t500 J . F ISH. RES. BOARD CAN. , VOL. 35 , 1978

MTLLER, M. C., exo J. E. Hossrp. 1976. RATE-TheToolik Lake program. Arctic Bulletin 2: 161-164.

O'Bnrer.r, W. J., lNo G. L. VrNyrno. 1978. Polymorphismand predation: The efiect of invertebrate pr-edation onthe distribution of two varieties of Dapltnia caritntu inSouth India ponds. Limnol. Oceanogr. 23: 452-460.

O'BnrcN, W. J., D. Krrrr-E, eNl H. Rrpssrx, 1978. Helmetsand invisible armor: structures reducing predation fromtactile and visual planktivores. Ecology. (In press)

P,lrNB, R. T. 1966. Food web complexity and species di-versity. Am. Nat. 100: 65-75.

RressEN, H, P., aNl W. J. O'BnrEN. 1978. Re-evaluation ofthe taxonomy of Dapltnia longirenis Sars 1862(Crustacea, Cladocera): Descript ion of a new morphfrom Alaska. Crustaceana (In press)

SrnrcrlEn, J. R. 1975. Swimming of planktonic C,-clopsspecies (Copepoda, Crustacea) : Pattern, movementsand their control, p. 599-613. In T. Y. T. Wu, C. J.

Brokaw, and C. Brennen [ed.] Swimming and flying innatule. Plenum Press, New York, N.Y.

Szt-,r.urn, L. 1965. The refuge ability of plankton animalsbefore models of plankton eating animals. Polski Arch.Hydrob io l . l3 : 89-95 .

Vrxvenn, G. L., eNl W. J. O'BnrsN. 1976. Effects of lightand turbidity on the reactive distance of bluegill(Lepomis macrocl irus). J. Fish. l{es. Boald Can. 33:2845-2849.

WrnNrn, E. E., eNo D. J. Her-r. 1974. Optimal foragingand the size selection of prey by the bluegill sunfish(Leponis macroclt ir t ts). Ecology 55 : 1042-1052.

Zennr, T. M., eNo W. C. Knnroor. 1975. Fish pledation onBosmina longirostris: Body size selection versus visibil-i ty select ion. Ecology 56: 233 237.

Zrnar, T. M., exo R. T. PetNp.. 1973. Species introcluct ionin a trooical lake. Science 182: 449-155.

Culture Studies on the Eftects from Fluoride Pollution onthe Growth of Marine Phytoplankters

Luls OrrvnrnaDeparttnent ol Botatry,, The University of British Columbia, Vancouver, B.C . V6T I W 5

Navar J. ANrra

Department ot' Fisheries and the Ettvironment, Fisheies and Marine Service,Pacific Environtnent Institltte, West Vatrcouver, B.C. V7V 1N6

AND THANA BTSATPUTNA,

Department ol Botany,TheUniversity ol Bri t ishColumbia,Vancouver,B.C.V6T 1W5

Olrvntnl, L., N. J. Aurn, lNo T. Brsllpurnl. 1978. Culture studies on the effects fromfluoride pollution on the growth of marine phytoplankters. J. Fish. Res. BoardC a n . 3 5 : 1 5 0 0 - 1 5 0 4 .

The autotrophic growth of 12 species of marine phytoplankters, from eight classes ofalgae, was tested on axenic cultures with NaF addit ions of 0-100 mgF/L. Al1 speciesshowed good growth without indication of toxicity or adaptation lag. The highest fluolideconcentration caused 25-307o growth-rate inhibition of a diatom, a dinoflagellate, and ahaptophyte; othel diatoms and species fi'om other classes of algae were virtually unaffected.It is hypothesized that the unexpected lack of toxicity from F ion may be due to theformation of innocuous complexes with one or more ions of seawater. The ecologicalinference is drawn that fluoride pollution may be readily tolerated by some marine phyto-plankton under nutrient-suffi cient conditions.

Key words: marine phytoplankton, growth in culture, fluoride-pollution effects

OLrvntn,l. L., N. J. ANur, eNo T. Brserpurne. 1978. Culture studies on the effects fromfluoride pollution on the growth of marine phytoplankters. L Fish. Res. BoardC a n . 3 5 : 1 5 0 0 - 1 5 0 4 .

Nous avons test6 la croissance autotrophe de 12 espdces de phytoplanctontes marinsappartenant e huit classes d'algues, en cultures zLx6niques avec addition de NaF i desconcentrations de 0 i 100 mgll- de F. Toutes les espEces croissent bien sans aucun signe

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Received February 14, 1978Accepted August 22, 1978

THe toxic effects of fluorine compounds, mainly in-organic, have been well established for a wide varietyof terrestrial organisms. In animals, both anatomic ab-normalities and physiological alterations were reportedto result from fluoride poisoning. The earlier literatuleon fluoride toxicity to animals and higher plants wasextensively reviewed by Eagers (.1969). In the case ofhigher plants, fluoride seemed to affect particularly themetabolism of growth (Ordin and, Skoe 1963) andrespirat ion (Lovelace and Mil ler 1967a, 1967b; Mil lerand Miller 1974). Typical toxicity symptoms at theultrastructural level were recently described for a bogmoss (Simola 1977).

Fluoride occLlrs naturally in seawater at levels of-1-2 mg/L, with an average sal inity-related rat io of6.75 x 10-i F/Cl for open seawaters (Greenhalgh andRiley 1961; Warner l97l); higher F/Cl rat ios of 8-9 X 10-; have been recorded for certain deep oceanicareas (Greenhalgh and Riley 1963). In addit ion to thisnatural occurrence, cases of chronic fluoride contamina-tion of coastal areas from industrial effiuents have beenrecently disclosed (Harbo et al. 1974), with l i t t le el lortmade to understand possible deleterious effects onaquatic ecosystems or marine l i fe (Moore 1971). Oursurvey of the literature revealed that fluoride effects onalgal growth have not been previously investigated.either for freshwater or marine forms. However, fluoridehas been tested from a purely biochemical point ofview as a metabolic inhibitor of respiration in speciesof Chlorella (see Sargent and Taylor 1972 and refet-ences cited therein).

Our present study reports the effects of fluoride con-centration on the growth rate in culture of 12 speciesof marine phytoplankters. representing eight taxonomicdivisions.

M aterials and methods - Axenic cultures of the 12 species,listed in Table 1 according to algal class, were grown photo-synthetically under aseptic conditions. Details of strainsources, their identification symbols, subsequent nomen-clattral revisions and taxonomic transfer from one algalclass to another as required, were recently summarized byAnt ia (1976) .

Crystalline NaF, giving concentrations from 0 to 100 mgF/L, was dissolved in a nutrient-enriched growth mediumbased on natural seawater of salinity adjusted to 26i1" inthe final medium. The endogenous F content of the mediumwas estimated as 1.05 mgll-, using a fluoride-specific ion-electrode determination method (Selig 1973). All other ex-

NorEs 15ol

de toxicit6 ou de d6calage d'adaptation. La teneur maximum en fluorure entlaine unediminution de croissance de 25 it 30Vo d'une diatom6e, d'un dinoflagell6 et d'unhaptophyte; les autres diatom6es et les espdces appaltenant d d'autres classes d'alguessont pratiquement inaffect6es. Nous 6mettons l'hypothdse que l'absence inattendue detoxicit6 de f ion F- est due i la formation de complexes inoffensifs avec un ou plusieursions de l'eau de mer. L'aspect 6cologique de ces observations est que cerlains phyto-planctontes marins peuvent facilement tol6rer la pollution par- fluorure dans des situationsoi les 6l6ments nutritifs sont suffisants.

ReEu le 14 f6vrier 1978Accept6 le 22 aoit 7978

perimental details were as described by Antia and Cheng(1915). Each growth experiment was perfotmed in tripli-

We were unable to test fluoride concentrations of 125mgll- and greater, because NaF failed to give solutions freefrom cloudiness or turbidity at these concentrations in theseawater medium. Since the solubility of NaF is known to69 -19 gF-/L in distilled water, we suspect that its appar-ent "saturation" solubility in seawater (between 100 and125mg F /L at 20'C) is due to precipitat ion of the highlyinsoluble MgF, and CaF, by interaction with the correspond-ing cations of seawater. The water solubilities of thefluorides derivable from the various seawater cations arelisted by Weast ( 197 1 ) .

Results -The effects of increasing concentrat ionsof fluoride were examined with respect to both thegrowth rate and possible adaptation requirement on first

exposure to the toxicant (Stockner and Antia 1976)-

The results (Table 1 ) revealed that even the highest

fluoride concentration caused little or no inhibitory ef-

fect on the exponential growth rate of 75ok of the

tested species. Furthermore, the preexponential lagperiods were invariably comparable to those of the

controls in all cases, suggesting that the algae did not

require significant adaptation to any level of added

fluoride (Fig. 1, Table 1).A certain degree of inhibition was observed in three

species of phytoplankters (Pavlova lutheri, Nitzschia

angularis, and Amphidinium carterl) when grown in

the presence of 100 mg F-lL. However, this inhibit ion

of growth rate never exceeded 25-30% of the cor-

responding controls, and there was no discernible alter-

ation in the preexponential lag period for these cases

(Fig. 1, Table 1). Furthermore, the inhibitory effect was

not accompanied by lethal signs of cell lysis after achiev-

ing maximum yield.The lower f luoride concentrat ions (( 10 mg/L)

seemed even to stimulate the growth of a few species(results not shown). This situation was part icularly

evident in the case of the cryptomonad Rhodomonas

/erus (see Table 1). Growth st imulat ion of this phyto-

plankter by fluoride addition ranged from 2O-30Vo

greater than the control and was consistently observed

at all concentrations tested up to the highest level. be-

coming most prominent at F- concentrat ion of 50 mg/ L

( s e e F i g . 1 ) .

Discussion - Our results show lhat under nutrient-

sufficient conditions, some marine phytoplankton can

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1502 J. FISH. RES. BOARD CAN., VOL. 35, 1978

Tnnlr l. Summary of fluoride concentration effects on phytoplankter growth.

ol Growth ratea (a) and preexponential lag period (6 days) fromadded fluoride concentration (mg/L) of

100502510

Species tested

ChlorophyceaeD unaliella tertiolec ta

PrasinophyceaePrasinocladus marinus

HaptophyceaePavlovq (Monoc hry sis) I ut heri

EustigmatophyceaeNannochloris oculata

BacillariophyceaeBelleroc hea polymorphaChaetoceros gracilisNitzschia angularis yar. ajfnisT ha I a s s io s ira fi uv iat i li sa

CryptophyceaeChroomonas salinaRhodomonas letts

DinophyceaeAmphidinium carteri

CyanophyceaeAgmenellum quadrupIic at nm

100

100

r00

101

92

7 1

100

9496789 1

100122

75

96

90

93

100

948998

102

l 0 l

104

1 1 0 3 10 1 0 0 01 1 0 7 12 9 4 2

108

O A

100

100

100100104109

o 7

122

1 0 3

1 1 0

106

79?b 2 74?b 2

r00

100100100100

100r00

r00

100

I0I2

I2

I2

I0I2

I2

0

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97r 1 9

90

r08

9 T1 3 1

96

103

aMaximum (exponential) growth rate expressed as percent of that obtained in control with no added fluoridebExcessive cellular clumping has cast doubt on the accuracy of these measurements.cNote that the name of this species was recently revised to T. weissfogii (Fryxell and Hasle 1972).

readily tolerate fluoride contamination of coastal waterswithout obvious adverse effects. In fact, it appears thatthis toleration extends to contamination levels approach-ing f fuor ide "sa tura t ion" in seawarer . Such con iamina-tion levels are far in excess of those presentlv knownin cases of f luoride pol lut ion (Moore 1971; Harbo etal. 19'14). We are thus led to infer that current levelsof fluoride pollution pose no immediate threat to theviability and growth of coastal phytoplankton providedsufficient nutrients are available. However. it must bepointed out that this investigation has not attempted toestablish phytoplankter toleration of fluoride contami-nation under nutrient-deficient conditions that couldbe more stressful; such studies are needed to arrive atecologically realistic pollution limits.

Some interesting questions are raised by our ob-servation that the marine phytoplankters tested cantolerate fluoride concentration as high as 100 mg/Lwithout obvious ill effects. Similarly, high fluoride levelswere also tolerated by the blue crab, Callinectes sapidus,despite considerable fluoride accumulation withrn mus-cular tissue (Moore l97l). Such toleration levels gen-erally exceed those known to be toxic to ten-estrialorganisms including man (Eagers 1969). Are therefactors in seawater that contribute to mitieation offluoride-ion toxicity to marine organisms? W-e suspectthat one such factor may be the degree of salinity in-volving the salt composition of seawater whereby the

added toxic fluoride ion is rendered unavailable andtherefore harmless by complexation with other com-ponents of seawater. According to Miller and Kester(1976), the ionic speciation of endogenous fluoridein seawater of sal ini ty 35%" at 25'C consists of 5OVo( F - ) , 4 7 V a ( M g F * ) , 2 . 1 % ( C a F + ) , a n d 1 . 1 % ( N a F 0 ) .It is not known to what extent this endogenous ionicequilibrium would be aflected by anthropogenic fluorideaddition to the seawater or by reduction of salinity fromoceanic to coastal or estuarine levels. Our suppositionthat the various ionic complexes are likely to be in-nocuous relative to F- toxicity also needs verification.At any rate, logistic considerations predict that ourhypothesized salinity-protection effect would be mini-mal at the lowest salinities tolerated by euryhalinespecies of phytoplankters, and we hope to extend ourstudies on fluoride toxicity to phytoplankton growthunder estuarine-salinity conditions. Similar studies onfreshwater phytoplankton, which might offer usefulclues to the salinity problem, have not been reported.

The small degree of growth stimulation of a fewspecies by low concentration (and in one case by eventhe highest concentration) of added fluoride raises an-other question concerning the possible beneflcial effectfrom this halogen on algal development. Could this ob-servation signify a fluorine requirement for optimalgrowth of certain microalgae? This question becomesparticularly relevant to the marine milieu, since fluorine

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o.6

o.5

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C. salina ,;:- '}-='o r

V:7' -- *irif.i R. lens

.^7,i.-..f.."-T:*r;.--l' ..-/'\

'i\, t3 F

.:)' ,/ \._.- loo F

. o F

I 503

P. lutheri =:.a^

A. quadruplicatum 0-100 F

o.2

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0 - 1 0 0 FB. polymorpha

N. oculata0 - 1 0 0 F

f--'o.5

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10 12 ',14 16 18

GROWTHo 2 4 6 I 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

PERIOD (days)Frc. 1. Growth of eight phytoplankter species (C/rroomonas salina; Rhodomonas lens; Dunaliella tertiolecta; Pavlova

Iutheri; Bellerochea polymorpha; Amphidiniunt carteri; Nannochloris ocul.ala; Agmenellum quadruplicatum) from first

exposure to sodium f luor ide at F (mg/L) concentrat ions as shown: O-O 0F; A" " 'L 25 F; C- ' -O 50F;

a - - - - 4 1 0 0 F .

is known to be a conservative (albeit minor) elementof seawater (Warner 1971). A bromine requirementwas reported for the optimal growth of a marine rhodo-phyte (Fries 1975) and iodine was shown to be anessential nutrient for a marine phaeophyte (Wooleryand Lewin 1973), but the possibility of a fluorine re-quirement for any phase of algal growth appears tohave been hitherto ignored. Carefully designed nutri-

tional studies, using artificial seawater lacking endoge-nous fluorine, are required to resolve this question.

Acknowledgments -The generous assistance of Z. Akhtarin making the growth measurements, of Dr J. A. J. Thomp-son in fluoride determinations, and of the National ResearchCouncil of Canada in providing financiai support (NRCGrant A-2288 ) is gratefully acknowledged.

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r504 J. FISH. RES. BOARD CAN.. VOL. 35. 1978

ANrre, N. I. 1976. Effects of temperature on the darknesssurvival of marine microplanktonic algae. MicrobialE c o l . 3 : 4 1 - 5 4 .

ANrte, N. J., eNo J. Y. CnENc. 1975. Cultur.e studies onthe effects from borate pollution on the growth ofmarine phytoplankters. J. Fish. Res. Board Can. 32:2487-2494.

EAGERS, R. Y. 1969. Toxic properties of inor-ganic flnor-inecompounds. Elsevier Publishing Co. Ltd., Amsterdarn,London, and New York. 152 p.

Fnres, L. 1975. Requirement of bromine in a red a|sa. ZeiI.Pflanzenphysiol. 7 6: 366-368.

Fnvxrr l , G. A., eNo G. R. Hesr-r. 1977. The genusThalassiosira: some species with a modified ring ofcentral strutted processes. Nova Hedwigia, Beih. 5.1:67-98.

GnerNrrlrcn, R., aNo J. P. Rruy. 1961. The determinationof fluorides in natural waters, with parliculal r.efer--ence to seawater. Anal. Chim. Acta 25: 179-188.

1963. Occurrence of abnormally high fluoride con-centrations at depth in the oceans. Nature 197: 371*372.

Hlnao, R. M., F. T. McCou,qs, eNo J. A. J. TnoupsoN.1974. Fluoride concentrations in two Pacific Coastinlets - an indication of industrial contamination. J.F ish . Res . Board Can. 31 : 1151-1154.

Lovrrecr, C. J., eNo G. W. MrnEn.1967a. In vitro eftecIsof fluoride on the tlicarboxylic acid cycle dehydro-genases and oxidative phosphorylation: part I. J.Histochem. Cytochem. 15: 195-201.

1967b. Histochemical investigations of the irr li r,t.reffects of fluoride on tlicarboxylic acid cycle dehy-drogenases from Pelargonium zonale: part lL J.Histochem. Cytochem. 1.5 : 202*206.

MTLLER, G. R., eNo D. R. Kr,srEn. 1976. Sodium fluorideion-pairs in seawater. Mar. Chem. 4: 67-82.

Mrrlrn, J. E., .q.No G. W. Mnrn. 1974. Effects of fluorideon mitochondrial activity in higher plants. Physiol.Plantarum 32: 115-121.

Moone, D. I . 197 1, . The uptake and concentration of fluorideby the blue crab, Callinectes sapidus. ChesapeakeSc ience 12 :7-73 .

OnorN, L., eNo B. P. Sron. 1963. Inhibition of metabolismin Avena coleoptile tissue by fluoride. Plant Physiol.38: 416-421.

SencrNr, D. F., exo C. P. S. Tlyr-on. 1972. The effect ofcupric and fluoride ions on the respiration of Chlorella.Can. J . Bot .50 : 905-907.

SErrc, W. 1973. Microdetermination of fluoride usingGran's plots. Mikrochim. Acta (Wien) 1973: 87-1 0 0 .

Stuore, L. K. 1917. The efiect of lead, cadmium, arsenate,and fluoride ions on the growth and fine structure ofSpltagnum nenToreum in aseptic culture. Can. I. Bot.55 :426-435.

Srocxxrn, J. G., eNo N. J. ANrr,l. 1976. Phytoplanktonadaptation to environmental stresses from toxicants,nutrients, and pollutants - a s'311i11g. J. Fish. Res.Board Can. 33: 2089-2096.

WenNsn, T. B, 1971. Normal fluoride content of seawater.Deep-Sea Res. 18: 1255-1263.

Wrrsr, R. C. [ed.] 1971. CRC Handbook of Chemistry andPhysics. 52nd edition. Chemical Rubber Co., Cleveland.Ohio. p. 862-8156.

Woorr;nv, M. L., ,,r,No R. A. LEwrN. 1973. Influence ofiodine on growth and development of the brown algaEctocarpus siliculosus in axenic cultures. Phycologia12: l3l-1,38.

The Hemolymph Bactericidin of American Lobster(H omarus &rnericanus) : Adsorption and Activation

Knrsuyosnr Monrl AND JAMES E. SrrwnnrDepartment ol Fisheries and the Environment, Fisheries and Environmental Sciences,

Halifax Laboratory, P.O. Box 550, Halilax, N.S. 83l 257

Mont, K., eNo J. E. Srrwenr. 1978. The hemolymph bactericidin of American lobster(Homarus americanus): adsorption and activation. J. Fish. Res. Board Can.3 5 : 1 5 0 4 - 1 5 0 7 .

The bactericidin of American lobster (Homarus americanus) hemolymph was ad-sorbed readily by representatives of two bacterial genera: a nonpathogen, Pseudotnonasperolens, and the lobster pathogen Aerococcus viridans (var.) homari. Activation of thebactericidin by factor(s) secreted by the hemocytes can occur before adsorption and alsoafter adsorption by bacteria. Although the lobster pathogen A. viridans (var.) homari isunaffected by the activated bactericidin, this lack of effect is not related to adsorptionsince this bacterium adsorbs the bactericidin as readily and completely as does thesusceptible nonpathogen.

Key words: lobster, Homarus americanus, plasma bactericidin, adsorption and activation,hemocytic activator, Pseudomonas perolens, Aerococcus yiridans (var.) homari

lNational Research Council Postdoctorate Fellow during this study. Present address: Department of Fisheries, Faculty ofAgriculture, Tohoku University, Sendai, Japan.

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