This pape r not to be cited without prior r eference to the

author s .

Intern2tional Council for the Explora tion of the sea

C. ~1 • 1 9 8 1/ F : 4 3 Mariculture Committee

Ab&tr act. ·~

Effects of c opper chloride on the eggs and

D larvae of Crassostrea gigas (Thwlberg) .

Preliminary results .

by

E. HIS and R. ROB ERT 0

Copper c hl oride is toxic to the E.'T!Ib t•yonic develop­

ment and D l a rvae of Cra ssostrea gigas &nd it affects t h e

g rowth of the veligers at low conce ntration E . •

At 50 p .p.b. t he embryonic devel opment is tot~lly

ri lsturb ed , while t h e more res istant D l a rvae show little

growth at the end of seven days . Therefore copper chl or·ide

i s less toxic than the o r g ano - metallic salts of tin, oth e r

compon e nts of antifouling paints.

Resume .

Le chlorure de cuivre empeche l e d e veloppement e m­

bryonnaire norma l chez Crassostrea gigas ; il affecte forte­

menc la crois&allc e des l a r ves D a de b o S SE:S concentrations.

0 E. HIS and R. ROBERT , I. S.T.P . H. ,- 63 Boulevard Deganne 33 1 20 ARCACHON (France)

Ainsi le d e veloppement embr yonnaire est tot.alement

perturbe d~s l a concentra t i on de 50 p .p.b. ; la croissanc e de

larves D sotunise s a 1 1 a ction du produi t d e s l es premi e res 2 4

heures, e s Jc fortement r a. l en t i e , au bout d e sept, jours d' ob s e r­

vat ions. Toutefois, l e chlorure de cuivre est mains toxique

que les sels organo- metalliques de l 1 et a in , qui sont eux aus s i

utilises d ans l a fabric 2tio n des p e i n ture s anti-salissure s.

1. Introducti on .

Important s patfall fluctuations o f the J a pane s e oys ter

Cr a ssostrea g i g as in some parts of the French coast, p c r t icul a r ­

ly in the Bay of Arc ach o n, have broug ht about experiments whic h

try to det e rm:i.IJ e t he effects of some antifouling compone nt s o n

t h e young vel i ger s o f t l1e J a panese oyster. Tributyl-tin ac ct ~t. e

(TBT), a compon ent of t in, c opp e r chloride (CuC1 2 ) is e l sa a

component of some an t i f ouling paints. We h a ve t h e refo r A t r ied to

e v a lua t e i t s effects o n the f e rtilizatio n, the e mbryoni c d e v e lon ­

ment an d on the g rowth o f D l .erv ae of C. g iga s . The toxic l evels

of this he a v y me tal s a lt u p on the e mbry os a nd y oung v e ligers of

Cra ssost r e a vir~inic a h a v e b e en d efined b y CA LABRRSE a n d 21 .

(1973) a nd He I NNES and CALABRESE (1979). Therefore, h'e h a ve

tried t o evalua t e t h e eff e c ts or c opper chloride on the l 2 rv~e

of C. g igc-s , with p a r t ic u l a r e mp h etsi s on t h e g ro,v·th o f th e v e l_i -

ger s .

2 . Mat e ri als and met h ods .

N .stural s e a ,,·ater obt a ined in bulk s e v e n miles of ~ slwr r-­

fr·om the Cap Fe rret p en i n s ul a , Arc achon, ·wa s u sed in these t r ::..C' l s .

3-

The Ee a w&tcr was diluted to 28 o/oo ~ith distil led wate r

a nd filtered through an 0,8 mic r o n s cellulose acetate filter .

The various copper chloride concentrations, r anging

f r om 0 to 500 p . p.b. ~ere prepa r ed by adding the required q u an­

t ity of copper chloride from a stock solution (SO mg .ml - 1 ) .

~o a lloKance was made for existing copper c oncentra tion in the

natural sea wa ter.

Oyst e r gametes were obtained by subjecting ripe

adul ts to cyclic t e mperature changes v a r y ing between 15 - 28°C . A spj:twning adult was placed in a 1 liter g lass beaker containing

filtered s ea water at 28 o/oo salinit y and ~mbient temperature

20-2 1° C. When sufficient eggs o r sperm was obtained , the adult

1va.s removed and the content of the beake r passed t ,hrough a

100 mi crons nylon screen to remove debris ; the eggs were

r e tained on a 32 microns sc r een . Aft e r wa shing with clean se a

water t ,he eggs werE: res uspende d in tv•o l.i t. el'~ [;l P f:.f' beaker s

a t a density of 30 eggs per ml. 1,5 ml of ~ense s u spension of

sper m \·'as added ·to evch beal~ er within h alf a n hour of 1 ib e r a ­

tion of the gameteE .

T"''o experiments v:er e m.::d e . Ex periment 1 exnmi ned

the e ffect of a r 2nge c f concentrations of copper c h loride

(0, 5 , 10, 25 , 50 <.nd 100 p.p.b.) on t ,h c f n't i1 j zc.ti~n of

eggs and on their subsequent developnent while experiment 2

examined the effect of copper chl o r ide (0 , 25, 50 , 100, 250

and 500 p.p.b . ) on 24 hours ol d D larvae which origin Bted from

eggs fertilized in n ormal sea water. Each tre atment w&s dupli­

c ated .

~~thin 24 hours of fertiliz£tion , the ~ l a rvae &fter

observation we r e adjus t ed to 3 l e rva e per m] . Keithcr antibio­

tics nor b ac tericides 11·e r e used bf'cc:use of t ,heir influence o n

g ro,,·th and mortality (HELM and JIITLLI CAN , 19 77 ; L E PENKEC et

PRIEUR, 1976). Th e cultures in two li ters h a rd glass beakers

":ere not aerated since turbulence is de tr imcnt<. l to the r-;r o,,·th

and s urvival of n ·1 a rv C:t C' i.n cnl turcs of smC~ ll volume (HELM a nd

1 ()'"'"') ) "' I - •

' -t -

The experiments lasted for seven days duri ng ~hich

time the water temper ature was kept at 26°C + 1°C. The l a rv a e

were fed with 100 cells per pl of Pnvlova lutheri at the r a n g e

concentration of 4 t o 6 cells X 10 per pl. The cultures Kere

changed daily when the larvae were passed through ~ 100 pm screen and retaine d on a 40 pm mesh screen and washed 1·: i t h

clean filtered se<1 , ... Tater~. The larvae \\'e re re suspend.ed i11 c 1 ec n

sea water with a fresh supply of food and copper chloride .

At each water change the behaviour of the la r vae was o~served

and a sample of 50 ali.v e J.arvae from each beaker (100 per t r e at ­

ment) was measured accross t he width o f the s hell_ (dorso - ventral

axis). Hortality a n d abnormality were assessed by c ount i n :'!. the

numb e r of dec.d larva e in a s a.mple of 100 i nviduals f r om ea.ch

beaker.

3. Results .

3.1. Experiment 1.

3.1.1. Success of fertil i z ation , emb r y0nic

d eve l opment an rl form ation o f D l a rvae .

Pr elimL'l cry t ests s hmved t ,h at fe r t i:!. i zCJt i on •.,·a s unsus­

cessfulJ at c oncentration~ of copper chloride above 50 p . p . b .

The a ims of this s eri e of e xperiments were to c on f irrn

the first ob ser vatio n s , to determin e mor e precisel y t h e effPc ts

of copp e r chl3ri dc on t h e e mbryon i c d e velopment and finally to

e st ablis h the effects on t he g rowth of t h e l arv~e .

After 2 4 h o urs , at a conce ntr ation o f 100 p . p . b . ,

65 % of the eggs r emain ed unfe rtilized . Of the fert ilized

eggs 5 % were st erroblastul ae and 30 % were t r ochopho r es of

which onl y 7 % app eare~ nor mal . On the second d a y a ll ~e r e

found decomposed , with a ver y few t r och oph o r es still r e ma ining .

The r a te of deve lopment of abnorma l larvae and t h e mortalit ~·

occuring when eggs were s ubj ected t o concentr a t i o ns of co p~Pr

chloride below 100 p.p . b . are shown in tabl e 1.

Afte r one and two days at 50 p.p.b. copper chloride

36 % and 95 % respectively of the larvae were abnormal . The

most eas ily det e ct ed and frequently seen abnorma] ities were an

enlarged velum nr irregular s hell shapes s uch as a concave hinge

o r inducted shell m~rgins . This treatment was discontinued cfter

t\,'0 days.

Up to 25 p.p . b . copper chloride, the proportion of

abnorma l larvae decrcesed with the concentration of copper

chloride. No s i gn ificant increase in t h e level of abnormality

was s een in any of the t reatments during the r e ma ining six days

of the experiment .

3.1.2. Effects on g rowt,h.

These r e sblt only concern the cultures that were

s ubjected to the conce ntrations of CuC1 2 below 50 p . p . b.

(figure 1). There were no significant differences in the size

of the veligers from the different cultures during the fit'st

24 hours , but after two days the larvae in the c o ntrol beakers

were significantly longer (P ( 0,05) than in those treated with

copper salt .

3 . 2. Experiment 2.

As in experiment 1, the rate of 50 p.p . b . of CuCl 2 r epresent s a limit above which the veligers quickly died and

below which sufficient larval g rowth was observed to allow

the formation of the curves i n figure 2. Th e massive de a th

r ate occuring to concentrations above 50 p.p.b. c a n be seen

as a correlation (table 2) . I n each case the behaviour of t he

veligers was abnormal . There was evidence of abnormal rotatio­

nal moveme n ts . The larvae remained a g r ey colour which shows

that there ' ''as considerable disturbance in the absorp"Lion of

food . There was no evidence of growt l1 (table 3).

6-

4 . Discussion .

Copper chloride is toxic to the embryonic d e v e ] o p­

me rrt of Crassostrea g i gas . Therefore the effects CJ.r e ( a ) un s ue­

c e ssf ull fertiliz ation a t 100 p . p . b ., (b) pronounced a bnorma ­

lities with a de l a y in t he formation of D larvae a t 50 p . p . b .

a nd a hig h percentage o f mal formed veligers at 25 p . p . b . The se

results a re cons i stent with t hose of Me INNES et a l (1 9 79 ) under

simi lar expe rime nta l c onditions. At a temperature of 2 5 ° c, salinity of 27,5 o / o o a nd c oncen~r ations of 0, 5, 10, 20 p . p . b .

of CuC1 2 t h e a v e r age percentages of abnorma l l a r vae a ft e r 48

hours are 1.4 %, 2 . 6 %, 4 . 4 % and 30 . 8 %. Althoug h the hig h e r

levels of abnormal ity obtaine d in our experimen t s a r e pr o bably

caused by the fact tha t t h e eggs were fertilized in th e p r esvncc

o f t his hea v y metal sa]t whe reas Me INNES and a l used eggs

which had alrea dy b een fe rtilized . Th e limit o f s e n s i t i v i ty

t o copper chloride i s reduc e d whe n the fertilization takes

pl ac e in the presen ce of the tox ica nt as we previous l y obser­

' ' ed during e xper iments ,.·i t h tributyle t i n 2.c e t a te (HIS :.:nd

ROB::RT 1980) .

I n e~pcriment 1 t he effect o f t h e copper chl oride

on the g r owt.h nf :) 1 .:- rv r>c of C • . '.! i gas ( f i g ure 1) a r e t .hcr ".:'f,.p·c

ch a r acte rised b y the im~ort2nt sl owing down of t h e g r owth f r om

t h e f irs t 48 hours . Then an increase i n the g r o wth r ate , of

which the slope is a di r ect r esult o f the co~centr at ion o f

CuC1 2 , was f oun d. The exi stence o f a sec o nd impo r tan t r educ ­

t i on in t he g r owth seems to sug~est that the h ea vy met. a l j s

not e l_i min a ted but~ r a t h e r accumulate d, \·.rhich confirms t h e

ob servations made by CO~~ O~ ( 1972 ) a n d b y BRYA N (19 69). Th e

r eviva l in g rm,•th ohs e r v e d after t he important r e duc t j on ··o n1 .. cJ

expl a in the t oler ance to the h e a vy met a l s a lt w1til a c e r t c> in

1 i mit o f sens i v ity which e v olves ~ith the oge of the l Erv2e .

I -

CALABRESE a nd DAVIS (1970), BRERETON et al (1973 ) BOYDEN et

al (1975 ) al l . state t h at t h e sen s ivity of the velige r s de ­

crea se with age .

In experiment 2, dur ing the seven days period nf

study, the cultures were ma i n tained without signs of massive

death rate for c o ncentrations lowe r than 50 p .p.b. Th e dif ­

ferent r ates that ..,,,ere noted Dr e s h own in table 2 es those of

He INNES a nd a.l. ( 19 79) ·r::hich show the s i milar r ates tes·ted

by these authors ~ith C!virgin ica. We therefore state that t h e

r esults obt a ined from C. gi::ras r esemble those f r om C. virg}:~i£.£

Howev er, mortality appear ed a day l a ter among the C. gig2s

larvae . This could be explained by the fact that t hese authors

u sed hig her concentr ations (30 and 60 p . p . b . ~,,hile we used

25 and 50) . Never t heless this delay in mortality also occured

at a hig her concentration 100 p.p.b . for t he J apanese oyster

compared with 90 f or the American oyster . C. g i ges woul d ther~ ­

fore seem sl.ightJy more r esi s tant .

In Experiment 2, t h e g r owth obtained from 25 p . p . b .

resembJ es the one {."~ot fro~·' Experiment 1. At 50 p . p . b . g r o;·:t h

increment prac ·ticall y .stopped 2.fte r 3 clc:-.ys and t .he appB r ent in­

crease in size at the e nd of t h e experiment was prob ably c? use~

by the el j mi nati o:' -:f ::-he s-·m2.J.lest l e>.rv 2.e from t.he cuJ tur l:' .

If we compa re the effects of copper chlori de to th2t

of tributyle tin acetate for the same experimental proce dure

(HI S a n d ROBERT 1980) ~e fin d that co~per chlor ide i nhibits

fer tj l.iz ation at t h e concentrat i on of 100 p . p . b . whe reas thi s

h appens at 25 with T B T.

At 25 p . p . b . of CuC1 2 l a r vae are formed as norm a l

but show a significant d elay in g r owth (no evide nc e of fe r t i­

li zation found at thi s concentrat ion with T B T ) .

, I

8-

At 10 p . p.b. a slowing down in t h e g r owth of the

velig e rs is still to be found, where as TBT pre vents the for ­

mation of D larva e even at t h i s weak r ate .

5 . Conclusions .

Because J a r vae g r ot·:n to spatfall ma.y no t achieve

goo d quality spat t h e r ate of 10 p g . l-l of c oppe r chloride

a~pears to be the limit of sensitivity for t h e embr yos a n d D

l arvae o f Crassostr e a g igas . Copper chloride is less toxic

than t h e org ano-metallic s a lts of tin, b u t c opper chloride is

fa r more solub le in sea water t h an the latter .

Fi n a lly , it must be sald that the r e are ver y few

dif fer enc e s b e tween the re s u lts obtained by Me INNES e nd

CALABRESE (1979) f r om Cr assostr e a vi r g i nic£ a nd those wh jch

we h a ve obtai ned f r om Crassostr ea gigas . Furthermore t h ese

author s h a v e shown thnt the veliger s of t h e Ame r i c a n oyster

directly exposed to copper c hl oride, wer e fu r t h er t·:ec.1 · e:1ec1

~ue to adverse environmental conditions of temper ature 2nd

sc:1lin i -ty .

Thi s r esearch was carrie d out with t h e financi al a id

of the Reg ion a] Dcpar t me.1t of t h e C. I . C. to ,,·hom we tw uld 1 i ke

to thank . The a u t hor s wish to tha n k Dr B. E. SP ENCER nnd Dr .R. H.

\~'ARt•!ICK fo r their u seful criticisms o f the ma uniscr ipt . We

a re g rateful to F i ss ~10RGAr~ wh o helped tdth th e t r enslc::.t ion .

- T A B L E 1 -

Age of the Copper Chlor ide concentra·tion ( p. p . b . ) I I

l a rv ae from I

fertilization 0 ,.. 10 25 50 :J

(days )

1 0,5 9 10 35 3(; (0) (0) ( 0! (0) ( 0)

- -- r-·

') L. 1 9 13 36 95

(0 , 5) ( 1) (2) ( 1 ) (0)

1--· - · 3 1 9 1 5 36 cultures

( 1 ) ( 1' 5) (2) ( 1 ) disc on-tinued

------

Table 1 Experiment 1 • The me a n perce t1tage abnormal l a rvae and mean percentage mor t a lity (in p a renthesis) occuring when Crassostr ea gigas embr yos were exposed to various concentrGtions of copper chloride during and after f e rtilization .

'! I

T !I D L E ..... .<... -

('.ge of the larvae from fertilization (days ) 9

Copp~r Chlor ide concentration (p . p . b . )

'"),. ·-) so 100 150 250 500

1

2

3

4

5

7

Tnble 2

/Ill

0 0 0 0 0 0 0

-0,5 0 3 2 , 5 1 4 88

1 1 3 8 6 70 100 (3,4) (10,7) (50 , 6 ) (77, 4 )

6,5 1 1 13 60 70 86 /Ill

8 16 26 91 Ill/ /Ill /Ill -·- -

10 18 45 !Ill Ill/ /Ill /Ill

Experime nt 2 . The me an percentGge mor t ality of Crass ostrea g i gas l arva e expose d to v a rious c oncentrations of coppe r chloride 24 hour s afte r ferti l.ization of the eggs. The val ues i.n p a renthesis 2r e the mean p erc entage mortalit~ · of Cr a s s ostrea virginic a larva e exposed to various concentrations of cop p e r c ltloride for 48 hour s u n der similar experiments conditions ( f rom Mac INNES Rnd C!ILAD~ESE 1979) .

Cultures discontinued.

~

0 I

- T A B L E 3 -------

f.geof the--larvae from Copper Chloride conc entrc tion ( p~ ~ . b .) fertilization (days )

---1

2

3

L].

5

Table 3

Ill/

100 150 250 500

62 ,74 ± 0 , 60 62 , 7-~ ± 0, 40 62 ,74 ± 0 , 40 62 , 74 + 0 , 40 -----------

65,02 ± 0,<)0 (13 , 78 :±: 0,52 62 , 45 ± 0, 47 63 , 35 ± 0 , 52 - ·

r. , 3 - J 0 (" ,). , ' J ;:: ' J..) 63 ,73 ± 0 ,55 62 , 62 ± 0 , 8 2 ///////////;'

6.~ , 54 ± o, 5 R ////////Ill/ l/l/1/////// /II/IIIII///

65 , 97 ± 0,2 5 / /III/II/II / ////////// ////////////

Experi.ment 2 ~ Me~n shell width ( pm ± 95 % c onfi dence interv2l) of Crassostre~ gi~a~ larvae g r own in variou s concentrations of Copper Chlori de .

Cultures discontin ued .

1-4 1-4

I

84

82

80

78 ,-....

E ~ 76

J: ~ 74 c -~ 72

-I -I 70 w J: 68 en z

66 ~ <( w :E 64 j

62

I 60

FIG . 1 :

I 1/

v 1 2 3 4

EXPERIMENT 1. Growth of larvae of Crassos t rea gigas at various

concentrations of copper chloride . The 95% confidence

limits of the mean size are shown.

.....

®

• ...

•

control

5 _pg. 1_,

1 0 _pg . 1-1

2 5 _pg. r,

5 6

TIME (days)

7 ...... N I

84 I

@ control 82 ~

( "! 25 .P9 . 1-1 80 J

---I' 50 .P9 . 1-1 .J

I E 78

~

~ :I: 76

J-c 74 -~

72 ..J _, w :I:

70

en 68

z <( w 66

:!: 64

62

60 1 2 3 4

FIG . 2 : EX PER I MEN T 2. Growth of 24 hours larvae of Crassostrea

gi gas at variou s concentra ti ons of copper chloride . The 95% conf idence

I imil s of the me an size are sh own .

T

T ..!.

1

1

I I I -

5 6 7

TIME (days)

1-'" w I