.. _ • 10

THIS PAPER NOT TO BE CITED WITHOUT PRIOR REFERENCE TO THE AUTHORS

International Council forthe Exploration of the Sea

CHEMICAL PRE-TREATMENT AS A TECHNIQUE TO IMPROVESEAWATER QUALITY IN BIVALVE HATCHERY WORK

Susan D. Utting and M. M. HelmMinistry of Agriculture, Fisheries and FoodFisheries Experiment StationConwy,' Gwynedd LL32 8UB, Wales

ABSTRACT

CM 1983/F:4Mariculture Committee

•

•

The value of EDTA, sodium metasilicate, alumina, magnesium trisilicate

and Fuller's earth in the pre-treatment of sea water to improve conditions

for the development of fertilized Pacific oyster (Crassostrea gigas) ova to

the veliger larva stage was investigated in aseries of 80 trials. Sea

water was treated with the compounds for 24 h before spawning and was

aerated during this period. Control trials used untreated sea water and

an artificial medium.

Most treatments were effective in improving the condition of sea

water but the extent of improvement 'varied considerably between trials.

Compared to untreated sea water, significantly better results were obtained

with EDTA at 1 mg 1-1 alo~e or in combination with 20 mg 1-1 sodium meta­

silicate, 20 mg 1-1 alumina, 50 mg 1-1 magnesium trisilicate or 150 mg 1-1

Fuller's earth. Possible modes of action of the compounds are discussed •

La valeur de l'EDTA, du m~tasilicate de sodium, de l'alumine, du tri­

silicate de magnesium et de la terre. a foulon dans le pre-traitement de

l'eau de mer pour ameliorer les conditions de developpement d'oeufs ~econ~es

d'huitres du Pacifique (Crassostrea gigas) jusqu'au stade yeligere a. e~e

exami~ee lors d'une serie de 80 essais. De l'eau de mer a. e~e trai~ee avec

les composes pendant 24 heures avant le frai, et a. e~e aeree au cours de

cette periode. Pour les essais de controle, on a fait usage d'eau de mer

non traitee et d'un milieu artificiel.

La plupart des traitements avaient efficacement ameliore la condition de. .

l'eau de mer mais le degr~ de cette amelioration ~ariait largement entre

essais individuels•.Par rapport. a l'eau de mer non trai~ee, des~esultats

nettement meilleurs ont. e~e obtenus avec l'EDTA a'1 ~g 1-1 seul ou en com­

binaison avec du ~etasi~icate d~ sodium.a.20 mg ~-1, de ~'a~umine a

20'mg 1-1 du trisilicate de magnesium a 50 mg 1-1 , ou de la terre a foulon

a 150 mg ~-1. D'eventue~s modesd'action des composes sont discut~s.

1

INTRODUCTION

The development of the delicate early stages in the life history.of

marine invertebrates in a laboratory or hatchery environment is frequently

retarded by what are thought to be adverse water quality conditions. This

phenomenon is weIl documented in the literature, e.g.i Wilson·(1951, 1981),

Davis (1953) ,Wilsonand Armstrong (1958), Loosanoff and Davis (1963),

MilIar and Scott (1968), Walne (1970) and Helm (1971). While theeffects

are readily demonstrable the underlying causes remain obscure, although a

range of naturally-occurring and man-made pollutants, substances leaching

from bottom sediments, the effects of.high turbidity and dense algae

blooms have been cited in the above references.

The most common manifestation of poor water quality in a bivalve

hatchery is the failure of fertilized ova of oviparous species to develop

normally to the straight-hinged, D-shaped veliger larva stage. This may

be because the phenomenon is evident at a particularly vulnerable stage.

In extreme cases development stops at the trochophore stage but more com­

monly a proportion of larvae develop with severely abnormal shell valves.

In larviparous species, e.g., Ostrea eduZis L., newly-liberatedlarvae

are unable to express their full growth potential, which is assessed as

growth performance in natural sea water compared with an artificial sea

water medium (Millarand Scott, 1968; Helm, 1971).

'MilIar and Scott (1968) showed that sea water with reduced ability

to support growth could sometimes be improved by pre-treatment with

Fuller's earth or magnesium trisilicate. They suggested that dissolved

substances were responsible on these occasions for impaired growth. This

theme has been continued at this laboratory to test the efficacy of a range

of chemical compounds in improving the condition of sea water to support

the development of fertilized ova of the Pacific oyster, Crassostrea gigas

Thunberg, to the D-larva stage. A total of 80 comparativetrials was

made during the period 1976 to 1978 inclusive and the results are reported

here.

METHODS

Preparation of sea waters

Test sampies of sea water were prepared 24 h before the start of

bioassays. Sea wate~ was collected in clean, borosilicate glass flasks

from the supply to the hatchery which is described in Wickins and·Helm

(1981)·. After adjusting the salinity to 280/00 ·with freshly distilled·

2

..

•

•

water, the sea water was filtered under a vacuum not exceeding 0.35 kg cm-2

through a Whatman GF/C filter paper. The filtrate was dispensed into

500 ml glass flasks containing the various pre-treatment agents under test

and then aerated from an oil and carbon-free source for 24h; the air

supply was filtered through a 0.3 ~m cartridge and humidified by bubbling

through distilled water so as to maintain the ~~linity of the test samples.

Chemical additives tested included the disodium salt of ethylene­

diaminetetraacetic acid (EDTA), sodium metasilicate (Na2Si03.9H20),

magnesium trisilicate (2 Mg03Si20), alumina (Al203) and Fuller's earth as

the commercial product Fulbent 570 (Laporte Industries Ltd). Fuller's

earth is a claymineral of the montmorillonite group with the general

formula AlFeO.67MgO.33Si4ol0 (OH)2NaCaO.35' Only the combination of EDTA

together with sodium metasi1icate at 1 mg 1-1 and 20 mg 1-1 respectively

... was tested in each trial. Treatments tested in the various trials together

with concentrations used are given later.

In all trials, controls were performed in filtered sea water at 280 /00

without chemical additives and in an artificial sea water developed by

Zaroogian et ale (1969) for the culture of Crassostrea virginica Gmelin

embryos and larvae. This was prepared by dissolving analytical grade

reagents in the recommended sequence in freshly distilled water to 280 /00.

A salinity of 280 /00 is the optimum for the development of Pacific oyster

embryos. Preparation was completed at least 24 h before bioassays and the

medium was aerated to stabilize pH to about 8.0. Once prepared the medium

could be stored in the dark at 20 to 22°C for up to 4 weeks without

deterioration.

Embryo bioassays

Adult oysters were artificially spawned by stripping gametes from the

gonads of oysters which had been held in laboratory tanks for 2 to 5 weeks

at 20°C in a through-flow of phytoplankton-enriched sea water (Walne and

Helm, 1974; Helm and Millican, 1977). Gametes were removed, after opening

the oyster, with sterile pasteur pipettes and those of each oyster trans­

ferred to .separate 1 1 beakers containing 200 ml of filtered sea water at

280 /00 and 24°C.

After stripping,the ova of 2 females were pooled and the concentra­

tion critically jcte:J:;mined usii1g a No<kl ZI3 Coulter. C:unter. Where neces­

sary the concentration was adjusted to about 4 000 ova ml-1 by the addition

of filtered sea water. Sperm from 2 males were pooled and the ova were

3

fertilized by adding at the rate of 2 ml of the resulting suspension at

10 to 20 million sperm ml-1 to 1 1 of ova. suspension while gently agita­

ting the beaker eontents. Sperm eoneentrations were determined using a

Model ZB Coulter Counter.. Fertilization was made within 1 h of gamete

stripping (Helm and Milliean,1977).

Aliquots of 1 500 fertilized ova were transferred to eaeh of aseries

of non-toxie, transparent polystyrene vials and the eontents made up to

30 ml with the appropriate test water to give 50 ova ml-1 • Eaeh treatment

within a trial was made in quadruplieate. After incubation for 24 h at

24°C the number of weIl formed D-larvae in duplicate 2 ml sub-samples

from 3 of the replicates for each treatment were counted using a binocu­

lar microseope. The fourth replicate was sampled only when there was

disparity in counts. Treatment results were expressed as the mean number

of D-larvae 2 ml-1 , effectively the percentage yield, and as net treatment

mortality (NTM).

[1 _ ( mean D-larvae 2 ml-1 in ~e:t.sea water )1.100mean D-larvae 2 ml- 1 in art1f1c1alsea water

RESULTS

EDTA and sodium metasilicate

•

Each of the 80 trials made between May 1976 and September 1978 com­

pared the development of fertilized Paeific oyster ova to D-larvae in

untreated sea water, the artificial medium and sea water treated with

1 mg 1-1 EDTA together with 20 mg 1-1 sodium metasilicate. This combina­

tion of pre-treatment additives proved to be generally effective in

improving seawater quality in early experiments. In 1978, 31 trials com-

pared EDTA at 1 and 2 mg 1-1 alone and in eombination with sodium meta- ~silicate at 10 and 20 mg 1-1 in a factorial arrangement.

Analysis of variance of D-larvae yields obtained in 1976 and 1977

showed a significant improvement inwater quality attributable to the

additivesto the extent that it supported, on average, as good conditions

for the development of embryos as the artificial medium (Table 1A). Sea­

sonal effects of the pre-treatment are shown in Figure 1. Improvement as

the result of pre-treatment was evident in almost all trials but the

extent of the improvement differed considerably between trials (Table 1B).

Differences between trials might have involved variations in the lipid

content of ova at the time of spawning. This possibility will be examined

fully elsewhere.

4

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•

The lowest yields of D-larvae in untreated water relative to the con­

trol artificial medium, which are shown as the highest NTM values in

Figure 1, occurred in late spring and summer coincident with intense phyto­

plankton blooms. Even relatively good natural sea water in the earlier

months of 1977 was improved for embryo development by pre-treatment so

that it frequently provided greater yields of D-larvae than the artificial

medium (negative NTM values) '.

Results of the 1978 trials comparing the effects of EDTA and sodium

metasilicate in the factorial arrangement are shown in Table 2. These

results were analysed by analys~s of variance. The mean yield in the arti­

ficial seawater controls to these trials was 57.2 D-larvae 2 ml-1 • These

trials clearly demonstrated that the most important component of the pre­

treatment was EDTA and that there was no advantage in increasing the con­

centration to 2 mg 1-1• The addition of 20 mg 1-1 sodium metasilicate in

the absence of EDTA resulted in a yield of D-larvae significantly greater

than that in sea water without additives. There was nb sig~ificant dif­

ference between 0 and 10 mg 1-1 sodium metasilicate or 10 and 20 mg 1-1

sodium metasilicate in the absence of EDTA. Yields not significantly

different from those in artificial sea water were provided by all treat­

ments which included EDTA at 1 or 2 mg 1-1 •

Alternative pre-treatments

AZumina: The value of alumina as apre-treatment agent was tested in 27

trials. Results shown in Table 3 compare yields of D-larvae in untreated

sea water and water treated either with 1 mg 1-1 EDTA, 20 mg 1-1 a1umina ­

the most effective concentration in a pre1iminary series of trials

(Figure 2A) - and a combination of 1 mg 1-1 EDTA with 20 mg 1-1 a1umina.

Treatment with alumina a10ne did not consistent1y improve water qua1ity

and no additive effect was observed with alumina in combination with EDTA.

EDTA alone was the most effective treatment.

Magnesium,trisiZicate: A preliminary series of 3 trials showed a concen­

tration of 50 mg 1-1 to be themost effective pre-treatment with this

agent (Figure 2B). Treatment at this concentration in combination with

EDTA at 1 mg 1-1 provided D-1arvae yields which were not significantly dif­

ferent from water samp1es pre-treated with the combined addition of

1 mg '1-1 EDTA and 20·mg 1-1 sodium metasilicate in 4 trials.

5

FuZZer's earth: Two trials tested the effeets of this agent at pre­

treatment eoneentrations ranging from 25 to 250 mg 1-1 (Figure 2C). In

both, the maximum effeet was at 150 mg 1-1• A further 7 eontrolled trials

eompared embryo development in sea water pre-treated with 1 mg 1-1 EDTA in

eombination with either 20 mg 1-1 sodium metasilieate or 150 mg 1-1

Fuller's earth (Table 4). Both pre-treatments brought about a signifieant

improvement in eonditions for D-larvae development with EDTA and Fuller's

earth generally providing the better results. However, it was found neees­

sary to filter water after treatment to remove the large quantity of sus­

pended Fuller's earth. While this was done with EDTA-sodium metasilieate

pre-treated samples in this series of trials to provide astriet eomparison,

it was not a neeessary step in the proeedure.

DISCUSSION

This series of trials shows that it is feasible to improve the eondi­

tion of natural sea water to support dcvclopment of Paeifie oyster embryos

to the D-larva stage by simple, inexpensive pre-treatment proeedures with

a number of ehemieal eompounds. In early trials it was found that the

eompounds exerted elose to their maximum effeet in 24 hand that aeration

of the sea water during this pre-treatment period was benefieial. Pre­

treatments almost invariably improved water quality but the extent of the

improvement between trials was ineonsistent.

Of the compounds tested EDTA as a single agent treatment at 1 mg 1-1

was the most effeetive. Overall, there was little advantage in using

other eompounds in eombination with EDTA but in some trials eombinations

provided the better results. Fuller's earth in eombination with EDTA was

notably valuable but had the disadvantage that it was neeessary to filter

the treated water before use.

Pre-treatment with EDTA alone or with sodium metasilicateis routinely

used at Conwy and now in some private hateheries during the early develop­

mental st~ges of a range of bivalve speeies. This treatment can improve

the ability of sea water to support the growth of veliger larvae but the

effects are less marked than during embryo development.

Various actions of the pre-treatment agents are suggested including

complexing of trace metals by chelating agents, e.g. EDTA and the adsorp­

tion'of metals and organic substances by the other compounds tested. The

calanoid copepod Euchaeta japonica exhibited improved survival in copper

contaminated sea water which was treated with various particulate and water

6

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•

soluble agents (Lewis et al., 1972). Successful treatments included clay

minerals, diatoms, ascorbic acid, sewage cfflucnts and aqueous extracts

of humic acids and certain soils. Substances such as clay minerals are

known to adsorb metals and also organic substances (Chester, 1956;

Siegel, 1966) while certain phytoplankton species concentrate metals

(Morris, 1971). Willey (1977) showed.the co-precipitation of insoluble

metal silicate compounds in metal enriched sea water in thc presence of

sodium metasilicatc. Compounds such as sodium metasilicate and Fuller's

earth may also act as flocculents removing sub-micron particulate material

from suspension.

One of the likely effects of successful treatments at Conwy is the

reduction of the conccntration of trace mctals, e.g. zinc and lead in the

ionized formi the Rivcr Conwy drains richly metalliferous strata in the

Snowdonia mountain range (Elderfield, 1971). This was demonstrated on two

occasions in 1978 when the metal ion concentration in untreated and treated

sea water was measured (Table 5). Pre-treatment of sea water with EDTA and

sodium metasilicate reduced the measurable level of zinc and lead. However,

there are times, particularly during and immediately following intense

phytoplankton blooms in spring and summer, at low fresh water inputs into

the estuary, when the mode of action of treatments which improve water

quality is not understood.

Reference to proprietary products does not imply endorsement by MAFF

or criticism of alternative products.

REFERENCES

CHESTER, R., 1956. Adsorption of zinc and cobalt on illite in sea water.Nature, Lond., 206: 884-886.

DAVIS, H. C., 1953. On food and feeding of larvae of the American oyster,C. virginica. Biol. Bull., Woods Hole, 104: 334-350.

ELDERFIELD, H., 1971. Some geochemical aspects concerning oyster rearingat Conwy, North Wales. Research Report, Applied GeochemistryResearch Group, Department of Geology, Imperial College, London,110 pp.

HELM, M. M., 1971. The effect of sea water quality on the laboratory cul­ture of Ostrea eduliD L. larvae. ICES C.M., 1971/K:28, 8 pp.

HELM, M. M. and MILLICAN, P. F.,1977. Experiments in the hatchery rear­ing of Pacific oyster larvae (CraSso3trea gigas Thunberg).Aquaculture, 11: 1-12.

7

LEWIS, A. G., WHITFIELD, P. H. and RAMNARINE, A., 1972. Some particulateand soluble agents affecting the relationship between metal toxi­city and organism survival in the calanoid copepod EUahaetajaponiaa. Mar. Bioi., 17: 215-221.

LOOSANOFF, V. L. and DAVIS, H. C., 1963. Rearing of bivalve mollusks.Adv. Mar. 'Biol., 1: 1-136.

MILLAR, R. H. and SCOTT, J. M., 1968. An effect of water qualitygrowth of cultured larvae of the oyster Ostrea eduZis L.perm. int. Explor. Mer, ~: 123-130.

on theJ. Cons.

MORRIS, A. W., 1971. Trace metal variations in sea water of the MenaiStraits caused by a bloom of Phaeoaystis. Nature, Lond., 233:427-428.

SIEGEL, A., 1966. Equilibrium binding studies of zinc-glycine complexesto ion-exchange resins and clays. Geochim. cosmochim. Acta., 30:757-768.

WALNE, P. R., 1970. Present problems in the culture of the larvae ofOstrea eduZis. Helgoländer wiss. Meeresunters, 20: 514-525.

WALNE, P. R. and HELM, M. M., 1974. The routine culture of the Pacificoyster (Crassostrea gigas) at Conwy during 1973. Shellf. Inf.Leafl., Fish. Lab., Burnham, (32), 10 pp.

WICKINS, J. F. and HELM, M. M., 1981. Sea water treatment. pp.63-128 In:'Aquarium Systems', (ed.) Hawkins, A. D. Academic press, London,452 pp.

WILL?Y, J. D., 1977. Coprecipitation of zinc with silica in sea water andin distilled water. Mar. Chem., 5: 267-290.

WILSON, D. P., 1951. A biological difference between natural sea waters.J. mar. biol. Ass. U.K., 30: 1-19.

WILSON, D. P., 1981. An experimental search for phytoplanktonic algaeproducing external metabolites which condition natural sea waters. ...J. mar. biol. Ass. U.K., ~: 585-607.

WILSON, D. P. and ARMSTRONG, F. A. J., 1958. Biological differencesbetween sea waters: experiments in 1954 and 1955. J. mar. biol.Ass. U.K., 12.: 331-348.

ZAROOGIAN, G. E., PESCH, G. and MORRISON, G., 1969. Formulation of anartificial sea water media suitable for oyster larvae development.Am. Zool., 9: 1144.

8

Table lA The mean yields of Crassostrea gigas D-larvae per 2 ml sampIein untreated sea water, sea water treated with 1 mg 1-1 EDTAand sodium metasilicate at 20 mg 1-1 and in artificial seawater in 16 trials in 1976 und 33 trials in 1977

Year

1976

1977

Untreatedsea water

31.8

34.7

Treatedsea water

59.0

50.8

Artificialsea water

70.9

53.5

16 trials

33 trials

Analysis of variance comparing treatments - 1976

•Source of variance

Between treatments

Residual

Sum of squares

12 857.18

17 674.11

df

2

45

Mean square

6 428.59

392.76

F ratio

16.37

p

< 0.001

Least significant difference between treatment means at 95% levelof probability 14.16

Analysis of variance comparing treatments - 1977

Source of variance

Between treatments

Residual

Sum of squares

6 832.79

47 196.24

df

2

96

Mean square

3 416.39

491.63

F ratio

6.95

p

< 0.01

•Least significant difference between treatment means at 95% levelof probability 10.80

Table IB Analysis of variance comparing treated and untreated sea waterand between trial effects in 1976 only

Source of variance

Between treatments

Between trials

Residual

Sum of squares

17 649.15

42 159.54

3 234.40

df

1

15

79

Mean square

17 649.15

2 810.64

40.94

F ratio

431.10

68.65

p

< 0.001

< 0.001

Table 2 The mean yields of Crassostrea gigas D-laryae per 2 ml sampIein untreated natural sea water and sea water treated with EDTAand/or sodium metasilicate in 31 trials in 1978. The mean yieldin artificial sea water in these trials was 57.2 D-larvae 2 ml~l

EDTA concentration, ppmSodium metasilicateconcentration, ppm

i I ~

o10

20

o

37.9

44.8

47.9

1

53.8

57.9

58.1

2

55.7

56.9

59.2

IIiII

, .

iI

Analysis of variance

Source of variation Sum of squares df Mean square F ratio P eBetween EDTA

9 153.33 2 4 576.67 14.82 < 0.001concentrations

Between sodiummetasilicate 1 480.15 2 740.08 2.40 n.s.concentrations

Interaction 351. 70 4 87.93 0.29 n.s.

Residual 83 373.02 270 308.79

Total 94 358.2 278

Least significant difference between treatment means at the 95% levelof probability 8.8

Table 3 The mean yields of Crassostrea gigas D-larvae per 2 ml sampIein untreated sea water and sea water pre-treated withA. 1 mg 1-1 EDTA, B. 20 mg 1-1 alumina, and C. a combinationof 1 mg 1-1 EDTA and 20 mg 1-1 alumina

Untreated ~reatment

sea water

36.4

A

53.1

B

40.4

C

55.7

Analysis of variance

Source of variance

Between treatments

Residual

Sum of squares

7 245

45 481

df

3

104

Mean square

2 415.0

437.3

F ratio

5.52

p

< 0.01

Least significant difference between treatment means at 95% levelof probability = 11.3

Table 4 Yields of D-larvae per 2 ml sample in untreated natural sea water(control) and sea water pre-treated with sodium metasilicate.at20 mg 1-1 and Fuller's earth at 150 mg 1-1 both in the presenceof 1 mg 1-1 EDTA

"

Trial Control EDTA + Na2Si03·9HZO EDTA + Fuller's earth

1 24.2 49.8 54.2

2 43.0 84.3 92.7

3 49.0 80.0 87.3

4 35.0 66.2 74.2

5 50.0 80.7 82.8

6 8.3 13.3 13.2

7 15.0 63.3 64.2

Mean 32.1 62.5 66.9

Analysis of variance

Source of variance

Between treatments

Residual

Sum of squares

5 045.59

9 803.64

df

2

18

Mean square

2 522.80

544.65

F ratio

4.632

p

<' 0.05

Least significant difference at 95% probability level 26.2

•

•

100

50

1976

~I'1 \I \

'd' ..~ , Rr \ \I \ ' ~

: \~! \.. I~0..., I ~\ I l

'6 0--0-_-<:/ 0

I',;, \

1 \,I \

\, \\, \ .\: ~ f,~ \ I '0

b... .".. I0- - """"'0--0

AJJM

\ R " ,.\ / , I " 9-0'\ / I \ I'b-_---O , I ' I

~ ''cl

1977

o

50

d-&­C-oE

-&- -50cQJ

E-&-

dQJ

;: 100

. -&­

QJ

Z

sAJJ

Mo nt hs

MA- 50'---_--L.__--L-__~__L___ ___J._____l...._

M

Figure 1 The yields of D-larvae relative to artificial seawater controlsin chemically untreated sea water (unbroken line) and sea watertreated with 1 mg 1-1 EDTA and 20 mg 1-1 sodium metasilicate(hatched line). Results are expressed as net treatment morta­lities and show seasonal variations in water quality for embryodevelopment in 1976 and 1977.

.. '

,.

100 ( A) 100 (8)

60

BO

40~I~

L-. 20E

N

o'---_---L ......

o 50 100Magnesium trisilicate mgl-01

O~---,----~

o 100 200Alumina, mgl-1

QJ

o>c-o

BO

40

60

20 L.....---.....&-__--L-__---J

o 100 200 300Fulbent 570 mgl-1

Io, 100coQJ

L

Figure 2 The mean"yield of Crassostrea gigas D-larvae per 2ml sampiein sea water pre-treated with:(A) alumina, at concentrations of 0-200 mg 1-1;(B) magnesium trisilicate, at concentrations of 0-100 mg 1-1 ;(C) Fulbent 570, at concentrations of 0-250 mg 1-1.95% confidence limits of yields are given.