samples was done under low light conditions on
board.
8.2. 400 µl was removed and placed into a second LSC vial containing 0.5
ml of β-phenethylamine. This sample was used for the determination
of total radioactivity in each sample (added activity).
8.3. The remainder was filtered through a 25 mm diameter GF/F filters. The
filters were placed into pre-labeled, clean glass liquid scintillation
counting vials (LSC vials).
9. 14
C SAMPLE PROCESSING
9.1. One ml of 2 M HCl was added to each sample vial (under the hood).
Vials were covered with their respective caps and shaken in a vortex
mixer for at least 1 hour with venting at 20-minute intervals. To vent,
the vials were removed from the shaker, and the cap opened (under the
hood). After shaking is completed, the vials were left open to vent
under the hood for an additional 24 hours.
9.2. Ten ml of Aquasol-II was added per vial (including vials for total 14
C
radioactivity) and the samples were counted in a liquid scintillation
counter. Samples were counted again after 2 and 4 weeks, before
discarding. Counts have not shown a consistent increase during the
first two weeks and become unstable between the second and the
fourth week. Counts per min (CPM) are converted to disintegration per
min (DPM) using the channels ratio program supplied by the
manufacturer (Packard Instrument Co.).

8. 8
10. CALCULATIONS
The 14
C method for the measurement of primary production in the sea has
been used for more than 45 years and most of the published methodologies
have been dealt with Steeman-Nielsen, E. (1952).
Sample activity (a)* total 12
CO2 (c)*12 (d)*1.05 (e)*K1. K2
PP (µgCL-1
hr-1
) = ----------------------------------------------------------------------------
Total activity added to the sample (b)
Where
(a) = Sample activity (minus back-ground), dpm
(b) = Total activity added to the sample (minus back-ground), dpm
(c) = Total concentration of 12
CO2 in the sample water, µmol/L (or µM)
(d) = The atomic weight of carbon
(e) = A correction for the effect of 14
C discrimination
k1 = subsampling factor (e.g. sample 50 ml, subsample 10 ml: k1=subsample
factor 50/10= 5)
k2 = time factor (e.g. incubation time 125 minutes: k2= 60/125= 0.48)
11. REFERENCES
Steeman-Nielsen, E. 1952. The use of radioactive carbon (14
C) for measuring
organic production in the sea. Journal du Conseil, 18, 117-140.