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respiration measurement the average individual wet weight was measured by blotting the collected material with a paper towel and weighing on a micro-balance. The ETS
activity was then measured using both the new and the traditional method of Owens and King 1975 using an incubation temperature of 218C for the latter method. A
conversion was made to the in situ temperature using the Arrhenius equation and a value
21
˚ for the energy of activation of 15 kcal mol
see Bamstedt, 1980. ETS activity, using the new method, was also measured on macrozooplankton from
the Barents Sea, collected during summer. Gentle collection with a conical net with a non-filtering cod-end was used. Single individuals of separate species were each placed
into a plastic capsule with 0.3 ml ETS reagent and frozen at 2228C. The material was thawed in the laboratory after 3.5 months and the animals picked up with forceps. The
animals were blotted on a paper towel and weighed see procedure above before being homogenised for ETS analysis. The weight of the animals might be negatively biased
due to loss of water during freezing. This might apply particularly to the hydromedusa, which has the highest water content.
Finally, ETS activity was also measured on POM particulate organic material at different depths in Kosterfjorden, western Sweden. Duplicate water samples of 2 l
volume were filtered through Whatman GF C filters, one was used for POM, the other one for ETS activity. POM was estimated as the weight loss from a dried 608C for 24 h
to an ashed sample 5008C for 24 h. ETS activity was analysed according to the standard procedure see above.
3. Results
3.1. Incubation time Fig. 2A The typical development of formazan in samples with biological material was well
described by a hyperbolic function, indicating that the substrate was gradually used up. An example is given in Fig. 2A, where two different sizes of Praunus flexuosus and eggs
of Cyclopterus lumpus were used. There was a slow but continuous increase also after 1 h incubation for large Praunus, whereas the small sample and the fish eggs showed only
marginal changes with prolonged incubation. The standardised incubation time was set at 60 min.
3.2. Sample preparation Fig. 2B The four different preparations of analytical samples showed rather high within-group
variability and an ANOVA did not reveal any significant treatment effects P . 0.05. However, the restricted results indicated that incubation of cell-free supernatant gave
somewhat lower variability, but also a lower average value than when using crude homogenate cf. I versus II and III versus IV and that use of INT during homogenisa-
tion gave the highest values cf. I and II versus III and IV. For practical reasons, especially during fieldwork, procedure I in Fig. 2B was selected as the standard
procedure for the method.
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Fig. 2. A Biological reduction of INT over time in three different homogenates, showing a logarithmically declining increase in absorbance formazan production. B Results for four different preparation methods for
the analytical sample of ETS activity. I, Crude homogenate and INT during homogenisation. II, Supernatant and INT during homogenisation. III, Crude homogenate and no INT during homogenisation. IV, Supernatant
and no INT during homogenisation. C Temperature dependence of the ETS activity expressed as absorbance.
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3.3. Incubation temperature Fig. 2C The formazan production over 1 h incubation increased with temperature, as shown
both for samples of Praunus flexuosus and samples of Mytilus edulis. The mussel samples increased by a factor of 3.9 from 10 to 408C, whereas the mysid samples
increased by a factor of 2.5 with the same temperature increase. Since the amount of biological material was not standardised, this does not mean anything. A sample
prepared from formalin-preserved Praunus did not show any increase with temperature,
Fig. 3. A ETS activity as absorbance in relation to the amount of biological material. B Effect of concentrating the produced formazan using different volume ratios of homogenate and extraction media.
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and therefore did not differ from the blank without biological material Fig. 2C. An incubation temperature of 408C was chosen as the standard.
3.4. Importance of the amount of biological material in the sample Fig. 3A In the test with different amounts of homogenised Praunus flexuosus there was no
sign of non-linearity Fig. 3A and the high coefficient of determination indicated that formazan production was solely determined by the amount of biological material. The
linearity in the relationship also makes it possible to adjust the concentration of
Fig. 4. A Effects of delayed photometric reading of the analytical samples stored at three different temperatures, approximately 222 frozen, 15 refrigerated and 1208C room temperature. B Effects of
storing live zooplankton before preparing the analytical samples. The ETS activity is expressed as mg O
2 21
21 21
sample , the respiration rate as mg O sample
h . All samples were the same size.
2
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formazan to a suitable level in the spectrophotometric reading by adjusting the final volume after extraction. The results on concentrating the formazan up to four times were
close to the theoretical value Fig. 3B and showed the suitability of the procedure. It is thus possible to increase the sensitivity of the method by concentrating the formazan in a
small final volume and using micro cells in the spectrophotometer.
3.5. Storage of samples Fig. 4A The three sets of processed samples of Praunus flexuosus crude homogenate
incubated and quenched did not show any significant trend with time t-test, P . 0.05 in
Fig. 5. Praunus flexuosus. Respiration rate versus ETS activity of individuals sampled at different times in summer autumn and either kept at in situ temperature or at a somewhat higher or lower temperature in
September. Average respiration ETS ratios are shown above the graph 6S.D..
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251 Fig. 6. Praunus flexuosus. Effects of different food and temperature treatments on the ETS activity. A,C ETS per individual versus individual wet weight.
B,D Weight-specific ETS activity versus individual wet weight. See Table 1 for an explanation of the groupings.
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all cases. Frozen, refrigerated and 208C samples showed average absorbance values of 0.40460.009 695 confidence interval, 0.39760.014, and 0.38960.019, respectively,
and the overlapping confidence intervals thus indicate no significant differences. Thus, once the samples have been quenched with the stop solution they can be stored for a
long time even at room temperature in airtight tubes which prevent evaporation.
3.6. Effects of delayed sampling of material Fig. 4B Samples over a 3-day period from the mixed zooplankton showed random variability
in ETS over time, and the slight negative slope was not significant t-test on the correlation coefficient, P . 0.05. The respiration rate measured on a few occasions
showed no clear trend with time and the variability was even higher than for ETS.
3.7. Effects of changes in environmental variables Figs. 5 and 6 The linear relationship between respiration rate and ETS activity of Praunus flexuosus
was fairly close, explaining 83 of the variability in the total material Fig. 5. Animals kept at a natural temperature showed significant differences between months, with
lowest R ETS ratios in August 2.31 and highest in September 4.53. A lowered or increased temperature in September gave a lower R ETS ratio than at the in situ
temperature, 3.39 and 2.53, respectively.
In the first set of experiments where temperature, food and light were manipulated I–IV, the results for the groups did not diverge much Fig. 6A,B and 81 of the total
variation in ETS was explained by its relation to body weight. The low temperature treatment showed the highest slope and lowest intercept and starving animals gave the
lowest slope and highest intercept Table 2. The second experimental series showed considerably more variability Fig. 6C,D, with the general regression equation Y 5
0.842
0.296X explaining 60 of the total variability in the data. However, by dividing the
material into two subgroups groups a–c and groups d and e, respectively the two separate regression equations explained 87 and 92 of the total variation, respectively.
Table 2
b
Regression parameters in power equations Y 5 aX describing the relationship between ETS activity and individual body wet-weight of Praunus flexuosus used in experiments where effects of changes in the
environment were studied. See Fig. 3A for plots and general regressions
2
Treatment group n
a b
r I
8 0.266
0.962 0.964
II 8
0.140 1.047
0.703 III
8 0.283
0.909 0.962
IV 8
0.502 0.715
0.891 a
8 0.278
0.900 0.819
b 8
0.466 0.783
0.957 c
8 0.401
0.817 0.875
d 7
0.045 1.282
0.917 e
7 0.181
0.873 0.968
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Fig. 7. Palaemon adspersus and Praunus flexuosus. A ETS activity versus individual wet weight. The material was divided into two subgroups, characterised by different activity weight relationships. B
Respiration rate versus individual wet weight. Same subgroupings as in A. The respiration ETS ratio also shows the subgrouping of small and large individuals.
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Fig. 8. Macrozooplankton from Kosterfjorden, Sweden. A Respiration rate versus individual wet weight. B ETS activity measured by the new method ETS versus individual wet weight. C ETS activity measured by
1
the standard method ETS versus individual wet weight. Groupings are the same in the three graphs and
2
represent crustaceans 1, semi-gelatinous species 2, and gelatinous species 3.
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Fig. 9. Ratios between the three metabolic measurements shown in Fig. 8.
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These subgroups represented two different acclimation periods Table 1. There were no consistent patterns of temperature effect within the two subgroups Fig. 6, Table 2.
3.8. Respiration and ETS activity in natural populations Figs. 7 –10 The two crustaceans Palaemon adspersus and Praunus flexuosus, showed variable
results both in respiration rate and ETS activity Fig. 7, but there was a clear separation
Fig. 10. Macrozooplankton from the Barents Sea. ETS activity and weight-specific ETS activity in relation to individual wet weight. The hydromedusa was not included in the regression equation due to its high water
content.
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257 Table 3
21 21
Average respiration rate mg O mg wet weight h
, ETS activity and R ETS ratio of 10 macrozooplankton species. ETS and ETS are the new and old
2 1
2 21
21 21
methods, respectively, with activity expressed as mg O mg wet weight and mg O mg
wet weight h , respectively. CV, coefficient of variation5S.D.
2 2
mean3100. In situ t indicates the calculated R ETS with ETS converted to in situ activity using the Arrhenius equation
2 2
Species n Wet weight mg
Respiration rate ETS
ETS R ETS
R ETS
1 2
1 2
Average CV
Average CV Average CV
Average CV Average CV
Average In situ t CV Boreomysis arctica
2 96.8 39.1
0.37 73.0
0.19 n 5 1
0.66 37.9
2.93 n 5 1
0.69 0.158
98.6 Meganyctiphanes
norvegica 8 160.6
59.2 0.27
30.0 0.25
28.4 1.06
34.7 1.09
28.9 0.26
0.052 35.4
Calanus finmarchicus 3 0.79
5.1 0.28
14.3 0.18
2.5 2.17
62.7 1.71
7.8 0.20
0.045 90.7
Chiridius armatus 1 2.7
0.40 0.21
1.69 1.91
0.24 0.055
Euchaeta norvegica 1 7.1
0.30 0.15
1.51 1.97
0.20 0.045
Metridia longa 3 1.4
10.8 0.37
6.8 0.18
16.7 2.62
7.6 2.12
11.3 0.14
0.032 13.3
Clione limacina 3 286.7
56.5 0.029
55.2 0.027
32.9 0.36
46.4 1.06
31.4 0.10
0.023 63.5
Pleurobrachia pileus 3 690.2
60.1 0.004
13.0 0.003
10.7 0.014
29.0 1.87
22.1 0.36
0.165 38.9
Sagitta elegans 2 23.5
4.2 0.057
29.8 0.047
20.2 0.64
34.4 1.29
47.6 0.10
0.023 60.0
Tomopteris helgolandica 1 83
0.049 0.035
1.02 1.39
0.05 0.011
Crustaceans six species 0.8–304
0.33 16.6
0.22 29.1
1.62 44.2
1.96 27.9
0.29 0.066
69.5 Semi-gelatinous
three species 23–456
0.045 32.0
0.035 34.0
0.67 49.1
1.25 13.5
0.08 0.018
35.6 Gelatinous one species
322–1140 0.005
0.003 0.014
1.87 0.36
0.082 Total material 10 species
0.8–1140 0.21
74.8 0.15
72.4 1.18
69.8 1.73
31.2 0.23
0.053 79.0
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into two groups, not defined by species. Thus, four individuals of Palaemon and six individuals of Praunus showed very low levels of respiration and ETS, but within-group
relationships similar to the group with higher rates cf. regression parameters in the two group equations, Fig. 7. Since the respiration rate and ETS activity varied synchronous-
ly, the R ETS ratio was rather stable, although there was also a diversification here Fig. 7C. One group could be defined by a body weight smaller than 100 mg, and this
showed an average R ETS ratio of 2.1360.05 95 confidence interval, n 5 29. The other group consisted of larger animals with an average R ETS ratio of 4.1360.36
n 5 10.
Ten species of macrozooplankton showed consistent patterns in respiration rate and ETS activity and when plotted against individual wet weight three subgroups, related to
their water content, could be separated Fig. 8. The crustacean group with lowest water content showed the highest activity level, the ctenophore group with highest water
content the lowest activity level, and between 79 and 98 of the variation was explained by the regression equations. There were no strong differences in variability
between the two ETS methods tested, although the new method gave consistently higher coefficients of determination Fig. 8B,C. Furthermore, differences between individuals
were usually more similar between respiration rate and ETS than between respiration
1
rate and ETS and the visual impression of the whole material indicated a closer
2
relationship between A and B than between A and C in Fig. 8. However, the ratios between respiration rate and ETS activity R ETS were variable for both ETS
methods and did not vary in synchrony, thus causing an increased variability in the ETS ETS ratio Fig. 9. The species-specific activities and ratios are summarised in
1 2
Table 3. Intraspecific variability was not very different for respiration rate and the two ETS methods. The new ETS method gave a range in CV of 3–52 with an average of
23, whereas CV for respiration rate ranged between 7 and 73 with an average of 32 and CV for the old ETS method ranged between 8 and 63, average 36.
However, the intraspecific variation in the ratio between respiration rate and ETS activity differed between the two methods, with CV using the new method showing a
range of 8–48 with an average of 25 and corresponding values with the old method of 13–99, average 57. The multi-species groupings gave a similar impression of
higher variability within groups for the old ETS method, 2.5 times higher for
Table 4
21
Average species-specific ETS activity mg O mg wet weight of Arctic macrozooplankton see Fig. 10 and
2
corresponding weight-specific respiration rate, calculated by applying appropriate R ETS conversion factors
1
from Table 3
Parameter Calanus
C . glacialis
C . glacialis
C . glacialis
Parathemisto Sagitta
Bouganvillea hyperboreus CV
CIV CV
ad. females libellula
elegans sp.
N 4
5 7
5 1
6 2
Average ETS 0.109
0.218 0.211
0.167 0.126
0.092 0.057
S.D. 0.027
0.023 0.112
0.028 0.064
0.001
ETS
Average respiration
0.186 0.372
0.361 0.286
0.247 0.119
0.107 S.D.
0.046 0.039
0.191 0.048
0.083 0.0018
Resp.
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Fig. 11. Vertical profile of particulate organic matter POM, ETS activity and specific ETS activity mg O
2 21
mg POM in Kosterfjorden, Sweden.
crustaceans, 2.6 times higher for semi-gelatinous species and 2.5 times higher for the total material 10 species.
The Arctic zooplankton showed a curvilinear relationship of ETS activity with body weight Fig. 10 with an exponent for the weight dependency of 0.61. By applying the
R ETS ratios for Calanus finmarchicus, crustaceans, Sagitta elegans and Pleurobrach-
1
ia pileus in Table 3 to Calanus spp., Parathemisto libellula, Sagitta elegans and Bouganvillea sp., respectively, the ETS activity can be converted into respiration rate
Table 4. The calculated respiration rate showed a range for the crustacean species of
21 21
0.19–0.37 mg O mg wet weight h
, Sagitta elegans being lower, and the weight-
2
specific rate for the medusa being lowest 0.11 mg O .
2
The vertical profiles of ETS and POM from Kosterfjorden showed a subsurface peak in both ETS and POM at 10 m Fig. 11. Below 25 m, POM increased and ETS
decreased slightly. The resulting ETS activity per mg POM showed a gradual decrease with depth over the full scale Fig. 11.
4. Discussion
