Dataset: SRiMP

Meteorological data are being continuously recorded by the SKiO weather station (http://weather.skio.usg.edu) which is located ~250 meters from the SRiMP sampling site. Actual meteorological data that include temperature, dew point, heat index, wind chill factor, relative humidity, barometric pressure, rainfall since midnight, wind direction, wind speed, peak wind gust, solar radiation, highest and lowest temperature since midnight, sunrise, sunset are utilized from the SKIO weather station using the SKIO Metric Mobile Weather application (weather.skio.usg.edu/metric/mini.html). In addition, historic meteorological data from an underground weather web page (http://www.wunderground.com/weatherstation/WXDailyHistory.asp?ID=KGASAVAN23) is included for a broader dataset.

Tidal stage (high, low and mid tide) and time determination has been extrapolated to local tide predictions by adding + 38 minutes to the data for the Tybee Lighthouse, Savannah River Entrance (station ID 8670892; 32.0333° N; 80.9017° W), which is located about 10 km from the SRiMP site. Corresponding heights of tidal stage are now recorded from Isle of Hope, Skidaway River (31.9833° N; 81.0500° W), located ca. 1.5 km from the SRiMP sampling site (http://tides.mobilegeographics.com/locations/2824.html).

Surface water temperature was measured weekly using a standard mercury thermometer (+- 0.1 ° C) as was surface salinity which used an AGE model 2100 salinometer from 1986 to 1996. From February 2004 until July 2011, water temperature, salinity, and dissolved oxygen (DO) were measured using a YSI Model 556 multi-probe system. From September 2011 until present, a multi-probe MANTA-2 (EUREKA Environmental Engineering, Austin, TX, USA) has been included to measure depths profiles of temperature, salinity, pH, conductivity, turbidity, in situ chlorophyll fluorescence and DO. The oxygen probe measurements are regularly (at least monthly) compared  and calibrated (if necessary) against the high-precision Winkler titrations using a  (Parsons et al., 1984; Hansen, 1999) and a Brinkmann Metrohm titrator. Also, monthly manual measurement of water surface temperature (standard mercury thermometer precision +- 0.1 °C), surface salinity (American Optical Refractometer; +- 0.2 psu or ppt), pH, and electrical conductivity (Hi 2550 pH/ORP & EC/TDS Meter, HANNA instruments, Smithfield, RI, USA) are standard protocol. Since early 2011, we have included weekly Secchi depth measurements in the SRiMP program.

From 1986 to 2011 the surface waters adjacent to the main dock of the Skidaway Institute of Oceanography were sampled using an acid cleaned bucket or a Niskin bottle at connective slack high and low waters on the same day. From 2011, water sampling of the Skidaway River Estuary is been performed at high tide in 1m depth using a Niskin bottle. Ctenophores are sampled at mid-tide the same day (method see below).

Sample water for the analyses of inorganic nutrients (PO4, Si(OH)4, NH4, NO3, NO2) is filtered through acid washed and pre-rinsed GFF filters using acid cleaned plastic syringes, and either analyzed fresh or stored at -20 °C in acid cleaned PE-bottles. Nutrient analyses were performed on automated procedures using a Technicon Auto Analyzer II; 0.1 µM precision (Gilbert and Loder 1977, Verity et al. 1993) while TDN was determined using persulfite digestion. DON was calculated as the difference between TDN and summed inorganic nitrogen (Solarzano and Sharp 1980, Valderrama 1981).
From 2011, dissolved nutrient concentrations [NO2/NO3, NH4, DON, PO4, Si(OH)4] are determined by contract with the nutrient analysis laboratory overseen by Dr. S. Joyce at the University of Georgia, Athens, GA. Continuing from mid 2011 to present, water samples for DIC and delta 13/12 C isotope ratios of 0.02 nylon filtered water samples is analyzed inhouse using a Mass Spectrometer (Thermo Scientific) in the lab of Dr. Jay Brandes (Skidaway Island Scientific Stable Isotope Laboratory, SISSIL). TDN and DOC concentrations of 0.2 µm filtered water samples are processed at SkIO by the lab of Dr. Aron Stubbins using a Total Organic Carbon Analyzer with a Total Nitrogen Measuring Unit (TOC-V and TNM-I, Shimadzu Scientific Instruments, Columbia, ML, USA).

Samples to determine the organic particulate fractions of carbon and nitrogen, i.e. POC and PON are filtered at low vacuum pressure (10 cm Hg) onto pre-combusted (450 °C for 4 h) 25 mm GF/F filters (Whatman). These filters are transferred to pre-combusted foil sheets and stored frozen at - 20 °C until analysis. In the past, filter samples were freeze-dried and combusted using a Fisons NA1500 NCS Series 2 CHNS analyzer and 2.5-Bis (5tert-butyl-2-benzo-oxazol-2-yl) thiophene (BBOT) standards (Verity 2002). Presently, POC and PON amounts and delta 13/12 C and 15/14 N isotope ratios are measured in house using a FLASH 2000 series CHNS/O elemental analyzer (Thermo Scientific) purchased in 2009.

Total viral abundance is determined by direct epifluorescence microscopic counting and quasi-automated image analysis (Shopov et a. 2000) using custom Skipper software (http://www.skipperimaging.com). Water samples are pre-filtered through 0.2 µm PC and then filtered onto 0.02 µm Anodisc filter and stained with SYBR Gold (Nobel and Fuhrman 1998).

Total bacteria abundance is determined by direct epifluorescence microscopic counting after staining with the DNA-specific fluorochrome DAPI following standard procedures (Porter and Feig 1980; Williams et al. 1998). Counting is facilitated by Bacteria data represent DAPI stained cells enumerated using epifluorescence microscopy, semiquasi-automated image analysis (Shopov et al. 2000) and custom Skipper software (http://www.skipperimaging.com) (Verity et al. 2006).

Total and fecal coliform bacteria concentration is determined using standard total coliform (method 9221B) and fecal coliform (method 9221E) procedures (Clesceri et al. 1998). Enterococci concentrations are determined using EPA method 1600.

Total and size fractionated (less and greater than 8 µm) phytoplankton chlorophyll-a concentrations are determined fluorometrically in house (lab of Dr. Jim Nelson) following the acetone extraction method as described by Parsons et al. (1984). From 2011, 0.2 µm and 8 µm PC filters are used for total and > 8µm fractions of chl-a analyses, respectively, following the acetone extraction overnight method as described for cellulose acetate filters in Nejstgaard et al. (2006) and compared to the chl-a in situ fluorescence readings of the MANTA-2 multiprobe.

Abundance and biomass of nanoplankton taxonomic groups are determined using true-color epifluorescence image analyzed microscopy. Water samples are fixed with glutaraldehyde and stained with proflavine-DAPI (Verity and Sieracki, 1993; Shopov et al., 2000). This protocol has been used since 1986 with the custom Skipper software (www.skio.usg.edu/?p=research/bio/veritylab/ip); (Verity  and Borkman 2010). Since 2011, the program Image Pro-Plus (Media Cybernetics) is utilized to determine auto- and heterotrophic organisms. In addition, a detailed assessment of the microplanktonic community composition (including ciliates) is mapped four times per year using classical microscopic sedimentation techniques (Utermöhl 1958) after Lugol's fixation. Currently, emphasis is focused on flagellates vs. diatoms as indicators of environmental quality, with attention to potential harmful algal groups.

Since 2012, a variety of flow cytometric instruments are utilized to quantify picoplankton and larger microplankton including diatoms and dinoflagellates in our SRiMP program. Picoplankton analysis is performed by Dr. Liz Mann using a FacsCalibur (BD Biosciences, San Jose, CA, USA) in house. Additionally, we employ a state of the art CytoSense benchtop flow cytometer equipped with an imaging system and wide (1.5 mm) flow-cell uniquely capable of rapid analysis of live cells including large and chain-forming species (up to 4 mm long) such as diatoms and dinoflagellates (CytoBouy b.v., Woerden, the Netherlands, http://www.cytobuoy.com) performed in house by Dr. Jens Nejstgaard.

During the first decade, dominant copepods (Acartia spp.) were collected at the same sample site in approximately 6 week intervals by net tows (153 µm nylon mesh net, diameter 30 cm, length 150 cm). The net was towed for 15 minutes during maximum flood tide and again during ebb tide. Volume filtered for each tow was calculated from a flowmeter (General Oceanics) mounted in the mouth of the net. The content of the cod end was preserved in 3 % buffered formalin and stored at room temperature until counting under a dissecting microscope. Since 2011, net tows (65 µm mesh size, diameter 30 cm, length 100 cm) are collected at the same sample site in weekly intervals to determine copepods + eggs (mainly Acartia tonsa) abundances. The net is towed from 5 m to the surface during maximum flood tide. The sample is preserved in 3 % formalin and stored in the refrigerator (4 °C) until counting under a dissecting microscope.

Ctenophores (Mnemiopsis leidyi and Beroe sp.) are quantified weekly to monthly. Samples are collected using a  0.5 m zooplankton net (153 μm) fitted with a TSK flowmeter for ctenophores and jellies, and hauled obliquely from near-bottom to the surface for 5 minutes repeated trips through the water column. Ctenophore/jelly samples are sorted live in the lab immediately. Depending on taxa, various morphometric and volumetric measurements are recorded. Morphometric measurements are facilitated using a zoom stereoscope, e.g. trunk length, lobe or bell diameter, body width.