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Effects of toluene on the survival, respiration, and adenylate system of a marine isopod
Volume 10/Number 4/April 1979 The ratio o f P C B s to Z D D T ranged a r o u n d unity in Areas C and D but was higher in Areas A and B. The ratio of D D D to Z D D T decreased going f r o m A r e a A (0.28) to B (0.19) to C (0.10) and to D (0.05). The residue levels seemed to increase with the length o f fish and the lipid content ( r = 0 . 6 0 for PCBs/lipids), but the n u m b e r o f data for each area was not e n o u g h for reliable conclusions. The same is true for seasonal variation, but it appears that the Spring values are the highest. The samples for this work have been collected by Messrs N. Tsimenides, C. Papaconstantinou, and Ch. Daoulas o f the Fisheries Research Section o f the Institute o f O c e a n o g r a p h i c and Fisheries Research, Athens.
Mzieu, C. (1976). Presence de diph~nylpolychlorbs chez certains poissons de l'Atlantique et de la M~diterran6e. Sci. Pdche, 258, 1-11. Franco, J. M. & Fernandez, M. J. (1976). Contaminacion pot PCB y
DDT en el littoral Espanol. In Mesa Redonda III, IIl Symposio "El Agua en la Industria" Madrid, Dec. 1976, pp. 49-67. Giam, C, S., Hanks, A. R., Richardson, R. L., Sackett, W. M. & Wong, M. K. (1972). DDT, DDE and polychlorinated biphenyls in biota from the Gulf of Mexico and Caribbean Sea - 1971. Pestic. Monit. J. 6, 139-143. Harvey, G. R., Miklas, H. P., Bowen, V. T. & Steinhauer, W. G. (1974). Observations on the distribution of chlorinated hydrocarbons in Atlantic Ocean organisms. J. mar. Res. 32,103-118. Holden, A. V. & Marsden, K. (1969). Single-stage clean-up of animal tissue extracts for organochlorine residue analysis. J. ChromatoL, 44, 481--492. Hopkins, T. S. & Coachman, L. K. (1975). Circulation patterns in Saronikos Gulf in relation to the winds. In: Intermediate Technical Report. Environmental Pollution Control Project, Athens. Revelante, N. & Gilmartin, M. (1975). DDT, related compounds and PCBs in tissues of 19 species of northern Adriatic fishes. Investigaci6n Pesq., 39, 491-509. Satsmadjis, J. & Gabrielides, G, P. (1977). Chlorinated hydrocarbons in striped mullet (Mullus barbatus) from Saronikos Gulf. Thalassographica, !, 151-154. Williams, R. & Holden, A. V. (1973). Organochlorine residues from plankton. Mar. Pollut. Bull., 4, 109-111.
0025-326X/79/04014)111 $02.00/0
Marine Pollution Bulletin, Vol. 10, pp. 111-115 i7) Pergamon Press Ltd. 1979. Printed in Great Britain.
Effects of Toluene on the Survival, Respiration, and Adenylate System of a Maline Isopod T O R G E I R B A K K E and H E I N R U N E S K J O L D A L Institute o f Marine Biology, University o f Bergen, N-5065 Blomsterdalen, N o r w a y
Median times to narcotization (ETs0) of Cirolana borealis Lilljeborg by various levels of toluene in seawater were determined. Narcotization quickly became irreversible, and the ETs0 values can be regarded as estimates of median lethal times. Respiratory rate, ATP concentration, and energy charge (E.C.) were not significantly affected by sublethal toluene levels which caused behavioural disturbance. At 12.5 ppm of toluene (nominal concentration; ETs0:69 h) ATP dropped gradually to less than 1% and E.C. to about 50% of the control values during 8 days, after which time all the individuals were dead. At 125 ppm neither ATP concentration nor E.C. changed significantly during 24 h although the animals were inactive after about 1 h and died within I00 h. Due to the high degree of stabilization of adenine nucleotide levels and E.C. during environmental changes, these parameters do not appear useful as general indices of stress in environmental pollution studies. Recently m a n y studies have dealt with the search for practical biological indicators o f stress in marine organisms caused by sublethal levels o f pollutants (Sprague, 1971; Reish, 1972; A n d e r s o n et al., 1974; Moore, 1976; A n d e r s o n , 1977; Percy & Mullin, 1977; and others), recognizing that acute toxicity tests are of limited value in judging effects o f pollutants on organisms in their natural environment. Since an organism m a y
be sensitive to more subtle changes of the environment than can be revealed by monitoring a suspected pollutant, efforts are made to find cytological, physiological, behavioural or other indices which reflect this sensitivity and which are easy to measure. The purpose o f the present w o r k was to investigate the sensitivity o f some metabolic features o f the isopod Cirolana borealis towards lethal and sublethal concentrations o f toluene, one o f the m a j o r a r o m a t i c h y d r o c a r b o n s in oil. The factors studied were respiratory rate, and absolute and relative concentrations o f adenine nucleotides. The effects o f petroleum hydrocarbons on respiratory rate have been studied in several invertebrates and in fish (Brocksen & Bailey, 1973; Gilfillan, 1973; H a r g r a v e & N e w c o m b e , 1973; A n d e r s o n et al., 1974), and the m o s t c o m m o n reaction to increased h y d r o c a r b o n levels seems to be an increase in oxygen c o n s u m p t i o n rate. The adenine nucleotides play an i m p o r t a n t role in most metabolic processes, and the adenylate energy charge ( E . C . = ( [ A T P ] + ½ [ A D P ] ) / ([ATP] + [ADP] + [AMP])) is a p r i m a r y factor regulating the activities o f catabolic and anabolic enzymes (Atkinson, 1969,1971). A l t h o u g h kept within the same narrow limits in a variety of organisms, cells in stationary, scenescent, or resting phase have been shown to have lower E.C. values than actively growing cells ( C h a p m a n etal., 1971; B a l l & Atkinson, 1975; Swedes etal., 1975). 111
Marine PollutionBulletin E.C. has been proposed as an index of pollutant stress in microbial populations (Wiebe & Bancroft, 1975), and has been used as an indicator of salinity stress in an estuarine mollusc (Anon., 1976). Little is known about the toxic effects of toluene and related aromatics on invertebrates. Hydrocarbons are generally lipophilic, and will preferentially associate with lipoidal structures such as the cell membranes (Roubal, 1974; Stegeman, 1977). The resultant effects are determined by the nature and extent of disturbance in the normal functions of the membranes, which again is determined by the types of hydrocarbons and organisms involved.
Materials and Methods Animals Specimens of Cirolana borealis were caught in fishbaited traps at 90 m in Skogsvaag (60°16'N, 05°06'E) and were kept in running seawater (temperature 8 - 1 0 ° C, salinity 33.5-34.50700). Individuals of approximately similar size (100-200 mg ash-free dry weight) were selected, and they were starved for one week prior to the experiments. Toluene Toluene and seawater were mixed in the proportion 5.5 to 10 000 by shaking vigorously with a Gallenkamp bottle shaker for 20 min. This saturated stock solution was kept in closed bottles at 10°C for at least 48 h before use, when the water phase was drained off and diluted to the desired concentration with seawater. The concentrations referred to in the text are nominal and calculated from a solubility of toluene in seawater of 400 ppm (N.A.S., 1975; p. 47). Experiments General procedure-Specimens to be exposed to toluene were kept in 800 ml of the desired toluene-water mixtures in 1 1. Pyrex beakers. These were partly closed with aluminium foil to reduce evaporation of toluene, and placed in running seawater (8-10°C). The medium was changed at least every second day and the animals were not fed during the experiments. ETso values-Groups of 15 individuals per beaker were exposed to 0, 0.0125, 1.25, 5.7, 12.5, 25, and 125 ppm toluene. Two replicate groups were exposed to each concentration. The number of inactive animals were counted in each beaker at regular intervals for 4 days. The data were treated according to the procedure of Sprague (1973) to give the ETs0 values (median effective time) for each of the concentrations causing inactivity. Recovery-Three groups of I0 specimens were exposed to each of the concentrations 125 and 12.5 ppm of toluene, while 3 control groups were kept in clean seawater. At intervals the activity of the animals were determined according to a scale from 5 to 1 : 5 - n o r m a l ; 4 - a c t i v e , but lying on back or side; 3 - o n l y slight movements of the limbs; 2 - i n a c t i v e ; 1 - o b v i o u s l y dead. Each time 2 individuals were selected at random 112
from each of the beakers and transferred to circulating, clean seawater. The activity of these individuals were then determined at intervals using the same activity scale.
Respiration-After 4 days exposure in the ETs0 experiment 5 or 6 pairs of individuals were picked at random from each of the concentrations 0, 0.0125, 1.25, and 5.7 ppm and transferred to 160-190 ml flasks with clean seawater. The flasks were immersed open in running seawater, and after 1 h gently flushed and stoppered. Four flasks without animals were included, and two of these were analysed immediately for oxygen by the standard Winkler method. The remaining flasks were analysed after 2 h. Volume and telson length of the animals were measured and the latter converted to ashfree dry weight by use of a regression based on 50 individuals from the same locality. Oxygen consumption rate for each pair of animals was calculated as yl 02 consumed per h and gram ash-free dry weight. Adenine nucleotides- Adenine nucleotides were extracted by dropping individuals into 40 ml of hot Trisbuffer (96°C, 0.02 M, pH 7.7) and macerating them with forceps. The extracts were analysed for ATP by the luciferase luminescence method and for ADP and AMP by the same method after enzymatic conversion to ATP (Chapman et al., 1971). The measurements were standardized by a linear regression between peak light emission and amount of ATP dissolved in Tris-buffer (0.02 M, pH 7.7). The details of the method have been given by Skjoldal & B~mstedt (1977). After 4 days, survivors of the ETs0 experiment were dropped into liquid nitrogen, freeze-dried, and weighed. The adenine nucleotides were then extracted from 2 or 3 groups from each experimental jar, with 3 individuals in each group. In another experiment 12 groups of 3 individuals in each were exposed to toluene. Six groups were kept in 125 ppm for 1, 2, 3, 4, 8, or 24 h, and 6 groups at 12.5 ppm for 0.5, 1, 2, 4, 6, or 8 days. Three control groups were kept in clean seawater for 1 h, 24 h, and 8 days. At the end of an exposure period the state of the animals was determined, and the adenine nucleotides extracted from single individuals. The amounts of adenine nucleotides are given per individual.
Results and Discussion Survival in toluene The behavioural response of Cirolana borealis when exposed to 5.7 ppm or higher levels of toluene was an immediate increase in swimming activity. Often the animals expelled the stomach content through the mouth, which is a common reaction in this species when handled. Next the animals would lie on their back or side with rapid movements of limbs and mouthparts. The activity gradually decreased to faint movements of the appendages, and eventually ceased completely. The median effective time to complete inactivity (ETs0) was investigated to give an idea of toxicity levels of toluene for C. borealis, as a basis for choice of
Volume 10/Number4/April 1979 TABLE 1 Cirolana borealis. Median effective time (ETs0) to inactivity for different nominal concentrations of toluene in seawater. Bars indicate no visible effects on any individual during 4 days of exposure.
Toluene concentration (ppm)
Median effective time (h)
0
0.0125 1.25 5.7 12.5
400 69
25 125
28 3
concentrations in the other experiments. Table 1 gives the computed ETs0 values for the different toluene levels. The highest concentration tested, 125 ppm, had a median effective time of 3 h, while 5.7 p p m had an ETs0 value of about 400 h. 1.25 and 0.0125 p p m caused no visible effects within 4 days. Recovery after various lengths of exposure to 125 p p m is shown in Fig. 1. One hour caused complete inactivity (a somewhat quicker narcotization than in the ETs0 experiment), but transfer to clean seawater resulted in total recovery within 12 h. Individuals exposed for 2 h showed partial recovery, but for the majority narcotization was irreversible as they died later. (Not shown in the figure.) After 3 and 4 h of exposure the majority died within 100 h, while longer exposure caused death of all individuals within 100 h. When exposed to 12.5 p p m for 18 and 30 h all the individuals showed behavioural disturbance, but none
:t I[ Ih
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became inactive, and all recovered completely (Fig. 2). After 63 h exposure 72°/o of the individuals were inactive. Two of the individuals recovered when transferred to clean water, the remaining died within 2 weeks, although some had recovered partially. After 99 h exposure 2 out of 12 individuals recovered, the others died. The individuals which showed complete recovery in the experiments were observed for 1 m o n t h afterwards and remained normally active. Since inactive specimens at both concentrations of toluene were able to recover, provided the state of narcosis was of short duration (less than 2 h at 125 p p m and about 30 h at 12.5 p p m as inferred from Figs. 1 and 2) the ETs0 values in Table 1 are not strictly equivalent to median lethal times (LTs0). However, the experiments showed that although none of the individuals were dead after the computed median effective times, most of them had been irreversibly affected and would die later. The ETs0 values can therefore be taken as conservative estimates of the corresponding LTs0 values.
Respiration There was a slight increase in mean respiratory rate in the animals exposed to 0.0125 and 1.25 ppm, and a decrease in those exposed to 5.7 p p m compared to the controls, but the standard deviations of the means were much too large to make these differences statistically significant (Table 2). Size variation within the groups was restricted (Table 2) and did not explain much of the variation in respiratory rates. Although 4 of the 10 individuals exposed to 5.7 p p m showed significantly reduced activity at the start of the oxygen uptake measurements, their respiratory rates were within the range of the control values. Thus a significant influence of toluene on behaviour was not accompanied by any marked change in respiration. One cannot exclude the possibility that respiration was affected during exposure since the measurements were made after transfer to clean water, but when the same method was applied by Anderson et al. (1974), it revealed effects of oil on the
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Fig. 1 Cirolana borealis. Activity of individuals during exposure to 125
ppm of toluene (e), and during subsequent transfer to clean seawater (O). The points represent the median activity of 6 individuals, and the vertical bars denote the range of variation. The activity scale is defined in the section on materials and methods. Exposure time is indicated on each graph. Note the logarithmic time scale.
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Fig. 2 Cirolana borealis. Activity of individuals during exposure to 12.5 p p m of toluene (e), and during subsequent transfer to clean seawater (O). See legend to Fig. 1 for further details. In the graph for 99 h exposure the points represent the median of 12 individuals.
113
Marine Pollution Bulletin
TABLE 2 Cirolana borealis. Mean oxygen consumption rates of pairs of individuals after exposure to different concentrations of toluene for 4 days. SD = standard deviation of the mean; n = number of measurements. Toluene Oxygen consumption rate concentration (ul O 2 h -1 g-l) (ppm) mean n SD
Individual weight (mg ash-free dry wt.) mean n SD
0 0,0125 1,25 5,7
183.38 165.27 158.19 164.37
181.00 184.80 210.40 166.64
6 6 5 5
56.10 54.27 68.80 25.65
12 12 10 10
40.47 13.11 20.49 52.07
TABLE 3 Cirolana borealis. ATP concentration and energy charge in groups of individuals exposed for 4 days to different concentrations of toluene. SD = standard deviation for n analyzed extracts, each from 3 individuals. Toluene concentration (ppm)
ATP (/tg mg -I dry wt.) mean n SD
Energy charge mean n SD
0 0,0125 1.25 5.7
0.34 0.35 0.42 0.49
0.67 0.63 0.65 0.69
6 5 6 6
0.04 0.06 0.10 0.13
5 5 6 6
0.05 0.08 0.07 0.09
al., 1974; Swedes et al., 1975). This is thought to reflect that E.C. is far more important in metabolic regulation than the ATP concentration (Swedes et al., 1975; Atkinson, 1971, 1976). The stabilization of E.C. when ATP decreases, is accomplished through degradation of AMP, thereby reducing the total adenine nucleotide pool (Chapman & Atkinson, 1973; Schramm & Lazorik, 1975). We observed a strong decrease in the total adenine nucleotide pool in the experiment shown in Fig. 3, leading to a less marked drop in E.C. The biochemical implications of this will be discussed elsewhere (Skjoldal & Bakke, 1978). Due to these properties of the adenylate system, ATP is expected to be a more sensitive parameter reflecting severe metabolic stress than is E.C. It is dangerous to generalize from experiments with one organism and one toxic substance. However, many studies, mostly with microorganisms and algae, have revealed a marked stabilization of the adenine nucleotide levels and E.C., despite drastic variations in the environment and in the degree of stress caused by substrate and nutrient depletion (see Niven et al., 1977, and references therein). We therefore think it justified to conclude that, due to the inherent nature of the adenylate system, concentrations of adenine nucleotides and E.C. are not suitable as general stress parameters in environmental
respiration of Penaeus aztecus postlarvae and of Mysidopsis almyra. The choice of temperature may also have influenced the response in C. borealis as Hargrave & Newcombe (1973) found a significant increase in respiratory rate by Littorina littorea at 18°C, but not at 4°C, when subjected to Bunker C oil. The measurements of C. borealis were carried out at 10°C, which was the approximate in situ temperature of the environment. Our results demonstrate that respiratory rate is not a sensitive indicator of stress caused by toluene in this species.
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A TP and energy charge Exposure for 4 days to toluene at 0.0125, 1.25 and 5.7 ppm had no significant effect on either the ATP concentration or E.C. (Table 3). Exposure to 12.5 ppm of toluene (ETs0 = 69 h; Table 1) led to a progressive decrease of ATP and E.C. during 8 days (Fig. 3). After 72 h ATP was markedly lower than the controls, while E.C. was only slightly reduced. After 8 days, when all the individuals were dead, ATP had dropped to less than 1% of the control values and E.C. was reduced to about 0.4. When exposed to 125 ppm of toluene (ET50 = 3 h; Table 1), the ATP content changed little during 24 h. E.C. showed a slight, but statistically insignificant, decrease during the same period (Fig. 4). These results show that neither ATP nor E.C. reflected the stress caused by sublethal concentrations of toluene. Furthermore, neither parameters had a better sensitivity than the inactivity criterion when exposed to 12.5 ppm, and they failed to reveal the quicker narcotization and death caused by exposure to 125 ppm. They are therefore not suitable as general indicators of toxic stress in C. borealis. E.C. is normally under strict regulation in the cells (Atkinson, 1971; Chapman et al., 1971), and can be stabilized despite changes in the ATP level (Dietzler et
114
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Fig. 3 Cirolana borealis. Individual content of ATP (A) and energy charge (B) during 8 days of exposure to 12.5 ppm of toluene. • individuals exposed to toluene; O-individuals kept in seawater without toluene. Each point represents one individual. I0 O8 o
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Cirolana boreafis. E n e r g y c h a r g e o f i n d i v i d u a l s d u r i n g 24 h o f exposure to 125 ppm o f toluene. Symbols are as in Fig. 3.
Volume 10/Number 4/April 1979
pollution studies. Since some specialized organisms have been shown to respond to environmental stress with a marked lowering in E.C. with little loss in viability (Ball & Atkinson, 1975; Skjoldal et al., 1977), this does not exclude the possibility of using E.C. as a stress parameter in special cases, where the responses of the particular organism(s) to the particular substance(s) have been thoroughly investigated. We thank Dr. J. Widdows, N.E.R.C. Institute for Marine Environmental Research, PLymouth, for correcting the English text, and the Institute of Physiology, University of Bergen, for providing facilities for doing the ATP analysis. Anderson, J. W. (1977). Responses to sublethal levels of petroleum hydrocarbons: are they sensitive indicators and do they correlate with tissue contamination? I n Fate and Effects o f Petroleum Hydrocarbons in Marine Organisms and Ecosystems, D. A. Wolfe (ed.), pp. 95-114. New York, Pergamon Press. Anderson, J. W., Neff, J. M. & Petrocelli, S. R. (1974). Sublethal effects of oil, heavy metals and PCBs on marine organisms. In Survival in Toxic Environments, M. A. Z. Khan & J. P. Bederka (eds.), pp. 83-121. New York, Academic Press. ~,non. (1976). Estuarine project. Progress report 1974-1976. CSIRO, Division of Fisheries and Oceanography. 83 pp. Atkinson, D. E. (1969). Regulation of enzyme function. A. Rev. Microbiol., 23, 47-68. ~tkinson, D. E. (1971). Adenine nucleotides as stoichiometric coupling agents in metabolism and as regulatory modifiers: the adenylate energy charge. In Metabolic regulation, H. J. Vogel (ed.), pp. 1-21. New York, Academic Press. Atkinson, D. E. (1976). Adaptations of enzymes for regulation of catalytic function. Biochem. Soc. Syrup., 41,205-223. Ball, W. J. & Atkinson, D. E. (1975). Adenylate energy charge in Saccharomyces cerevisiae during starvation. J. Bacteriol., 121, 975 -982. Brocksen, R. W. & Bailey, H. T. (1973). Respiratory response of juvenile chinook salmon and striped bass exposed to benzene, a watersoluble component of crude oil. In Joint Conference on Prevention and Control of O i l Spills, pp. 783-792. American Petroleum Institute, Washington, D.C. Chapman, A. G. & Atkinson, D. E. (1973). Stabilization of energy charge by the adenylate deaminase reaction. J. biol. Chem., 248, 8309-8312. Chapman, A. G., Fall, L. & Atkinson, D. E. (1971). Adenylate energy charge in Escherichia coli during growth and starvation. J. Bact., 108, 1072-1086. Dietzler, D. N., Lais, C. J., Magnani, J. L. & Leckie, M. P. (1974). Maintenance of the energy charge in the presence of large decreases in the total adenylate pool of Escherichia coli and concurrent changes in
glucose-6-P, fructose-P 2 and glycogen synthesis. Biochem. biophys. Res. Commun., 60, 875-881. Gilfillan, E. S. (1973). Effects of seawater extracts of crude oil on carbon budgets in two species of mussels. In Joint Conference on Prevention and Control o f Oil Spills, pp. 691-695. American Petroleum Institute, Washington, D.C. Hargrave, B. T. & Newcombe, C. P. (1973). Crawling and respiration as indices of sublethal effects of oil and a dispersant on an intertidal snail Littorina littorea. J. Fish. Res. Bd Can., 30, 1789-1792. Moore, M. N. (1976). Cytochemical demonstration of latency of lysosomal hydrolases in digestive cells of the common mussel Mytilus edulis, and changes induced by thermal stress. Cell Tissue Res., 175, 279-287. N.A.S. (1975). Petroleum in the marine environment. National Academy of Science, Washington, D.C. Niven, D. F., Collins, P. A. & Knowles, C. J. (1977). Adenylate energy charge during batch culture of Beneckea natriegens. J. gen. MicrobioL, 98, 95-108. Percy, J. A. & Mullin, T. C. (1977). Effects of crude oil on the locomotory activity of arctic marine invertebrates. Mar. Pollut. Bull., 8(2), 35-40. Reish, D. J. (1972). The use of marine invertebrates as indicators of varying degrees of marine pollution. In Marine Pollution and Sea Life, M. Ruivo (ed.), pp. 203-207. FAO, London, Fishing News (Books) Ltd. Roubal, W. T. (1974). Spin-labeling of living t i s s u e - a method for investigating pollutant-host interaction. In Pollution and Physiology o f Marine Organisms, F. J. Vernberg & W. B. Vernberg (eds.), pp. 367-379. New York, Academic Press. Schramm, V. L. & Lazorik, F. C. (1975). The pathway of adenylate catabolism in Azotobacter vinelandii. Evidence for adenosine monophosphate nucleosidase as the regulatory enzyme. J. biol. Chem., 250, 1801-1808. Skjoldal, H. R. & Bakke, T. (1978). Relationship between ATP and energy charge during lethal metabolic stress of the marine isopod Cirolana borealis. J. biol. Chem., 253, 3355-3356. Skjoldal, H. R. & B,~mstedt, U. (1977). Ecobiochemical studies on the deep-water pelagic community of Korsfjorden, western Norway. Adenine nucleotides in zooplankton. Mar. Biol., 42, 197-211. Skjoldal, H. R., Kumazawa, S. & Mitsui, A. (1977). Dark hydrogen production and adenylate energy charge of tropical marine blue-green algae. A bstr. A. Meet. Am. Soc. Microbiol. Sprague, J. B. (1971). Measurement of pollutant toxicity to f i s h - I I I . Sublethal effects and "safe" concentrations. WaterRes., 5,245-266. Sprague, J. B. (1973). "The ABC's of pollutant bioassay using fish", biological methods for the assessment of water quality. A S T M STP, 528, 6-30. American Society for Testing and Materials. Stegeman, J. J. (1977). Fate and effects of oil in marine animals. Oceanus, 20(4), 59-66. Swedes, J. S., Sedo, R. J. & Atkinson, D. E. (1975). Relation of growth and protein synthesis to the adenylate energy charge in an adeninerequiring mutant of Escherichia coil J. biol. Chem., 250, 6930-6938. Wiebe, W. J. & Bancroft, K. (1975). Use of the adenylate energy charge ratio to measure growth state of natural microbiological communities. Proc. natn. Acad. Sci. U.S.A., 72,2112-2115.
115
Mzieu, C. (1976). Presence de diph~nylpolychlorbs chez certains poissons de l'Atlantique et de la M~diterran6e. Sci. Pdche, 258, 1-11. Franco, J. M. & Fernandez, M. J. (1976). Contaminacion pot PCB y
DDT en el littoral Espanol. In Mesa Redonda III, IIl Symposio "El Agua en la Industria" Madrid, Dec. 1976, pp. 49-67. Giam, C, S., Hanks, A. R., Richardson, R. L., Sackett, W. M. & Wong, M. K. (1972). DDT, DDE and polychlorinated biphenyls in biota from the Gulf of Mexico and Caribbean Sea - 1971. Pestic. Monit. J. 6, 139-143. Harvey, G. R., Miklas, H. P., Bowen, V. T. & Steinhauer, W. G. (1974). Observations on the distribution of chlorinated hydrocarbons in Atlantic Ocean organisms. J. mar. Res. 32,103-118. Holden, A. V. & Marsden, K. (1969). Single-stage clean-up of animal tissue extracts for organochlorine residue analysis. J. ChromatoL, 44, 481--492. Hopkins, T. S. & Coachman, L. K. (1975). Circulation patterns in Saronikos Gulf in relation to the winds. In: Intermediate Technical Report. Environmental Pollution Control Project, Athens. Revelante, N. & Gilmartin, M. (1975). DDT, related compounds and PCBs in tissues of 19 species of northern Adriatic fishes. Investigaci6n Pesq., 39, 491-509. Satsmadjis, J. & Gabrielides, G, P. (1977). Chlorinated hydrocarbons in striped mullet (Mullus barbatus) from Saronikos Gulf. Thalassographica, !, 151-154. Williams, R. & Holden, A. V. (1973). Organochlorine residues from plankton. Mar. Pollut. Bull., 4, 109-111.
0025-326X/79/04014)111 $02.00/0
Marine Pollution Bulletin, Vol. 10, pp. 111-115 i7) Pergamon Press Ltd. 1979. Printed in Great Britain.
Effects of Toluene on the Survival, Respiration, and Adenylate System of a Maline Isopod T O R G E I R B A K K E and H E I N R U N E S K J O L D A L Institute o f Marine Biology, University o f Bergen, N-5065 Blomsterdalen, N o r w a y
Median times to narcotization (ETs0) of Cirolana borealis Lilljeborg by various levels of toluene in seawater were determined. Narcotization quickly became irreversible, and the ETs0 values can be regarded as estimates of median lethal times. Respiratory rate, ATP concentration, and energy charge (E.C.) were not significantly affected by sublethal toluene levels which caused behavioural disturbance. At 12.5 ppm of toluene (nominal concentration; ETs0:69 h) ATP dropped gradually to less than 1% and E.C. to about 50% of the control values during 8 days, after which time all the individuals were dead. At 125 ppm neither ATP concentration nor E.C. changed significantly during 24 h although the animals were inactive after about 1 h and died within I00 h. Due to the high degree of stabilization of adenine nucleotide levels and E.C. during environmental changes, these parameters do not appear useful as general indices of stress in environmental pollution studies. Recently m a n y studies have dealt with the search for practical biological indicators o f stress in marine organisms caused by sublethal levels o f pollutants (Sprague, 1971; Reish, 1972; A n d e r s o n et al., 1974; Moore, 1976; A n d e r s o n , 1977; Percy & Mullin, 1977; and others), recognizing that acute toxicity tests are of limited value in judging effects o f pollutants on organisms in their natural environment. Since an organism m a y
be sensitive to more subtle changes of the environment than can be revealed by monitoring a suspected pollutant, efforts are made to find cytological, physiological, behavioural or other indices which reflect this sensitivity and which are easy to measure. The purpose o f the present w o r k was to investigate the sensitivity o f some metabolic features o f the isopod Cirolana borealis towards lethal and sublethal concentrations o f toluene, one o f the m a j o r a r o m a t i c h y d r o c a r b o n s in oil. The factors studied were respiratory rate, and absolute and relative concentrations o f adenine nucleotides. The effects o f petroleum hydrocarbons on respiratory rate have been studied in several invertebrates and in fish (Brocksen & Bailey, 1973; Gilfillan, 1973; H a r g r a v e & N e w c o m b e , 1973; A n d e r s o n et al., 1974), and the m o s t c o m m o n reaction to increased h y d r o c a r b o n levels seems to be an increase in oxygen c o n s u m p t i o n rate. The adenine nucleotides play an i m p o r t a n t role in most metabolic processes, and the adenylate energy charge ( E . C . = ( [ A T P ] + ½ [ A D P ] ) / ([ATP] + [ADP] + [AMP])) is a p r i m a r y factor regulating the activities o f catabolic and anabolic enzymes (Atkinson, 1969,1971). A l t h o u g h kept within the same narrow limits in a variety of organisms, cells in stationary, scenescent, or resting phase have been shown to have lower E.C. values than actively growing cells ( C h a p m a n etal., 1971; B a l l & Atkinson, 1975; Swedes etal., 1975). 111
Marine PollutionBulletin E.C. has been proposed as an index of pollutant stress in microbial populations (Wiebe & Bancroft, 1975), and has been used as an indicator of salinity stress in an estuarine mollusc (Anon., 1976). Little is known about the toxic effects of toluene and related aromatics on invertebrates. Hydrocarbons are generally lipophilic, and will preferentially associate with lipoidal structures such as the cell membranes (Roubal, 1974; Stegeman, 1977). The resultant effects are determined by the nature and extent of disturbance in the normal functions of the membranes, which again is determined by the types of hydrocarbons and organisms involved.
Materials and Methods Animals Specimens of Cirolana borealis were caught in fishbaited traps at 90 m in Skogsvaag (60°16'N, 05°06'E) and were kept in running seawater (temperature 8 - 1 0 ° C, salinity 33.5-34.50700). Individuals of approximately similar size (100-200 mg ash-free dry weight) were selected, and they were starved for one week prior to the experiments. Toluene Toluene and seawater were mixed in the proportion 5.5 to 10 000 by shaking vigorously with a Gallenkamp bottle shaker for 20 min. This saturated stock solution was kept in closed bottles at 10°C for at least 48 h before use, when the water phase was drained off and diluted to the desired concentration with seawater. The concentrations referred to in the text are nominal and calculated from a solubility of toluene in seawater of 400 ppm (N.A.S., 1975; p. 47). Experiments General procedure-Specimens to be exposed to toluene were kept in 800 ml of the desired toluene-water mixtures in 1 1. Pyrex beakers. These were partly closed with aluminium foil to reduce evaporation of toluene, and placed in running seawater (8-10°C). The medium was changed at least every second day and the animals were not fed during the experiments. ETso values-Groups of 15 individuals per beaker were exposed to 0, 0.0125, 1.25, 5.7, 12.5, 25, and 125 ppm toluene. Two replicate groups were exposed to each concentration. The number of inactive animals were counted in each beaker at regular intervals for 4 days. The data were treated according to the procedure of Sprague (1973) to give the ETs0 values (median effective time) for each of the concentrations causing inactivity. Recovery-Three groups of I0 specimens were exposed to each of the concentrations 125 and 12.5 ppm of toluene, while 3 control groups were kept in clean seawater. At intervals the activity of the animals were determined according to a scale from 5 to 1 : 5 - n o r m a l ; 4 - a c t i v e , but lying on back or side; 3 - o n l y slight movements of the limbs; 2 - i n a c t i v e ; 1 - o b v i o u s l y dead. Each time 2 individuals were selected at random 112
from each of the beakers and transferred to circulating, clean seawater. The activity of these individuals were then determined at intervals using the same activity scale.
Respiration-After 4 days exposure in the ETs0 experiment 5 or 6 pairs of individuals were picked at random from each of the concentrations 0, 0.0125, 1.25, and 5.7 ppm and transferred to 160-190 ml flasks with clean seawater. The flasks were immersed open in running seawater, and after 1 h gently flushed and stoppered. Four flasks without animals were included, and two of these were analysed immediately for oxygen by the standard Winkler method. The remaining flasks were analysed after 2 h. Volume and telson length of the animals were measured and the latter converted to ashfree dry weight by use of a regression based on 50 individuals from the same locality. Oxygen consumption rate for each pair of animals was calculated as yl 02 consumed per h and gram ash-free dry weight. Adenine nucleotides- Adenine nucleotides were extracted by dropping individuals into 40 ml of hot Trisbuffer (96°C, 0.02 M, pH 7.7) and macerating them with forceps. The extracts were analysed for ATP by the luciferase luminescence method and for ADP and AMP by the same method after enzymatic conversion to ATP (Chapman et al., 1971). The measurements were standardized by a linear regression between peak light emission and amount of ATP dissolved in Tris-buffer (0.02 M, pH 7.7). The details of the method have been given by Skjoldal & B~mstedt (1977). After 4 days, survivors of the ETs0 experiment were dropped into liquid nitrogen, freeze-dried, and weighed. The adenine nucleotides were then extracted from 2 or 3 groups from each experimental jar, with 3 individuals in each group. In another experiment 12 groups of 3 individuals in each were exposed to toluene. Six groups were kept in 125 ppm for 1, 2, 3, 4, 8, or 24 h, and 6 groups at 12.5 ppm for 0.5, 1, 2, 4, 6, or 8 days. Three control groups were kept in clean seawater for 1 h, 24 h, and 8 days. At the end of an exposure period the state of the animals was determined, and the adenine nucleotides extracted from single individuals. The amounts of adenine nucleotides are given per individual.
Results and Discussion Survival in toluene The behavioural response of Cirolana borealis when exposed to 5.7 ppm or higher levels of toluene was an immediate increase in swimming activity. Often the animals expelled the stomach content through the mouth, which is a common reaction in this species when handled. Next the animals would lie on their back or side with rapid movements of limbs and mouthparts. The activity gradually decreased to faint movements of the appendages, and eventually ceased completely. The median effective time to complete inactivity (ETs0) was investigated to give an idea of toxicity levels of toluene for C. borealis, as a basis for choice of
Volume 10/Number4/April 1979 TABLE 1 Cirolana borealis. Median effective time (ETs0) to inactivity for different nominal concentrations of toluene in seawater. Bars indicate no visible effects on any individual during 4 days of exposure.
Toluene concentration (ppm)
Median effective time (h)
0
0.0125 1.25 5.7 12.5
400 69
25 125
28 3
concentrations in the other experiments. Table 1 gives the computed ETs0 values for the different toluene levels. The highest concentration tested, 125 ppm, had a median effective time of 3 h, while 5.7 p p m had an ETs0 value of about 400 h. 1.25 and 0.0125 p p m caused no visible effects within 4 days. Recovery after various lengths of exposure to 125 p p m is shown in Fig. 1. One hour caused complete inactivity (a somewhat quicker narcotization than in the ETs0 experiment), but transfer to clean seawater resulted in total recovery within 12 h. Individuals exposed for 2 h showed partial recovery, but for the majority narcotization was irreversible as they died later. (Not shown in the figure.) After 3 and 4 h of exposure the majority died within 100 h, while longer exposure caused death of all individuals within 100 h. When exposed to 12.5 p p m for 18 and 30 h all the individuals showed behavioural disturbance, but none
:t I[ Ih
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became inactive, and all recovered completely (Fig. 2). After 63 h exposure 72°/o of the individuals were inactive. Two of the individuals recovered when transferred to clean water, the remaining died within 2 weeks, although some had recovered partially. After 99 h exposure 2 out of 12 individuals recovered, the others died. The individuals which showed complete recovery in the experiments were observed for 1 m o n t h afterwards and remained normally active. Since inactive specimens at both concentrations of toluene were able to recover, provided the state of narcosis was of short duration (less than 2 h at 125 p p m and about 30 h at 12.5 p p m as inferred from Figs. 1 and 2) the ETs0 values in Table 1 are not strictly equivalent to median lethal times (LTs0). However, the experiments showed that although none of the individuals were dead after the computed median effective times, most of them had been irreversibly affected and would die later. The ETs0 values can therefore be taken as conservative estimates of the corresponding LTs0 values.
Respiration There was a slight increase in mean respiratory rate in the animals exposed to 0.0125 and 1.25 ppm, and a decrease in those exposed to 5.7 p p m compared to the controls, but the standard deviations of the means were much too large to make these differences statistically significant (Table 2). Size variation within the groups was restricted (Table 2) and did not explain much of the variation in respiratory rates. Although 4 of the 10 individuals exposed to 5.7 p p m showed significantly reduced activity at the start of the oxygen uptake measurements, their respiratory rates were within the range of the control values. Thus a significant influence of toluene on behaviour was not accompanied by any marked change in respiration. One cannot exclude the possibility that respiration was affected during exposure since the measurements were made after transfer to clean water, but when the same method was applied by Anderson et al. (1974), it revealed effects of oil on the
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Fig. 1 Cirolana borealis. Activity of individuals during exposure to 125
ppm of toluene (e), and during subsequent transfer to clean seawater (O). The points represent the median activity of 6 individuals, and the vertical bars denote the range of variation. The activity scale is defined in the section on materials and methods. Exposure time is indicated on each graph. Note the logarithmic time scale.
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Fig. 2 Cirolana borealis. Activity of individuals during exposure to 12.5 p p m of toluene (e), and during subsequent transfer to clean seawater (O). See legend to Fig. 1 for further details. In the graph for 99 h exposure the points represent the median of 12 individuals.
113
Marine Pollution Bulletin
TABLE 2 Cirolana borealis. Mean oxygen consumption rates of pairs of individuals after exposure to different concentrations of toluene for 4 days. SD = standard deviation of the mean; n = number of measurements. Toluene Oxygen consumption rate concentration (ul O 2 h -1 g-l) (ppm) mean n SD
Individual weight (mg ash-free dry wt.) mean n SD
0 0,0125 1,25 5,7
183.38 165.27 158.19 164.37
181.00 184.80 210.40 166.64
6 6 5 5
56.10 54.27 68.80 25.65
12 12 10 10
40.47 13.11 20.49 52.07
TABLE 3 Cirolana borealis. ATP concentration and energy charge in groups of individuals exposed for 4 days to different concentrations of toluene. SD = standard deviation for n analyzed extracts, each from 3 individuals. Toluene concentration (ppm)
ATP (/tg mg -I dry wt.) mean n SD
Energy charge mean n SD
0 0,0125 1.25 5.7
0.34 0.35 0.42 0.49
0.67 0.63 0.65 0.69
6 5 6 6
0.04 0.06 0.10 0.13
5 5 6 6
0.05 0.08 0.07 0.09
al., 1974; Swedes et al., 1975). This is thought to reflect that E.C. is far more important in metabolic regulation than the ATP concentration (Swedes et al., 1975; Atkinson, 1971, 1976). The stabilization of E.C. when ATP decreases, is accomplished through degradation of AMP, thereby reducing the total adenine nucleotide pool (Chapman & Atkinson, 1973; Schramm & Lazorik, 1975). We observed a strong decrease in the total adenine nucleotide pool in the experiment shown in Fig. 3, leading to a less marked drop in E.C. The biochemical implications of this will be discussed elsewhere (Skjoldal & Bakke, 1978). Due to these properties of the adenylate system, ATP is expected to be a more sensitive parameter reflecting severe metabolic stress than is E.C. It is dangerous to generalize from experiments with one organism and one toxic substance. However, many studies, mostly with microorganisms and algae, have revealed a marked stabilization of the adenine nucleotide levels and E.C., despite drastic variations in the environment and in the degree of stress caused by substrate and nutrient depletion (see Niven et al., 1977, and references therein). We therefore think it justified to conclude that, due to the inherent nature of the adenylate system, concentrations of adenine nucleotides and E.C. are not suitable as general stress parameters in environmental
respiration of Penaeus aztecus postlarvae and of Mysidopsis almyra. The choice of temperature may also have influenced the response in C. borealis as Hargrave & Newcombe (1973) found a significant increase in respiratory rate by Littorina littorea at 18°C, but not at 4°C, when subjected to Bunker C oil. The measurements of C. borealis were carried out at 10°C, which was the approximate in situ temperature of the environment. Our results demonstrate that respiratory rate is not a sensitive indicator of stress caused by toluene in this species.
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A TP and energy charge Exposure for 4 days to toluene at 0.0125, 1.25 and 5.7 ppm had no significant effect on either the ATP concentration or E.C. (Table 3). Exposure to 12.5 ppm of toluene (ETs0 = 69 h; Table 1) led to a progressive decrease of ATP and E.C. during 8 days (Fig. 3). After 72 h ATP was markedly lower than the controls, while E.C. was only slightly reduced. After 8 days, when all the individuals were dead, ATP had dropped to less than 1% of the control values and E.C. was reduced to about 0.4. When exposed to 125 ppm of toluene (ET50 = 3 h; Table 1), the ATP content changed little during 24 h. E.C. showed a slight, but statistically insignificant, decrease during the same period (Fig. 4). These results show that neither ATP nor E.C. reflected the stress caused by sublethal concentrations of toluene. Furthermore, neither parameters had a better sensitivity than the inactivity criterion when exposed to 12.5 ppm, and they failed to reveal the quicker narcotization and death caused by exposure to 125 ppm. They are therefore not suitable as general indicators of toxic stress in C. borealis. E.C. is normally under strict regulation in the cells (Atkinson, 1971; Chapman et al., 1971), and can be stabilized despite changes in the ATP level (Dietzler et
114
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Fig. 3 Cirolana borealis. Individual content of ATP (A) and energy charge (B) during 8 days of exposure to 12.5 ppm of toluene. • individuals exposed to toluene; O-individuals kept in seawater without toluene. Each point represents one individual. I0 O8 o
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Cirolana boreafis. E n e r g y c h a r g e o f i n d i v i d u a l s d u r i n g 24 h o f exposure to 125 ppm o f toluene. Symbols are as in Fig. 3.
Volume 10/Number 4/April 1979
pollution studies. Since some specialized organisms have been shown to respond to environmental stress with a marked lowering in E.C. with little loss in viability (Ball & Atkinson, 1975; Skjoldal et al., 1977), this does not exclude the possibility of using E.C. as a stress parameter in special cases, where the responses of the particular organism(s) to the particular substance(s) have been thoroughly investigated. We thank Dr. J. Widdows, N.E.R.C. Institute for Marine Environmental Research, PLymouth, for correcting the English text, and the Institute of Physiology, University of Bergen, for providing facilities for doing the ATP analysis. Anderson, J. W. (1977). Responses to sublethal levels of petroleum hydrocarbons: are they sensitive indicators and do they correlate with tissue contamination? I n Fate and Effects o f Petroleum Hydrocarbons in Marine Organisms and Ecosystems, D. A. Wolfe (ed.), pp. 95-114. New York, Pergamon Press. Anderson, J. W., Neff, J. M. & Petrocelli, S. R. (1974). Sublethal effects of oil, heavy metals and PCBs on marine organisms. In Survival in Toxic Environments, M. A. Z. Khan & J. P. Bederka (eds.), pp. 83-121. New York, Academic Press. ~,non. (1976). Estuarine project. Progress report 1974-1976. CSIRO, Division of Fisheries and Oceanography. 83 pp. Atkinson, D. E. (1969). Regulation of enzyme function. A. Rev. Microbiol., 23, 47-68. ~tkinson, D. E. (1971). Adenine nucleotides as stoichiometric coupling agents in metabolism and as regulatory modifiers: the adenylate energy charge. In Metabolic regulation, H. J. Vogel (ed.), pp. 1-21. New York, Academic Press. Atkinson, D. E. (1976). Adaptations of enzymes for regulation of catalytic function. Biochem. Soc. Syrup., 41,205-223. Ball, W. J. & Atkinson, D. E. (1975). Adenylate energy charge in Saccharomyces cerevisiae during starvation. J. Bacteriol., 121, 975 -982. Brocksen, R. W. & Bailey, H. T. (1973). Respiratory response of juvenile chinook salmon and striped bass exposed to benzene, a watersoluble component of crude oil. In Joint Conference on Prevention and Control of O i l Spills, pp. 783-792. American Petroleum Institute, Washington, D.C. Chapman, A. G. & Atkinson, D. E. (1973). Stabilization of energy charge by the adenylate deaminase reaction. J. biol. Chem., 248, 8309-8312. Chapman, A. G., Fall, L. & Atkinson, D. E. (1971). Adenylate energy charge in Escherichia coli during growth and starvation. J. Bact., 108, 1072-1086. Dietzler, D. N., Lais, C. J., Magnani, J. L. & Leckie, M. P. (1974). Maintenance of the energy charge in the presence of large decreases in the total adenylate pool of Escherichia coli and concurrent changes in
glucose-6-P, fructose-P 2 and glycogen synthesis. Biochem. biophys. Res. Commun., 60, 875-881. Gilfillan, E. S. (1973). Effects of seawater extracts of crude oil on carbon budgets in two species of mussels. In Joint Conference on Prevention and Control o f Oil Spills, pp. 691-695. American Petroleum Institute, Washington, D.C. Hargrave, B. T. & Newcombe, C. P. (1973). Crawling and respiration as indices of sublethal effects of oil and a dispersant on an intertidal snail Littorina littorea. J. Fish. Res. Bd Can., 30, 1789-1792. Moore, M. N. (1976). Cytochemical demonstration of latency of lysosomal hydrolases in digestive cells of the common mussel Mytilus edulis, and changes induced by thermal stress. Cell Tissue Res., 175, 279-287. N.A.S. (1975). Petroleum in the marine environment. National Academy of Science, Washington, D.C. Niven, D. F., Collins, P. A. & Knowles, C. J. (1977). Adenylate energy charge during batch culture of Beneckea natriegens. J. gen. MicrobioL, 98, 95-108. Percy, J. A. & Mullin, T. C. (1977). Effects of crude oil on the locomotory activity of arctic marine invertebrates. Mar. Pollut. Bull., 8(2), 35-40. Reish, D. J. (1972). The use of marine invertebrates as indicators of varying degrees of marine pollution. In Marine Pollution and Sea Life, M. Ruivo (ed.), pp. 203-207. FAO, London, Fishing News (Books) Ltd. Roubal, W. T. (1974). Spin-labeling of living t i s s u e - a method for investigating pollutant-host interaction. In Pollution and Physiology o f Marine Organisms, F. J. Vernberg & W. B. Vernberg (eds.), pp. 367-379. New York, Academic Press. Schramm, V. L. & Lazorik, F. C. (1975). The pathway of adenylate catabolism in Azotobacter vinelandii. Evidence for adenosine monophosphate nucleosidase as the regulatory enzyme. J. biol. Chem., 250, 1801-1808. Skjoldal, H. R. & Bakke, T. (1978). Relationship between ATP and energy charge during lethal metabolic stress of the marine isopod Cirolana borealis. J. biol. Chem., 253, 3355-3356. Skjoldal, H. R. & B,~mstedt, U. (1977). Ecobiochemical studies on the deep-water pelagic community of Korsfjorden, western Norway. Adenine nucleotides in zooplankton. Mar. Biol., 42, 197-211. Skjoldal, H. R., Kumazawa, S. & Mitsui, A. (1977). Dark hydrogen production and adenylate energy charge of tropical marine blue-green algae. A bstr. A. Meet. Am. Soc. Microbiol. Sprague, J. B. (1971). Measurement of pollutant toxicity to f i s h - I I I . Sublethal effects and "safe" concentrations. WaterRes., 5,245-266. Sprague, J. B. (1973). "The ABC's of pollutant bioassay using fish", biological methods for the assessment of water quality. A S T M STP, 528, 6-30. American Society for Testing and Materials. Stegeman, J. J. (1977). Fate and effects of oil in marine animals. Oceanus, 20(4), 59-66. Swedes, J. S., Sedo, R. J. & Atkinson, D. E. (1975). Relation of growth and protein synthesis to the adenylate energy charge in an adeninerequiring mutant of Escherichia coil J. biol. Chem., 250, 6930-6938. Wiebe, W. J. & Bancroft, K. (1975). Use of the adenylate energy charge ratio to measure growth state of natural microbiological communities. Proc. natn. Acad. Sci. U.S.A., 72,2112-2115.
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