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, "D" or "L" for darkor light bottle, and depth, respectively. (e.g.: "L145 L1"; "W224 D6")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 "14C pipettor").3. Pipette an additional 500 microL of water from the 6 dark bottles using the"DCMU pipettor" (the dark bottles are used as a t = 0 control).4. Using the "DCMU pipettor," add 500 microL DCMU to the dark bottles tokill the phytoplankton. (always done before 14C addition.) It isimportant not to contaminate "light" bottles with DCMU! Darkbottles are labeled, and are used only as dark bottles.5. Using the "14C pipettor," 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, "TOT" ,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 "light" or "dark"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.