Factors Affecting the Algicidal and Algistatic Properties of Copper

G. P. FITZGERALD AND S. L. FAUST

Hydraulic and Sanitary Laboratories, University of Wisconsin, Mladison, WVisconsin

Received for publication 22 February 1963

ABSTRACT

FITZGERALD, G. l'. (University of Wisconsini, Madison)AND S. L. FAUST. Factors affectinig the algicidal and al-gistatic properties of copper. Appl. Microbiol. 11:345-351. 1963.-Data from laboratory studies are presentedto show that, whereas five different sources of copperappear to be equally effective as toxic agents for algae,the medium in which toxicity tests are carried out has agreat influence on the toxicity of copper. A technique ofsubculture is described for determining whether a con-centration of a chemical is algicidal, algistatic, or nontoxicin action against a specific alga, and demonstrations aregiven of tests with algae illustrating each class of action.

Concern about the use in many publications of theterm "algicide" when dealing with copper sulfate has ledto a short review of the algicidal versus algistatic prop-erties of copper sulfate.

Copper sulfate and copper products have beeii used forthe control of algae since the early 1900's (Moore andKellerman, 1904) with apparent success (American WaterWorks Association, 1950; Derby and Townsend, 1953;Hale, 1954; Bartsch, 1954). However, one should be awareof the selectivity in the toxicity of copper sulfate to algae(Domogalla, 1926; Hale, 1954; Maloney and Palmer,1956). The selectivity in the toxicity of copper sulfate isconsidered by some (Maloney and Palmer, 1956) to bedue to the formation of insoluble copper complexes undercertain culture conditions, but receint studies have indi-cated that copper is just as toxic to cultures of algae whenthe copper is in a soluble form as when it is precipitated.

This report will demonstrate the results of normaldilution toxicity tests (Fitzgerald, Gerloff, and Skoog,1952; M\aloney anid Palmer, 1956) and some of the factorsthat might influence the toxicity of copper compounds inthese tests. Also, tests with different types of algae willbe used to demonstrate situations in which copper can beshown to kill algae (algicidal effect), prevent the growthof algae (algistatic effect), or be relatively nontoxic.

MATERIALS AND METHODS

The algae used are maintainied on agar slanits in theUniversity of Wisconsin algae culture collection. Thiscollection has algae representing the lake bloom-producingblue-greeni species and planktonic diatom species, as well

as several blue-green and greeni algal species which wereobtained from unsuccessfully chemically treated ponds orswimming pools. For use in toxicity tests, the alga to bestudied was cultured in either Allen's (1952) or Gorham's(Hughes, Gorham, and Zehnder, 1958) liquid culturemedia. The amount of algae inoculated into toxicity testculture flasks was the approximate equivalent of 1,500,000cells per ml of Microcystis aeruginosa Wis. 1036 for repre-sentatives of the planktonic blue-green algae and theapproximate equivalent of 300,000 cells per ml of Chlorellapyrenoidosa Wis. 2005 for the testing of blue-green andgreen algae of swimming pools.A bacteria-free isolation of one of the green algal species

(Wis. 1125) from a swimming pool was obtained by usinga modification of the technique suggested by McDaniel,Middlebrook, and Bowman (1962). In the present pro-cedure, a sample of the algae from a dense culture wasplaced in sterile medium, treated for 1 hr with the algicideExalgae L. C. (Inertol Co., Inc., Newark, N.J.) at aconcentration of 10 mg per liter, centrifuged from thetreating solution, washed with sterile medium, and thenstreaked on agar plates of Allen's (1952) culture mediumplus 1 % glucose. Isolated algal colonies that appeared tobe free from bacteria after 7 days of incubation were thenstreaked on Tryptone glucose extract agar (AmericanPublic Health Association, 1960), and bacteria-free colonieswere isolated for use in toxicity tests.The selection of the medium in which toxicity tests

are to be carried out is dependent upon the species ofalgae to be used and the factors of the environment to beinvestigated. Allen's (1952) medium is a very hard watermedium of pH 7; Gorham's (Hughes et al., 1958) me-dium, a moderately hard water medium, normally has apH of 9, but with suitable buffering (McLachlin and Gor-ham, 1961) or by aeration with 0.5 % CO2 in air can bemaintained at pH 7.Normal dilution toxicity tests determine the minimal

concentration of a chemical which will inhibit or kill analgal culture. These tests were carried out by adding to aseries of inoculated culture medium flasks (25 ml per50-ml Erlenmeyer flask) increasing amounts of distilledwater solutions of the chemical to be studied. Preliminarytests were usually carried out to determine the generalrange of concentrations of the chemical that are toxic tothe algal species employed. The inoculated toxicity testflasks were maintained in a constant-temperature (24 +

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FITZGERALD AN-D FAUST

2 C) continuous-light (150 ft-c) culture room. The resultsof the tests can be recorded as visual estimates of thedegree of inhibition of the growth of the algae after differentperiods of treatment (Palmer and N\Ialoney, 1955) or thecultures can be filtered through glass-fiber pads (H. ReeveAngel & Co., Inc., New York, N.Y.), the pads dried, andphotographs taken of comparative pads. The latter pro-cedure was used in the present report.Whether a toxic coincenitration of a chemical is algicidal

or algistatic can be determined by removing a sample ofthe algae from the environment of the toxic chemical anidplacing it in a suitable culture medium (Fitzgerald andFaust, Water Sewage Works, in press). If the sampled algaehave absorbed a toxic concenltration of the chemical in-volved, subcultures of the alga will not revive, and the con-ceintration of chemical tested can be considered to be algi-cidal after the treatment time tested. If the chemical actsonly as an algistat, samples of the algae which have beenplaced in sterile culture medium will revive and start togrow within 1 or 2 weeks. In the tests reported here, 1-mlsamples of toxicity test cultures were removed after differ-ent periods of treatment anid placed in 25 ml of sterileculture medium; these subcultures were incubated in theculture room for suitable periods of time.

Analysis for the copper content of the stock solutionsand some of the toxicity test flasks was carried out bythe Cuprethol methods (American Public Health Associa-tion, 1960). The comparative amount of soluble copperin solutions was determined by analysing the amount ofcopper in unfiltered samples as compared with samplesfiltered through glass fiber pads (Fitzgerald and Faust,J. Environ. Health, in press).

RESUTLTSComparison of different sources of copper. A inumber of

tests have indicated that the toxicity of copper is in-dependent of the source of copper when inorganic saltsare compared. In the field of commercial copper algicidepreparations for swimming pools, the source of copper is

CrrAxo IIj)n, 1/2 ppm 1iI 2 pr 3X

2:;Citli Ae-i(f ( C t

(Npexrsc

FIG. 1. Relative toxicity of diferent copper products to Chlorella

usually anl organiic chelated form of copper in order toprevent the precipitation of the copper in hard waters(Fitzgerald, 1960; Fitzgerald and Faust, J. Environ.Health, in pr-ess). Tests were therefore carried out to deter-mine the relative toxicity to C. pyrenoidosa Wis. 2005 inAllen's medium of copper sulfate, a mixture of 1 partcopper sulfate anid 2 parts citric acid, and three commer-

cial sources of copper [Algeeclear, Wallace & TiernamInc., Belleville, N.J.; Cuprose (Texas), W. R. Henderson& Co., Houstoni, Texas; anid Cuprose (Nalco), NalcoChemical Co., Chicago, Ill.]. Analysis of stock solutionsof the chemicals to be tested (25 mg per liter) showed thefollowing actual copper concenitrations presenit (in mg ofCu per liter); copper sulfate, 6.0; copper sulfate-citricacid mixture, 6.3; Algeeclear, 4.0; Cuprose (Texas), 4.2;and Cuprose (Nalco), 4.2. The results of a typical toxicitytest with these sources of copper are presented in Fig. 1,a photograph of the algae from the toxicity test flaskswhich had been filtered through glass-fiber pads after 7days of treatment or contact time.

It is evident from these data the copper sulfate (CuS04-5H20) is toxic to Chlorella at a concentration of 2 mg per

liter (2 ppm) or more after 7 days of contact time. Thepresence of citric acid appears to have had nIo effect oni thetoxicity of the copper sulfate, since this mixture was alsotoxic at 2 mg per liter (2 ppm). The three commercialproducts were approximately equal in toxicity in this test,a conicenitration of 3 mg per liter (3 ppm) being required toprevent the growth of Chlorella.When one compares the actual concentrations of copper

in the stock solutions of the chemicals and the concentra-tions of the chemicals required to prevent the growth ofChlorella under these test conditions, it is evident thatcopper from these different sources appears to be equallytoxic to Chlorella.Inasmuch as the reason for the use of chelated forms of

copper in algicides for swimming pools is to prevent theloss of the copper from solution and because tests haveshown that a mixture of copper sulfate and citric acid andthe commercial sources of copper tested are subject toattack by bacteria anid a resulting loss in solubility of thecopper in a few days time (Fitzgerald and Faust, J. En-viroii. Health, in press), comparative toxicity tests were

carried out with a green algal species (Wis. 1125; isolatedfrom a swimming pool) under conditions free from bac-teria. The toxicities of autoclaved solutions of coppersulfate and of a mixture of 1 part copper sulfate and 2 partscitric acid were compared. A photograph of the algaefiltered from these test solutions after 12 days of treat-ment is presenited in Fig. 2.

Analyses of the comparative amounts of copper in un-

filtered and filtered samples of the actual toxicity testsolutions of Gorham's medium at pH 7 (+0.3) indicatedthat at both the start of the test and after 12 days oftreatment only 8 %'o of the copper from copper sulfate was

soluble, whereas 78% of the copper from the mixture ofpyrenoidosa Wis. 2005 after 7 (lays of culture on Allen's medium-.

346 APPL. 'AlICROBIOL.

copper sulfate and citric acid was in a soluble form.

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OLl,(19AICIJ)A, AND1) ALGISTATIC 11RO10ERTIES 01 COPPER

C>nt rol 2.0 ppn I ( ,().Va 1 )[)t.1 l (.} I}} >> 1}

_s .. .s\ f ¢.:' . :. .- .. : \w..(N '1' .Itls .

-.4,fvs1,'ale<ti

Citric AI-id7;ti(S . ..)

FI(. 2. Comparison of the toxicity of insoluble and soluble copper to a green algal species (Wis. 1125; isolated from a swimming pool)culturedfor 12 days urnder bacteria-free conditions on. Gorhant's mediumt (-Fe).

Therefore, the results of the toxicity test (Fig. 2) in-dicate that wheii copper is present in the culture as aprecipitate, it is just as toxic to this alga as when it ispresent in a soluble form.

Effect of the culture medium on the toxicity of copper.Toxicity tests were carried out in modifications of thedilute, alkaline medium of Gerloff, Fitzgerald, and Skoog(1950) by Fitzgerald et al. (1952), by Palmer aind Maloney(1955), by Maloney and Palmer (1956), and by Fitzgerald(1959). Allen's (1952) neutral, but more concentrated,medium was used by Fitzgerald (1959, 1960). The choiceof the medium to use for toxicity tests is usually limited bythe species of algae to be tested inasmuch as some speciesdo not grow as well in some media as in others. To demon-strate that the culture medium in which toxicity tests arecarried out affects the results, comparative tests of thetoxicity of copper sulfate were made with the green algaC. pyrenoidosa Wis. 2005 and the blue-green alga 111.aeruginosa Wis. 1036.The toxicity of copper sulfate (CUSO4 * 5H20) was com-

pared in Chlorella cultures in Allen's (1952) medium, inGorham's medium (Hughes et al., 1958), and in Allen'smedium in which a chelated form of iron (McLachlin andGorham, 1961; ASM medium) was added in place of theusual ferric chloride. The iron of the latter medium waschelated by the addition of 7.4 mg per liter of disodiumethylenediaminetetraacetate (EDTA). The iron of Gor-ham's medium is ferric citrate with the addition of 1 mgper liter of EDTA. The results of the tests are presentedin Fig. 3 as a photograph of the filtered test solutionsafter 5 days of treatment.

It is evident from these data that 1 mg per liter (1 ppm)of copper sulfate is toxic to Chlorella in Allen's mediumafter 5 days, whereas 2 mg per liter are required in Gor-ham's medium, and 8 mg per liter in Allen's medium inwhich EDTA has been added. The 1 mg of EDTA perliter in Gorham's medium had only a slight effect on thetoxicity of copper sulfate, increasing the required con-

centration for toxicity from 1 to 2 mg pei liter. The 7.4

mg of disodium EDTA per liter in the Allen's medium withASM-Fe increased the required concentration of coppersulfate from 1 to 8 mg per liter.

Similar results were obtained in other tests in which thetoxicity of copper sulfate to Chlorella was determined ineither Allen's or Gorham's media which had different sourcesof iron (ferric chloride, ferric citrate, ferric citrate plus 1mg per liter of EDTA, or ferric chloride plus 7.4 mg perliter disodium EDTA) substituted for the usual sourcesof iron.The results of tests comparing the toxicity of copper

sulfate to Hl. aeruginosa Wis. 1036 in Gorham's mediumwhich had the above four sources of iron substituted forthe usual source of iron are presented in Fig. 4.

Ajlle I(sM diikn

(n.ruI 1.'t })1 l1,x~ 2ppm p lml plo

_s~~~~~~~Gwxm Med. n

C# nt~ l _ _I flT 12 pun mn:. ..

2 ppmf

2. lip

A.len s AlMcdium (+ASM '.)

(Contri I ppnll 2 I 4 pim-i_.ill,1111,11

FIG. 3. Effect of the culture medium on the toxicity of coppersulfate to Chlorella pyrenoidosa Wt'is. 2005 after culture foor 5days.

347VOL. II], 961(3;

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348 FITZGERALD AN\D FAIUST

These data iindicate that coiisiderably less copper sulfateis required for toxicity to JIicrocystis then to Chlorella.There appears to be no effect oni the toxicity of coppersulfate wheni the iron of Allen-'s medium (ferric chloride)or Gerloff's medium (ferric citrate) is uised, a concentra-tion of 0.05 mg per liter (0.05 ppm) beiiig toxic. However,the EDTA added wheni the ironi of Gorham's medium(1 mg of EDTA per liter) or ASAI medium (7.4 mg ofdisodium EDTA per liter) was employed was sufficientto ineutralize the toxicity of at least 0.3 mg of coppersulfate per liter (0.3 ppm).

Irollr Sol Ir c(w

Aileis le

(:;erldM's l'F

(.Xorhisis Fe

ASMI Fe

(:nt:rl 0.0125 ppml1

APPL. AIICR{OBIOL.

It is apparenit from these data that carefutl conisidera-tion must be made in selecting a medium for toxicitytests, especially when the toxicity of copper compounidsis to be tested.

Algicidal properties of copper sutlfate. To be algicidal,a concentrationi of a chemical must kill the treated cultureof algae. To determine whether the algae of the treatedculture are actually dead or their growth is merely in-hibited, stubcultures into sterile meditum should be made.The results of studies oni the toxicity of copper sulfate tothree bloom-forminig blue-green algae, 1I. aerugin.osa Wis.

0.02.5 0)m(.05 )pm 0.1 pptaill 0).2 ppinX pon.sst

i'°i: :::::t ................... ::..........

J............ ..............

............... :....... ..........

FIG. 4. Effect of the cultuire medium on the toxicity of copper sulfate to Microcystis aeruginosa Wis. 1036 after culture for 5 days WI Gor-ha nt's itedium.

Origintl Cultures

.lIi('o(yltis Z iQie(Wis. I0 .)

.lnabq'aa ire1inlt 4sW\-i. 11l:IS)

tO.f1;l vifll-t

(J0 IPf

I rld/15 ml Subcultures

0.2 ppm

0.l IP!Pll 0.2 ppm

Contrl 0.05 ppm 0.2 ppm

0.1 pPi 0.2 ppm00ttiehia ch'tudala)(Wi4 .105)2)

Plankton D)iutmon(Wis. 1131f)

Con t rol 0.05 ppilt

CO tr1-ol 0.2 pp}I)

0.1 ppm:t

0.3 pp1)m 0.4 pp)10

Utxntl ()l__

K +,.9+,+ ;\.19' Ft a: a.-¢.<-.>;d[X9 9 9sj9 . ..e ..9 5 0&-\t9)99Ri9 a9..,a9 s+:):e a-+ owX

5,f 9g.at °tffls=

0.05 ppml

0.) .))l

0. 1 ppnm0.Co)ntrol3 0.2 ppm> 0.3 Xppm6 0.4 pP:mt0a

FIG. 5. Algicidal properties of copper suilfate (CullSO45H20). Suibcultutres were ii.ade after tereat tent foi 3 days. Resuilts shoun (liae afterincuibation for 14 days.

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ALGICIDAL AND ALGISTATIC PRO1EIRTIES 01' Cl'01IER 349

Cont rol

Control

2 )j1>sl

i h ~~~~~~~~~~~~~~~

* : : . .. . . , ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~... .....:.:.:.. . . ,-.,... .. ... ...... . .... . < ..

Oi-ig;li.l LTreatedl Cultulres (20} ti|\ uaeu re)* . ..............~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.......... : 8 )tXtll. ...... tS )pttl ~~~~~~~~~~~~~~~~~~~...... .: ....i: :: .. .. ........ .. .: ::: ...... ................. ......:............

......

..

2 p)xyt1

FIG. 6. Algistatic effect of copper sulfate (CUS04-5H20) on Chlorella pyrettoidosa H4'is. 2005.

10o., .abaena circinalis Wis. 1038, and Gloeotrichiaechitntdata Wis. 1052, anid to a planiktoniic diatom (Wis.1134) are presented in Fig. a.The tests were carried out inl Gorham's medium in

which ferric chloride was substituted for the usual sourceof it-on. After 3 days of treatment, 1-ml samples wereplaced in 25 ml of sterile medium. All cultures were har-vested by filtration through glass-fiber pads after 14 daysof inicubation.The pads from the originially treated cultures indicated

tlhat 0.05 mg per liter (0.05 ppm) of copper sulfate w-astoxic to Jlicrocystis and Gloeotrichia, 0.1 mg per liter wastoxic to Anabaena, anid 0.4 mg per liter was toxic to thediatom. The results of the subcultures indicated that nonieof these algae grew up in stubcultures from cultures thathad beeni treated with apparently lethal concentrationisof copper sulfate, although unitreated control subculturesor stubcultures from sublethal concenitrations of coppersulfate did revive.

Inl the case of these algae, therefore, copper sulfate canibe conisidered to be algicidal at the concentrationis andconitact times tested.

Algistatic properties qf copper7 sulfatc. It was noted byPalmer anid Maloniey (1953) anid Fitzgerald (1959) thatcultures of algae treated with certain chemicals appearto die at first, but theni recover after further incubation.This has been frequently observed in toxicity tests withcopper sulfate; as the inlcubationi time iniereases, cultutresthat appeared to be dead iecover alid grow in time tomuchl the same density as untreated control cultures.That this recovery of certaini treated algal cultures is dueto the fact that the cells of the algae are not dead but theirgrowth is temporarily inlhibited was demonstrated withtests oIn the toxicity of copper sulfate to Allen's mediumcutltures of Chlorella anid a blue-greeni alga referred to asthe "black algae" of swimming pools, Phormidium i71-uindatum Wis. 1093. Stubcultures were made of the cul-

tures treated with differenit concentratiolis of copper sul-fate after 14 and 5 days, respectively. The original culturesand subcultures were harvested for photographs after 20days of culture (Fig. 6 anid 7).

It is evidenit from the data preseinted that 4 and 8 mgof copper sulfate per liter (4 anid 8 ppm) were sufficient tobe apparently lethal to these two species of algae. Theresults of the subcultur es, however, indicate that con-centrations as high as 24 mg per liter were insufficient toprevenit the growth of these algae when they had beentreated for 14 or 5 days and theni samples were removedto sterile, unitreated medium.Copper sulfate would be coinsidered to be algistatic

in effect in the case of algae represented by these species.Algae resistant to copper sulfate. Bussy (1950) mentioned

that some species of algae from swimming pools were founidto be relatively resistanit to treatments with copper sul-

.... ..Oriintdl Cultux *::

'Contro4. 4 ppli 81S t 'ul: 18 p1mv, 20 ppm 24 ppin

I0C000CSubmeltu-dred I ml,l o2 ml) After 3 Days Treatiamt

*cOItroI 'V {F)rtl 24 }pm

FIG. 7. Algistatic properties of copper suilfate (CUS04 5H20) toPhormuidiutm inundatums W is. 1093 after 20 days of culture.

N-OL. 11 X 1963 349

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FITZGERALD AND FAUST APPL. MICROBIOL.

Miten's Mledium

4 pl-Ma 8 Pp)mI 20 ppmn 24 Ippm 30 pp

G orliamit's M hedimni

Cloitt rol

Cu. 'Oi'.5H,0.

Exalgae L. C.

Algimilyem-4

8 ppIlm 20 ppmi 24 ppm 30 ppill

I ppni .2 ppin

.s...!i..

IppSIll 2 ppil

3 ppm *5 ppm1

3 ppt 5 ppmIn

00FIcG. 8. Resistance of a swiminminq-pool alg(a (Peatrsall blue-green, Wi8. 1129) to (opper sulfate.

fate. To demonstrate this fact, toxicity tests in Alleni'smedium and Gorham's medium (with ferric chloride as thesource of iron) were carried out with a blue-green alga(Wis. 1129) isolated from a swimming pool. Concentra-tions of copper sulfate (CuSO4. 5H20) up to 30 mg perliter were tested in either medium, as well as concentra-tions of 1 to 5 mg per liter of the algicides, Exalgae L. C.and Algimycin AIT-4 (Great Lakes Biochemical Co.,Milwaukee, Wis.). After 13 days of treatment, the cultureswere harvested for pictures (Fig. 8).

It can be seen from these data that a concentrationi ofcopper sulfate of 30 mg per liter (30 ppm) was not sufficientto do more thani cause a slight inhibition of the growth ofthis alga in either Allen's or Gorham's medium. In coIn-trast, a concentration of the two commercial algicides of3 mg per liter (3 ppm) appeared to be lethal. It is thereforeevident that whereas a species of algae may be resistantto the action of one type of toxic chemical it does not neces-

sarily mean it will be resistant to other toxic chemicals.

DIsCUSSION

Data presented in this repor-t demonstrate that, whereasdifferent sources of copper appear to be equally effectiveas toxic agents for algae, the constituents of the mediumthe tests are carried out in can alter the results of toxicitytests to a marked degree. The effect caused by differentmedia on the toxicity of copper to algae is apparentlyrelated to the source of iron used in the media. The effectof EDTA onl the toxicity of copper and chromium was

discussed by Morgan anid Lackey (1958). The results ofthe present tests seem to indicate that approximatelyeach part of EDTA in a medium can neutralize the toxicityof one part of copper sulfate.

The importance of the use of chelated forms of copper

foi the control of the growth of algae should not be assumedto be niullified by the facts that the different sources ofcopper were approximately equal in toxicity and, as was

poinited out by FX itzgerald anid Faust (J. Environ. Health,in press), that bacteria could attack various sources ofcopper anid cause the copper to be precipitated. By know-ing how loing a product will mainitain sufficient copper inlsolution for the preven-tioin of the growth of algae, one

could make additions of the chemical frequently enooughto replace that which was lost from solution.

It is interesting to note that the studies carried out inbacteria-free media indicated copper was just as toxicwhen in a precipitated form as wheni soluble. This factseems to be in contradiction to the suggestion of Maloneyand I'almer (1956) that the selectivity in the toxicity ofcopper to algae is due to the formation of insoluble formsof copper in certain solution-s. The fact that the species ofalgae most sensitive to the toxicity of copper, the bloom-forming blue-greeni algae, appear to be affected by copperunder the most ideal conditionis for the precipitation ofcopper also tends to lead one to believe that the selectivityof toxic action of copper for different algal species is prob-ably a function of processes withini the algal cells and niotdue to the chemistry of the surrounding water.The need to know whether a coincentration of a chemical

will be algicidal or algistatic in action is of prime impor-tance if the chemical involved can be readily removed fromthe environiment of the algae by precipitation or adsorp-tion onto filter media, debris, etc. From the data presented,it is evident that the planktonic blue-green algae and thediatom tested were killed by the conicentrations of coppertested. Thus, even if the excess of copper in an environ-

350

Control

CUSOX 35 ii

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ALGICIDAL AND ALGISTATIC PROPERTIES OF COPPER

ment, such as a lake, becomes insoluble and settles outof the water body, the algae that had been treated woulddie and decompose, as has been noted to be the case in thesuccessful treatments of lakes and reservoirs (Moore andKellerman, 1904; American Water Works Association,1950; Derby and Townsend, 1953; Hale, 1954; Bartsch,1954). Other algae that were either resistant to the con-centration of chemical applied (Moyle, 1949; Domogalla,1926) or were only inhibited in their rate of growth by theconcentration of applied chemical (algistatic effect) wouldbe able to grow as soon as the precipitated chemical waslost from their immediate environment.

In the case of swimming pools, in which insoluble prod-ucts will be filtered from solution and products that areadsorbed onto the filter media will be lost from solution(Bussy, 1950; Fitzgerald, 1960, 1962, 1963), algistaticchemicals must be maintained at all times, whereasalgicidal chemicals need be replaced only so often as re-quired to kill the successive growths of algae.The techniques described for determining whether a

chemical is algicidal or algistatic in action against a specifickind of algae can be used to evaluate chemicals for suchuse and the conditions under which they are most effective.

It should be pointed out that in the case of copper com-pounds the range in toxic action can vary from algicidalactivity at relatively low concentrations (0.05 to 0.4 mgof CUSO4- 5H20 per liter), or algistatic activity at concen-trations of 2 to 24 mg of CUSO4 * 5H20 per liter with certainalgae, to situations in which the growth of algae is onlyslightly inhibited by a concentration of copper sulfate ashigh as 30 mg per liter. However, the relative resistanceof the different algae to copper compounds has not beenshown to be related to the amount of chemical necessaryfor toxic action in the case of other types of toxic chemicals.

ACKNOWLEDGMENT

This research was supported in part by a research grantfrom the National Institutes of Health, U.S. Public HealthService.

LITERATURE CITED

ALLEN, M. B. 1952. The cultivation of Myxophyceae. Archi. Mlikro-biol. 17:34-53.

AmERICAN PUBLIC HEALTH ASSOCIATION. 1960. Standard methodsfor the examination of water, sewage, and industrial wastes,11th ed. New York.

AMERICAN WATER WORKS ASSOCIATION. 1950. Water quality andtreatment, 2nd ed., p. 109-124.

BARTSCH, A. F. 1954. Practical methods for the control of algaeand water weeds. Public Health Rept. (U.S.) 69:749-757.

BUSSY, I. J. 1950. Growth and control of algae in open-air swim.ming pools. Comm. Zwembaden T.N.O. Rept. No. 1, p. 1-87.

DERBY, R. L., AND F. W. TOWNSEND. 1953. Reservoir treatment byimproved methods. Water Sewage Works 100:211-216.

DOMOGALLA, B. P. 1926. Treatment of algae and weeds in lakes atMadison. Eng. News-Record 97:950-954.

FITZGERALD, G. P. 1959. Bactericidal and algicidal properties ofsome algicides for swimming pools. Appl. Microbiol. 7:205-211.

FITZGERALD, G. P. 1960. Loss of algicidal chemicals in swimmingpools. Appl. Microbiol. 8:269-274.

FITZGERALD, G. P. 1962. Bioassay for algicidal chemicals in swim-ming pools. Water Sewage Works 109:361-363.

FITZGERALD, G. P. 1963. Field tests on the duration of algicides inswimming pools. J. Environmental Health 25:319-325.

FITZGERALD, G. P., G. C. GERLOFF, AND F. SKOOG. 1952. Studieson chemicals with selective toxicity to blue-green algae.Sewage Ind. Wastes 24:888-896.

GERLOFF, G. C., G. P. FITZGERALD, AND F. SKOOG. 1950. Theisolation, purification, and culture of blue-green algae. Am.J. Botany 37:216-218.

HALE, F. E. 1954. The use of copper sulfate in the control of mi-croscopic organisms. Phelps Dodge Refining Corp., NewYork.

HUGHES, E. O., P. R. GORHAM, AND A. ZEHNDER. 1958. Toxicity ofa unialgal culture of Microcystis aeruginosa. Can. J. Micro-biol. 4:225-236.

McDANIEL, H. R., J. B. MIDDLEBROOK, AND R. 0. BOWMAN. 1962.Isolation of pure cultures of algae from contaminated cultures.Appl. Microbiol. 10:223.

McLACHLIN, J., AND P. R. GORHAM. 1961. Growth of Microcystisaeruginosa Kutz. in a precipitate-free medium buffered withtris. Can. J. Microbiol. 7:869-882.

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