Hydrogen peroxide as a potential algicide for oscillatoria rubescens D.C.

Wat. Res. Vol. 20, No. 5, pp. 619~23, 1986 Printed in Great Britain. All rights reserved

0043-1354/86 $3.00+0.00 Copyright © 1986 Pergamon Press Ltd

H Y D R O G E N PEROXIDE AS A POTENTIAL ALGICIDE FOR OSCILLATORIA RUBESCENS D.C. G. BARROIN and MAURICETTE FEUILLADE Institut de Limnologie, INRA, 75 Avenue de Corzent, 74203 Thonon, France (Received September 1985)

Abstract--The algicidal properties of hydrogen peroxide (H202)for Oscillatoria rubescens were evaluated at different concentrations between 0 and 7 ppm. The toxicity threshold, under laboratory conditions, was about 1.75 ppm depending on culture density. Higher concentrations of H202 destroyed more than 90% of biliproteins and carotenoids and nearly 50% of chlorophyll, whereas they had no noticeable effect on Pandorina morum, a chlorophyte tested for comparison. This differential sensitivity is discussed on the basis of chemical and physical pigment fragility. Mixed continuous culture experiments suggest a possible shift from blue-greens to volvocales following the use of H202 as an algicide. Key words--algicides, hydrogen peroxide, Oseillatoria rubescens, Pandorina morum, eutrophication

INTRODUCTION Because of its oxidizing properties, potassium permanganate is used to eliminate odors and sulfides from different sources and to remove iron and manganese (Ailing, 1963; Welch, 1963; Willey and Jennings, 1963; Hughes, 1971). Its use to control problem-causing algae has already been evaluated (Fitzgerald, 1966; Kemp et al., 1966): K M n O 4 is particularly useful where algal species are unknown or where several species with different susceptibilities to algicides are present. Algicidal activity of potassium permanganate results from its strong oxidizing action. One of its advantages is that it reacts to form manganese oxide, a biologically inert residue. Thus, there is no residual toxicity of treated water, as is the case with other algicides. Hydrogen peroxide (H202) is another strong oxidizing agent totally devoid of any harmful byproduct, since it decomposes into water and oxygen. It is used for waste water and tip leachate treatment to eliminate odors, sulfides, organic matter and filamentous bacteria (Cole et al., 1973, 1974, 1976; Putz, 1976; Interox-Chimie, 1981; Fraser and Tytler, 1983). The action of hydrogen peroxide on filamentous bacteria has been attributed to the erosion of protective polysaccharide coatings, cleavage of cell chains and destruction of holdfasts. In practice, dose levels lie between 10 and 52 ppm of H202 depending on microbial growth density (Fraser and Sims, 1983). Soares (1980) evaluated its effects on reduced sediments and in-fauna, and Barroin (1980) used it to improve the quality of hypolimnetic water and sediments of a small experimental lake: as a side effect, phytoplankton community exhibited short-lived disturbances (Balvay, 1981), suggesting to Kay et al. (1982) that H202 would be a potential algicide. In fact, in that experiment, biological modifications

reported by Balvay were thought to be due essentially to the intense sediment and water stirring up created by gaseous oxygen release resulting from H202 decomposition. Barroin and Feuillade (1978) have shown that H202 is toxic for Oscillatoria rubescens at active oxygen concentrations similar to those found by Kemp et al. (1966) and Fitzegerald (1966) who used K M n O 4 : l . 5 p p m H202 induced filament sedimentation, which reveals a physiological stress, but did not cause any obvious modification of pigment content or growth potential whereas 7.7ppm H20: destroyed 30% of the chlorophyll a, 90% of the carotenoids, 100% of the phycobilins and 100% of the growth potential. Kay et aL (1982) found that, under field conditions, 9.8 ppm H202 reduced chlorophyll levels of a dense bloom of Anabaena sp. to 20% of controls in the presence of catfish (5 ppm were sufficient in their absence); toxicity thresholds under laboratory conditions were between 6.8 and 10.2 ppm for Ankistrodesmus sp., below 3.4ppm for Raphidiopsis sp. and below 1.7 ppm for Microcystis sp. This indicates a higher sensitivity of cyanophytes as compared to chlorophytes. The purpose of this study is two-fold: first, to evaluate the lower algicidai concentration of H202 for O. rubescens; secondly, to investigate if the apparently selective action of H202 on O. rubescens, supposedly due to the destruction of typical bluegreen pigments can result in the elimination of the cyanophyte in favour of a more desirable chlorophyte, for example, Pandorina morum, studied by Kemp et al. (1966), which is likely to grow under similar environmental conditions as O. rubescens.

619

MATERIAL AND M E T H O D S

Two studies were carried out. The objective of the first was to determine algicidal effect of H202 on O. rubescens

620

G. BARROIN and MAURICETTE FEUILLADE

(series A) and to compare O. rubescens response with that of Pandorina morum (series B). It consisted of short-term batch cultures experiments. The second used continuous cultures on the basis of longer experiments requiring specially designed cyclostats (Feuillade and Feuillade, 1979). Its objective was to evaluate the ability of H202 to eliminate the cyanophyte from a mixed culture of the two species. In order to avoid any interference from bacteria or fungi growth, all the experiments were carried out under strict axenic conditions and only results of experiments exhibiting a total axeny are presented. Experimental material Test organisms. O. rubeseens D.C. and P. morum Borg. were supplied from axenic continuous cultures grown in separate 2.241. stocking cyclostats, under identical conditions: culture medium = 50% ASMj (Gorham et al., 1964), pH increased to 8.0 by the addition of Na2CO 3 (40 mg 1-1) after autoclaving; flow rate = 19 ml h-I; temperature = 20°C; illumination = 9 W m -2 (1.500 Ix) light/dark cycle: 18/6 h. Hydrogen peroxide. From a 3.5 g 1-~ solution prepared by extemporaneous dilution of the 35% (w/w) commercial product in deionized water, appropriate amounts were used to obtain desired final concentrations expressed in ppm of H202. [For concentrations in ppm of the 35% (w/w) commercial product multiply by 2.86.] Batch cultures experiments 50 ml aliquots were drawn from the stocking cyclostat and introduced in culture tubes with the necessary amounts of H202 solution to obtain final concentrations of 0, 0.35, 0.70, 1.05, 1.40, 1.75, 3.50, 5.25 and 7.00 ppm for series A and 0, 1.40, 1.75, 2.62, 3.50 and 5.25 ppm for series B. After stirring, the tubes were incubated at 20°C under a 9 W m - : illumination and stirred again after 5 and 24 h. After 29 h algal growth was estimated as well as optical density, pigment concentration, axeny and growth potential. The experiments were carried out in triplicate for series A and duplicate for series B. Continuous cultures experiments O. rubescens and P. morum were cultivated in similar conditions in two separate 1 1. cyclostats sown from the stocking cyclostats. After 2 weeks of cultivation, growth having reached steady-state, the contents of the two cyclostats were mixed so that each contained about 6000 filaments of O. rubescens and 6000 colonies of P. morum (o.d.-~ 0.2). One cyclostat received the necessary amount

t

Table 1. Characteristics of the control batch cultures Series A

Series B

O. rubescens

Abs. value

O. rubescens

% Tot. pigm,

Abs. value

404 307

22.5 17

530

551 1792

Opt. dens.

% Tot. pigm.

morum

232 250

21 22.5

972 489

29.5

292

26

31

338

30.5

100

1112

0.429

Chl.-a (pg 1-1) Carot. (# g I- ~) PE (/zgl -t) PC-APC (/zgl -~) Tot. pigm. (#gl -~)

0.316

0.128

100

of H202 so as to obtain a final concentration of 1.75 ppm, the other remaining as control. The two cyclostats were monitored during 2 weeks for growth and axeny. Analytical methods Algal growth was estimated by microscope counting of filaments for O. rubescens and colonies for P. morum. Optical density was evaluated at 650 nm. Pigment concentration was determined on algal cells collected by filtration through Whatman GF/C fibreglass filter and preserved by freezing. Chlorophyll a and carotenoids were extracted by ultrasonic disintegration in 90% acetone followed by a 15 h contact in darkness at 4°C. After filtration through a Whatman GF/C fibreglass filter, the filtrate was analysed according to Parsons and Strickland (1963). Biliproteins were extracted by three successive freezing-thawing cycles in phosphate buffer (pH 7.0). After centrifugation (48,000g, 4°C, 5h) the supernatant was analysed according to Bennett and Bogorad (1973). Absence of bacteria and fungi was immediately checked by direct microscope observation and confirmed by counting colonies developed on appropriate media (Nutrient Broth, Nutrient Agar, Yeast Extract) sown from tested cultures and incubated at 28°C. Growth potential was evaluated by sowing an aliquot of each culture on two different media, Gorham's ASMI and Zehnder's Z (Staub, 1961), and observing algal growth during about 1 month. RESULTS AND DISCUSSION

Batch cultures experiments (Figs I and 2) Results are expressed in percent o f the control values o b t a i n e d w i t h o u t H202. A t first sight, hydrogen peroxide effect o n O. rubescens appears to be a threshold effect m o r e t h a n a p r o p o r t i o n a l one; con-

ii

a O.O. • Chl • Cor o PC, v Pe

e-

0.00

P.

l

r

T

1.75

3.50

5.25

HzOz p p m Fig. 1

--~' 7.00

Hydrogen peroxide as a potential algicide for Oscillatoria rubescens

100,

--Or aoo • Chl • Car o Pc v Pe

\x~q \

~3

5O

0 00.0

621

75

3.50

3 25

H202 ppm

Fig. 2 sequently it is sensitive, especially to experimental conditions. All these conditions are exactly the same for O. rubescens in the two series, except the initial filament density which is higher in series A as indicated by optical density and total pigments values (Table 1). This results in a difference of the pigment distribution: low density culture induces higher individual cell illumination and therefore slightly promotes protection pigments, i.e. carotenoids, to the detriment of collecting pigments, i.e. chlorophyll a, phyco- and allophycocyanins and phycoerythrin. This results also in a difference of sensitivity between the two series of batch cultures experiments: the lower is the filament density, the more sensitive is the culture. This is noticeable through optical density measurements as well as pigment analysis and growth potential estimation. Concentration of pigments are the obvious and most readily determined changes effected by H202. But, although many organisms possessing carotenoids have been found to be sensitive to photo-oxidative reactions when deprived of this pigment, photo-oxidative death and bleaching of photosynthetic pigments are separable phenomena (Abeliovich and Shilo, 1972). Other cell constituents and mediative substances, and consequently physiological-biochemical processes may be affected by H20: action, inducing changes in photosynthetic activities and growth potential. The second fact appearing on Figs 1 and 2 is that pigments have not the same response to H20,_. Biliproteins are the most affected, in a nearly identical manner. Their destruction starts at the lowest concentration tested, i.e. 0.35ppm, whereas carotenoids begin to be destroyed only at 1.4 ppm and chlorophyll a at 1.75 ppm. This differential sensitivity concerns also the percentage of destruction: the highest H202 concentration destroys more than 90% of biliproteins and carotenoids and hardly 50% of chlorophyll a, the percentage of destruction being slightly higher at lower initial filament density (series B). Such a difference can be explained by chemical structures: biliproteins, and to a lesser extent carotenoids molecules, are linearly structured, consequently easier to

destroy compared to chlorophyll, of which molecular structure is more compact. The last thing evident on Fig. 2 is that all the H202 concentration tested are without any effect on P. morum whether it concerns optical density or pigments concentration or even growth potential, in spite of the fact that P. morum is present at a lower density, which is supposed to increase its sensitivity. The difference of reaction between P. m o r u m and O. rubescens pigments, especially unexpected for carotenoids, can be due to the fact that in the latter as in other prokariotic algae the photosynthetic apparatus does not segregate into discrete organelles and has a direct connexion with the plasma membranes of the cell, whereas in P. morum, as in other eukariotic algae the photosynthetic apparatus is partitioned from the cytoplasm by a pair of limiting membranes giving rise to a discrete cell organelle, the chloroplast, which protects the pigments against external incursions. Olofsson proposed that "this physiological difference lies at the root of differences in growth response characteristics for the two groups 10 5

~o~ ~ .o~

10 4

~o

~o ~

/'

~e~e

\°

c

8

10

o

Pm

•

O.r

3

t0 2

--

without

---

with H202

~,

o 0

1

2

3

4

5

6

7

8

Time (days]

Fig. 3

9

t0

H202

622

G. BARROINand MAURICETTEFEUILLADE

of organisms and is spccifically responsible for associated nutrient transport characteristics" (Olofsson, 1980). Continuous culture experiments (Fig. 3)

H:O2 (1.75 ppm) totally destroys O. rubescens after few hours. Microscopic observation reveals that filaments have broken up into small pieces of 10-70 # m size. This result is consistent with those of batch culture experiments where this concentration appears only as a critical value by considering that H20 2 efficiency is inversely related to filament density and that continuous cultures are conducted at cell density of the same order of magnitude than naturally encountered, i.e. half that of batch cultures. O. rubescens elimination allows P. morum to grow more intensively than in the control. However, even in the control O. rubescens disappears after 9 days. This spontaneous elimination cannot be attributed to a difference of initial growth rate since the experiment has been designed to get, before cultures mixing, about the same generation time and even slightly higher for O. rubescens (3.6 days) than for P. morum (3.3 days). It cannot result from a competition for essential nutrient since PO4-P and NO3-N concentrations in effluents are well in excess, respectively 2 and 10 mg 1-~. The most likely explanation lies in an inhibitory effect of P. morum on O. rubescens growth: release of algal growth inhibiting substances is well documented and P. morum is known for producing extracellular antibacterial substances (Harris, 1971), which is not without interest, considering the supposed affinities between cyanophytes and bacteria. CONCLUSIONS H202 concentrations around l.Sppm have a deleterious effect on O. rubescens: growth potential is destroyed as well as pigments, especially biliproteins and carotenoids, chemically more destructible than chlorophyll a. H202 efficiency is threshold shaped and the threshold value is inversely related to culture density. Concentration ten times higher is totally harmless for P. morum whose pigments are physically better protected inside chloroplasts. This differential response allows, under experimental conditions, to replace the cyanophyte by the chlorophyte. As a side result it appears that P. morum is able alone to eliminate O. rubescens, at least in experimental conditions. Further laboratory experiments are needed to confirm the specificity of H202 detrimental effect on other cyanophytes. Finally, by directly experimenting on a lake previously treated for external and internal nutrient loading, field experiments will show if effectively O. rubescens or other cyanophytes can be eliminated by H202 application and for what concentration. The following questions must also be answered: how do natural conditions modify the sensitivity of the algal population and the fate of the algicidal

substance, with its economical consequences; how the whole ecosystem will react to H202 application even at homeopathic concentrations, with its ecological consequences. Acknowledgement--We are grateful to Mrs Barraud, Mrs

Menthon and Mr Colon for their technical help and to two unnamed reviewers for comments on draft of this paper.

REFERENCES

Abeliovich A. and Shilo M. (1972) Photooxidative death in blue-green algae. J. Bact. 111, 682-689. Ailing S. F. (1963) Continuously regenerated greensand for iron and manganese removal. J. Am. Wat. Wks Ass. 55, 749-752. Balvay G. (1981) Quelques consrquences biologiques du traitement d'un lac avec du peroxyde d'hydrogdne sur la biocrnose planctonique. Wat. Res. 15, 691-696. Barroin G. (1980) Lake treatment with hydrogen peroxide. In Hypertrophic Ecosystems (Edited by Barica J. and Mur L. R.), pp. 267-294. Junk, The Hague. Barroin G. and Feuillade M. (1978) Effects of hydrogen peroxide on Oscillatoria rebescens. Rapport interne, Station d'Hydrobiologie Lacustre, Thonon. Bennett A. and Bogorad L. (1973) Complementary chromatic adaptation in filamentous blue-green alga. J. cell. Biol. 58, 419-435. Cole C. A., Ochs L. D. and Funnell F. C. (1974) Hydrogen peroxide as supplemental oxygen source. J. Wat. Pollut. Control Fed. 46, 2579-2592. Cole C. A., Paul P. E. and Brewer H. P. (1976) Odor control with hydrogen peroxide. J. Wat. Pollut. Control Fed. 48, 297-306. Cole C. A., Stamberg J. B. and Bishop D. F. (1973) Hydrogen peroxide cures filamentous growth on activated sludge. J. War. Pollut. Control Fed. 45, 829-836. Feuillade J. and Feuillade M. (1979) A chemostat device adapted to planktonic Oscillatoria cultivation. Limnol. Oceanogr. 24, 562-564. Fitzgerald G. P. (1966) Use of potassium permanganate for control of problem algae. J. Am. War. Wks Ass. 58, 609-614. Fraser J. A. L. and Sims A. F. E. (1983) Tip leachate treatment using hydrogen peroxide. War. Pollut. Control 82, 243-255. Fraser J. A. L. and Tytler N. (1983)Operational experience using H202 in landfill leachate treatment. E~. War. Treat. J. 149-156. Gorham P. R., McLachlan J., Hammer U. T. and Kim W. K. (1964) Isolation and culture of toxic strains of Anabaena flos-aquae (Lung) de Breb. Verh. Int. Verein. Limnol. 15, 796-804. Harris D. O. (1971) Growth inhibitors produced by the green algae Volvocaceae. Arch. Mikrobiol. 76, 47-50. Hughes J. S. (1971) Pond management: key to fish farming success. Am. Fish. Farm. 2, No. 10, 10-15. Interox-Chimie (1981) Le peroxyde d'hydrogdne dans le traitement des effluents municipaux. Eau Ind. 53, 55-59. Kay S. H., Quimby P. C. Jr and Ouzts J. D. (1982) H202: a potential algicide for aquaculture. Proceedings o f the 35th Annual Meeting of the Southern Weed Science Society, Atlanta, Ga, pp. 275-289.

Kemp H. T., Fuller R. G. and Davidson R. S. (1966) Potassium permanganate as an algicide. 3". Am. War. Wks Ass. 58, 255-263. Olofsson J. A, (1980) The role of microlayers in controlling phytoplankton productivity. In Hypertrophic ecosystems (Edited by Barica J. and Mur L. R.), pp. 83-93. Junk, The Hague.

Hydrogen peroxide as a potential algicide for Oscillatoria rubescens Parsons T. R. and Strickland J. D. H. (1963) Discussion of spectrophotometric determination of marine plant pigments with revised equations for ascertaining chlorophylls and carotenoids. J. Mar. Res. 21, 155-163. Putz M. (1976) Geruchsfreihaltung von Tiefenwasserableitungen durch Wasserstoffperoxid. Osterreich. Abwasser Rundschau 3, 39-42. Soares de Assis L. F. (1980) Le traitement au perhydrol (H202) d'un s6diment r6duit; une approche de laboratoire de son impact sur la macrofaune endobenthique

623

(tubificides). Th6se doctorat de 36me cycle, Universit6 Claude Bernard, Lyon. Staub R. (1961) Ern/ihrungsphysiologisch-aut6kologische Untersuchungen an der planktischen Blaualge Oscillatoria rubescens D.C. Schweiz Z. Hydrol. 23, 82-198. Welch W. A. (1963) Potassium permanganate in water treatment. J. Am. Wat. Wks Ass. 55, 735-741. Willey B. F. and Jennings H. (1963) Iron and manganese removal with potassium permanganate. J. Am. War Wks Ass. 55, 729-734.