Journal of Microbiological Methods 9 (1989) 297 - 308
Elsevier M I M E T 00310
Urea turnover in a coastal marine sediment measured by a 4C-urea short-term incubation Bente Aa. L u n d a n d T. H e n r y B l a c k b u r n Department of Ecology and Genetics, University of Aarhus, Aarhus, Denmark (Received 5 March 1988; revision received 23 January 1989; accepted 25 January 1989)
Summary A method is described for the determination o f urea turnover in marine sediments. 14C-urea was injected into sediment cores and 14CO2 and the total 14C content were measured in a time-course incubation (29, 72, 109 and 141 min). The method is a true-tracer technique as the addition of urea does not significantly alter the ambient concentration. Urea was not b o u n d or adsorbed to the sediment, within the time span o f the incubation, indicating that it was a free porewater pool of urea that was turned over. The turnover rate of urea was calculated by means of three different isotope-dilution models. Model I described nonsteady-state conditions and was the valid model for this study. Models II and III were both steady-state models and were invalid. The urea pool was assumed to change linearly with time in Model I and to remain constant in Models II and Ill. The 14C-urea pool was predicted to decrease exponentially in Models I and II and to decrease linearly in Model III within a short time span in the beginning of the incubation. The turnover rate was slightly overestimated, 21%, on an areal basis, when Model 1I was applied and severely overestimated, 33 %, when Model III was applied. We suggest macro fauna to be responsible directly and/or indirectly for a major part of the urea production and urea to be the main source for N H g mineralization, in the study area.
14C; Isotope dilution; Sediment; Urea; Urea turnover
Introduction The importance of urea in the water column of marine environments has been reported in numerous publications during more than two decades. Remsen  was one of the first to recognize urea as a potentially important N source in his study on the distribution of urea in coastal and oceanic waters. Most attention has been on urea as a N source for phytoplankton [e.g., 2 - 6 ] . These studies have established that urea is available and utilized in quantities comparable to inorganic N sources and that urea is turned over rapidly. Urea-turnover times in the tropical Atlantic Ocean have been Correspondence to: B.Aa. Lund, Department of Ecology and Genetics, University of Aarhus, Ny Munkegade, 8000 Aarhus C, Denmark.
0 1 6 7 - 7 0 1 2 / 8 9 / $ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
298 reported to be < 24 h . The role of zooplankton in the excretion of urea is well documented . Dag et al.  reported that urea can account for 53 °7o of total N excreted by copepods in the Peru upwelling system. On average, urea and amino acids represent 20°7o of the total N excreted by zooplankton . There are only few reports on the role of pelagic bacteria in the metabolism of urea . One study from the Baltic Sea revealed that the rate of bacterial urea hydrolysis can be comparable to, or shorter than, those reported from phytoplankton uptake studies . In pure cultures, evidence for a carrier-mediated energy-dependent uptake of urea has been illustrated in Alcaligenes eutrophus H16 and Klebsiella pneumoniae M5al . Kaltwasser et al.  demonstrated that the external N H 2 concentration reduced urease activity in a variety of aerobic bacteria. There is limited evidence for the production and excretion of urea from bacteria [14-15]. A study by Satoh  demonstrated that bacterial decomposition of dead phytoplankton was possibly an important source of urea in natural waters. Urea can, furthermore, be produced by bacteria as a byproduct in the biosynthesis of the osmoregulating agent putrescine, with 1 mol urea produced and excreted.mol putrescine synthesized -1 [16-17]. Stanier et al.  suggested that putrescine can play an important role in assuring a constant intracellular ionic strength in bacteria. Urea has been considered to be a potentially important N source in freshwater sediments, i.e., Jones et al.  found that urea was a significant intermediate in the generation of NH~-. To our knowledge, there is only one study, performed in anoxic Long Island Sound sediments, that examines the importance of urea in marine sediments . However, the amount of urea in the interstitial water ( 0 - 1 6 ~M) was insignificant compared to the amount of dissolved N H g (100-10000 tzM). The main purpose of the present study was to develop a method for the measurement of urea-turnover rates in marine sediments. Materials and Methods Samples were collected in Norsminde Fjord during April 1988, with Plexiglas tubes (20 cm, 2.6 cm diameter). The Plexiglas tubes were provided with self-sealing injection ports of silicone rubber at 1-cm intervals . After sampling, the sediment cores were kept dark, at the in situ temperature (4 °C). Processing for urea concentrations, sediment characteristics and 14C-urea incubations was performed within a few hours after sampling.
Sediment character&tics Sediment characteristics were measured from three cores (2.6 cm diameter, 12 cm). The cores were fractionated into 2-cm segments. The specific density of each segment (10.62 cm 3) was determined from the wet weight. The porewater content was determined as the weight loss from flesh sediment dried at 105 °C for 24 h. Sediment porosity was calculated as the specific density multiplied by the water content. The macrofaunal biomass was determined from four cores (3.6 cm diameter, 12 cm). The cores were fractionated into 2-cm segments and each segment was washed
299 through a 1-mm sieve screen. Subsequently, the fresh weight of animals was determined and expressed as mg fw.cm -3.
14C-urea-turnover~injection technique After the overlying water of the cores had been carefully removed, 10 #1 tracer (3.95 nCi. #1-1, Amersham Radiochemical Center) was injected in a line through the core at 1-cm depth intervals. The enrichment of the ambient urea pool was on the average 12_+1o70.Five cores were injected for each time point in a time course (29, 72, 109 and 141 min) and incubated in the dark at the in situ temperature. After the given times, the cores were sectioned into 2-cm segments, transferred to 5 ml 2.5% (w/v) N a O H (final pH ~ 14) and mixed thoroughly in order to trap the CO2 and stop the incubation. The samples were centrifuged at 2000×g for 10 min and the supernatant frozen for later radioisotope analysis.
Recovery of 14C in samples (14C-urea + 14C02) 250 #1 supernatant was transferred to 8 ml solvent EE (1:7 v/v mixture of ethanolamine and ethyleneglycolmonomethylether) to which 10 ml scintillation fluid (10.88 g P P O + 0.272 g POPOP/2.5 1 toluene) was added. The 14C activity was measured in a Packard 2200 CA Tri-Carb liquid scintillation analyzer with an automatic efficiencytracing DPM software program. Standards were treated in the same way as samples. D P M . cm-3 of sediment was calculated from the porewater content and the amount of N a O H added.
Urea turnover The 14C02 produced by the turnover of 14C-urea was displaced from the alkaline porewater extracts by acidification (1 ml 5 N H2SO 4, giving a pH = 1) in a closed vessel (Fig. 1). The C02 evolved upon acidification was trapped in 200 #l phenylethylamine (PEA) absorbed on two (1.5 × 10 cm) strips of Whatman 3 filter-paper suspended over the sample. The filters were fastened to the rubber stopper that sealed the vessel. The method used is a modification of already existing methods for the collection of 14C [21-27]. A test was performed to determine the diffusion time necessary for a 100°70 recovery of 14CO2 on filters. 100 #1 HI4CO~ (0.03 #Ci. 100/z1-1, 56 mCi .mmol -l, Amersham Radiochemical Center) and 1 ml 5 N H2SO 4 were added to 1, 2 and 3 ml of a 3.7:5 (v/v) mixture of porewater and 2.5 % (w/v) N a O H and allowed to diffuse for L 2, 4 and 24 h. Following diffusion, the filters were transferred to 8 ml solvent EE. The further treatment was as described above. To calculate the diffusion effectivity, 100 #l I-I14CO3 (0.03 #Ci.100 #1-1) was added directly to 8 ml solvent EE, together with filter-paper, PEA and the scintillation fluid. The recovery of 14CO2 on filters is shown in Table 1 for different volumes of sample. Small volumes of sample (1, 2 and 3 ml) showed a 100% recovery of 14CO2 within a few hours ( 2 - 4 h) and the 14CO2 remained on the filters for at least 20 h after the completion of diffusion. The time necessary for a 100°70 recovery was almost independent o f the sample volume diffused (1- 3 ml). The amount of 14CO2 diffused in the test and true experiments was < 0.05 and 0.01070, respectively, of the unlabelled porewater CO 2- diffused along with the label. The recovery of 14CO2 could, therefore, be expected to be 100%.
L •, ~ l . - -
crn - - ~ , , ,
Fig. 1. Diffusion vessel. To rubber stopper were fastened two (1.5 cm, 10 cm) pieces of Whatman 3 filterpaper to which were added 200 tzl phenylethylamine (PEA). 1 ml 5 N H2SO 4 was added to glass vessel (3.7 cm i.d., 7 crn). Vessel was closed immediately after addition of sample. CO 2 was allowed to diffuse onto filter-paper at room temperature.
Turnover experiments: 1 ml alkaline-porewater extract was acidified and CO2 was allowed to diffuse onto the PEA-containing filter-paper, overnight, at room temperature. D p m . c m -3 of sediment was calculated from the porewater content and the amount of N a O H added to the sediment. The °7o laC-urea left in the urea pool was calculated from the 14CO2 data, assuming that 14C was present in the sediment only as CO2 and urea. Net changes in the pool size of urea were determined from a time course incubation: 0, 20, 80, 121 and 163 rain. The overlaying water of the cores (2.6 cm diameter, 12 cm) had been removed carefully at time zero. After each given time, four cores were sec-
Diffusion time (h)
Recovery of 14CO2 (%) 1 ml a
2 ml a
3 ml a
2 4 24
101 98 99
100 100 101
96 100 103
a Volume of sample diffused.
301 tioned into 2-cm segments, transferred to 5 ml urea-deficient artificial seawater and mixed thoroughly. The samples were extracted with seawater to obtain enough sample for the later urea analysis. The samples were centrifuged 10 min at 2000×g and the supernatant immediately frozen for later analysis. Urea was assayed by the diacetylmonoxime method described in Price and Harrison . The turnover rate of urea was calculated by means of three different isotope-dilution models to evaluate the error made by using a nonvalid model. Since Glibert et al.  and others already have made extensive reviews of isotope-dilution models, the three models will only be discussed briefly. Model L" a nonsteady-state model. The model applied for this situation is in principle that of Blackburn . The pool size of urea, Pt, changes linearly with time, t, with the rate of change in the urea pool, C, as the slope and the initial pool size, Po, as the intercept:
If the urea pool can be assumed to change linearly with time, the model predicts: ln(R) = ln(Ro)- (d/C) ln[(C, t + Po)/Po],
where R is the %o 14C-urea in the sample at a given time, - (d/C) the slope of the plot and the natural logarithmn of the % 14C-urea initially injected, ln(Ro), the intercept. The d, which in the Blackburn study  represented the gross-production rate, is in this example the turnover rate o f urea as the % 14C-urea was calculated from the measured production of 14CO2.Model IL" a steady-state model where the pool size of urea is assumed to remain constant and the ~4C-urea pool to decrease exponentially with time. The turnover-rate constant, - k , is the slope of a semilogarithmic plot of % 14C-urea vs. time and the actual turnover rate of urea - k . u r e a - p o o l size. Model III: a steady-state model where the pool size of urea is assumed to remain constant and the ~4C urea pool to decrease linearly with time, provided that the incubation time is short enough to ensure isotope dilution to be negligible. Results
Sediment characteristics A summary of sediment specific density, water content and macrofauna biomass is shown in Table 2. The specific density increased almost linearly from the sediment surface down to 12 cm whereas the water content decreased with sediment depth. The upper 4 cm of the sediment was dominated by bivalves, with the greatest biomass in the upper 2 cm. Macrofauna was not observed in the 4 - 6 - c m fraction. Polychaetes were the dominating group of macrofauna in the 6 - 1 2 - c m stratum.
Recovery of 14C in samples (14C-urea + 14C02) The total recovery of 14Cfrom the sediment could be considered to be 100% in all fractions during the time span of incubation (data not shown). On an earlier occasion, cores were injected for the determination of 14C-urea recovery at time zero. These results demonstrated a 100%0 recovery of 14C-urea. The amount o f 14CO2in the 14C-
302 TABLE 2 S E D I M E N T SPECIFIC DENSITY, P O R E W A T E R C O N T E N T A N D M A C R O F A U N A L BIOMASS Depth (cm)
Specific density ( g . c m - 3)
Porewater content ( m l - g - i)
Bivalve biomass (mg f w - c m - 3)
Polychaete biomass (mg f w . c m - 3)
O- 2 2- 4 4- 6 6- 8 8-10 10-12
1.44 1.57 1.65 1.77 1.77 1.95
0.55 0.43 0.31 0.30 0.30 0.24
_+ 0.04 + 0.09 _+ 0.05 + 0.04 _+ 0.05 + 0.04
+_ _+ +_ _+ + _+
0.04 0.01 0.05 0.02 0.02 0.03
2.58 2.82 1.84
9.2 c c
Incubation time, min
l n l ( C - t + Po)/Po)
40 20 0 i
Incubation time, min
Incubation time, min
Fig. 2. (a) Change in urea pool with time, slope, gives rate of change, C, in pool. (b) Plot of ln(R) against ln[(C, t + Po)/Po]. Slope gives turnover rate, d, divided by rate of net change in urea pool, C. (c) Change in 14C-urea pool with time, slope o f this plot is turnover-rate constant, - k . (d) Plot of 1 4 C O 2 production vs. incubation time.
a b c d e
0.27 +_ 0.03
_+ + + + ± ±
-16.6 -15.5 - 8.2 - 8.6 1.4 -20.3 0.4
5.9 13.2 5.1 2.8 2.9 9.5
0.72 0.31 0.91 0.76 0.07 0.60
_+ + + + _+ ±
26.9 26.2 11.7 23.4 17.4 16.3 0.9
10.7 23.0 4.1 7.8 37.2 7.7
( n m o l . c m - 3 . d - 1)
Model I ratea
0.91 0.95 0.93 0.99 0.95 1.00
U r e a - t u r n o v e r r a t e c a l c u l a t e d f r o m M o d e l I. S q u a r e d c o r r e l a t i o n c o e f f i c i e n t f o r l i n e a r r e g r e s s i o n o f In(R) vs. l n [ ( C . t + P o ) / P o ] . U r e a - t u r n o v e r r a t e c a l c u l a t e d f r o m M o d e l II. U r e a - t u r n o v e r r a t e c a l c u l a t e d f r o m M o d e l III. I n t e g r a t e d u r e a - p o o l size g i v e n in m m o l . m - 2 a n d i n t e g r a t e d r a t e s in m m o l . m - 2 - d 1.
1.0 0.5 1.6 0.3 0.3 0.7
+ ± +_ + + +
3.0 1.9 1.8 2.4 1.7 2.9
0- 2 2- 4 4- 6 6- 8 8-10 10-12
Urea pool ( n m o l . c m - 3)
SIZE AND RATES OF UREA TURNOVER
TABLE 3 UREA-POOL
11.7 13.0 9.7 13.5 9.7 6.5
+__ 1.4 + 1.1 __. 1.3 + 1.1 + 1.3 + 0.5 0.96 0.98 0.95 0.98 0.95 0.98
_+ _+ _+ + + _+
12.1 6.3 5.9 4.5 3.3 4.7 2 . 9 _+ 0.3
35.0 25.0 17.5 31.9 16.4 18.6
M o d e l II rate c ( n m o l . c m - 3. d - l)
+ + _+ _+ ± +
26.8 15.5 9.6 13.4 6.1 3.8 3.2 _+ 0 . 7
55.0 34.2 21.4 26.3 12.3 13.0
M o d e l III rate d ( n m o l . c m - s . d - 1)
304 urea used for injection and as standard was insignificant ( < 2 parts per thousand) compared to the amount o f 14C-urea. The 14CO2 background was, nevertheless, always subtracted. Urea turnover
The pool size o f urea varied between 1.7 and 3.0 nmol N . c m -3 in the different sediment strata, with the highest concentration in the 0 - 2 , 6 - 8 and 1 0 - 1 2 - c m fractions (Table 3). The net change in the urea pool with time is shown in Fig. 2a and the rates of net changes, C, for the different sediment layers in Table 2. Data from the 0 - 2-cm fraction are presented in Fig. 2a-d, the other fractions behaved similarly. The net change in the pool size was linear with time for each fraction as assumed by the nonsteady-state Model I. The squared correlation coefficients were > 0.60 in the 0 - 2, 4 - 6, 6 - 8 and 1 0 - 1 2 - c m fractions. The squared correlation coefficients were, however, poorer in the 2 - 4 and the 8 - 10-cm fractions (Table 3). There was a significant decrease in the pool size of urea with time in all sediment strata, except in the 8 - 10-cm layer where the pool
g o E
Biomass. mg fw cm-3
Biomass, mg fw cm-s Fig. 3. Turnover rate of urea, based on nonsteady-state Model I, vs. fresh weight of macrofauna. Turnover rate for 4 - 6 - c m sediment fractions, without macrofauna, is included in both figures. (a) Strata dominated by bivalves and (b) strata dominated by polychaetes.
305 size increased slightly with time. In Model I, the urea-turnover rate was calculated from a plot of ln(R) vs. ln[(C.t + Po)/Po], Fig. 2b, r E=0.91. The turnover rates and the squared correlation coefficients for the plots are given in Table 3. The turnover rate was decreased from the surface down to 6 cm and increased to a second maxima in the 6 - 8 - c m stratum. The average turnover time of the urea pool was 2.9 h. In Model II, the rate constant - k (ll.7-d -l, ra=0.96, Fig. 2c) was calculated from a plot of ln(14C-urea) against time. The rate constant varied between 6.5 and 13.5 d -1 (r E> 0.95, Table 3). The turnover rate showed the same tendencies with depth as the rates calculated by the valid Model I. The integrated ~ 0 - 1 2 cm Model II-based rate was, however, slightly higher (2.9 m m o l - m - 2 . d -1) than the Model I-based value (2.4 m m o l . m - 2 . d - 1 ) . In Model III, the production of 14CO2 was assumed to increase linearly with time within the first 29 min of incubation. The production of 14CO2relative to the amount of lgC-urea initially injected in the cores is shown in Fig. 2d. When the nonvalid Model III was applied to the data the turnover rates showed the same tendencies with sediment depth as the rates derived by the two other models. The overestimation of the actual turnover rate was, however, larger than the Model II-derived rate, T 0 - 1 2 cm = 3.2 m m o l . m -2. d - I (Table 3). The turnover rate of urea, based on Model I, was significantly correlated, r E= 0.6, with the biomass of bivalves in the upper 6 cm of the sediment (Fig. 3a) and with the polychaete biomass in the 6 - 1 2 cm stratum (Fig. 3b, rE = 0.7). Discussion There are several methods for the collection of 14CO2for radiotracer analysis [e.g., 21-27]. The method used has an advantage over the Ansb~ek and Blackburn  and the Lidstrom and Somers  methods for carbonated particulates. In using only the porewater extracts for 14Canalysis, we avoid acid addition to carbonated sediments which causes vigorous CO2 production. Furthermore, the counting efficiency, without using the automatic efficiency-tracing D P M correction program, was greater for samples containing filter-paper + P E A (93%, data not shown) than the efficiency Brown  obtained in a test experiment with PEA. Brown  pointed out that a low counting efficiency can be due to masking effects by the filter-paper, that the complex of P E A with CO2 has a low solubility in toluene, and that P E A darkens on storage with a resultant increase in quenching. The solubility problem may have been overcome in our experiments by adding the filter-paper strips to solvent EE, soluble in toluene, before the addition of the toluene-based scintillation fluid. All the abovementioned problems were, however, overcome by use of an automatic efficiencytracing D P M correction program for the Packard 2200 CA Tri-Carb liquid scintillation analyzer. The counting efficiency proved to be 100% by use of the program (data not shown). The method is a true-tracer technique. Because of the relatively large urea-pool sizes in marine sediments, it is possible to add the tracer in concentrations much smaller than the concentrations in the sediment. The isotope additions were, in the present study, on the average 12_+1%, within the limits normally considered to be the maximal addition in tracer studies . Urea was not adsorbed or in other ways bound to the sediment during the time span
306 of incubation as demonstrated by the 100% recovery of urea in the extracted porewater. The porewater pool could, therefore, be considered as a free pool available for bacterial degradation and/or turnover by urease in the sediment. If bacteria were responsible for at least part of the urea degradation, it can be concluded that the urea-C was not incorporated into bacterial cells, proved by the 100% recovery of total 14C during the time span of incubation. The 100°70 recovery also implies that the 14C was present only in the urea and the CO 2 pools in the sediment, suggesting that 14C label was not reexcreted in another form. This validates the use of the ~4CO2 production as a measure for the amount of ~4C-urea hydrolyzed. Despite the fact that the pool size of urea normally is 1 0 - 20 times lower than that of N H ~ (Lund and Blackburn, unpubl, data) the present study illustrated that the flow through the urea pool was dynamic as the average turnover time of the urea pool was 2.9 h. We propose urea to be the major source of a m m o n i u m in the study area. Based on similarities between the study area and an area in the Bering Sea (Blackburn and Lund, unpubl, data), we anticipate an ammonium-mineralization rate of 3 0 - 4 0 n m o l - c m -3 .d -~. Compared to an average urea-turnover rate of 27 n m o l N . c m - 3 . d -1, urea could be the most important source for a m m o n i u m production. Since it was only the production of CO 2 that was measured, the fate of the NHgproduced from urea hydrolysis is unknown. There is, however, some evidence from the literature on a preferential uptake of NH~- as N source when both sources are available [12-13]. Furthermore, soil studies demonstrate that extracellular urease is present in a wide variety of soil types, bound to soil colloids and/or dissolved in porewater [e.g., 3 2 - 36]. Petit and coworkers  found that ~ 60% of the total urease activity was extracetlularly bound and that the remainder was composed of intracellular and extracellular-unbound enzyme. It has been shown that some ureolytic bacteria hydrolyze urea to maintain an elevated extracellular p H . The hydrolysis of urea and following excretion of N H 3 is accompanied by a considerable increase in p H since 2 mol N H 3 are formed per mol of urea decomposed. The correlation between the turnover rate of urea and the biomass of macrofauna indicate that the macrofauna might have been responsible for the production of urea: directly by excretion and/or indirectly by irrigation of the sediment. For different species of macrofauna, it has been shown that urea can consitute a considerable amount of the total NH~- + urea excreted by the animals (Lund and Henriksen, unpubl. data). The macrofauna could not, however, be the only source for urea production since the turnover rate of urea was also significant in the layer without macrofauna. There was considerable horizontal heterogeneity in urea-turnover rates and the urea pools. H o p k i n s o n  concluded that horizontal heterogeneity is high in bioturbated sediments, explained by patchiness of sediment macrofauna. We find this explanation to be plausible as the study area was bioturbated. The patchiness of sediment fauna can also explain the nonsteady-state conditions observed in the cores during incubation, provided that the fauna were the main cause for the production of urea; i.e., the incubation conditions might have led to the observed decrease in the urea pool with time, by exclusion and/or inactivation of the urea producers. Having this in mind, there is no discrepancy between the higher correlation coefficients obtained for the plots of In(R) vs. time compared to those for the plots of Pt vs. time. The organisms and/or enzymes responsible for the turnover of urea can, due to their size, be expected to be
307 more h o m o g e n e o u s l y distributed t h a n the producers. T h e B l a c k b u r n n o n s t e a d y - s t a t e M o d e l I 0 proved to be a useful tool for the calcul a t i o n o f u r e a - t u r n o v e r rates in the present study. The m o d e l was valid because the urea p o o l c o u l d be considered to change linearly with time in all sediment fractions. The p o o r correlation in the 8 - 1 0 - c m s t r a t u m was due to a slope close to zero. The net c h a n g e was, however, significantly different from zero. The p o o r correlation coefficient (0.31) o b t a i n e d in the 2 - 4 - c m fraction could n o t be explained. T h o u g h a n o n s t e a d y - s t a t e model, u n q u e s t i o n a b l e , is the correct m e t h o d to use for i n c u b a t i o n s where the urea pools change with time, M o d e l II o n l y slightly overestm a t e d the actual t u r n o v e r rate o f urea. We, therefore, conclude that Model II can be used to estimate the t u r n o v e r rate in situations where the change in pool size is n o t m e a s u r e d a n d can be expected to be within the limits that we have measured. At this point, it is i m p o r t a n t to stress that, even t h o u g h the correlation coefficients for the plots, from which the t u r n o v e r c o n s t a n t s were calculated, were highly significant, they do n o t prove M o d e l II to be correct. Like, e.g,, Glibert et al.  we also emphasize the i m p o r t a n c e o f time-course m e a s u r e m e n t s . Since the p r o d u c t i o n o f 14CO2 could be a s s u m e d to change linearly with time d u r i n g the first 29 m i n o f i n c u b a t i o n , the error in use o f Models II a n d III was expected to be almost the same. The severe overest i m a t i o n o f the t u r n o v e r rate, by use o f Model III, m u s t have been due to the lack o f precision i n h e r e n t in a single t i m e - p o i n t m e a s u r e m e n t .
Acknowledgements We t h a n k Stig B a c h m a n n Nielsen for excellent technical assistance; a n d Erik Lorestein a n d Kaj H e n r i k s e n for helpful c o m m e n t s o n the m a n u s c r i p t .
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