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THULIUM BIOACCUMULATION BY THE SHORECRAB CARCINUS MAENAS COLLECTED FROM

THE FRENCH COASTS OF THE CHANNEL : ASTRUCTURAL, ULTRASTRUCTURAL AND

MICROANALYTICAL STUDY BY SECONDARY IONMASS AND X RAY SPECTROMETRY

C. Chassard-Bouchaud, P. Hallegot, M. Meignan

To cite this version:C. Chassard-Bouchaud, P. Hallegot, M. Meignan. THULIUM BIOACCUMULATION BY THESHORE CRAB CARCINUS MAENAS COLLECTED FROM THE FRENCH COASTS OF THECHANNEL : A STRUCTURAL, ULTRASTRUCTURAL AND MICROANALYTICAL STUDY BYSECONDARY ION MASS AND X RAY SPECTROMETRY. Journal de Physique Colloques, 1984,45 (C2), pp.C2-541-C2-544. �10.1051/jphyscol:19842123�. �jpa-00223792�

JOURNAL DE PHYSIQUE

Colloque C2, supplgment a u n02, Tome 45, fgvr ie r 1984 page C2-541

THULIUM BIOACCUMULATION BY THE SHORE CRAB CARCINUS MAENAS COLLECTED

FROM THE FRENCH COASTS OF THE CHANNEL : A STRUCTURAL, ULTRASTRUCTURAL

AND MICROANALYTICAL STUDY BY SECONDARY ION MASS AND X RAY SPECTROMETRY

+ L C. Chassard-Bouchaud , P . ~ a l l e g o t ~ and M. ~ e i g n a n '

' ~ a b o r a t o i r e de Zoologic, BioZogie e t PhysioZogie des ~ r g a n i s m e s Marins, Universite' Pierre e t Marie Curie, 4 , place Jussieu, 75230 Paris Cedex 05, France L ~ b o r a t o i r e de Biophysique, FacuZte' de Me'decine (Fr. P. GaZZe), 6 , rue du Ge'ne'ral SarraiZ, 94000 CrdteiZ, France

R&sum&. Deux m6thodes d e microanalyse o n t d t d ut i l is6es : l a spec t rographie d e s rayons X h I'Cchelon d e s microscopes optique e t 6 lec t ron ique et I'dmission ionique secondaire h l 'dchelon du microscope optique, pour mont re r que d e s Carcinus maenas r6col tds d e Novernbre 1982 i Janvier 1983, dans 8 s ta t ions d e s c 6 t e s d e l a Manche (de Boulogne h Roscoff), ainsi q u e d e s animaux contamin& expCrimentalement, accumulent 169 Tm.

L e s i t e principal d e re ten t ion est I 'exosquelet te et les organi tes cibles sont l es lysosomes qui cont iennent d e s microgranules A h a u t e t e n e u r e n Tm associ6 i P. Les hdmocytes macrophages jouent un r61e i m p o r t a n t dans l a cap ture , l e t ransport , l e s tockage et I 'excrdtion d e cette t e r r e rare.

C.maenas appara i t c o m m e un sys tgme biologique accumulant Tm qui, prdsent dans J 'environnement marin l 1 6 t a t d e t races , est s tock6 et concent rd sous f o r m e insoluble par l e crabe.

Abstract. Two microanalyt ical methods h a v e been used: X r a y emission a t t h e light and e l e c t r o n microscopes levels and secondary ion emission a t t h e light microscope level t o show t h a t Carc inus maenas co l lec ted f r o m November 1982 t o January 1983, in 8 s ta t ions of t h e channel c o a s t s (from Boulogne t o Roscoff) and exper imenta l ly conta- mina ted samples, b ioaccumula te 169 Tm.

The major re ten t ion s i t e is t h e exoskeleton and t h e t a r g e t organelles a r e t h e lyso- somes which conta in microprec ip i ta tes with high level of T m assoc ia ted with P. Macro- phage haemocytes play a n impor tan t p a r t in ingestion, t ranspor t , s t o r a g e and excre t ion of t h e r a r e ear th .

C.maenas appears a s a biological sys tem accumula t ing Tm which, p resen t in t h e mar ine envi ronment a t t r a c e level, is s to red and concent ra ted under a n unsoluble f o r m by t h e crab.

Thulium is a r a r e e a r t h which consists of severa l rad ioac t ive and o n e s tab le i so tope 169 T m (100 % natura l abondance). It ' is known t o occur among phosphogypsum and T i 0 2 industr ial was tes which a r e notably released in to t h e Seine Estuary. T m was pre- viously d e t e c t e d in diseased f ish ( I ) co l lec ted in t h e Channel , b u t nothing i s known about i t s metabolism.

1. MATERIALS Samples of Carc inus m a e n a s (L) (Crus tacea r iecapoda) w e r e co l lec ted f r o m November 1982 t o January 1983, in 8 s ta t ions of t h e French c o a s t s of t h e Channel: Boulogne, Dieppe, Baie d e Seine, Ouis t reham, Poin te d e Barfleur, C a p d e l a Hague, Granvil le and Roscoff . C r a b s ranged in width f r o m 3 t o 4 c m and w e r e a t interrnolt s t a g e C4. For exper imenta l contamination, c r a b s w e r e co l lec ted f r o m t h e At lan t ic Ocean. They w e r e exposed t o a Tm chlorure solution a t a concent ra t ion of 10 ppm during a period f rom 1 t o 30 days.

2. SAMPLES PREPARATION AND MICROANALYTICAL METHODS. 2.1. Microanalysis a t the light microscope level. Sof t t issues w e r e dissected, f ixed in Carnoy liquid, embedded in paraff in and sec t ioned at 7 pm. Sections w e r e deposited

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842123

C2-542 JOURNAL DE PHYSIQUE

e i t h e r on mylar s l ides and carbon c o a t e d (for X ray microanalysis) oron p u r e gold spec imen holders (for ana ly t ica l ion microscopy) and deparaff ined. F o r hard t issues (exoskeleton),

bulk spec imens t h e s u r f a c e of which has been carefu l ly polished, w e r e used ; t h e evapo- ra t ion of a meta l l i c grid was necessary t o avoid charging e f fec t s . Microanalysis w e r e per formed with t w o ins t ruments :

- a CAMECA MS 46 Microprobe equipped with a transmission l ight microscope and 4 wavelength dispersive s p e c t r o m e t e r s (quartz, LIF, P E T and KAP crystals) .

- an analytical ion Microscope: CAMECA IMS 300, equipped wi th a n e l e c t r o s t a t i c deflector . The s a m p l e s u r f a c e i s spu t te red by a primary beam of a c c e l e r a t e d ions (02c) and t h e secondary ions a r e e m i t t e d f rom t h e s u r f a c e of t h e sect ion. The resolution a t t h e s u r f a c e of t h e image i s I prn for a n i m a g e a r e a 250 pm in d iameter . The ana ly t ica l images a r e obtained d i rec t ly on t h e sc reen o r on t h e photographic plate.

2.2.Microanalysis a t the electron microscope level. For e lec t ron microscopy and e lec t ron probe X r a y microanalysis, t issues w e r e dissected, f ixed in glutaraldehyde and ncn osmicated; u l t ra th in sec t ions w e r e mounted o n T i grids, unstained and carbon coa ted . Microanalysis w e r e per formed with a CAMEBAX equipped with a conventional t ransmission e lec t ron microscope and 4 wavelength dispersive s p e c t r o m e t e r s (ODPB, TAP, P E T and LIF crystals). The following opera t ing conditions w e r e used: 45 kV acce le ra t ing voltage, 150 nA probe c u r r e n t and 500 n m probe d iameter .

3. RESULTS. Crabs co l lec ted in s i tu f rom t h e Channel and c r a b s exper imenta l ly contamina ted a r e shown t o c o n c e n t r a t e thulium. 3.1. Light microscope level results. Main t issues a n organs w h e r e 169 Tm w a s detecte'd a r e , in order of decreas ing con~en t ra t ion :~xoske le ton , gill, muscle and digest ive gland. 3.1.1. Ion emission microanalysis: 169 Tm values normalized t o 40 ~ a + a r e given in fig. 1 for samples of every co l lec t ion si te; t h e s e ra t ios show t h a t t h e highest values w e r e d e t e c t e d in Ouistreham c r a b s while decreas ing values occur red e a s t w a r d and west- ward. Ion images obtained f r o m gill c u t i c l e ( f ig31 show t h e Tm local concentrat ion. Macrophagehaemocytes which occur in various t issues and organs w e r e invest igated. Ion s p e c t r u m obtained f r o m these macrophages and ion images (fig. 5 ) show t h a t they a r e overloaded with Tm. 3.1.2. MS 46 Microprobe microanalysis. S p e c t r a obtained f r o m t h e d i f f e r e n t t issues (f ln.1 conf i rm t h e ion emission microanalvsis d a t a . 3 . z Electron microscope level results. In t h e c u t i c l e (fig. 7 ) e lec t ron dense microgra- nules w e r e shown t o conta in high levels of T m associated with P. Within gill epi thel ium, digest ive gland and macrophage haemocytes , lysosomes (fig. 6 ) exhibited microprecipi- t a t e s which w e r e shown t o consist of T m associated with P. ( f i g . 8 ) .

4. DECUSSION AND CONCLUSION. Repor t s on distr ibution and ro le played by r a r e e a r t h e l e m e n t s in mar ine organisms a r e s c a r c e and poorly s t a t e d (2). Our presen t d a t a show t h a t C.maenas appears a s a biological sys tem accumulat ing Tm which, p resen t in t h e mar ine environment at t r a c e level , is uptaken, s to red and concent ra ted in t h e form of a n unsoluble phosphate by t h e c r a b , mainly within i t s exoskeleton. These results demons t ra t ing t h e exoskeleton a s t h e main s t o r a g e t issue mus t b e re la ted t o previous repor t s where high c o n t e n t s of l an thanum, another r a r e e a r t h , occur in f ish bone s t r u c t u r e s and in prawn exoskeleton (2). In t h e Mammals too, t h e nearly def in i te re ten t ion s i t e of t h e lanthanides i s t h e ske le ton (3). Macrophages play a n impor tan t p a r t in Trn ingestion, t ranspor t , s to rage and excre t ion , probably according t o t h e s a m e mechanism which was previously demons- t r a t e d for uranium in t h e crayfish (4). A t t h e u l t ras t ruc tura l level , lysosomes appear a s t a r g e t organelles of Tm concentrat ion: t h e s e d a t a a r e in a g r e e m e n t with our previous results showing t h e intralysosomal U concent ra t ion in C.maenas. Anyhow, in t h e presen t c a s e of T m concent ra t ion , sphero- c rys ta l s (=calcium phosphate granules) which a r e usually known in t h e c rab , for the i r abi l i ty of concent ra t ing many m e t a l s and radionuclides such a s U f o r ins tance (51, d o not s e e m t o a c c u m u l a t e t h e r a r e ear th . Trn detoxica t ion processes happen v ia t h e macro- phages which a c c u m u l a t e t h e e l e m e n t and via t h e exoskeleton which is shed at every ecdysis.

In t h e disposal of industr ial was tes in t h e sea , t h e abil i ty of mar ine organisms such a s Crus tacea , of concent ra t ing t h e r a r e e a r t h e l e m e n t s must b e considered and f u r t h e r invest igat ions a r e required on o ther spec ies of economic in te res t .

(1) CHASSARD-BOUCHAUD C., Electr.Microsc. 3 (1982) 405. (2) KAMEDA K., J.Rad.Res. - 2 (1962) 89. (3) MASSE R., in Radionuclide, Metabolism and Toxicity, e d i t e d by P.GALLE and

R. MASSE, Masson, Par i s (1982) 143. (4) CHASSARD-BOUCHAUD C.,C.R.Acad.Sc. Paris, 294 (1982) 919. ( 5 ) CHASSARD-BOUCHAUD C.,Mar.Pol.Bul. E, 4 (1983) 133.

Sources of partial support for this work were CNRS (GRECO Manche) & INSERM (SC 27). The technical assistance was provided by F. Kleinbauer, F . Escaig and S. Halpern. Material was, in part, provided by D. Calmet.

%.l.Geographical repartition of Thulium bioaccwnulation fTm/Ca ratios) in C.maenas. Secondary ion emission microanalysis.This map shows that the highest values were dp- tected in Ouistreham sampZes,whiZe decreasing vaZues occumd eastward and westward.

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%.g. X ray emission Tm & P spectra (Carnebax) obtai- ned from the microprecipi- tates of the fig. 6.

Fig. 2. 23iVa7 $on image obtained from a gill section,E,epithelium,~,cuticle (M X 150). Fig.3. 169 Tm ion image obtained from the same area as in the fig.2.C,cuticle which - is sZown tq be the Tm accumulation site (arrows), (M X 150) a.z.40Ca ion image obtained from a nervous tissue ( N I section showing several ma-

fig. 4. It shows that macro-

Fzg.7.EZectron micrograph showing !?m microprecipitates(ar- -- rowsjwithin the gill cuticZefClE,epitheZ. .iVbn osmic. & unsi 3 0 0 ~ 6 T,

a . 5 X ray emission Tm & P spectra ( MS 46 mi- croprobel obtained from the macrophage haemo- cytes of the fig.5.

( M X 6000 ' I . 4