Sampling Methods

Water Sampling

One of the objectives of the RMP is to evaluate if water quality objectives are met at the sampled stations. Therefore, the sampling and analysis methods have to be able to detect substances below these levels. In order to attain the low detection levels used in the RMP (see Appendix B), ultra-clean sampling methods were used in all sampling procedures (Flegal and Stukas, 1987; EPA Method 1669, 1995).

Water samples were collected approximately one meter below the water surface using peristaltic pumps. The sampling ports for both the organic chemistry and trace element samplers were attached to aluminum poles that were oriented up-current from the vessel and upwind from equipment and personnel. The vessel was anchored and the engines turned off. Total (or near-total) and dissolved fractions of Estuary water were measured for trace elements. Particulate and dissolved fractions were measured for trace organics, and totals were calculated.

The RMP used the polyurethane foam plug sampler to collect water for trace organics analyses during the first four years of the Program (Risebrough et al., 1976; de Lappe et al., 1980; 1983) and began to phase in a new, modified, commercially available XAD resin extraction sampler in 1996, beginning with side-by-side comparisons of both sampling systems. Results for 1996 trace organic contaminants in water are still based on the foam-plug sampler, whose operation is described in the Methods sections of previous Annual Reports (SFEI, 1994; 1995; 1996).

XAD resins have been used throughout the world to measure synthetic organic contaminants in both water and air (Infante et al., 1993). The custom-manufactured AXYS system (AXYS Environmental Systems, Ltd., Sidney, B.C.) consists of a constant-flow, gear-driven positive displacement pump, ¹/₂ inch Teflon^® tubing, 1 µm glass fiber particulate filter enclosed in a cartridge, and two parallel Teflon^® columns filled with XAD-2 resin with a particle size range of 300­900 µm. The flow rate was approximately 1.5 liters per minute. The intercomparison results between the foam-plug and resin samplers will be described in detail in the 1997 Annual Report.

For trace metals, water samples were collected using a peristaltic pump system equipped with C-Flex tubing in the pump head. Sample aliquoting was conducted on deck on the windward side of the ship to minimize contamination from shipboard sources (Flegal and Stukas, 1987). Filtered water samples were obtained by placing an acid-cleaned polypropylene filter cartridge (Micron Separations, Inc., 0.45 µm pore size) on the outlet of the pumping system. Unfiltered water samples were pumped directly into acid-cleaned containers. Prior to collecting water, several liters of water were pumped through the system, and sample bottles were rinsed five times before filling. The bottles were always handled with polyethylene-gloved "clean hands". The sample tubing and fittings were acid-cleaned polyethylene or Teflon^®, and the inlets and outlets were kept covered except during actual sampling. Samples were acidified within two weeks in a class 100 trace metal laboratory, except for chromium samples, which were acidified and extracted within an hour of collection.

Samples for conventional water quality parameters were collected using the same apparatus as for trace metals; however, containers were only rinsed three times, and the "clean hands" procedure was not necessary.

Water samples were collected for toxicity tests using the same pumping apparatus as for the collection of the trace organic samples, but were not filtered. Five gallons of water were collected and placed in ice chests for transfer at the end of each cruise day to the testing laboratory. Two field blanks were collected each cruise by filtering (0.45 µm) water from the Bodega Marine Laboratory known to be non-toxic.

Sediment Sampling

Sediment sampling was conducted using a modified van Veen grab with a surface area of 0.1 m². The grab is made of stainless steel, and the jaws and doors are coated with Dykon^® (formerly known as Kynar^®) to achieve chemical inertness. All scoops, buckets, and stirrers used to collect and homogenize sediments were also constructed of Teflon^® or stainless steel coated with Dykon^®. Sediment sampling equipment was thoroughly cleaned prior to each sampling event.

A sub-core of sediment was removed for measurement of porewater ammonia. Then, the top 5 cm of sediment were scooped from each of two replicate grabs and mixed in a bucket to provide a single composite sample for each station. Aliquots were split on board for each analytical laboratory, for archive samples and for sediment toxicity tests. The quality of grab samples was ensured by requiring each sample to satisfy criteria concerning depth of penetration and disturbance of the sediment within the grab (see the 1996 Quality Assurance Project Plan available from SFEI).

Bivalve Bioaccumulation Sampling

Bivalves were collected from uncontaminated sites and transplanted to fifteen stations in the Estuary during the wet season (February through May) and the dry season (June through September). Contaminant concentrations in the animals' tissues and the animals' biological condition (expressed as the ratio of dry weight and shell cavity volume) were measured before deployment (referred to as time zero or background samples) and at the end of the 90­100 day deployment period. Since the RMP sites encompass a range of salinities, three species of bivalves were used, according to the expected salinities in each area and the known tolerances of the organisms. The mussel Mytilus californianus was collected from Bodega Head and stored in running seawater at the Bodega Marine Laboratory until deployment at the stations west of Carquinez Strait, which were expected to have the highest salinities. Mytilus californianus will survive exposure to salinities as low as 5 ppt (Bayne, 1976). Oysters (Crassostrea gigas) were obtained from Tomales Bay Oyster Company (Marshall, California) and deployed at moderate-salinity sites closest to Carquinez Strait and in the extreme South Bay. Crassostrea gigas tolerates salinities as low as 2 ppt. The freshwater clam Corbicula fluminea was collected from Putah Creek, moved to UC Davis for depuration, and deployed at sites with the lowest salinities. Corbicula fluminea tolerates salinities from 0 ppt to perhaps 10 ppt (Foe and Knight, 1986). The effects of high, short-term flows of freshwater on the transplanted bivalves west of Carquinez Strait were minimized by deploying the bivalves near the bottom where density gradients tend to maintain higher salinities. All bivalves were kept on ice after collection and deployed within 24­48 hours.

Because of the unavailability of clams at Lake Isabella, the RMP's traditional reference site, clams were collected from Putah Creek, conditioned at a pond and fed by Davis well water. Survival during deployment was also measured. Composites of tissue were made from 40­60 individual bivalves from each site before and after deployment for analyses of trace contaminants.

Within each species, animals of approximately the same size were used. Mussels were between 49­81 mm shell length, oysters were between 71­149 mm, and clams were 25­36 mm. One hundred and fifty oysters and 160 mussels and clams were randomly allocated for deployment at the appropriate sites, with the same number being used as travel blank (time zero) samples for analysis of tissue and condition before deployment. At each site, oysters were divided among five nylon mesh bags, and mussels and clams were divided among four nylon mesh bags.

Moorings were associated with pilings or other permanent structures. Mooring installation, bivalve deployment, maintenance, and retrieval were all accomplished by SCUBA divers. The deployed samples were checked approximately half-way through the 90-day deployment period to ensure consistent exposure. Moorings and nylon bags were checked for damage and repaired, and fouling organisms were removed.

Upon retrieval, the bags of bivalves were placed into polyethylene bags and taken to the surface. On the vessel, the number of dead organisms was noted, with 20 percent of the live organisms being allocated for condition measurement, and the remainder being equally split for analyses of trace metal and organic compounds. Bivalves used for trace organic analyses were rinsed with reagent grade water to remove extraneous material, shucked using a stainless steel knife (acid-rinsed) and homogenized (until liquefied) in a combusted mason jar using a Tissumizer or Polytron blender. Bivalves used in trace element analyses were shucked with stainless steel knives, gonads were removed, and remaining tissue was rinsed with ultrapure water and placed in an acid cleaned, plastic-coated, glass jar. The sample was then homogenized (until liquefied) using a Brinkmann homogenizer equipped with a titanium blade.

Based on findings by Stephenson (1992) during the RMP Pilot Program, bivalve guts were not depurated before homogenization for tissue analyses, although gonads were removed from organisms for trace metal analyses. Stephenson (1992) found that, with the exception of lead and selenium, no significant differences were found in trace metal concentrations between mussels depurated for 48 hours in clean Granite Canyon seawater before homogenization and undepurated mussels. However, sediment in bivalve guts may contribute to the total tissue contaminant concentration.

Analytical Methods

Conventional Water Quality Parameters

Samples for dissolved nutrients were analyzed using the Lachat QuikChem 800 System Nutrient Autoanalyzer (Ranger and Diamond, Lachat Instruments, 1994). The QuickChem methods used were: 31-114-27-1 for silicates, 31-107-06-1 for ammonia, 31-107-04-1 for nitrate/nitrite, and 31-115-01-3 for phosphate. Chlorophyll and phaeophytin were measured using a fluorometric technique with filtered material from 200 ml samples (Parsons et al., 1984). Shipboard measurements for temperature, salinity, pH, and dissolved oxygen content were made using a hand-held Solomat 520 C multi-functional chemistry and water quality monitor. Dissolved organic carbon (DOC) was measured using high-temperature catalytic oxidation with a platinum catalyst (Fitzwater and Martin, 1993). Total suspended solids (TSS) was determined using method 2540D in Standard Methods for the Examination of Water and Wastewater (Greenberg et al., 1992)

A Sea-Bird SBE19 Conductivity, Temperature, and Depth probe (CTD) was used to measure water quality parameters at depths throughout the water column. CTD casts were taken at each site during water and sediment sampling. At each site, the CTD was lowered to approximately one meter below the water surface and allowed to equilibrate to ambient temperature for 3 minutes. The CTD was then lowered to the bottom at approximately 0.15 meters per second, and raised. Only data from the down-cast were kept. Data were downloaded onboard the ship, and processed in the laboratory using software supplied by Sea-Bird.

The CTD measures temperature, conductivity, pressure, dissolved oxygen, and backscatter at a sampling rate of two scans per second. These data were edited and averaged into 0.25 m depth bins during processing. Also during processing, salinity (based on conductivity measurements), oxygen, time, and depth (based on pressure) were calculated. Although the CTD data are not detailed in this report, SFEI maintains these data in its database.

Trace Elements

In water, total and dissolved (0.45 µm filtered) concentrations of mercury, arsenic, selenium, chromium, copper, nickel, lead, silver, and zinc were measured. Mercury, arsenic, and selenium samples were obtained from the same field sample. The mercury sub-samples were photo-oxidated with the addition of bromium chloride, and quantified using a cold-vapor atomic fluorescence technique. Arsenic and selenium were analyzed by hydride-generation atomic absorption with cryogenic trap preconcentration based on a method described in Liang et al. (1994) and Cercelius et al. (1986).

The chromium samples were collected separately. The suspended particulates underwent hydrofluoric acid digestion, and the dissolved chromium was co-preciptated with a ferrous hydroxide scavenger (Cranston and Murray, 1978). Chromium was quantified by graphite furnace atomic absorption spectrometry (GFAAS).

The remaining trace elements in water were measured using the APDC/DDDC organic extraction and preconcentration method (Bruland et al., 1985; Flegal et al., 1991) and then quantified by GFAAS.

Results for cadmium, chromium, copper, nickel, lead, silver, and zinc were reported by the laboratory in weight/weight units (µg/kg). For use in this report, those values are reported as µg/L, without taking account of the difference in density between Estuary water and distilled water. This difference was not taken into account because it was much less than the precision of the data, which was on the order of 10%. In some instances, dissolved metal concentrations are reported as higher than total (dissolved + particulate) metal concentrations. This is due to expected analytical variation in the methods of analysis, particularly at concentrations near the detection limits. Such results should be interpreted as no difference between dissolved and total concentrations, or that the total fraction of metals is in the dissolved phase.

Sediments were digested with aqua regia to obtain "near-total" concentrations of aluminum, silver, cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc (Flegal et al., 1981). The metals were quantified by inductively coupled plasma atomic emission spectrometry (ICP-AES) or by inductively coupled plasma mass spectrometry (ICP-MS). The method chosen for RMP sediment analysis is comparable to standard EPA procedures (Tetra Tech, 1986) but does not decompose the silicate matrix of the sediment. Because of this, any element tightly bound as a naturally occurring silicate may not be fully recovered.

Bivalve tissue samples were digested with aqua regia to obtain near-total concentrations of trace elements similar to techniques used in the California State Mussel Watch Program (e.g., Flegal et al., 1981; Smith et al., 1986) and consistent with the RMP Pilot Program (Stephenson, 1992). The trace metals were quantified on ICP-AES or ICP-MS. Hydride generation coupled with atomic absorption spectroscopy was used to quantify arsenic. Mercury was quantified using a cold-vapor atomic fluorescence technique, and selenium was quantified using the methods of Cutter (1986). Butyltins were measured following NOAA Status and Trends Mussel Watch Project methods described in NOAA Technical Memorandum NOS/ORCA/CMBAD71 vol. IV. This technique involves extracting the sample with hexane and the chelating agent tropolone and measuring the butyltin residues by capillary gas chromatography. Concentrations are expressed in total tin per gram of tissue dry weight.

Trace Organics

For water samples, the foam plugs and filters containing the particulate fraction were spiked with extraction surrogates. ECD surrogates consisted of PCB 103 and PCB 207 for the first fraction, and Pentachloronitorobenzene for Fractions 2 and 3. The MSD surrogate consisted of deutereated acenaphthalene. The foam plugs were eluted using the methods described in previous Annual Reports. The XAD columns used in the intercomparison tests were eluted in reverse with methanol and methylene chloride in a method similar to the filter cartridges. The separate extracts were then combined and separated into three fractions. Extraction methods were based upon standard EPA and AXYS extraction protocols.

The extracts were subjected to Florisil column chromatography resulting in three fractions, a PCB/aliphatic, a pesticide/aromatic fraction, and a polar third fraction, which contains diazinon and other polar pesticides. Chlorinated hydrocarbons (CH) were analyzed on a Hewlett Packard 6890 capillary gas chromatograph utilizing electron capture detectors (GC/ECD). The quantitation internal standards utilized for the CH analysis were dibromo-octafluorobiphenyl (DOB) for Fractions 1 and 3, and DOB or PCB 209 for Fraction 2. Analyte concentrations were corrected for surrogate losses prior to reporting. PAHs were quantified in the F-2 fraction by analysis on a Hewlett-Packard 6890 capillary gas chromatograph equipped with a 5971A mass spectral detector (GC/MS). A 2 µL splitless injection was chromatographed on a DB-5 column and analyzed in a single ion monitoring (SIM) mode. The quantitation internal standard utilized for the PAH analysis when samples were at 100 µL was hexamethyl benzene (HMB). DOB was used as an internal standard for diazinon.

Sediment samples were analyzed based on the methods followed by NOAA's Status and Trends Program. Samples were freeze-dried, mixed with kiln-fired sodium sulfate, and soxhlet-extracted with methylene chloride. Surrogate standards were added prior to extraction to account for methodological analyte losses. ECD surrogates consisted of PCB 103 and PCB 198. The extract was concentrated and purified using EPA Method 3611 alumina column purification to remove matrix interferences. Tissue samples were homogenized and macerated, and the eluate was dried with sodium sulfate, concentrated, and purified using a combination of EPA Method 3611 alumina column purification and EPA Method 3630 silica gel purification to remove matrix interferences. PAHs and their alkylated homologues in both sediment and tissue extracts were quantified by gas chromatography mass spectrometry (GC/MS) in the selected ion monitoring mode (SIM) with a temperature-programmable gas chromatograph with a 30-m long 0.32-mm internal diameter fused silica capillary column with DB-5MS bonded phase. Surrogates for PAHs consisted of naphthalene-d8, acenaphthene-d10, phenanthrene-d10, chrysene-d12, and perylene-d12. Chlorinated hydrocarbons are quantified in both sediment and tissue extracts via high-resolution capillary gas chromoatorgraphy using electron-capture detection (GC/ECD). For the first time in 1996, dual-column confirmation on 30-m long, 0.25-mm internal diameter fused silica capillary columns with DB-5 and DB-17 bonded phase, was conducted. Data from the two columns were combined by SFEI to generate the values listed in the Annual Report. Some analytes included in the 1996 analyte list coeluted on both columns and could not be reported as single congeners.

Aquatic Bioassays

Water column toxicity was evaluated using a 48-hour bivalve embryo development test and a seven-day growth test using the estuarine mysid Mysidopsis bahia. The bivalve embryo development test was performed according to ASTM standard method E 724-89 (ASTM, 1991). The mysid test was based on EPA test method 1007. Larval Mytilus sp. were used in both sampling periods. The mysid growth and survival test consisted of an exposure of 7-day old Mysidopsis bahia juveniles to different concentrations of Estuary water in a static system during the period of egg development and was used during both sampling periods. Appropriate salinity adjustments were made for Estuary water from sampling stations with salinities below the test species' optimal ranges. Reference toxicant tests with copper chloride and potassium dichromate were performed for the bivalve and mysid tests, respectively. These tests were used to determine if the responses of the test organisms were relatively consistent over time.

The salinities of the ambient samples and the control/diluent (Evian spring water) were adjusted to 5 ppt using artificial sea-salts (Tropic Marin). The test concentrations were 100%, 50%, and control, each with eight replicates, and with 20 larvae per replicate. Waste, dead larvae, excess food, and 80% of the test water were siphoned from the test chambers daily, and general water chemistry parameters of dissolved oxygen, pH, and salinity were recorded before and after each water change.

Sediment Quality Characteristics

Sediment size fractions were determined with a grain-size analyzer based on x-ray transmission (Sedigraph 5100). Total organic carbon was analyzed according to the standard method for the Coulometrics CM 150 Analyzer made by UIC, Inc. This method involves measurements of transmitted light through a cell. The amount of transmitted light is related to the amount of carbon dioxide evolved from a combusted sample. Spectrophotometric analyses of sulfides in sediment porewater were performed using a method adapted from Fonselius (1985) with variations from Standard Methods (APHA, 1985).

Sediment Bioassays

Two sediment bioassays were used: a ten-day acute mortality test using the estuarine amphipod Eohaustorius estuarius exposed to whole sediment using American Society for Testing and Materials (ASTM) method E 1367 (ASTM, 1992), and a sediment elutriate test where larval bivalves were exposed to the material dissolved from whole sediment in a water extract using ASTM method E 724-89 (ASTM, 1991). Elutriate solutions were prepared by adding 100 g of sediment to 400 ml of Granite Canyon seawater, shaken for 10 seconds, allowed to settle for 24 hours, and carefully decanted (EPA and COE, 1977; Tetra Tech, 1986). Larval mussels (Mytilus sp.) were used in both sampling periods, where percent normally developed larvae was measured.

Bivalve Condition and Survival

The condition of bivalves is a measure of their general health following exposure to Estuary water for 90­100 days. Measurements were made on subsamples of specimens before deployment and on the deployed specimens following exposure. Dry weight (without the shell) and the volume of the shell cavity of each bivalve was measured. Bivalve tissue was removed from the specimens and dried at 60^o C in an oven for 48 hours before weighing. Shell cavity volume was calculated by subtracting shell volume of water displaced by a whole live bivalve less the volume of water displaced by the shell alone. The condition index is calculated by taking the ratio of tissue dry weight and the shell cavity volume.

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