Methods

14C Production Methods <br/>The approaches for estimating primary production in the study lakes using 14C incubations differed slightly between the three research programs, but all resulted in a similar estimate of daily epilimnetic pelagic production (mmol C m-3 d-1). In NTL lakes, integrated samples of water from the surface of the lake to the bottom of the epilimnion were collected between 2007 and 2013 using a 1.5 inch PVC tube approximately every two weeks during the open water season (first described in these lakes by Adams et al. 1993). Samples were labeled with inorganic 14C in the form of NaHCO3 and then incubated in the lab for 3-hr across a range of light intensities with additional dark bottles to correct for non-uptake sorption of 14C at ambient epilimnetic water temperature. The resultant photosynthesis-irradiance (P-I) data was used to derive P-I curves by fitting a 3-parameter photosynthesis light-inhibition model (Platt et al. 1980) to these data. The P-I curves were coupled with concurrent, high-frequency photosynthetically active radiation (micromol m-2 s-1; PAR) measurements and water column light extinction data (m-1) to estimate daily primary production (mmol C m-3 d-1) in both Sparkling and Trout Lake. Over this time period, the availability of data for 14C production varied due to sporadic sample contamination and equipment failures.<br/>The approaches for estimating primary production in the study lakes using 14C incubations differed slightly between the three research programs, but all resulted in a similar estimate of daily epilimnetic pelagic production (mmol C m-3 d-1). In NTL lakes, integrated samples of water from the surface of the lake to the bottom of the epilimnion were collected between 2007 and 2013 using a 1.5 inch PVC tube approximately every two weeks during the open water season (first described in these lakes by Adams et al. 1993). Samples were labeled with inorganic 14C in the form of NaHCO3 and then incubated in the lab for 3-hr across a range of light intensities with additional dark bottles to correct for non-uptake sorption of 14C at ambient epilimnetic water temperature. The resultant photosynthesis-irradiance (P-I) data was used to derive P-I curves by fitting a 3-parameter photosynthesis light-inhibition model (Platt et al. 1980) to these data. The P-I curves were coupled with concurrent, high-frequency photosynthetically active radiation (micromol m-2 s-1; PAR) measurements and water column light extinction data (m-1) to estimate daily primary production (mmol C m-3 d-1) in both Sparkling and Trout Lake. Over this time period, the availability of data for 14C production varied due to sporadic sample contamination and equipment failures.<br/>The approaches for estimating primary production in the study lakes using 14C incubations differed slightly between the three research programs, but all resulted in a similar estimate of daily epilimnetic pelagic production (mmol C m-3 d-1). In NTL lakes, integrated samples of water from the surface of the lake to the bottom of the epilimnion were collected between 2007 and 2013 using a 1.5 inch PVC tube approximately every two weeks during the open water season (first described in these lakes by Adams et al. 1993). Samples were labeled with inorganic 14C in the form of NaHCO3 and then incubated in the lab for 3-hr across a range of light intensities with additional dark bottles to correct for non-uptake sorption of 14C at ambient epilimnetic water temperature. The resultant photosynthesis-irradiance (P-I) data was used to derive P-I curves by fitting a 3-parameter photosynthesis light-inhibition model (Platt et al. 1980) to these data. The P-I curves were coupled with concurrent, high-frequency photosynthetically active radiation (micromol m-2 s-1; PAR) measurements and water column light extinction data (m-1) to estimate daily primary production (mmol C m-3 d-1) in both Sparkling and Trout Lake. Over this time period, the availability of data for 14C production varied due to sporadic sample contamination and equipment failures.<br/>

Methods

Nutrients were added to Peter and Tuesday lakes to cause algal blooms. Details on nutrient additions (start/end dates, loading rates, N:P ratios) are described in Wilkinson et al. 2018. (Ecological Monographs 88:188-203). Methods are described in Pace et al. 2021 (Limnology and Oceanography linked below), Wilkinson et al. 2018 (Ecological Monographs 88:188-203), and Pace et al. 2017 (Proceedings of the National Academy of Sciences USA 114: 352-357). These publications including supplements should be consulted for details.<br/>Nutrients were added to Peter and Tuesday lakes to cause algal blooms. Details on nutrient additions (start/end dates, loading rates, N:P ratios) are described in Wilkinson et al. 2018. (Ecological Monographs 88:188-203). Methods are described in Pace et al. 2021 (Limnology and Oceanography linked below), Wilkinson et al. 2018 (Ecological Monographs 88:188-203), and Pace et al. 2017 (Proceedings of the National Academy of Sciences USA 114: 352-357). These publications including supplements should be consulted for details.<br/>Nutrients were added to Peter and Tuesday lakes to cause algal blooms. Details on nutrient additions (start/end dates, loading rates, N:P ratios) are described in Wilkinson et al. 2018. (Ecological Monographs 88:188-203). Methods are described in Pace et al. 2021 (Limnology and Oceanography linked below), Wilkinson et al. 2018 (Ecological Monographs 88:188-203), and Pace et al. 2017 (Proceedings of the National Academy of Sciences USA 114: 352-357). These publications including supplements should be consulted for details.<br/>Nutrients were added to Peter and Tuesday lakes to cause algal blooms. Details on nutrient additions (start/end dates, loading rates, N:P ratios) are described in Wilkinson et al. 2018. (Ecological Monographs 88:188-203). Methods are described in Pace et al. 2021 (Limnology and Oceanography linked below), Wilkinson et al. 2018 (Ecological Monographs 88:188-203), and Pace et al. 2017 (Proceedings of the National Academy of Sciences USA 114: 352-357). These publications including supplements should be consulted for details.<br/>

Methods

General: Bade, D., J. Houser, and S. Scanga (editors). 1998. Methods of the Cascading Trophic Interactions Project. 5th edition. Center for Limnology, University of Wisconsin-Madison, and Cary Institute of Ecosystem Studies, Millbrook, NY.

Methods

Detailed field and laboratory protocols can be found in the Cascade Methods Manual, found here: https://cascade.limnology.wisc.edu/public/public_files/methods/CascadeManual1998.pdf
POC, PON and DOC: 1. 100 - 300 ml (Typically ~200mL for PML, 150 metalimnion and 75 – 100 for the hypolimnion) of lake water from each depth was filtered through 153 um mesh to remove large zooplankton. Water was then filtered through a precombusted 25mm GF/F filter (0.7 um pore size) at less than 200 mm Hg pressure. Filters were placed in drying oven at 60 C to dry for at least 48 hours. 20mL of filtered water was stored in a scintillation vial and acidified with 200uL of 2N H2SO4 for DOC analysis. Blank samples for POC and DOC were prepared with deionized water to control for contamination. All samples were sent to the Cary Institute of Ecosystem Studies for analysis.

Methods

Data were collected from two lakes, Sparkling Lake (46.008, -89.701) and Peter Lake (46.253, -89.504), both located in the northern highlands Lake District of Wisconsin and the Upper Peninsula of Michigan over a 10 day period on each lake in July and August of 2007. Refer to Van de Bogert et al. 2011 for limnological characteristics of the study lakes.Measurements of dissolved oxygen and temperature were made every 10 minutes using multiple sondes dispersed horizontally throughout the mixed-layer in the two lakes (n=35 sondes for Sparkling Lake and n=27 sondes for Peter Lake). Dissolved oxygen measurements were corrected for drift.Conductivity, temperature compensated specific conductivity, pH, and oxidation reduction potential were also measured by a subset of sensors in each lake. Of the 35 sondes in Sparkling Lake, 31 were from YSI Incorporated: 15 of model 600XLM, 14 of model 6920, and 2 of model 6600). The remaining sondes placed in Sparkling Lake were 4 D-Opto sensors, Zebra-Tech, LTD. In Peter Lake, 14 YSI model 6920 and 13 YSI model 600XLM sondes were used.Sampling locations were stratified randomly so that a variety of water depths were represented, however, a higher density of sensors were placed in the littoral rather than pelagic zone. See Van de Bogert et al. 2012 for the thermal (stratification) profile of Sparkling Lake and Peter Lake during the period of observation, and for details on how locations were classified as littoral or pelagic. In Sparkling Lake, 11 sensors were placed within the shallowest zone, 12 in the off-shore littoral, and 6 in each of the remaining two zones, for a total of 23 littoral and 12 pelagic sensors. Similarly, 15 sensors were placed in the two littoral zones, and 12 sensors in the pelagic zone.Sensors were randomly assigned locations within each of the zones using rasterized bathymetric maps of the lakes and a random number generator in Matlab. Within each lake, one pelagic sensor was placed at the deep hole which is used for routine-long term sampling.Note that in Sparkling Lake this corresponds to the location of the long-term monitoring buoy. After locations were determined, sensors were randomly assigned to each location with the exception of the four D-Opto sensor is Sparkling Lake, which are a part of larger monitoring buoys used in the NTL-LTER program. One of these was located near the deep hole of the lake while the other three were assigned to random locations along the north shore, south shore and pelagic regions of the lake. Documentation: Van de Bogert, M.C., Bade, D.L., Carpenter, S.R., Cole, J.J., Pace, M.L., Hanson, P.C., Langman, O.C., 2012. Spatial heterogeneity strongly affects estimates of ecosystem metabolism in two north temperate lakes. Limnology and Oceanography 57, 1689-1700.

Methods

Study sitesWe sampled surface waters of 31 lakes in the Northern Highland Lake district of Wisconsin and the Upper Peninsula of Michigan during July and August of 2000 (Table 1). The lakes were chosen to span wide and orthogonal ranges in DOC and TP concentrations and for their close proximity to the Trout Lake Station in Vilas county, Wisconsin. The order in which the lakes were sampled was randomized.Limnological samplesLimnological samples were collected for each lake at 0.5 m depth as follows. DOC samples were collected as the filtrate through Whatman GForF filters, and were analyzed on a Shimadzu model 5050 high temperature TOC analyzer. Color was also measured from this filtrate as absorbance at 440 nm on a Spectronic Genesys 2 spectrophotometer using 10 cm quartz cuvettes. Chlorophyll a was collected by filtering 200 ml of lake water, and then freezing filters for at least 24 hours, followed by methanol extraction for 24 hours. Fluorescence was determined before and after acidification to correct for pheopigments. Total phosphorus was analyzed on a Lachat autoanalyzer after persulfate digestion of a whole water sample. DIC and was measured on a Shimadzu GC-8AIT (TCD detector) gas chromatograph. DIC was determined from the headspace of acidified samples, which was injected into the GC. pH was measured using an Orion digital pH meter with automatic temperature compensating electrode. Temperature and dissolved oxygen profiles were measured using a YSI temperatureordissolved oxygen meter. Spot measurements of surface water DO were made on quadruplicate samples, using Winkler titrations as described in Bade and others (1998).BuoysWe deployed a buoy that sampled dissolved CO2, DO, water temperature, photosynthetically active radiation (PAR), and wind speed for 2-4 days on each lake. All water measurements were made at a depth of 0.5 m. Wind speed was measured one meter above the lake, using an RM Young model 03001, and PAR was measured 10 cm above the lake surface using a Li-Cor model 190SA quantum sensor. Electronic control and data collection were managed by a Campbell Scientific CR10X data logger. DO and water temperature were measured with a YSI model 600-XLM sonde fitted with a Rapid Pulse oxygen probe (model 6562) and temperature sensor. The sonde was attached to the buoy at the opposite end from the CO2 equilibration chamber (described below).We measured dissolved CO2 independently from DO. We equilibrated a closed loop of atmospheric gas in an equilibration chamber submerged to 0.5 m. The equilibrated gas volume was about 234 ml. We recirculated gas for the last 10 minutes of every 30 minute period, with a flow rate of about 9 ml s-1. A pump exchanged lake water every minute during equilibration. Equilibrated gas was diverted to the IRGA, equipped with a 14 cm sample cell for lakes with CO2 concentration under 2000 ppm or a 5 cm sample cell for lakes with CO2 concentration between 2000-20000 ppm. Following analysis of equilibrated gas, solenoids were activated to route atmospheric gas (taken 10 cm above the water) for CO2 analysis.Time series data are included for 25 of the lakes.Additional detail of the methods available in Hanson et al. (2003)Hanson, P. C., D. L. Bade, S. R. Carpenter, and T. K. Kratz. 2003. Lake metabolism: Relationships with dissolved organic carbon and phosphorus. Limnol. Oceanogr. 48: 1112-1119.

Methods

CHLOROPHYLL a ANALYSISEQUIPMENT: Film canistersTurner 450 Fluorometer fitted with:1. Quartz-halogen lamp2. Emission filter -SC6653. Excitation filter -NB44047mm Whatman GForF filters12 x 75 mm disposable glass culture cuvettes (Do not reuse cuvettes!)1-5 mL Oxford pipettorFinnpipette Stepper Pipetter with 5 mL tiptimestimesNOTEtimestimes-Change filters with fluorometer off! (Remember that chlorophyll analysis filters are different from APA analysis filters.)-Make sure Fluorometer has been calibrated for chlorophyll a (see Fluorometer Calibration for Chlorophyll a Analysis).REAGENTS: 100percent Methanol, spectrophotometric gradeCAUTION - wear gloves whenever you use methanol.0.1 N HCLEthidium Bromide Stock 3 standard (40microM solution)PROCEDURE:A. Filter water samples from each of the 6 light-depths onto a 47 mm GForF filter.1. Filters have a grid side and a smooth side. Place filter smooth side up.2. Shake sample bottle well before filtering (do this after the DIC sample has been taken from the same bottle.)3. For each depth, filter enough water so there is a faint color on the filter. For our lakes this ranges between 100-300ml. Record the volume filtered. Make sure you filer at less than 200 mm Hg pressure.4. Rinse filter towers and filters with DI water, place filters in labeled film canisters and place in freezer. Labels should include lake, date, and depth ID.5. If measuring edible chlorophyll as well, repeat steps 1-4 above, but first filter the sample through 35 microm mesh. (This has not been done since 2001, inclusive.)B. Extraction - DO IN DIM LIGHT and WEAR GLOVES!!1. Remove one tray of film canisters from the freezer. Extract chlorophyll by adding 25 mL 100percent MeOH to each film canister. If using re-pipettor, verify dispensed volume. (Record extraction volume if different from 25 mL.) Note the extraction time for each group of samples.2. Re-cap and place canisters in refrigerator to extract for exactly 24 hours (in the dark).3 Repeat steps 1 and 2 for all trays that have been in the freezer more than 24 hours.C. FluorometryCalibration of the fluorometer using a chlorophyll standard is typically performed at the beginning of the field season, or when a bulb is changed. Calibration using Ethidium Bromide is done at the beginning of each sample set.1. Insert correct filters in fluorometer while fluorometer is off. (Emission filter -SC665, Excitation filter -NB440), and warm it up for 1 hour .2. TURN LIGHTS OUT. Chlorophylls must be read in low light and samples must be kept cool. Do not remove film canisters from the refrigerator until you are ready to process the samples.3. Place clean cuvettes into a labeled rack (12 cuvettes per rack). Remove one lake-day of film canisters from the refrigerator.4. Place Ethidium Bromide Stock 3 standard into fluorometer and record reading on datasheet. Then, turn the span knob until the reading is 908. Record this on the datasheet.5. Shake film canister, remove the lid, and rinse the pipette tip with 2.5 mL of the sample. Then remove 2.5 mL of sample and place in cuvette.times Repeat for all film canisters.6. Pipette 2.5 mL of 100percent methanol into a cuvette for the blank and use it to zero the fluorometer. Choose a gain and turn the zero knob until the fluorometer reads 000. You must zero the machine every time you change gains.7. Remove the first sample cuvette from the rack, wipe with a Kimwipe, and place in fluorometer. Record the gain and the fluorescence before acidification, Fb. Repeat for all 12 cuvettes in the rack. Readings should be between about 200 and 1000. If not, adjust the gain and re-zero.8. Acidify each cuvette with 100 microL 0.0773 N HCl using the repeating pipetter and mix (hold the top of the cuvette securely, then &quot;thump&quot; the bottom several times). Check for condensation on the outside of the cuvettes, and wipe with a Kimwipe if necessary. Wait about 1 min from the acidification of the first cuvette.9. Record the fluorescence after acidification for all 12 cuvettes. VERY IMPORTANT: Make sure you read the Fb and Fa values for each sample on the same gain.10. Remove a new lake-day batch of film canisters from the refrigerator and repeat steps 3-9.times if particulate matter is present, centrifuge sample for 10 min. and use supernatant.D. Clean Up: DO THIS UNDER THE HOOD!1. Dump methanol solution from cuvettes and film canisters into a metal tray. Place the film canisters and lids in a separate tray. Position them in one layer on the tray with their openings facing up. Leave the trays under the hood overnight to evaporate the methanol.REFERENCES:Marker, A.F.H., C.A. Crowther, and R.J.M. Gunn. 1980. Methanol and acetone as solvents for estimating chlorophyll a and phaeopigments by spectrophotometry. Arch. Hydrobiol. Beih. Ergebn. Limnol 14: 52-69.Strickland, J.H. and T.R. Parsons. 1968. A practical handbook of seawater analysis. Fish. Res. Brd. Can. Bulletin 167.pp. 201-206.Holm-Hansen, O. 1978. Chlorophyll a determination: improvements in methodology. Oikos 30:438-447.

Methods

Our first goal was to understand the relationship between primary productivity and species richness for several groups of freshwater organisms. By species richness, we mean the number of species observed in a lake over a number of years. It is useful to have several years of observations because the number of species observed varies from year to year. We chose the total list of species (the asymptote of the collectors curve) as our index of species richness. The lakes studied as part of the U.S. Long-Term Ecological Research (LTER) Program are particularly valuable because they have been studied for two decades, and complete species lists exist for many kinds of organisms in these systems. LTER lake sites occur in northern and southern Wisconsin and northern Alaska (Toolik Lake). However, because there are fewer than 15 LTER lakes (and only seven with measured rates of primary productivity), we increased sample size by including data from additional well-studied lakes of similar size, but which span a greater range of primary productivity (see Table 1). These lakes have been studied for several years, and estimates of annual primary productivity exist for each lake. Some well-studied lakes were not included, such as those which lacked much of the crucial data, or lakes that were unusually turbid or saline. For example, Lake Okeechobee (Florida, USA) is turbid and exhibits a wide range of productivity levels, depending on the part of the lake sampled, while Marion Lake (British Columbia, Canada) has a flushing rate of only a few days (W. E. Neill, personal communication). Sampling design and protocol are not standardized among studies of lakes. For example, species identifications were done by different people, sampling period was quite variable, and the number of samples per lake was variable. Such heterogeneity reduces the accuracy and precision of relationships between productivity and species richness.Primary productivity.&mdash;Pelagic primary productivity (PPR) can be measured by the 14C method (Vollenweider 1974). This method gives a close approximation to gross primary productivity (GPP), but because some of the fixed carbon is respired quickly, the value obtained is less than GPP (Fee et al.1982). Point values of PPR are then integrated by depth and area to produce estimates of whole-lake annual primary productivity per cubic meter or square meter.Lake primary productivity is fundamentally different than productivity measured in other biomes (e.g., grasslands, forests). The 14C method measures available (gross) primary productivity more than utilized (net) production, which is what is normally measured in terrestrial systems. The 14C method is also a fairly direct measure of productivity, compared to the proxy methods (e.g., nutrient loading, biomass, climate, soil fertility) used in many studies.Sampling protocols for aquatic organisms.&mdash;Sampling protocols differed among taxonomic groups and lakes (e.g., Downing and Rigler 1984). For example, phytoplankton samples are taken by capturing (at most) a few liters of lake water, either from a specific depth or with a sampler that integrates water across a range of depths. Zooplankton are usually sampled by vertical tows (i.e., raising a net through the water column). Both zooplankton and phytoplankton samples are typically taken from the center of the lake, although replicate samples at different locations may be taken from larger lakes. Planktonic organisms are much smaller than the sampling device, and hundreds to hundreds of thousands of organisms are typically captured in a single sample. In contrast, aquatic macrophytes are sampled using quadrats and rake samples, or simply based on a walk around the lake, while fish are sampled using a variety of nets andoror electroshocking equipment. Criteria for species lists.&mdash;Species lists for fish, macrophytes, and pelagic phytoplankton, rotifers, cladocerans, and copepods were obtained from the literature and from unpublished data. We avoided lists restricted to only dominant or common species, and thus included only lists that were exhaustive. Few lakes had species lists for all six groups of organisms. However, we included any lake that had an estimate of the average annual primary productivity and had lists for at least three taxa.We standardized this database by developing criteria for inclusion of species in analyses. Phytoplankton lists included all prokaryotic and eukaryotic photosynthetic phytoplankton for which there were abundances of more than one organism per milliliter (a criterion also used by Lewis 1979). We included all nonsessile species caught in open water as pelagic rotifers. For the crustacean zooplankton (cladocerans and copepods), we followed the criteria of Dodson (1992). Species lists of macrophytes included all submerged, floating, or emergent species of flowering plants, including Typha, sedges, grasses, and duck weed. We did not include Isoetes or macroalgae such as Chara and Nitella as macrophytes. The fish list included all species reported from the lake, including introduced taxa. Fish species reported to occur in the watershed, but not in the lake (as in Pearse1920) were not considered part of the lakes biota.

Methods

14C-PRIMARY PRODUCTIVITYEQUIPMENT:Field:PPR float with line and clips for hanging bottles at selected depthsPPR field boxes containing:18, 60 mL reagent bottles wor glass stoppers, plus extra bottles and tops.2, 100-1000 microL automatic pipettor and several tipslab gloves and plastic apronplastic bag for used gloves and pipette tipsextra clips for hanging bottleswater pumpLab:6-tower vacuum filter apparatus4.7 cm Whatman GForF filtersScintillation vials with labeled caps (label includes lake, day, &quot;D&quot; or &quot;L&quot; for darkor light bottle, and depth, respectively. (e.g.: &quot;L145 L1&quot;; &quot;W224 D6&quot;)REAGENTS:10 microCi 14C-bicarbonate (2 microCi mL-1)Dichlorophenol-dimethyl urea (DCMU) saturated solution0.1 N HCLScintillation fluor (Biosafe)PROCEDURE:(Observe safe radioisotope handling techniques at all times during this analysis!)A. In lab (morning, before going to the field)1. Transfer 14C from ampule to labeled scintillation vial with a disposablepipettor (under the fume hood). Put scintillation vial in the field box.2. Replenish supply of lab gloves, pipette tips, and DCMU in the field box.3. Remember to include the PPR float and the field boxes (check contentswith list) in the items loaded into the field vehicle(s).106B. In field1. Rinse and fill 3, 60 mL BOD bottles with water from each depth,corresponding to 100, 50, 25, 10, 5 and 1percent of surface irradiance. Avoidgetting air bubbles in bottles. TRY TO KEEP BOTTLES IN THE DARKAS MUCH AS POSSIBLE.2. Pipette 250 microL of water from each bottle (using the &quot;14C pipettor&quot;).3. Pipette an additional 500 microL of water from the 6 dark bottles using the&quot;DCMU pipettor&quot; (the dark bottles are used as a t = 0 control).4. Using the &quot;DCMU pipettor,&quot; add 500 microL DCMU to the dark bottles tokill the phytoplankton. (always done before 14C addition.) It isimportant not to contaminate &quot;light&quot; bottles with DCMU! Darkbottles are labeled, and are used only as dark bottles.5. Using the &quot;14C pipettor,&quot; Pipette 250 microL of 14C into each of the 18 bottles,starting with the dark bottles to ensure there is enough isotope forcontrols.Summary of subtractions and additions:light bottles dark bottlesremove 250 microL 750 microL (250 plus 500)add DCMU - 500 microLadd 14C 250 microL 250 microL6. Replace stoppers and invert bottles 2 or 3 times to mix. Ensure thatstoppers are well-seated, so they don t come out. It often helps to twist thestopper as you push it into the bottle.7. Suspend bottles at appropriate depths for incubation. Record incubationstart time.8. AFTER 6 HRS: Remove bottles from water and place in carrying caseuntil ready to filter (filtering should be done promptly after removal ofbottles from water). Record incubation finish time. (Incubations usuallygo from ~9:30am-3:30pm)C. In lab (afternoon)1. Have readya. Flask used only for collecting 14C waste107b. Filter towers equipped with 4.7 cm GForF filters. Separate towersshould be used for light and dark (DCMU) bottles.c. Scintillation vials, with caps labeled for all samples.d. A full squirt bottle of 0.1 N HCl and a full squirt bottle of Milli-QTURN OFF THE LIGHTS - THE REST OF THE PROCEDURE SHOULD BEDONE IN DIM LIGHT!2. Prepare 3 totals:a. Add 10 mL scintillation solution (Biosafe) and 100 microL 1 N NaOHto 6 vials (label on cap should include lake, day, &quot;TOT&quot; ,anddepth id).b. Remove 250 microL from one of the light bottles from each depth andadd it to the proper vial. These vials are for calculating the totalamount of 14C added to the bottles.c. Tightly cap the total vials and put aside for later analysis with thescintillation counter3. Samples (process samples in the designated 14C fume hood):a. Empty the entire BOD bottle into the appropriate &quot;light&quot; or &quot;dark&quot;filter tower. Record volume if entire bottle is not filtered.b. Once the sample has filtered completely, rinse the bottle with asquirt of 0.1 N HCl, and filter this rinse. Then rinse the bottlewith water and filter this rinse. Rinse tower with 0.1N HCl, andthen finally with Milli-Q.c. Remove filter by folding it in quarters and place it at the bottom ofthe appropriate scintillation vial. Filter should be compact enoughin the bottom of the vial to be completely covered by thescintillation fluor (which fills half of the vial).d. Dry at 60-70degreeC for 24 hours.e. After drying filters, add 10 mL liquid scintillation solution to vialsand count in scintillation counter (see Scintillation CountingProcedure).D. Clean up:1081. When all samples have been filtered, squirt some acid down the last towerin the line to rinse. When the acid has been pumped out of the line, ventthe tower to expel all liquid. Lift towers to drain completely.2. Rinse BOD bottles and caps three times with hot tap water.3. Radioactive waste goes into a carboy marked and reserved for radioactivewaste. timestimesNOTEtimestimes The total radioactivity in each carboy must beknown; Record the date when 14C is initially put in the carboy and the datewhen the final amount of 14C is put in the carboy.4. Empty the remaining amount of 14C from the scintillation vial taken intothe field into the radioactive waste carboy. Discard the vial in the dryradioactive waste bag.5. Record the amount of radioactivity used in the isotope log book.CALCULATIONS:Use the SYSTAT command file CALCPPR.CMD to calculate primaryproductivity according to the following equation:mg Ctimesm-3timesh-1 =(CPMs - CPMb) times (VincorVfil) times (A) times (1.05)(DPMt) times (Eff) times (T)where:CPMs = counts per minute for sampleCPMb = counts per minute for DCMU controlVine = volume (mL) incubatedVeil = volume (mL) filteredA = total C in sample (in mg Corm3), calculated from sample alkalinity1.05 = isotope discrimination factorDPMt = disintegrations per minute of total amount of 14C added to each bottleEff = efficiency of scintillation fluor calculated from internalstandards for each sampleT = length of incubation (h)2. Use the method in Appendix III, along with measurements of solar radiationtimes andlight extinction from the weekly light profiles, to calculate daily production of thephotic zone and the mixed layer (see Carpenter et al., 1986).times see Pyranograph Method109REFERENCES:Carpenter, S.R., M.M Elser and J.J. Elser. 1986. Chlorophyll production, degradation,and sedimentation: Implications for paleolimnology. Limnol. Oceanogr. 31:112-124.Strickland, J.D.H., and T.R. Parsons. 1968. A practical handbook of seawater analysis.Bull. Fish. Res. Board Can. 167:267-279.Legendre, L., S. Demers, C.M. Yentsch, and C.S. Yentsch. 1983. The 14C method:Patterns of dark CO2 fixation and DCMU correction to replace the dark bottle.Limnol. Oceanogr. 28: 996-1003.

Methods

see abstract for methods desciption