Heavy metal complexation in polluted molluscs. III. Periwinkles (Littorina littorea), cockles (Cardium edule) and scallops (Chlamys opercularis)

Chem.-Biol. Interactions, 23 (1978) 227--231

227

© Elsevier/North-HollandScientific Publishers Ltd.

HEAVY METAL COMPLEXATION IN POLLUTED MOLLUSCS. III.PERIWINKLES (LITTORINA LITTOREA ), COCKLES (CARDIUM EDULE) AND SCALLOPS (CHLAMYS OPERCULARIS) A L A N G. H O W A R D

and G R A H A M

NICKLESS*

Department of Chemistry, Universityof Southampton, Southampton S09 5NH. *Department of Inorganic Chemistry, University of Bristol, Cantock's Close, Bristol, Avon

(United Kingdom) (ReceivedMarch 10th, 1978) (Revision receivedMay 15th, 1978) (Accepted May 21st, 1978)

SUMMARY The accumulation of cadmium, zinc and copper by environmentally contaminated periwinkles, cockles and scallops has been studied by gel permeation chromatography of water-soluble extracts of the shellfish. Three distinct low mol. wt. (less than 3000) zinc complexes were separated from periwinkle extracts. Copper and cadmium were present as a single low mol. wt. species which is apparently responsible for the elevated copper levels in

Littorina littorea.

INTRODUCTION

In the first two parts of this series, investigations into the nature of cadmium, zinc and copper in environmentally polluted oysters and limpets have been reported. Two different mechanisms of metal retention have been identified. Copper and cadmium are accumulated by the limpet (Patella vulgata) in conjunction with a protein which is apparently similar to the mammalian cadmium binding protein, metallothionein [1] and the cadmium binding protein recently found in the mussel [2]. Much of the zinc and copper accumulated by oyster~ however, is associated with low tool. wt. complexes, possibly of the betaine homarine and/or the amino acid taurine [3,4]. The organisms reported to date represent species exhibiting high metal concentration factors. In this paper we present details of studies into the nature of cadmium, zinc and copper accumulated by environmentally polluted periwinkles, cockles and scallops. These shellfish generally have lower concentration factors than those exhibited by oysters and limpets, and thus provide samples possibly demonstrating different concentration mechanisms.

228 EXPERIMENTAL Samples L i t t o r i n a littorea were obtained from Sand Point in Somerset (contaminated with cadmium, zinc and copper), Restronguet Creek in Cornwall (copper contaminated) and Brighton in Sussex (relatively u n c o n t a m i n a t e d ) . C h l a m y s opercularis were obtained from P l y m o u t h in Devon (relatively uncontaminated). Cardium edule were obtained from Shoeburyness, Essex (relatively uncontaminated). S a m p l e p r e p a r a t i o n and analysis Tissue extracts were obtained by extraction with phosphate b u f f e r (pH 7.0) and were separated by gel permeation c h r o m a t o g r a p h y on a 1 m × 2.6 cm column of SelShadex G-75 and a 90 cm × 2.6 cm column of Sephadex G-25 (Pharmacia). Eluate fractions were analysed for trace metals, compounds giving rise t o a positive ninhydrin colour reaction and homarine. F u r t h e r details o f the techniques used can be obtained from refs. 4 and 5.

RESULTS Mollusc samples were extracted and portions of the fractions obtained during extraction were analysed for cadmium, zinc and copper. The results o f these analyses are given in Table I. Samples o f the mollusc extracts (approximately 0.1 g) were dissolved in 1 ml o f doubl y distilled water and were separated on Sephadex G-75. 5 ml fractions o f the eluate were collected and analysed for cadmium, zinc and TABLE I DETAILS OF TRACE ELEMENT CONCENTRATIONS FOUND IN MOLLUSC SAMPLES. THE FIGURES IN BRACKETS SHOW THE PROPORTION OF METAL WHICH WAS EXTRACTED BY PHOSPHATE BUFFER Sample

Species

Origin

A

L. littorea

Restronguet

B

L. littorea

Brighton

C

L. littorea

Sand Point

D

C. edule

Shoeburyness

E

C. opercularis

Plymouth

Metal Concentration (ppm, wet wt.) (Extraction efficiency(%))

Cd

Zn

Cu

2.2 (68) 1.5 (48) 7.3 (--) -(--) 0.9 (8.2)

44.9 (4) 12.0 (39) 25.2 (23) 13.8 (46) 78.5 (II)

142 (46) 14.4 (59) 46.6 (29) 0.8 (64) 4.0 (23)

229 copper. The sample elution was monitored by measurement of the ultraviolet absorbance of the fractions at 250 and 280 nm. An example chromatogram, obtained of Littorina (Brighton) and typical of chromatograms of all three species, is given in Fig. 1.

.......

,_~'~./-.

2"0 Abs"

i ~"~ ~ i~ ~ i

~; \!

•

0.2

=Cu

...........

=70

~ ~. ~x!

!~..: !~ \

ppm-

Absorbance (250 nm)

---

~ \ ,,\

c. 0'1ppm Zn

~ ~ £

~.~.~.~.

~.

~"

%.

./"

~'~ ~ x~',.~ ~;% ~ "~. f .. ~. 30

"~'~,.

/./ / : 60 Fraction

,% •

"%. % "%.

..-~z-.

I 90

number

lnce,250nm,

Fig. ] . E x ~ p l e

2"0"

~p~atlon

on Sephadex G-75 - Littorina

I

i j~,~

- Cd

0.1

~/11

0'5 0.1-

p~

littorea (Brighton).

!L.II!

iI1

~'

,, ~X''-~

/v,I z l , ,I

...........

=211

---,---.---

= CU

I!,,

s/

I

;\ ~

I I

I ~

0'05 mg/ml" h0marine

- homarine ............ ninhydrinpositive substances

3O

i

*

60 Fraction number

~

90

Fig. 2. Low mol wt. fractionation of Littorina littorea (Sand Point) extract on Sephadex G-25.

230 The low mol. wt. fractions of the extracts were investigated b y chromatography on a column of Sephadex G-25. 2 ml fractions of the eluate were collected, analysed for cadmium, zinc, copper, homarine and "ninhydrinpositive" substances. Examples of the chromatograms which were obtained are given in Figs. 2--4. DISCUSSION

Separations of mollusc extracts on Sephadex G-75 have failed to give any t'~.

f~

,

'~

i

i

!

i

Cu

.~

I

2. 0

i#~

:

i!

2' 0

"i

ppm-

O. I mg/ml • homarine

..... homarine ............ Zn . . . . . = Cu

Ii ~i ~'t

,/

i I I .' I I

;"t

\ ~-~ ..........

.~

',J--I

30

~ \

;-.,

,

%.

60 Fraction number

90

Fig. 3. S e p a r a t i o n o f c o p p e r c o n t a m i n a t e d Littorina o n

S e p h a d e x G-25.

r.~

0.1

Cockle

0.1

II

r\ ~!

i I

......

I~ ~ ~l

I~

0.1 ppm-

i ~ ~ l I ~ l ~ ~

Cu • I

~ ,'

tt

Cd

..........

=

78

. . . . .

*~Cu

,!A

! I

,,, I I

I'0

"~ 0-0! ppm Cu

Scallon

I I | I

I/~

/~ I ~ I ~ i ~ Z ~ I *,

~0

x~

!

60 Fraction number

I 90

Fig. 4. Metal d i s t r i b u t i o n in l o w tool. wt. f r a c t i o n s o f cockle a n d scallop e x t r a c t s .

231 indication of the presence o f metallothionein-type proteins in the cockles, periwinkles or scallops studied. Further chromatography o f these extracts on Sephadex G-25 have shown marked similarities in the retention behaviour o f copper and cadmium. Both metals are present, within the separation range of Sephadex G-25, as single, small molecular size complexes. It is this complex which is apparently responsible for the accumulation of elevated levels of copper in Littorina littorea (Fig. 3). Three distinct zinc complexes were separated from periwinkle extracts from Sand Point. This situation is similar to that obtained from contaminated Crassostrea gigas [ 4 ] , b u t the elution volumes are n o t comparable for the t w o species. Although it is difficult on structural grounds to propose a role of homarine in the complexation of the trace metals, elution of this c o m p o u n d frequently occurs together with the elution of copper and zinc (Fig. 3). It is well known that cadmium, zinc and copper are all able to form stable complexes with certain amino-acids in aqueous solution [ 6 ] . However, until full structural investigations can be carried out on the low tool. wt. complexing agents, their nature must remain a matter of conjecture. REFERENCES

1 A.G. Howard and G. Nickless, Heav~ metal complexation in polluted molluscs. I. Limpets (Patella vulgata and Patella intermedia), Chem.-Biol. Interact., 16 (1977) 107. 2 F. No~l-Lambot, Distribution o f cadmium, zinc and copper in the mussel Mytilus edulis. Existence of cadmium binding proteins similar to metallothioneins, Experientia, 32 (1976) 324. 3 T.L. Coombs, The nature of zinc and copper complexes in the oyster, Ostrea edulis, Mar. Biol. 28 ( I 9 7 4 ) 1. 4 A.G-. Howard and G. Nickless, Heavy metal complexation in polluted molluscs II. Oysters (Ostrea edulis and Crassostrea gigas), Chem.-Biol. Interact., 17 (1977) 257. 5 A.G. Howard and G. Nickless, The analysis o f the betaine homarine by high pressure liquid chromatography, Anal. Biochem., 76 (1976) 377. 6 Stability constants of metal-ion complexes. The Chemical Society Special Publication No. 17, London 1964.