Zooplankton oxygen consumption and nutrient release in relation to species composition, animals size and enviromental conditions in the Baltic Sea during May and August

This paper not to be cited without prior reference to the authors International Council for the C.M. 1992/L:21 Exploration of the Sea Session V Zooplankton oxygen consumption and ñutrient reléase in relation to species composition, animáis size and environmental conditions in the Baltic Sea during May and August by L. PosteU), S. Hernández Leon^), M. Gomez^, S. Torres^), U. Mikkatl),and A. Portillo Hahnefeldl) 1) Institut fiir Ostseeforschung (10W) an der Universitat Rostock, Seestr. 15, D-O-2530 Wamemünde, Germany 2) Facultad de Ciencias del Mar, Aptdo. 550, Las Palmas de G.C., Canary Islands, Spain KEY WORDS: zooplankton oxygen consumption, ñutrient reléase, size classes, composition, environmental conditions, Baltic Sea, spring 1988, 1990, 1991, sommer 1988,1990 ABSTRACT: Zooplankton metabolism in terms of oxygen consumption and ñutrient reléase (ammonia, phosphate) were measiu'ed in the Baltic Sea, a températe área with high envirormiental changes both in space and in time. Plankton of the surface layer were analysed with balance measurements in 4 size classes between 50 and 1000 nm during spring in 1988, 1990 and 1991, in summer 19^8 and 1990 as well. The use of electrón transport system (ETS), and the Glutamate Dehydrogenase (GDH) activity as indicators for respiration and ammonia reléase respectively, enlarged the data density and made a three dimensional resolution available (May 1990, 1991). Data are in the range of the latitudinal dependend magnitude. They reflect slight interannual, more seasonal and regional aspects. Animáis size, temperature, food concentration, and species composition influence the specific rates. METHODS: Oxygen consumption, inteqireted as respiration, and phosphate and ammonia reléase (excretíon) were measured in the Baltic Sea, mostly from Mecklenburg Bight to the northem Gotland Sea, sometimes from the transitition área (Skagerrak / Kattegat) to the Gulf of Finland (Fig.l). Determination were carried out by means of balance or "bottle" methode (Omori and Dceda, 1984). Samples were carefiíl coUected m the surface layer down to the thermocline using a WP-2-net, equiped with a special cod end to prevent, that animáis get dry. After dark adaptation of about 3 to 5 hours in máximum, under oxygen saturated conditions, using water from the same station, the plankton was fractionated in 4 size classes (55 - 100, 100 - 200, 200 - 500, 500 - 1000 ^im), washed carefuUy with fíltered seawater to clean it from nutrient partióles, and transfered in 11 bottles. Bottles were incúbate now in a container, cooled by sea surface water, at a rotating wheel (2. 5rpm). At least one control bottle with the same fíltered, and oxygen saturated seawater is to add, to be able to calcúlate the difference of oxygen and nutrients of the bottles with and without zooplankton after the 6 to 15 hours lasting incubation periode. The enrichment of animáis in comparison to in situ conditions is about 20 to 1000 times, depending from the temperature. Finally oxygen, and nutrients will be determined, the zooplankton stored in buffered formaline, to analyse the species composition, the dry mass, using conversión factors as recommended by BMB (1985). Dry mass is needed to calcúlate the specifíc metabolic rates. For the calculation of in situ rates the in situ biomass is to determine in the above mentioned size classes. To be sure, that all the different size classes are quantitatively collected, nets of 55 jim, 100 ^m and 200 ^m mesh size were used for that purpose . From the 200 jim net the two larges fractions were produced. With the same nets plankton for the determination of metabolic rates by means of enzymatic activity (ETS, GDH) were catched in 3 depth levéis (surface -thermocline, thermocline - halocline, halocline - bottom). Size fractionation and storage in liquid nitrogen were done immediately. ETS means Electron Transport System activity and is in relation to respiration, GDH means Glutamate Dehydrogenase, to be in relation to ammonium reléase. The single steps are described in a flow diagramm (Fig. 2). The ETS assay was runned in the laboratory according to Packard (1969, 1971), Owens and King (1975) and modifíed by Kenner and Ahmed (1975). The samples were homogenized in teflon-glass at O - 4 C using a phosphate buffer 0.1 M, pH = 8 containing Tritón X-100 (Sigma Chem. Co.) for the solubilization of the enzymes (Owens and King, 1975). An aliquot of the crude homogenate was incubated for 20 minutes at 12 •€ in darkness conditions in the presence of NADH, NADPH, succinate and an artificial electrón acceptor, the tetrazolium salt INT (Biomedical Lab.). After this time, the reaction is stopped with a solution containing phosphoric acid and formaline and centrifugued between O - 4 -C for 10 minutes at 4000 rpm. The reaction colour was measured at 490 nm with a turbidity base line at 750 nm, and respect to a blank without substrates and treated as the sample. For the INT coefficient factor, the valué of 1.42 was taken firom the molar absorption coefficient obtained from Kenner and Ahmed (1975). Final activity valúes were recalculated for the "in situ" temperature using the Arrhenius equation and an activation energy of 15 Kcal/mol.-C (Packard et al., 1975). Biomass as proteins was determined according to the method of Lowry etal. (1951), using Bovine Sero Albumin (BSA, Sigma Chem. Co.) as standard. The modification of the Lowry method by Peterson (1977, 1983) was used for the samples with low protein content. The GDH assay was runned in accordance with Bidigare and King (1981) and Bidigare et al. (1982), including certain modifications: One milliliter of crude homogenate (same as ETS assay) is diluted in Tris buffer, pH = 8.6 and centrifíigued (O - 4 -C) for 10 minutes at 4000 rpm. In a Perkin-Elmer 551-S UVA^S spectrophotometer equipped with an 1 cm water-jacketed cuvette thermostatized at 12 •C, an aliquot of 0.5 mi of the supematant is placed in the presence of NAD and ADP. When absorbance lecture at 340 nm is stable, the assay is runned by adding glutamate, and changes in absorbance were recorded continuously for 2 minutes. The slope is proportional to GDH activity. Total volume in the cuvette was 3.0 mi and NAD, ADP and glutamate concentrations were 1.2, 2 and 40 mM, respectively (Bidigare et al., 1982). RESULTS: 1. Methodological aspeets: In general there is a body mass - temperature dependency of metabolic rates. A coincidence with calculated rates in magnitude should be a quality mark of the own measurements, because 700 to 1200 observations are the basis of the Ikeda's (1985) equations. Using May and August 1988 as an example a satisfying correspondence is to observe: May August in vitro temperature spec. oxygen consumptíon per individuum Hm^ O2/ Ind.* h spec. PO4 reléase per individuum pM / Ind. * h spec. NH4 reléase per individuum pM / Ind. * h observation calculation observation calculation observation calculation 7.50 c 24.64 +/- 10.8; N=25 10.82 +/- 2.07; 15.17 +/- 7.76; N=27 4.85 +/- 1.22; 105.32 +/- 92.32; N=24 70.71 +/- 16.54; 18.3°C 15.06 +/- 7.76; N=16 17.99 +/. 1.67; 6.90 +/- 6.15; 6.25+/- 0.41; 57.94 +/- 50.38; N=14 112.57 4-/- 11.16; In May, after or during the spring bloom periode, the observed valúes are higher, than the calculated. In August there is the opposit case, indicating, that temperature is one influence, but the nutritive basis probably of higher priority. Next table includes results of correlations of 6 data groups, the comparisons (1) of oxygen consumption and nutrients reléase ontheraw datalevel, (2) of ETS - and GDH - activity on the raw data level, (3) of dry mass specific oxygen consumption and nutrients reléase rates (raw data devided by biomass), (4) ofprotein specifícenzymatic activity, (5) of (1) and (2), (6) of (3) and (4), using the May '91 data as the basis: raw data: 02 vs. P04 02 vs. NH4 P04 VSNH4 raw data: ETS vs. GDH dry mass specific rates: O2 vs. PO4 O2 vs. NH4 PO4 vs. NH4 protein specific ETS vs. GDH raw data: O2 vs. ETS NH4VS. GDH biomass specific O2 vs. ETS NH4VS. GDH r = 0.8309 r = 0.9475 r = 0.9035 r = 0.6730 r = 0.6582 r = 0.4904 r = 0. 5425 r = -0.0577 r= -0.0476 r= 0.3353 r= -0.0225 r= -0.0937 p<0.00l p< 0.001 p< 0.001 p< 0.001 p< 0.001 p < 0.05 p < 0.05 N=21 N=21 N=21 N = 23 N = 23 N = 21 N = 21 N = 21 N = 21 N = 21 N = 21 N = 21 Correlations were to expect in all the cases. The significant coincidence between the raw data is a satisfying indication for their quality. This is trae for both, the data of bottle methode and thoseofthe enzymatic determinations . The reason for the lesser correlation of the dry mass specific rates are probably the calculated biomass data. This is worse in the case of protein specific ETSand GDHactivities. Not to miderstand is the missing correlation between the raw oxygen consumption and the ETS activity on the same level. That biomass specific rates of both groups in the last two rows don't match does finally not wounder. So the link between the two methods, shown in Figure 2, is not active now. Both data sets have to be seperately used. 2. Size classes: The smaller organisms show an about 7 times higher respiration rate compared with the largest group (F¡g.3). The mean valúes are from August 1990. The columns indícate the standard deviation. 3. Vertical structure: The determination of the oxygen consumption and the ammonia reléase in terms of en2ymatic activity in all the depth levéis allows to estímate the average vertical structure of metabolic rates.. This will be done as apercentage in comparison to the surface layer in May 1990 and 1991. There were no remarkable differencies between the both years, between ETS and GDH, especially in the group of protein specifíc rates. Larger vertical gradients are to observe in the in situ - group. The metabolic rate decreases from the surface layer successive downward by aproximately 30% per depth level. The ETS results from 1991 are presented in the next table as an example: surface layer (up to thermocline) intermedíate layer (thermocline to halocline) bottom layer (halocline to bottom) surface layer (up to thermocline) intermedíate layer (thermocline to halocline) bottom layer (halocline to bottom) ETS spec. 1000 - 500 ^im 100 143 101 in situ ETS 1000 - 500 ^m 100 61 25 500 - 200 ^m 100 85 69 500 - 200 \xm 100 33 17 200 - 100 }im 100 79 80 200 - 100 nm 100 65 31 total mean 100 102 83 total mean 100 53 24 4. Regional Patterns: Figure 4.1 includes environmental parameters, like temperature, salinity, chlorophyll averaged for the surface layer, above the thermocline, and the diy mass of all size fractions, the dry mass of the most important class between 100 and 200 ^ini size, and the frequency of the main taxonomic groups within this class. The data are regional averaged, one to ten valúes are the basis. SK&KG means Skagerrak and Kattegat área, MB Mecklenburg Bight, AS Arkona Sea, BS Bomholm Sea, S&CG southem and central Gotland Sea, and NG Northern Gotland Sea (c.f. Fig.l). Figure 4.2 includes biomass specific metabolic rates, oxygen consumtion, ammonia and phosphate reléase, ETS and GDH activity, and the assimilation number (primary productivity divided by chlorophyll concentration). Figure 4.3 includes the in situ rates respectively.The averages are the same like in Figure 4.1. In athe most cases a trend is visible, characterizing the late spring situation in the Baltic Sea, with lower temperatures in the northem part, where the metabolic active centre is located during that time, May 1991. The seasonal succession seems to be orientated from the transitition área of the Baltic Sea to the northem Gotland Sea. The in situ oxygen consumption and the phosphate reléase show a clear decreasing tendency, in accordance with the biomass patterns in Fig.4.1. 5. Seasonal and interanual variation To discribe these variability data from two seasons, May and August, are available, from at least two years. In Table 1 (Annex) the mean situation is included for in situ dry mass, incubation temperature, in situ frequency of Copepods and of Cladocerans, the dry mass specific and the zooplankton in situ rates of respiration and remineralisation, partly of different size classes. Comparing the May situation 1990 was significant warmer, than 1988 and 1991. Also the frequency of Copepods / Cladocerans is different. The biomass of the larger organisms is more developed in 1990, than in the other two years. The seasonal cycle started earlier in that year. This has no signifícant influence on the biomass specific metabolic rates, if they are averaged for the whole Baltic. But it has an input on the in situ rates, in dependency to the biomass. Remarkable differences are to observe in the biomass specific rates, if May and August will be compared. During August the plankton composition is quit different in comparison to May, the temperature. also. 8 It is to conclude, that seasonal variability is more signifícant than interannual. Temperature may have an effect, but nutrition and species composition of zooplankton is also important. Changes between 8 to 12_C (May 88, 91 / May 90) have a slight influence to the specifíc rates. A comparison with data collected in 1989 in the subtropical Atlantic show more signifícant differences, but on the basis of a 10 K temperature change! In that case a specifíc oxygen consumption rate of about 13 mm^ 02 /mg dry mass * h in the Atlantic is to compare with 5.55 from August 88. This is areIationof2.3. (From Ikeda (1985) a factor of 2.8 is to expect, if an área of 21° N (Atlantic) and another of 50° N (Balite Sea) will be compared. ) LITERATURE CITED: BIDIGARE, R.R. and F.D. KING, 1981. The measurement of glutamate dehydrogenase activity in Praunus flexuosus and its role in the regulation of ammonium extretion. Comp. Biochem. Physiol., 70 B: 409 - 413. BIDIGARE, R.R., F.D. KING and D.C. BIGGS, 1982. Glutamate dehydrogenase (GDH) and respiratory electron-transport-system (ETS) activities in Gulfof México zooplankton. J. Plankton Res., 4: 895 - 911. HERNROTH, L. & H. VIL JAMA A (Eds.), 1979. Mesozooplankton biomass assessment. BMB Publ. No. 6 . I5pp. IKEDA, T., 1985. Metabolic rates of epipelagic marine zooplankton as a fimction of body mass and temperature. Mar. Biol.. 85: 1 - 11. KENNER, R.A. & S.I. AHMED, 1975a. Measurements of electrón transport activities in marine phytoplankton. Mar. Biol.. 33: 119 - 127. LOWRY, P.H., N.J. ROSENBROUGH, A.L. FARR & R.J. RANDALL, 1951. Protein measurement with a Folin phenol reagent. J. Biol. Chem.. 193: 265 - 275. OMORI, M. & T. IKEDA, 1984. Methods in marine zooplankton ecology. John Wiley & Sons. New York, Chichester, Brisbane, Toronto, Singapore. 332 pp. OWENS, T.G. & F.D. KING, 1975. The measurement of respiratory electrón transport system activity in marine zooplankton. Mar. Biol., 30: 27 - 36. PACKARD, T.T., 1969. The estimation of the oxygen utilization rate in seawater from the activity of the respiratory electrón transport system in plankton. Ph. D. Thesis, Univ. Washington, Seattie. 115 pp. PACKARD, T.T,, 1971. The measurement of respiratory electrón transport activity in marine phytoplankton. J. Mar. Res., 29: 235 - 244. Enzvmology. Vol. 91. Academic Press, pp 95 - 119. ACKNOWLEDGMENT: We would like to thank the crew of RV "A.v.Humboldt", of RV "Prof.A.Penck" for their help, the Chemical Department of the Institute of Baltic Sea Research, Wamemünde, for many nutrient analyses, Anneli Postel and Heide Sandberg for the numerous zooplankton determinations, the lOW Data Centre, providing temperature, salinity, chlorophyll data, and assimilation numbers, and the BMFT, because the measurement of 1991 were supported by this Ministry (Contract No.:03F0030A). N = mg dry tnass-m'' í incubatíoo temperature/oC frequency of Copcpods/ % frequency of Gadocerans Oxycen coosomption dry mass !^)ecific/ mm' Oi-mK"'-h'' in situ/ mm' Oj-mg'-h'' ', Pbosphate reléase dry mass spedfíc/ nM-mj-'-ít-' in situ/ : >íM-m-'-d' t \ Afflmonia reléase ; dry mass speciñd nM-mg-'-h-' 1 in situ/ ;iM-m'-d' . Size fraction//im May '88 26 27 7,5 76 16 9,12 7,24 5,40 3,50 34,68 42,66 >200 4,19 Í2,T~ 43 46 13,93 1.4 4,30 0,43 53,48 5,38 May'90 16 19,50 __ 4.32 _ - 1 4Ú 10 13 41 12 23 4,77 8,95 32,33 2.23 0,93 E - 4.56 3,12 7,65 21,70 1,46 0.87 r'-"2,76 16,51 29,76 38,65 7,73 3,08 E » 16,19 1000500200100500 200 100 55 >20Ó •" • May'91 12 4.77 8.59 '"8;6 So 34 11___22__ 8.16 5,18 0,93 1,07 19,26 3 0 8.20 3,70 E-.6.5 3,78 1.92 0.43 0.40 2,19 1,01 E - 2.03 30,48 17.35 3,49 3,58 22,67 10,48 E = 18.44 1000600500 200 >20b 200__100_ 1,61 0 0 18,28 0,71 5.01 0,19 23,06 0,89 100_55 _ Aug. '88 16 100 18,3 Mi 70 5.55 __13,37 2,55 5741 21,00 53,61 >200 _J,86 31" 67 , 5,26 1,29 0,65 0,14 24.66 5.25 1000500 65.37 18.2 29 70 6.29 8,48 ' 1,49 2.34 20,08 31,50 500200 >200 Aug. '90 5 12.51 29 69 11,61 3,98 T m 15 30 2,60 0,79 E - 3,38 15,25 4,64 E - 43.38 200lOQ I 09 41 57 39,68 1,64 4,06 0,11 71,70 1,99 10055. _ Table: Cotnparísons of mean valúes of environmental conditíons. zooplankton biomass. frequency ofCopepods and PhyHopods. specific and in situ mctabolic rates in May I988.I990. I99I and in August I988.I991.