Mechanisms of delayed ozone toxicity to bluegill Lepomis machrochirus rafinesque

EnvironmentalPollution(SeriesA) 22 (1980)229-239

MECHANISMS OF DELAYED OZONE TOXICITY TO BLUEGILL LEPOMIS MACHROCHIR US RAFINESQUE

MICHAEL H. PALLER & ROY C. HEIDINGER

Fishery Research Laboratory and Department of Zoology, Southern Illinois University, Carbondale, Illinois 62901, USA

ABSTRACT

Ozone causes gross damage to the gills. Extensive fungal infections often followed exposure to ozone, suggesting epithelial damage. An ,abrupt' decrease in blood serum osmolality was detected infish exposed to ozone. Mortality occurred when osmolalit~ dropped approximately 50 mOsm below initial values.

INTRODUCTION

Ozone is a powerful disinfectant, currently employed in European water treatment plants, which may replace chlorine in similar US plants. Although more costly than chlorine, ozone is biologically attractive because it is ephemeral--disappearing rapidly from treated water--in contrast to chlorine, which is relatively persistent. Although the effects of chlorine on fishes are fairly well documented, much less is known about ozone toxicity (Jolley et al., 1978). Only a few authors have investigated the manner in which ozone kills fishes. Hubbs (1930) attributed the toxicity of ozone to nascent oxygen but failed to indicate the mode of lethal action. Rosenlund (i975) found that exposure to ozone causes destruction of gill tissue. In the most thorough study to date, Block et al. (1978) concluded that ozone impairs both respiration and osmoregulation, presumably by damaging the gills. These conclusions were reached after ob_serving an increase in plasma osmolality and other pathological changes in white perch Morone americana exposed to ozone- in salt water, a hyperosmotic environment. This paper describes the effects of ozone on blood serum osmolality of the bluegill Lepomis macrochirus in freshwater, a hypoosmotic environment. 229 Environ. Pollut. Ser. A. 0143-1471/80/0022-0229/$02-25 © Applied Science Publishers Ltd, England, 1980 Printed in Great Britain

230

MICHAEL H. PALLER, ROY C. HEIDINGER METHODS AND MATERIALS

The dynamic bioassay method was employed since ozone undergoes rapid degradation. The bioassay facility consisted of an ozone generator, two mixing columns and two raceways. Ozone was produced with a Welsbach T-23 ozonator by exposing compressed air to an electric arc. The quantity of ozone produced could be controlled by varying the voltage used by the ozonator. Tygon tubing was used to transport ozone from the generator to a mixing column--a vertically situated I183-cm section of 15 cm PVC pipe acting as a counter-current exchanger. A continuous flow of charcoal-filtered City water was ozonated and conveyed to the experimental raceway. A second mixing column, similarly designed, was used to mix water with either pure oxygen or compressed air. Water from this mixing column was conveyed to a second raceway where control experiments were conducted. The wooden raceways, 236 cm long and 30 cm wide, were covered by two coats of epoxy paint and filled to a depth of 12 cm. Water from the mixing columns entered at the head of each raceway at approximately 1.9 litres/min and flowed out from a standpipe at the opposite end into a sewer. Ozone concentrations were determined spectrophotometrically (Schechter, 1973). Bluegill averaging 4-0 g were the experimental animals. They were well acclimated to laboratory conditions and appeared to be healthy. Six groups of ten bluegill were exposed to six different ozone concentrations for 24h 1(24-h bioassay). Five other groups, each containing fifteen bluegill, were subjected to six 30-min periods of exposure to ozonated water at 8-h intervals (40-h bioassay). Lastly, we attempted to expose a group of fifteen bluegill to low ozone concentrations for six weeks. At the conclusion of each trial the surviving fish were placed in aquaria for further observation (see Paller & Heidinger, 1979, for detailed information about procedures used in these studies). Certain phenomena observed during these investigations led to a follow-up study to investigate the effect of ozone on the osmolality of the fishes' blood serum. Only the experimental raceway was used. Five compartments of plastic screen, 20 cm long and 6 cm wide, were placed in the raceway and arranged in parallel so that each was equidistant from the water inlet. This design ensured that separate groups of bluegill held in each compartment would be exposed to similar ozone concentrations. Four fish were placed in each of the five raceway compartments. Water containing ozone was passed through the raceway for 10h; four fish were removed from one compartment every 2 h and examined. The group of fish was selected at random. Examination consisted of the determination of blood serum osmolality on a Wescor 5100 vapour pressure osmometer. Blood samples were obtained by severing the caudal peduncle and collecting the forming blood drop in a heparinised capillary tube. After centrifuging for 5 min at 6000 rpm in a clinical centrifuge, the capillary

DELAYED OZONE TOXICITY TO BLUEGILL FISH

231

tube was severed, the portion containing the red blood cells discarded and the serum blown out of the remaining portion onto a Wescor sample disc. After being thoroughly infiltrated, the sample disc was placed within the test chamber of the osmometer. To prevent evaporation and contamination, the fish were blotted dry and the sample discs were exposed to the atmosphere for as brief a period as possible. Osmolality values are considered accurate to within +__2 mOsm. A control experiment was conducted in the same facility, similarly employing twenty fish with groups of four fish removed at 2-h intervals and examined for changes in osmolality. The ozone generator was not activated during the control run. The significance of changes in osmolality, as well as distinctions between experimental and control groups, were evaluated by multiple regression analysis (Program DP linear). In a separate experiment the gills of!five bluegilli werelexamined following six hours of exposure to ozone. The gills of these fish were compared with those of five bluegill not exposed to ozone. Gills were removed, placed in a drop of water on a depression slide, examined microscopically and photographed.

RESULTS

Delayed mortality Many fish which survived the immediate effects of ozone in the 24- and 40-h bioassays (see Paller & Heidinger, 1979 for LCsos) succumbed to a severe fungal infection 2 to 7 days later (Table 1). Fungus usually appeared within 2 or 3 days after the fish were removed from the experimental facility and returned to the original holding tank or placed into other aquaria. The infection spread rapidly over the body with mortality occurring within several days after its initial appearance. At the t~me of death, most of the surface of the fish was covered by a massive growth of fungal hyphae. In no instance did a control animal develop this condition. Ozone concentrations fluctuated considerably during the first week of the sixweek experiment--from less than 0.01 ppm to 0-06 ppm. Due to lethal peaks such as 0.06 ppm, all fish died within 7 days. Approximately 50 ~ of the fish became covered with an extensive growth of fungi even while remaining within the experimental facility; no control fish developed this condition. Although ozone concentrations varied greatly, there was at least a trace amount of ozone present at all times. In addition, although concentration peaks appeared to affect the fish adversely, they did not retard the spread of fungal infection, once established. Approximately 50 of the fish were heavily infected before death. Control animals remained healthy throughout the experiment. Changes in serum osmolality Blood serum osmolality was measured after varying periods of exposure to a

232

MICHAEL H. PALLER, ROY C. HEIDINGER TABLE 1 OCCURRENCE OF DELAYED MORTALITIESOF BLUEGILL FOLLOWINGTHE 24- AND140-hBIoASSAYS Ozone (ppm)

Number of mortalitiesat termination of trial

Number of delayed mortalities after 2-7 days

24-h experiment* 0"00 0'01 0'02 0"05 0'06 0'07 0'09

0

0

0 0 1 6 I0 9

0 0 9 4 -1

40-h intermittent experimentb

0.00 0.03 0.12 0.23 0.39 0.78

0 0 1 5 9 t5

0 0 7 10 6 --

° Ten bluegill per trial (mean weight = 3.82g), mean temperature = 10°C, mean pH = 7-1, mean alkalinity = 62 ppm CaCO a. b Fifteen bluegill per trial (mean weight = 2.49 g), mean temperature = 10"8°C, mean pH = 7.3, mean alkalinity = 50ppm CaCO 3. m e a n c o n c e n t r a t i o n o f 0 . 1 7 p p m o f ozone. E x p o s u r e to o z o n e was c o r r e l a t e d with a r a t h e r a b r u p t decrease in o s m o l a l i t y with values stabilising a r o u n d 2 2 5 m O s m (Table 2), at which p o i n t m o r t a l i t i e s began o c c u r r i n g ; two in the 8-h e x p o s u r e g r o u p a n d two m o r i b u n d fish in the 10-h e x p o s u r e g r o u p . T h e c o l o u r o f the b l o o d serum also c h a n g e d f r o m clear to slightly pinkish with increasing d u r a t i o n o f exposure. In c o n t r a s t , o s m o l a l i t y in the c o n t r o l g r o u p showed a lesser a n d t e m p o r a r y decline for the first 6 h, then r e t u r n e d to n e a r n o r m a l levels in the latter stages o f the e x p e r i m e n t (Fig. l). N o control animals died. Trend analysis (Table 3) indicated that the distinction between e x p e r i m e n t a l a n d c o n t r o l g r o u p s was very significant a n d caused b y a difference between the rates o f c h a n g e in o s m o l a l i t y characteristic o f the two groups. This t r e n d is e l u c i d a t e d by a g r a p h i c c o m p a r i s o n (Fig. l) which illustrates t h a t o s m o l a l i t y d r o p s m o r e r a p i d l y with increasing d u r a t i o n o f e x p o s u r e in the e x p e r i m e n t a l groups. Effects upon gill morphology A s e p a r a t e e x p e r i m e n t indicated that e x p o s u r e to o z o n e (0.09 p p m ) c a u s e d the d e s t r u c t i o n a n d loss o f epithelial cells covering the gill filaments. D a m a g e was severe a n d led to e x p o s u r e a n d d e s t r u c t i o n o f lamellae a n d frequent loss o f the entire tip o f

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233

TABLE 2 CHANGES IN BLOOD SERUM O~MOLALITY OF BLUEGILL EXPOSED TO 0-17ppm OF OZONE, DEMONSTRATED BY THE REMOVAL, AT SUCCESSIVE 2-h INTERVALS, OF GROUPS OF FISH FROM WATER CONTAINING OZONEa

Group No. b

Duration of exposure (h)

Mean osmolality (mOsm)

Standard deviation (mOsm)

Experimental groups 1 2 3 4 5 6c

0 2 4 6 8 10

277 261 244 226 226 225

4"25 6.68 8"79 4"92 15.17 1-23

Control groups l 2 3 4 5 6

0 2 . 4 6 8 10

276 273 267 258 262 267

2'11 3"5 11'22 6.76 1.26 2"08

Temperature = 11 °C, pH = 7.1, alkalinity = 58 p p m CaCO3. Four bluegill per group (mean weight = 4.0 g). c Since accurate osmolality values could not be obtained from dead specimens, the mean value was derived from only two fish in this group. °

280 2:'0 1

A

I .01 0 0

240.

0

230-

"

220-

B |

210 0

!

,

,

I

l

I

I

2

4

6

8

10

12

HOURS OF EXPOSURE Fig. I. Association between change in blood serum osmolality of bluegill (mean weight - 4.0g) and exposure to 0. ! 7 p p m of ozone (temperature = 11 °C, pH --- 7.1, alkalinity = 58 p p m CaCO3). Control groups (A) and experimental groups (B) are depicted. Data points represent group means. Vertical bars represent standard error for each group.

234

MICHAEL H. PALLER, ROY C. HEIDINGER TABLE 3

TREND ANALYSIS OF THE RELATIONSHIP BETWEEN EXPOSURE TO OZONE AND CHANGE IN SERUM OSMOLALITY

Variables: x(I) = osmolality x(2) = linear trend x(3) = quadratic trend x(4) = cubic trend x(5) = distinction between experimental and control groups x(6) = linear interaction x(7) = quadratic interaction x(8) = cubic interaction N = 40 (20 experimental, 204control) ° P = 0.05 Models, Restrictions, R2s and F Ratios: Full model: x(l) = a(O)u + a(2)x(2) + a(3)x(3) + a(4)x(4) + a(5)x(5) + a(6)x(6) + a(7)x(7) + a(8)x(8) + E R 2 -- 0.7861 Significance of linear trend: Null hypothesis: a(2)=O Restricted model: x(1) = a(O)u + a(2)x(2) + a(4)x(4) + a(5)x(5) + a(6)x(6) + a(7)x(7) + a(8)x(8) + E R z = 0'3876 F = 70-7807

Non-directional probability = 0.0000

Significance of quadratic trend: Null hypofhesis: a(3) --- 0 Restricted model: x(1) --- a(O)u + a(2)x(2) + a(4)x(4) + a(5)x(5) = a(6)x(6) + a(7)x(7) + a(S)x(8) + E R 2 = ;0-7723 F = 2.4362

Non-directional probability = 0.1268

Significance of cubic trend: Null hypothesis: a(4) = 0 Restricted model: x(1) = a(O)u + a(2)x(2) + a(3)x(3) + a(5)x(5) a(6)x(6) + a(7)x(7) + a(8)x(8) + E R2 ~

0'1162

F = 2'5837

Non-directional probability = 0-1162

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235

T A B L E 3--contd.

Significance of group distinction: Null hypothesis: a(5)=0 Restricted model: xtl) = atO)u + a(2)x(2) + a(3)x(3) + a(4)x(4) + a(6)x(6) + a(7)x(7) + a(8)x(8) + E R" = 0.5377 F = 44-1191

Non-directional probability --- 0.0000

Significance of linear interaction: Null hypothesis: at6) = o Restricted model: x(l) = a(O)u + a(2)x(2) + a(3)x(3) + a(4)x(4) + a(5)x(5) + a(7)x(7) + a(8)x(8) + E R 2 = 0.6782 F = 19.1631

Non-directional probability -- 0.0001

Significance of quadratic interaction: Null hypothesis: a(7) = 0 Restricted model: x(l) = a(2)x(2) + a(3)x(3) + a(4)x(4) a(5)x(5) + a(6)x(6) + a(8)x(8) + E R 2 -----0"7830 F = 0"5362

Non-directional probability - 0.4685

Significance of cubic interaction: Null hypothesis: a(8) = 0 Restricted model: x(1) = a(O)u + a(2)x(2) + a(3)x(3) + a(4)x(4) a(5)x(5) + a(6)x(6) + a(7)x(7) + E R z --- 0.9730 F--- 0.0012

Non-directional probability = 0.9730

° The sixth group was dropped from both the experimental and control groups due to mortality in the experimental group which created unequal sample sizes.

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MICHAEL H. PALLER, ROY C. HEIDINGER

Fig. 2.

Fig. 3.

Photomicrograph of the gills of normal bluegill fish.

Photomicrograph of the gills of bluegill fish exposed to 0.09 ppm of ozone for 6 h.

DELAYED OZONE TOXICITY TO BLUEGILL FISH

237

the filament. These effects were primarily confined to the distal portion of the filament, leaving the base relatively unscathed (Figs 2 and 3). Gills of experimental fish also appeared to be bleached and lacking in blood. None of these effects was observed in control fish.

DISCUSSION

This and other studies (Rosenlund, 1975; Block et al., 1978) indicate that ozone damages the gills severely, Since respiratory gases are exchanged at the surface of the lamellae (Lagler et al., 1962), damage to the lamellar epithelium and underlying capillaries can cause anoxia. Osmoregulation may be impaired since the gills are sites of active ion absorption and secretion (Lagler et al., 1962). Also important in this respect are possible changes in gill tissue permeability which could permit a more rapid flux of water and salts through the gills by diffusion. The skin and mucous layer also contribute to osmoregulation by largely preventing the movement of water and salts through the body surface (Lewis, 1970; Van Oosten, 1957; Parry, 1966). Disruption of this permeability barrier can result in an influx of water and loss of salts in freshwater fishes. Lewis (1970) has demonstrated that damage to the integument of golden shiners Notemigonus crysoleucas causes a fatal reduction of osmotic pressure accompanied by bacterial and fungal infections. If intact, the mucous layer and integument prevent infection by opportunist pathogens. Fungus frequently occurs as a secondary infection in wounds and sites of bacterial invasion--areas in which the external barriers preventing infection have been disrupted (Reichenbach-Klinke & Landolt, 1973). Similarly, Paller & Heidinger (1979) found that exposure to ozone frequently caused delayed mortalities associated with extensive fungal infections covering the body surface. This suggests damage to the skin and mucous layer~damage which can lead to a flow of water and ions through the unprotected body surface, causing a reduction of plasma osmolality as observed in this study. Mortalities occurred after a blood serum osmolality decrease of 50mOsm. Similarly, Lewis (1970) observed that mechanically damaged golden shiners died after osmolality dropped approximately 45 mOsm below normal. It is possible that an osmotic imbalance caused by damage to the integument, especially the mucoid layer and gills, is an important factor contributing to mortalities induced by acute exposure to high ozone concentrations. This type of severe change in osmolality should be distinguished from temporary stress-induced haemodilution which appears to be a normal physiological response (Mazeaud et al., 1977). The control fish in the osmolality experiment experienced a less drastic and reversible decline in osmolality which was possibly due to stress caused by handling. The fact that both ozone and chlorine are powerful oxidants suggests a possible

238

MICHAEL H. PALLER, ROY C. HEIDINGER

similarity in their modes of action upon fishes. The effects of chlorine are in some respects comparable with those of ozone. Like ozone, chlorine causes physical damage to the gills (Doudoroff, 1957; Ellis, 1937) and impairs osmoregulation (Block et al., 1978). Dent (1974) reported discoloration of the skin of channel catfish intermittently exposed to chlorine, which suggests damage to the integument. There are also possible differences between ozone and chlorine toxicity. Several researchers (Ellis, 1937; Grothe & Eaton, 1975) indicate that chlorine and/or chlorine derivatives are capable of passing through epithelial membranes and exerting toxic effects within the body. Grothe & Eaton (1975) have demonstrated that exposure to chloramines induces striking elevations ill methaemoglobin concentration which can cause death by anoxia. Block et al. (1978) observed that plasma sodium and potassium levels changed significantlyin chlorine-exposed fishes but remained constant in fish exposed to ozone. This suggests a difference between the effects of ozone and chlorine on Na +- K ÷-ATPase activity (Block et al., 1978). Perhaps the principal difference between ozone and chlorine toxicity is that chlorine and/or chlorine compounds are capable of entering the body while the primary effects of ozone are mainly confined to the body surface. Ozone appears to cause severe damage to all external tissues. This can result in disruption of respiration, osmoregulation and possibly excretion, resulting either in death or in the development of various sublethal secondary pathological effects. The change in colour of the blood serum from clear to pinkish with duration of exposure to ozone may be an example of this. A change in osmotic concentration of the blood could have led to a haemolysis of the erythrocytes in v i v o - - t h u s releasing haemoglobin into the blood--or so weakened the erythrocytes that they were easily ruptured during centrifugation. Ozone is capable of causing severe tissue destruction because it is a very strong oxidant---considerably stronger than chlorine (Rosenthal, 1974). Therefore, ozone can probably cause considerably greater damage to exposed body surfaces than chlorine. Block et al. (1978) observed that exposure to ozone was associated with a significant decrease in gill protein while the latter was not significantly affected by exposure to chlorine under comparable conditions. The preceding evidence suggests that ozone toxicity is largely irreversible. Additionally, it is probable that if sufficient ozone is present to cause the death of any fish, the remaining fish will have suffered gross tissue damage and further mortalities and/or disease can be anticipated. Moreover, since the infections observed by us were probably caused by opportunistic saprophytic fungi, it is probable that environments supporting these organisms are more likely to induce disease in damaged fishes. In terms of future ozone bioassays, it is evident that results may be misleading unless delayed mortalities are fully reported. Although more toxic than chlorine, ozone retains an important advantage as a wastewater treatment: it is a short-lived compound which is readily removed from treated water, while chlorine and chlorine derivatives are considerably more refractory.

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REFERENCES BLOCK, R. M., BURTON, D. T., GULLANS, S. R. & RICHARDSON, L. B. (1978). Respiratory and osmoregulatory response of white perch (Morone americana) exposed to chlorine and ozone in estuarine waters. In Water chlorination environmental impact and health effects, ed. by R. L. Jolley, H. Gorchev and D. H. Hamilton, Jr., 351-60. Michigan, Ann Arbor Science Publishers, Inc. DENT, R. J. (1974). Effects upon fishes of a periodic flushing of electrical power plant boiler tubes with chlorine. Unpublished MA thesis, Carbondale, Southern Illinois University. DOUOOROFF, P. (1957). Water quality requirements of fishes and effects of toxic substances. In The physiology of fishes, ed. by M. E. Brown, Vol. 2. 403-30, New York, Academic Press. ELLIS,i . i . (1937). Detection and measurement of stream pollution. U.S. Bur. Fish. Bull., 22, 365-437. GROTHE, D. R. & EATON,J. W. (1975). Chlorine-induced mortality in fish. Trans. Am. Fish. Soc., 104, 800-2. HUBBS, C. L. (1930). The high toxicity of nascent oxygen. Physiol. Zool., 3, 44~60. JOLLEY,R. L., GORCHEY,H., HAMILTON,D. H. Jr. (1978). Water chlorination environmental impact and health effects,,Michigan, Ann Arbor Science Publishers, Inc. LAGLER,K. F., BARDACH,J. E. & MILLER,R. R. (1962). Ichthyology, New York, John Wiley & Sons. LEWIS,S. D. (1970). The effect of salt solutions on osmotic changes associated with surface damage to the golden shiner, Notemigonous crysoleucas. Unpublished PhD dissertation. Carbondale, Southern Illinois University. MAZEAUO,M. M., MAZEAUO,F. & DONALDSON,E. M. (1977). Primary and secondary effects of stress in fish: Some new data with a general review. Trans. Am. Fish. Soc., 106, 201-12. PALLER, M. H. & HEIDINGER,R. C. (1979). The toxicity of ozone to the Bluegill. Environ. Sci. & Hlth, A14(3), 169-93. PARRY, G. (1966). Osmotic adaptations in fishes. Biol. Rev., 41,392-444. REICHENBACH-KLINKE,H. & LANDOLT,M. (1973). Fish pathology, Neptune City, T. F. H. Publications. ROSENLUNO,B. (1975). Disinfection of hatchery influent by ozonation and the effects of ozonated water on rainbow trout. In Aquatic applications of ozone, ed. by W. J i Blogoslawskiland R. G. Rice, 59--69. Syracuse, The International Ozone Institute, Inc. ROSENTH^L,H. (1974). Selected bibliography on ozone, its biological effects and technical applications. Fish. Res. Bd Can. Tech. Rep., No. 456. SrmCH~a, H. (1973). Spectrophotometric method for determination of ozone in aqueous solutions. Water Res., 7, 729-39. VAN OOSTEN,J. (1957). The skin and scales. In The physiology offishes, ed. by M.E. Brown, 207-44, New York, Academic Press.