Received May 1, 1975Accepted August 20, 1975

Tne presence of high levels of arsenic in somemarine animals has been recognized since thework of Cox in 1925. More recent investigationsinto the environmental biochemistries of mercuryand arsenic have revealed similarities in themetabolism of the two elements (Wood 1974).Any assessment of the environmental hazard ofarsenic now hinges on the characterization andtoxicology of its me abolites (Penrose 1974).

As early as 1.926, Chapman was able to showdifferences in chemical and biological propertiesof inorganic arsenic and the arsenic-containingextracts of marine animals. His work and that ofCoulson et al. (1935) demonstrated that the largequantities of arsenic in marine crustaceans (often100 ppm or higher) were rapidly and totallyeliminated by human consumers, whereas a pro-portion of inorganic arsenic consumed by thesame subjects was retained indeflnitely. Lunde(1969) postulated the existence of organoarseniccompounds in fish based on the ion exchange be-havior of extracted arsenic. It has been demon-strated in this laboratory that all of the arsenic inwitch flounder (Glyptocephalus cynoglossas) and

Printed in Carada (J3819)Imprim6 au Canada (J3819)

Biosynthesis of Organic Arsenic Compounds in Brown Trout (Salmo trutta)

W. R. PENnoss

Department of the Environment, Fisheries and Marine Sewice, Biological Station,St. John's, Nfld. AIC IAI

PeNnost, W. R. f 975. Biosynthesis of organic arsenic compounds in brown troul(Solmo trutta\.J. Fish. Res. Board Can. 32:2385-2390.

Radioactive, inorganic arsenic was administered to brown trout (Salz o trutta) orally andbyintramuscular injection. The orally administered arsenic appeared in the tissues in an altered formthat is presumed organic; this organoarsenical was also found in the gastrointestinal contents.The injected arsenic appeared in the tissues in inorganic form and was only slowly converted toorganic form; meanwhile, large amounts of inorganic and organic arsenic were detected in thebile. These findings are consistent with biosynthesis of the organic arsenic compound within thegastrointestinal tract.

PrNnosn, W. R. 1975. Biosynthesis of organic arsenic compounds in brown trout (Salmo trutta).J. Fish. Res. Board Can. 32:2385-2390.

Nous avons administr6 par voie orale et par injection intramusculaire de I'ars6nic inorgani-que radioactif dans des truites b runes (Salmo trutta). L'ars6nic administr6 par voie orale apparaitdans les tissus sous une forme alt6rde qu'on pr6sume organique; ce compos6 organo-ars6nical setrouve 6galement dans les contenus gastro-intestinaux. L'ars6nic inject6 apparait dans les tissussous une forme inorganique et ne se transforme que.lentement en forme organique; entre temps,de fortes quantit6s d'ars6nic inorganique et organique se trouvent dans la bile. Ces constatationssont en aicord avec la biosynthdse du compos6 d'ars6nic organique i I'intdrieur du tractussastro-intestinal.

Regu le 1 mai 1975Acceot6 le 20 aoot 1975

the shrimp Pandalus borealis exists as a single com-pound, identical in both species, that is cationic,hydrophilic, and nonvolatile (W. R. Penrose un-published data). The structure of this compoundis unknown, but based on its chemical propertiesit cannot be one of the known forms of environ-mental arsenic: methylarsines (Challenger 1945;Wood 1974), methanearsonic or dimethylarsinicacids (Braman and Foreback 1973), or inorganicarsenic.

A potential source of the arsenic found in fishis sediment particles that may be ingested withfood. Sediments rarely contain less than 4 ppm ofarsenic (Penrose 1974). We have demonstratedthat arsenic leaches readily from sediments intosea water (Penrose et al. 1975) and ought todo so under conditions existing in the digestivetract of a fish. I therefore examined the abilityof fish to convert inorsanic arsenic into otherforms of the element.

Materials and Methods

SpncrueNs

Brown trout (Salmo tutta) were trapped in QuidiVidi Lake in St. John's, Nfld., and maintained inrecirculated fresh water on a diet of beef liver.

2385

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2386 J, FISH. RES. BOARD CAN.. VOL. 32(12), 1975

Animals of 200-300 g were selected for these ex-periments.

Cnsvrrcers

Carrier-free "1A.s-arsenic acid was purchased fromAmersham-Searle Ltd.; thin{ayer chromatography ofeach lot revealed that 80-90% of 'nAs was present asarsenate and the remainder as arsenite. A slight ex-cess of solid NaHCOg was added to dilutions beforeadministration to neutralize the HCI present. Protosoltissue solubilizer and Aquasol liquid scintillationcounting fluid were purchased from New EnglandNuclear. Silica gel thin-layer plates (Macherey-NagelSIL G-25) were obtained from Brinkmann Instru-ments Limited. A solvent system of methanol:aceticacid (9:1) adequately separated arsenate (R/ 0.8),arsenite (0-0.15), and the natural organoarsenic com-pound isolated from witch flounder muscle (0.35).

BtosyxrgBsls ExpsnlMpxts

Fish were inspected for weight and normal appear-ance and removed to a separate tank. Oral adminis-tration was accomplished by injection of I ml ofradioarsenic solution through the pharynx with al-ml tuberculin syringe. Intramuscular injections of0.10 ml were made with a 22-gauge, 37-mm needleinto the muscle 2 cm below the dorsal fin. Injectionswere always made on the left side and muscle sam-ples removed from the right.

Fish were fed daily during experiments. At appro-priate times, fish were dried with a towel and bloodremoved by cutting into the heart. They were thenkilled by decapitation. Bile was removed with asyringe and fine needle before removal of the liver.Feces and stomach contents were removed and mixedthoroughly together before sampling. Muscle sampleswere removed from 2 cm below the dorsal fin.

MEesunelrsNr op Rlorolcrrvrry

Total radioactivity in tissues was measured by im-mersing a weighed amount of tissue (about 0.1 g) in2 ml Protosol in a polyethylene scintillation vial.After 24-48 h at 44 C with occasional agitation,10 ml Aquasol and two drops acetic acid were added,and the radioactivity counted in a scintillation spec-trometer,

The proportion of organic to total arsenic was esti-mated by a method similar to that described byLunde (1969). A weighed amount (0.2-1.0 g) of t issuewas homogenized with 4.0 ml 107o sulfosalicylicacid. After centrifugation (5 min at 7000 X e),0.20ml of the supernatant liquid was mixed with 3 mlwater and 10 ml Aquasol. A second 0.20 ml ofsupernatant liquid was applied to a 4.5 X 50-mmcolumn of Dowex 1-X8 or Amberlite CG-400 (200-400 mesh, hydroxide form) in a Pasteur pipette. Thecolumn was washed with 3 ml water into a scintilla-tion vial and 10 ml Aquasol added. The ratio of thecount rates in the second sample to those in the firstis the proportion of nonanionic arsenic (i.e. neitherarsenite nor arsenate) to total arsenic. Appropriate

controls confirmed that arsenite and arsenate in thesamples were retained by the anion exchanger. Totalradioactivity extracted by sulfosalicylic acid wasalways 80-95Vo of that determined by Protosolsolubilization. No attempt was made to wash theremaining activity from the pellet.

Where necessary, count rates were corrected fordecay using a half-life of 17.7 days. The efficiency ofcounting was monitored by the channels ratiomethod; corrections were not found to be necessaryat any time, however, presumably due to the highenergy of the "nAs beta particles.

Plnrrar PuRrFrcATroN oF NoNANroNrc ARsENrcCovrpouNos nnolr Muscrp

Muscle from witch flounder or trout was homog-enized with an equal weight of water. To this homog-enate was added an equal volume of water and 0.05vol of l00Vo (wt,zvol) trichloroacetic acid withstirring. After centrifugation for 10 min at 7500 x g,the clear supernatant was decanted. To this wasadded 0.025 vol of 12 x H,SOI, and the mixture wasextracted twice with 0.2 vol of benzene. The benzeneextracts were discarded. The aqueous phase was neu-tralned to a phenolphthalein endpoint and appliedto a cation exchange column (Dowex 50-X8, 20-50mesh, H-form, 22 x 180 mm). The column waswashed with 200 ml water and eluted with 3% fuol/vol) ammonium hydroxide. Fractions containingphenolphthalein color were pooled and evaporated todryness in vacuo at 60 C. Arsenic concentrations invarious fractions were determined as described bvPenrose et al. (1975).

Results

In the first experiments, small doses of TaAs-

arsenic acid (1 p,,E;20 pCi per flsh) were adminis-tered intramuscularly or orally. The total tissueactivities and ratios of "organic" (nonanionic) tototal radioarsenic were determined (Table 1).Arsenate introduced intramuscularly appearedrapidly (4.5 h) in blood, muscle, and liver, vir-tually all in anionic form. Only slowly did theproportion of organic arsenic increase. Whenadministered orally, on the other hand, the radio-activity appeared more slowly and almost en-tirely in organic form.

Safety considerations arising from the amountof radioarsenic that had to be adrninistered toeach fish meant that each set of data in Table 1(one route of administration'and one time point)represents only one fish. The experiment wasrepeated twice at appropriate intervals with qual-itatively similar results. Figure 1 shows the dy-namics (for liver only) of arsenic conversion ina longer term experiment.

Based on the results of this first experiment, alikely site of conversion of the arsenic to non-anionic form was the lumen of the trout gut (see

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PENROSE: ORGANOARSENIC BIOSYNTHESIS

Tasu l. Conversion of inorganic to organic arsenic by(Salmo tutta) after two modes of administration. Dose250 + 50-g fish (2.25 x 107 cpm ?aAs).

2387

brown trout1000 ng per

Time Tissue

Injected

Concn Percent(ne/g)" nonanionic

Oral

Concn Percent(ng/g)" nonanionic

4 . 5 h

12h

24h

4 8 h

BloodMuscleLiver

6 .671 . 1 02. 80

2 .030.540 .74

3 .461 .051 . 8 0

0 . 1 31 . 3 70 .76

0 . 87 . 55 . 7

6 . 535 .17.6

12.76537 .

0 .200 . 1 81 . 0 8

0 . 3 70 .990 .61

0 . 3 72 .O31 . 4 2

828493

969784

9297

105

BML

BML

BML

26.

60uNanograms As per gram wet weight.

Discussion). Initial trials to test this hypothesisinvolved a) incubation of gut contents with ?4As-

arsenate, b) incubation of isolated, ligated gastro-intestinal tracts from fish with arsenate introducedinto the lumen, and c) isolation of strains ofaerobic bacteria from trout intestines and screen-ing for arsenic methylation. A1l these approachesfailed. (If methanogenic bacteria are involved,this would be expected, because these organismsrequire anaerobic conditions for growth. (Becausethese specialized techniques [Hungate 1969] werenot available, the investigation was continued em-ploying only the whole animal.)

The livers and gastrointestinal contents of troutfed tracer (carrier-free) doses of 74As-arsenate

were sampled after 24 and 72 h. The results ofone experimen't are given in Table 2. The gutcontents in the four fish contained variable butsignificant proportions of "organic" radioarsenic.

Injected arsenic is eventually converted partlyto the "organic" form. If conversion occurs solelyin the gut, it is necessary that injected inorganicarsenic be excreted into the lumen. Excretion ofarsenic via the bile is es,tablished in mammals(Klaassen 1974); therefore, an experiment wasconducted to determine the presence of injectedinorganic arsenic in the bile of trout. Six fish wereadministered 1,5 pci each of carrier-free arsenicacid intramuscularly and killed after various inter-vals. The quantities of bile recoverable were inthe range 0.05-0.20 ml; these were mixed withonly 1.00 rnl of lOVo sulfosalicylic acid beforedetermination of total and "orsanic" radioarsenic.

The flgures in Table 3 clearly demonstrate thatarsenic is rapidly excreted into the bile in bothinorganic and organic forms. In the two fishsampled at 5 h most of the arsenic is inorganicand at 24 h most is organic, but in both cases theconcentration in the bile is a significant propor-tion of that in the blood. For specimens 1, 3, 4,and 5, the calculated concentration of organicarsenic in bile is higher than in the blood, whilethat of inorganic arsenic is lower. (Fish 2 hadtoo low a concentration of organic arsenic ineither blood or bile to provide a meaningfuldatum.) Organic arsenic appears to be excretedagainst a concentration gradient; while inorganicarsenic is definitely excreted, the process is notnecessarily an active one.

At ,this time, the identity of the anionic arsenicfound in the blood of fishes I and 5 was con-firmed to be inorganic. A small quantity of theacid extract was chromatographed on silica thin-layer plates with sodium arsenate and sodiumarsenite. The two inorganic compounds werevisualized by spraying with ammoniacal l%silver nitrate. The plate was scraped and countedas described in ,the legend to Fig. 2. AU of theradioactivity in fish 1 and most of that in fish 5cochromatographed with arsenate.

Cnlne.crBnrzATroN oF NoNeNroNIc AnsBNlcColrpouNo

Witch flounder muscle (95 g, containing 25ppm arsenic) was processed as described in

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J. FISH. RES. BOARD CAN.. VOL. 32(IA. 1975

Materials and Methods. lolVo of the arsenicoriginally present in the muscle was recovered inthe pooled fractions from the ion exchange step.Inorganic arsenic was no,t retained by the cationexchange resin.

From a trout that had been siven an oral dose

of 10 pCi TaAs-arsenate 24 h previously, 63 gmuscle tissue was recovered. The trout muscledid not contain detectable amounts of arsenicwhen analyzed chemically. The yield of radio-activity eluted with ammonia from the cationexchange resin was 9OVo, indlcaling that the

Ttw-n 2. Organic arsenic in gastrointestinal contents and liver of trout fedTaAs-arsenate.

Liver G. I. contents

Fish Time cpm/gPercent

nonanionic cpmlgPercent

nonanionic

I2J

4

24h1 i

7272

13800115201488019940

e6%7 1 %e6%e8%

2081601 689802294048300

1'ro7

\)07

16%1\o7

T2I5Hllz=

froo-

r3zl

13 roDAYS

Ftc. 1. Form of arsenic in the livers of browntrout (Salmo trutta) administered 10 pCi "1As-arsenate by the intramuscular and oral routes. Livertissue was treated as described. Counts per minuteshown are for I g of liver tissue. Each pair of points(total and organic) represents a single fish.

300

300

DISTANCE MOVED (CM)

FIc. 2. Thin-layer chromatography of cationic frac-tion of radioarsenic extracted from trout muscle.Extracts were streaked on 20 X 20-cm Silica GelG plates and overlaid with a sample of the witchflounder (Glytocephalus cynoglossus) arsenic com-pound. After development, 1-cm zones were scribedonto the adsorbent layer, and the layer was scribedlaterally in half. One half of each 1-cm zone wasscraped into a liquid scintillation counting vial; theother half was ashed for arsenic determination. Sol-vent systems used were (A) methanol:acetic acid(90:10) and (B) propanol:ammonia (50:1). Theorigin is marked as O; SF, solvent front; GA, loca-tion of witch flounder arsenical compound.

trJF

=ElrJa 600(/,Fzo(J

INJECTED

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PENROSE: ORGANOARSENIC BIOSYNTHESIS

Taglr 3. Excretion of injected inorganic arsenic into the digestive tract.For clarity, proportions and concentrations are tabulated separately.

/o1aAs in nonanionic form

2389

Fishno.

Time(h) Blood Bile G.I. contents Liver

12JA

56

55

. A

. A

7272

0 . 7 20 . 4 46 . 3 05 . 3 6

1 8 . 32 4 . 1

5 . 3 5o ,23

80. 388 .048.3no bile

-0 .471 1 . 044.95 7 . 98 1 . 79 8 . 5

9 . 7 48 . 3 0

34.728.9i < 1

6 2 . 1

Fishno.

Time(h)

Concn total ?aAs (cpm/g)

Blood Bile G.I. contents Liver

I23

56

55

1 A

1 A

7272

1088404264044000235002400024400

22000140001700032509900

no bile

86401200

35200142064605080

2712010840I 876083609600

10500

nonanionic arsenic was similar to that in witchflounder at least in its hydrophilic and cationicproperties. ThinJayer chromatography revealedthat two compounds were present in the eluate,neither of which was identical to that isolatedfrom witch flounder (Fig. 2).

Discussion

Very little is now known of the metabolism ofarsenic outside of the simple alkylations per-forrned by certain bacteria, fungi, and yeasts(Challenger 1945; McBride and Wolfe l9j l ;Cox and Alexander 1973). Lunde has observedtransformations of arsenic in unicellular algae(1973) and rainbow trout (1,972). The productsof hese transformations he described as "orsano-arsenic" compounds. but there is as yet no- evi-dence that the compounds actually contain car-bon-arsenic bonds. The term "orsanic" musttherefore be applied cautiously whein describingthese compounds.

Lunde (1972) administered radioactive inor-ganic arsenic to trout by addition to the waterand by feeding. Arsenic was only weakly ab-sorbed from water and rapidly lost on return toclean water. He observed, as I have. that arsenicfed to the fish is converted to "organic" form.The present work has demonstrated the follow-ing: a) arsenic introduced via the gastrointestinaltract was much more efficiently converted to theorganic form than arsenic introduced systemi-cally; b) nonanionic arsenic appeared within 1day in the gastrointestinal tract of fish fed inor-ganic arsenic; the nonanionic arsenic seemed to

be selectively absorbed; c) both inorganic andnonanionic arsenic were excreted in the bile. Thepotential therefore exists for the cycling of ar-senic between the body and the lumen of theintestine. The apparently selective absorption ofnonanionic arsenic insures that the proportionof arsenic in the tissues will shift towards thenonanionic form, regardless of the route of ad-ministration. These findings are consistent withthe hypothesis that the conversion to "organic"form is mediated by the intestinal flora.

Acknowledgments

The diligent assistance of M. J. Hayward and J.Vallis was vital to the completion of this work.

Bnau,qN, R. S., ,qNo C. C. Fonenrcr. 1973. Methylatedforms of arsenic in the environment. Science 182:1247-1249.

CHrr-rercEn, F. 1945. Biological methylat ion. Chem.R e v . 3 6 : 3 1 5 - 3 6 1 .

CHrpvaN, A. 1926. The pre sence of compounds of arsenicin marine crustaceans and shell fish. Analyst 5l:548-563.

Cout-soN, E. J., R. E. Rrrr.rrNcroN, AND K. M. LyNcs.193-5. Metabolism in the rat of the naturally occurringarsenic of shrimp as compared with arsenic trioxide. J.Nutr. l0: 255-270.

Cox. D. P., aNo M. ALpxnNnE,n. 1973. Production oftr imethylarsine gas from various arsenic compoundsby three sewage fungi. Bull. Environ. Contam. Tox-icol.9: 8zt-88.

Cox. H. E. 1925. On certain new methods for the determi-nation of small quantities of arsenic, and its occur-rence in urine and fish. Analyst 50: 3-13.

Hurqcerr., R. E. 1969. A roll tube method for the cultiva-t ion of str ict anaerobes. o. l l7. In J. R. Norris and

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2390 J. FrSH. RES. BOARD CAN.. VOL. 32(12), 1975

D. W. Ribbons, [ed. ] Methods in microbio logy. Vol .38. Academic Press, Inc. , New York, N.Y.

KrresseN, C.D.1974. Bi l iary excret ion of arsenic in rats,rabbits and dogs. Toxicol. Appl. Pharmacol. 29:447-45'7.

LuNoe, G. 1969. Water soluble arseno-organic com-pounds in mar ine f ishes. Nature 224: 186-187.

1972. The absorption and metabolism of arsenicin fish. Reports on Technological Research Concern-ing Norwegian Fish Industry Vol . 5, No. 12, Directo-rate df Fisheries, Bergen, Norway.

1973. The synthesis offat and water soluble ar-seno organic compounds in marine and limnetic algae.Acta Chem. Scand. 27: 1586-1594.

McBnron, B. C. , aNo R. S. WorpB. 1971. Biosynthesis of

dimethylarsine by methanobacterium. Biochemtstryl0: 4312-431'7.

PENRosE, W. R. 1974. Arsenic in the marine and aquatic

environments: Analysis, occurrence and significance.CRC Crit. Rev. Environ. Control 4: 465-482.

PsNnosn, W. R., R. Br-acr, AND M. J. Hlvwano. 1975'

Limited arsenic dispersion in sea water, sediments,and biota near a continuous source' J. Fish' Res.

Board Can. 32: 1275-1281.

Wooo, J. M. 19'74. Biological cycles for toxic elements inthe environment, Science 183: 1049-1052.

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