Degradation of benzoic acid in the terrestrial crustacean, Oniscus asellus

DEGRADATION OF BENZOIC ACID IN THE TERRESTRIAL CRUSTACEAN, ONISCUS ASELLUS EDWARD NEUHAUSER,CHARLESYOUMELLand ROY HARTEENS~IN School of Biology, Chemistry and Ecology, Science and Forestry,

State University College of Environmental Syracuse, New York 13210, U.S.A.

(Accrgfrd

20 Octohrr

1973)

Summary-The rate of respiration of radioactive CO, from fasting Oniscus asellus L. during 7.5 days was qualitatively similar for ring-labeled and carboxyl-labeled benzoic acid. The rate of respiration of ring-labeled benzoic acid during 7 days was quantitatively similar for isopods that were fed throughout, and had received DO9 fig benzoic l-t4C acid with or without a “load” of 3Opg of unlabeled benzoic acid. The “loaded” animals displayed a qualitative difference in respiring greater quantities of CO, at night vs day. At 15°C in July, 24.4 per cent of the radioactivity from an injected dose of benzoic l-r4C acid was respired over a 7 day period; 1.3 per cent was excreted; I.9 per cent was present as carbonates; 5.4 per cent was ether-extractable, of which 48 per cent was chromatographically accountable as benzotc acid; and 56.7 per cent of the label was retained in the body. Ring-labeled carbon from benzoic acid was incorporated into the tissues of the isopod. An analysis of a hydrolyzate from the soluble cellular fraction showed at least six identifiable amino acids and four unidentifiable components.

INTRODUCTION

Fallen leaves contain large amounts of aromatic compounds (Smith, 1960). Intact wood consists of a very high percentage of lignin, the decay of which results in the release of various aromatic substances (Sarkanen and Ludwig, 1971). Aromatic compounds usually are designated as xenobiotics insofar as animal life is concerned, since the animal is not equipped enzymatically to use the carbon atoms as precursors of protoplasm or as a source of energy. Organic xenobiotics usually are transformed within vertebrates into ionic substances (Williams, 1959) that are excreted by renal excretory mechanisms which are partially understood. In insects, the only other extensively studied taxon, organic xenobiotics usually are transformed into either ionic substances or glucosides (Smith, 1968) both of which are excreted through as yet largely unknown mechanisms. The complete catabolism of organic xenobiotics is attributed almost entirely to microbes and fungi. Several mechanisms of aromatic degradation are known (Dutton and Evans, 1969; Gibson, 1968; Hayaishi and Nozaki, 1969). Soil invertebrates are beneficial organisms to soil because of their activities in triturating fallen leaves. decaying wood and soil per se and because of their dominant role over all other organisms in homogenizing and opening up aeration channels in the soil. However, little is known regarding their capability of oxi101

dizing organic materials of soil. particularly the aromatic components. In this paper we show that benzoic acid is not xenobiotic to the terrestrial isopod; that this organism utilizes. rather than excretes, benzoic acid; that the isopod is able to cleave the aromatic nucleus of benzoic acid and may therefore be capable of utilizing other aromatic substances. We do not attribute the metabolism of benzoic acid per se to the isopod inasmuch as microbial symbionts may initiate ring scission.

MATERIALS AND METHODS

Specimens of Oniscus a.sce//u.s L. were obtained from beneath decaying wood near the College, and were used within one day. Benzoic l-14C acid (ring-labeled), with specific activities of 2.05 and 3.26mC/mM and benzoic 7-14C acid (carboxyl-labeled), with a specific activity of 4.81 mC/mM, were purchased from New England Nuclear, Boston, Mass., and [i4C]carboxyl-labeled Lalanine, L-aspartic acid, L-serine, and L-phenylalanine from Amersham-Searle Corp., Arlington Heights, Illinois. The experiments were designed to investigate the following: (1) the rates at which benzoic acid is metabolized under specified conditions, using respiratory CO, as a criterion; (2) the disposition of the radioactivity of ring-labeled benzoic acid, and especially the percent-

102

EDWAKD

NI~IIHACISEK. CHARLES YOUM~LLSIIK~ Rev HARTKNST~IN

hours Fig. I. Rate of metabolism of benroic I-14C acid (0) and benzoic 7-lJC (0) in 0niscu.s usrlhs exposed at room temperature (about 20°C) to normal diurnal photo variations in October. Each point represents an average countsjmin for four isopods. Each isopod was injected with 14,000 counts/ min of the ring-labeled or 254,200 counts/min of the carboxyl-labeled acid.

age of unreacted

benzoic acid, after 7 days of metabolism; (3) the ability of the isopod to incorporate aromatic carbon into protoplasm, looking at the soluble protein fraction of the isopod as a criterion; and (4) whether fed animals arc more active in metabolizing benzoic acid than fasted animals, and whether either of these organisms can break down trace quantities of radioactively labeled benzoic acid equally rapidly in the presence or absence of a “load” of unlabeled benzoic acid. Each isopod in each experiment was administered 1 ~1 of a solution by injection with a 10~1 Hamilton syringe into the coelomic cavity between two tergites midway along the thorax. The solution contained radioactive benzoic acid in 0, I2 M NaHCO, and 0.12 M KHCO,, with or without 0.24 M unlabeled benzoic acid, brought to pH 7.0 with cone HCl or 10 N NaOH under a glass electrode. The isopods which received the high concentration of unlabeled bcnzoic acid arc referred to as “loaded-isopods”. Following injection, each isopod was placed separately into a 19 x 65mm flask. The flasks were either exposed to diurnal photo fluctuations at room temperature (1%23-C) or placed in the dark in a constant temperature cabinet at 15”C, as indicated. The flask contained either a filter paper floor wetted with lo/t1 of water, or 1 g of washed and ignited sea sand wetted with 200~1 of water. About 1cm* of decaying maple leaf was added to flasks which contained what we refer to as fed isopods. Fasting isopods were not allowed to eat during the interval of examination. Each of the flasks was sealed with a No. 2, one-hole rubber stopper equipped with glass tubing which held a 0.5 x 3.0cm flag of filter paper with 0.01 ml 20 P; NaOH. New stoppers and flags replaced the old ones at intervals indicated under “Results and Discussion”.

Counting was done with a Model 3375 Packard TriCarb Scintillation Spectrometer or Nuclear Chicago Unilux I (Model 6850) with spectrafluor PBD in toluene (data in Fig. 1) or 0.4 g PPO, 5 g POPOP. 6.5 ml monoethanolamine. 500 ml toluenc. plus methanol to 1000 ml (all other data). All data are reported as counts/min. No correction was made for efficiency, which was constantly about 85 per cent with the TriCarb and about 80 per cent with the Nuclear Chicago Unilux I. Quenching was not noticed in any of our procedures. In seeking data on the disposition of the radioactive carbon of ring-labeled benzoic acid over a 7-day metabolic period, five kinds of measurements were made: (1) Respiratory CO? for the 7-day reaction period. (2) Carbonate and bicarbonate CO,. These data were obtained by trapping CO1 after adding 1 ml of concentrated HCl to each of seven flasks containing an isopod that had been oven-dried at 80-C. (3) Unreacted benzoic acid. Groups of seven isopods were homogenized in a mortar with a pestle in 4ml water. The homogenate was transferred quantitatively with two-washes of water into a large test tube. aciditied with I ml concentrated HCl, and extracted three times with 10 ml ethyl ether. Some samples of the ethereal extract were counted directly. Other samples were first treated with excess NaOH, dried, and taken into water. (Preliminary runs with known quantities of the radioactive compound in water resulted in about 90 per cent recovery of the benzoic acid.) To determine what percentage of the ethereal extract was unreacted benzoic acid, samples were spotted on Baker-Flex Silica Gel 1B and chromatographed with the following solvent systems: benzene-acetic acid-water (2: 2: 1); butanol-acetic acid-water (12: 3: 5); and water-cone HLS04 (9: 1). The thin layer strips were cut into I cm pieces, placed resin-side up in scintillation vials, and counted. (4) Residue in the aqueous homogenate of the isopods. Samples of this residue were counted directly. (5) Leaf and fecal material. These materials were extracted with water and ether, as in (3) above, and counted. In seeking information on incorporation of ring carbon into protoplasm, 40 isopods were injected, each with about 65,000 counts/min benzoic l-“YY acid. After 3 days, 25 very active isopods (1.25 g total) were homogenized in 40 ml water with a mortar and pestle. The homogenate was centrifuged at 65.000 rev/min for 30 min in a Spinco L2-65B Ultracentrifuge. The supernatant was acidified with trichloroacetic acid and recentrifuged. Siekevitz’s method (1952) was used to cleanse the precipitate ofextraneous radioactivity. The washed proteinaceous material was hydrolyzed with 6 N HCl at 100 C overnight. The acid was evaporated at 120 C and the residual amino acids were dissolved in 25’>,, methanol. A light brown solution was obtained. After decolorizing it by passage through a @5 x 5cm column of Norit, the material was concentrated by gentle heating. A sample with about 1.3 x lo5 counts,

Degradation

of benzoic

min was chromatographed in two directions on 22 x 22 cm Whatman No. 1 paper with (1) butanol: acetic acid:water (4: t:5), and (2) 75’1, (w/v) phenol: ammonium hydroxide (200: 1). The chromatograms were cut into strips I cm wide and counted with a Packard 7201 Scanner, which also integrated the arca under the peaks, thus providing quantitative data on the unknowns. The chromatograms were reconstructed to determine R, values. Radioactive samples of L-alanine. L-aspartic acid. L-serine. and L-phznylalanine were chromatographed separately to obtain reference R, values.

3600

3200

i I : I

103

acid in a crustacean

i i\

\ I \ t q’\\&

0-

24

46

RESULTS

1 / ‘\ -t 1‘\ + 72

96

120

‘4.‘L-( 144

166

hours Fig. 2. Rate of metabolism of benzoic l-‘T acid in fed and loaded Oniscus clsrl[us in the dark at 15°C in July. -Each point and bar represents \- counts/min 5 SE for 14 isopods that weighed 94.7 + 16 mg and were injected with 44,OOOcounts/min. Data were gathered concurrently with those of Fig. 3. Dark bars: 10 p.m. to 10 a.m.

3600 3200

2400

48

72

96

120

144

166

I-“C acid in fed and at 15°C in July. Each point and bar represent .Tcounts/min f 1 SE for 14 isopods that weighed 92.7 + 16.3 mg and were injected with 44.000 counts/min. Data were gathered concurrently with those of Fig. 2. Dark bars: 10 p.m. to 10 a.m. Fig. 3. Rate of metabolism

non-loaded

of benzoic

Oniscus usellu.s in the dark

DISCC'SSION

Each of the isopods exuded a droplet of fluid upon being injected. We examined this situation to determine what proportion of radioactivity was represented in the exudate. Eight loaded and eight non-loaded isopods were injected, each with 48.000 counts,‘min. The droplets were taken up into filter paper through capillary action and counted. An average of 793 counts;min or 1.65 per cent of the dose was found for the loaded isopods; 750 countsimin or I.56 per cent for the nonloaded. In all of the experiments the exudate was allowed to remain at the site of its formation. In experiments conducted in the fall or spring there was very little mortality, usually from zero to about IO per cent. In the summer. however, isopods brought in from the field and subjected to injection exhibited high mortality. Accordingly, tests were run to determine optimum conditions of temperature and moisture. The best condition for survival was found to be 1 g of sand with 200 ~1 of water at 15°C. A survival rate of 90 per cent was found over a 7-day period of starvation among 120 animals that were sham injected, injected with a load, or injected with a non-load quantity of the bicarbonate solution. The data reported in Figs. 2 and 3. and Tables 3 and 4 are based on groups of 20 isopods. The survival rate ranged from 65 to 95 per cent in these experiments. and 100 per cent for the experiment reported in Fig. 1.

Rute

24

AND

of metaholi.wl

qf herlzoic

wit/

The rate-patterns for cleavage of carbon atoms I and 7 are qualitatively the same. These patterns. which were observed to be the same on five separate occasions (three summer. one fall, one spring). are shown. with data from one experiment, in Fig. I. From this figure, in which the animals were fasted throughout. and from Figs. 2 and 3. in which the animals were fed, it is clear that the rate of catabolism increased initially after injection of the label, reached a maximum after about a day and a half, and then declined to a lower rate. It is likely that the initial high rate is due to the

104

EDWARD N~UHALwt.

Table 1. Disposition

Product

Total amount

MLLL and

Distribution (counts/min’7 isopods)

CO2

injected

*Seven isopods

Yw

of the radloactivc carbon of henroic tion into O,~i.wr.\ c~~//k\*

or treatment

Respiratory CO2 Carbonate, bicarbonate Ethereal extract Residue in organisms Leaf and fecal material

CHARLIT

76.039 59X0 16.971 176.377 4335 279.702

Rev I-“C

HARTLNSTIIN

acid 7 da)s after mjec-

Distribution (“,, of total Injected) 24.4

1.9 5.3 56.7 1.3 89.7

3 13.195

were injected each with 44.741 counts

mln in .luly. 1973.

cleavage of carbon atoms I and 7 in the initial phase of metabolism, before incorporating thcsc carbon atoms into other metabolites. Control flasks were run with each set of experimental flasks. Isopods were omitted, and the filter paper floor or sand was injected with the test label. Periodic analysts of “respiratory” CO2 over the interval of the experiments rcvcalcd onl~ hackground counts. So too, whenever death occurred, trivial to background counts were measured in the flag-traps of the dead organism. In Fig. 2 it may be noted that loaded-isopods expelled greater amounts of radioactive CO, at night, compared to daytime. after the second day. This phenomenon coincides with Cloudsley-Thompson’s (1956) observation that terrestrial isopods display greater locomotory activity at night. vs day. Failure to see this cyclic activity in Fig. 3, where non-loaded isopods were examined, suggests that the amount of benzoic acid prr SCJin the organism had reached a critically low level. where ditrerences could not be discerned.

of ring-labeled benzoic acid was recovered from each of 26 individual earthworms over a reaction period of 3 days. Only trivial amounts of radioactivity were found in the fecal pellets and uneaten leaves. The datum shown in Table I represents the combined counts from ethcreal and aqueous extracts. In conJunction with the experiment reported in Fig. I. in which the animals fasted throughout the cxperimcntal period, and therefore excreted vcrv few pellets, 2.3 per cent of the ring-labeled radioact&ity and 0.05 per cent of the carboxyl-labeled radioactivity were found in the excrement. It is of considerable interest that very little injected benzoic acid W;IS rccovcrcd in the feces (Table I : preceding data in conjunction with Fig. 1). Vertebrates convert ncarlq all administered benzoic acid into one or another peptide derivative or glucuronate (Williams. 1959). Various invertebrates also arc capable of li>rming excretory peptides from aromatic acids (Smith. 1968).

The data in Table 1 are representative of data obtained on three separate occasions with injections of different quantities of material at different times of the year. From this table it may be seen that nearly 25 per cent of the radioactivity was expelled from loaded-isopods as CO,. another 50 per cent was retained in the body, partly in the form of carbonate and bicarbonate, and only 5.4 per cent was extractable with ether. Correcting this latter quantity, by considering the extraction efficiency. results in a value of about 6 per cent. The chromatographic analyses revealed that X7 per cent, 72 per cent, and 48 per cent of the radioactivity coincided with the R, value for benzoic acid in the benzcnc. butanol and sulfuric acid systems, respectively. Taking the smallest of these numbers. we find that 3 per cent of the administered benzoic acid WIS recovered unchanged from the isopod after a 7-day rcaction period. This value is in sharp contrast to work conducted in our laboratory on Lu~~~hric~r.s ~crrc.s//‘is L.. where M. L. Gustcly (personal communication) found that less than I per cent of about 400.000 counts.:min

The data in Table 2 show that the ring carbon of bcnloic acid WIS incorporated into proteins within the isopod. It should be stated hcrc that these data refer strictly to the soluble fraction of the organism. They were not obtained through an examination of the particulatc fraction. which is comprised of microbes, as well as undisrupted isopod cells and organelles. However. although the data provide strong evidence that the aromatic carbon of benzoic acid was incorporated into the isopod’s tissue, there is no reason to exclude the possibility that the initial oxidative steps. through which ring sclssion occurs. are due to microbes. On :I quantitative basis. ncarlq half of the metaholitcs could be identified 21s one amino acid or another. Fibc of thcsc amino acids (aspartic. serine. alanine, argininc and prolinc) ordinarily arc prcscnt along with glycinc and glutamic acid LIS the dominant amino acids in the pool of free amino acids of (‘rustacca (Hartenstein. 1970). Gamma amino-butyric acid. which also was identified, may be dcrivcd from glutamic acid. Of the dominant free amino acids in Crustacea. only gly-

Degradation

of benzoic

105

acid in a crustacean

Table 2. R, values and quantitative data on probable amino acids derived from benzoic I-‘VY acid in Oniscus asrllus L R, value Butanol: Phenol: acetic acid ammonia 31 31 43 18 44 68 36 62 67 75 83

tine could not be located

Metabolite

identified

as a radioactive

on the basis of R, values alone

substance

in

the hydrolyzate. Metabolism ofbenzoic isopods, fed vs,fasted

16.6 1.o I.7 20.3 3.3 4.3 3.6 10.3 4.6 34.2 1.7

Aspartic acid Serine Alanine Histidine* arginine* lysine* Gamma amino butyric acid Phenylalanine Proline* Unidentifiable “a” Unidentifiable “b” Unidentifiable “c” Unidentifiable “d”

18 32 50 77 84 83 93 11 40 40 56

* Metabolites

Percentage of total radioactrvity chromatographed

mid in louded vs non-loaded

From Table 3 it may be noted that no significant differences exist between the means of respiratory radioactive CO2 over a 48 h period for any of the four groups examined. However, in several (but not all) preliminary runs, the fasted isopods disposed of larger quantities of radioactive ring-carbon than fed isopods. A question arose regarding the confirmability of one kind of result or another. Accordingly, the experiment reported in Table 3 was repeated 5 days later under otherwise nearly identical conditions. The results are shown in Table 4. For these data, but not those of Table 3, the variance was not homogeneous when tested according to a procedure by Bartlett (Snedecor, 1956). Moreover, neither a square-root transformation nor a log transformation resulted in data amenable to

statistical analyses. We cannot account for the high variance seen here. where the standard deviation for two of the four groups exceeded the mean. We have noted such variation in several experiments on benzoic acid. not reported herein. Also. data on the release of gaseous ammonia (Wieser et al., 1969) and the consumption of oxygen, especially from May through July (Phillipson and Watson, 1965) are characterized by high variances that cannot at present be ascribed to some underlying causes. The experiments reported in Tables 3 and 4 were carried out in July. The test specimens were not selected at random, in view of the dependency of metabolic rate on size (Phillipson and Watson. 1965). We purposely selected large specimens (each about 100 mg). Injections were thus facilitated. and the metabolic rates per total organism were higher. However, no attempt was made to discriminate sex or gravidity, though few males were available in nature at the time of our experiments and about 90 per cent of the females were gravid. In spite of our inten-

Table 3. Respiration of radioactive CO, from benzoic 1-r4C acid 48 hours after injection into fed or fasted Oniscus aselltrs with or without a load of unlabeled benzoic acid*

Treatment 1 Loaded,

fed

2 Loaded, fasted 3 Non-loaded, 4 Non-loaded,

fed fasted

n

Weight (mg) .F i S.D.

16 13 17 17

97 100.3 91.6 92.2

+ i: + +

13.6 12.9 13.2 11.9

Countsjmin .Y 6245 5615 4859 5343

sumsof Source of variation Total Treatments Individuals

* Each isopod

d.f.

squares

62 381.071.069 3 16,434.854 59 364.636,2 I5 F,_“s = 2.76; F = 5,478,284/6,180,274 = 0.886 was injected with 44.000 countsimin

for 48 h period S.D. 292 I 1149 243 1 3018 Mean square

5,478,284 6.180,274

106

EDWARD NEUHAUSER.CHARLIS YOUMELLand ROY HAK~CNSMN

Table 4. Respiration ofradioactive CO2 from benzoic I-‘*C acid 48 hours after inJection into fed or fasted Oniscus u.srllus with or without

Treatment I 2 3 4

Loaded. fed Loaded. fasted Non-loaded. fed Non-loaded, fiisted * Each isopod was injcctcd

II

Weight (mg) 2 + S.D.

IX 17 IX 18

96.6 98.0 97.0 97.5

capucity,for

&grading

henzoic

Counts!min \-

+ 13.1 If: 14.0 f 14.0 f 13.1

650 372 1514 4451

benzoic

acid*

for 48 h period S.D. 147 2x9 I829 1990

with 44.000 countsimin.

tional effort to obtain isopods as uniformly similar as possible, there was great variation in some groups of data, such as those in Table 4, but not Table 3. A comparison of these tables shows that the loaded isopods reported in Table 4 were less active metabolically than the non-loaded, indicating that their degradative enzymatic capacity had been saturated. In addition, the non-loaded fasted isopods of Table 4 were nearly three times more active than the non-loaded fed isopods, indicating a need for a source of energy by the fasted animals. More experiments. at various times of the year, paying close attention to such variables as sex and weight, need to be performed in order to determine the conditions under which large or small amounts of benzoic acid are catabolized, where the release of CO, is used as a criterion. Enzymatic

a load of unlabeled

ucid

It is too early to obtain a “best estimate” for the rate at which benzoic acid may be degraded in terrestrial isopods, as Phillipson‘and Watson (1965) were able to do with regard to oxygen consumption per unit weight of isopod per unit time. However. a crude estimate may be gleaned from Fig. 2 and Tables 3 and 4 with respect to “loaded fed isopods”. From these sources of data, using respiratory CO, over a 48 h period. converting the data to disintegrations/min, considering the ratio of 30:0.09 /lg for unlabeled to labeled benzoic acid, and assuming the unlabeled form is reacted with the same probability as the labeled form, values of 43, 43 and 4.6 /lg of benzoic acid are obtained per isopod per 2-day period. For the three examples chosen these values represent minimum amounts of benzoic acid degradation, since the calculations are based on respiratory CO?. rather than total ring degradation. 0. usellu,s consumes about 5 ;tl of O,/mg day-’ (Phillipson and Watson, 1965 and our unpublished observations), and it metabolizes foodstuffs from decaying sugar maple leaf at an equivalency of about 1 per cent of its live body weight day-’ (Hartenstein. 1964). The complete combustion of I PM benzoic acid requires an equivalency of 7.5 {IM or 168 ,~l 0,. The values of 4.6 and 43 pg henzoic acid (from the preceding paragraph) represent 0.038 and 0.356~~ benzoic acid which would require 6.4 and 59.7 ~1 O2 for their combustion. Rounding off the weight of the isopods to IOOmg, and using Phillipson and Watson’s “best esti-

mate” of oxygen consumption, 0. adus consumes 1000 ~1 per 2 days. From this figure and the volumetric equivalents (6.4 and 59.7 $1 of benzoic acid degradation, it follows that 0.6 and 6 per cent of the animal’s total caloric budget was attributable to benzoic acid. Based

on metaholizahle

weights

of solids

from

decay-

ing sugar maple leaves (about 2000 pg/lOO mg animal per 2 days), values of 0.23 and 2.6 per cent of the animal’s caloric consumption were due to the metabolism of benzoic acid. These are, of course, approximate values based on limited sampling. Also, it should be stressed that they are based on metabolism over a 2day period and on data for respiratory CO,. Considering that very little of the injected dose of benzoic acid was eliminated in the feces (four separate observations) it is safe to assume that much larger percentages of henzoic acid were metabolized. The radioactive carbon represents only l/7 of the total carbon in benzoic acid. Terrestrial isopods are very abundant in nature (about 200-400/m’ : Stockli, 1946). Future research may reveal that they are important to soil, not only for their contribution to the physical degradation of decaying leaves and wood, but perhaps also for a potential role in the metabolic degradation of certain aromatic compounds. REFERENCES Cloudsley-Thomson

J. L. (1956) Studies in diurnal rhythms. VII. Humidity responses and nocturnal activity in woodlice (Isopoda). J. LJYP.Bid. 33, 576-582. DLITTON P. L. and EVANS W. C. (1969) The metabolism of aromatic compounds by Rhodop,srudot~~o~~a.s palustris. Biochcm. J. 113, 525- 536. GIBSON D. T. (1968) Microbial degradation of aromatic compounds. Scrcr~cr N.Y. 161, 1093-1097. HAR’rENsTrxN R. (1964) Feeding. digestion, glycogen, and the environmental conditions of the digestive system in Onisctrs usell~rs. J. Insect Physiol. 10, 623- 632. HARTENSTEIN R. (1970) Nitrogen metabolism in non-insect arthropods. In Cou~purutiw Biochcvnistrq’ of Nitrogen Mt~tcrholi,sm (J. W. Campbell. Ed.) pp. 299-387. Academic Press, New York. HAYAISHI 0. and NO~AKI M. (1969) Nature and mechanisms of oxygenases. Science N. Y. 164, 389- 396. PHILLIPSON J. and WATSON J. (1965) Respiratory metabolism of the terrestrial isopod 0ni.scu.s a.sceflus L. Oikos 16, 78-87.

SARKANEN K. U. and LUI~WIC; C. H. (1971) Lignins

Occw-

Degradation

of benzoic

WMY~, Forr~tation, Structurc~ rrnd Raactions. Wiley-Interscience. N.Y. SIEKEVITZ P. (1952) Uptake of radioactive alanine in vitro into the proteins of rat liver fractions. J. Viol. C&l. 195, 549-565. SMITH I. (1960) Chrornutoyruphic and Electrophowtic Techttiqurs. Vol. I, pp. 308 354. Heinemann. London. SMITH J. N. (196X) The comparative metabolism of xenobiotics. Ad1>.Cornp. Ph~\iol. Biochrm. 3, 173-232. SNFDIXNR G. W. (1956) Statistical Methods. pp. 2X5-297. Iowa State College Press, Ames. Iowa.

acid in a crustacean STOCKLIA.

dung

der

107

(1946)Die biologische Care

und

der

Komponente Nahrstoffpufferung.

der Vere-

Schwriz. Landwirt.sch. Monatsh. 24, .V 19. WIESI-K W.. SCHW~ILFKG. and HAKTENSTEINR. (1969) Pat-

terns in the release of gaseous pods. Orw~lqia 3, 390-400. WILLIAMS R. T. (1959)

John Wiley. N.Y.

ammonia

Dctouiurioc

by terrestrial

Mecha~tisms.

iso-

2nd edn.