Marine Em'ironmental Research 8 (1983) 241-254

Physiological Responses of a Marine Fish Exposed to Chlorinated Seawater at Concentrations Near Its Avoidance Threshold J. E. Hose, W. Hunt and R. J. Stoffel VANTUNA Research Group, Department of Biology, Occidental College, Los Angeles, California 90041, USA (Received: 24 March, 1982)

ABSTRACT To determine if avoidance of chlorinated seawater by fish resulted in physiological protection from toxicity, studies were carried out which assessed (a) changes in the routine o.vygen consumption rate and (b) the ability of treated fish to successfully compete with untreated conspecifics for a limited food resource. Temperate marine damselfish, Chromis punctipinnis, were exposed to stepwise increasing levels of chlorinated seawater in a behaviour chamber and avoided total residual oxidant (TRO) levels greater than O.15-0.16 mg/litre. Cumulative exposures ranged from the equivalent of 0.38-5.23min at 1.0mg/iitre TRO. One day after exposure, routine oxygen consumption rates were decreased by 25 to 45% from preexposure rates and were correlated with the cumulative oxidant exposure. One month post exposure, respiration rates returned to pre-treatment levels. This transient depression of the respiratory rate did not affect survival or growth of chlorine-treated fish which were forced to compete with untreated conspecifics for a restricted food supply.

INTRODUCTION Exposure o f fish to chlorinated seawater has been shown to interfere with normal respiratory and osmoregulatory function (Block et al., 1978). The toxic actions o f chlorine-produced oxidants presumably affect energy 241 Marine Environ. Res. 0141- 1136/83/0008-0241/$03"00 ~ Applied SciencePublishers Ltd, England, 1983. Printed in Great Britain

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metabolism and:or produce damage to the gill filaments, impeding both oxygen and ion transport (Middaugh et ai., 1977, Capuzzo, 1979). Haemoconcentration, haemolysis and decreased plasma pH values of white perch (Morone americana) resulted from exposure to chlorine (Block et al., 1978). Depressed oxygen consumption rates were documented in killifish (Fundulus heteroclitus) and larval lobsters, and a concomitant reduction in the growth rates of lobster larvae was evident after exposure to chlorinated seawater (Capuzzo et al., 1977: Capuzzo, 1977). However, some fish have been shown to actively avoid chlorinated waters both in the laboratory and in the environment (Schumacher & Ney, 1980: Giattina et al., 1981; Hose et at., in press). Such avoidance may mitigate the toxic effects of chlorine-produced oxidants. Thus, studies in which fish have been subjected to chlorine (that is, without the opportunity to move into non-chlorinated areas) may not be representative of actual environmental situations (Block, 1977) such as the point chlorination of an open body of water. Therefore, it is of interest whether the avoidance response of fish to chlorinated seawater actually results in physiological protection from toxicity. To this end, the respiratory function, growth and mortality of a southern California damselfish, Chromis punctipinnis, after exposure to subthreshold concentrations of chlorinated seawater (<0.20mg/litre TRO) were studied. Immediate and delayed effects of chlorine-produced oxidants on the routine oxygen consumption rate of C. punctipinnis were investigated to determine if recovery of normal respiratory function was possible. In a second experiment, fish exposed to chlorinated seawater were held with untreated fish and fed a restricted ration. Mortality and growth were monitored to determine if chlorine-treated fish could successfully compete with untreated conspecifics for a limited resource. MATERIALS AND METHODS Fish

Juvenile Chromispunctipinnis (1 to 15 g wet weight) were collected in King Harbor Marina, California, USA, by SCUBA divers using gill nets. Fish were directly transported to laboratory holding facilities and acclimated to their preferred temperature of 20°C (Shrode et al., 1982) for at least 1 week.

Fish responses to chlorine

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Determination of routine oxygen consumption rates Groups of eight fish were placed into individual 1-1itre flasks in a constant temperature differential respirometer (Shrode & Gerking, 1977). Seawater (33-34%0 salinity) maintained at 20 °C was circulated through flasks during a 4-h acclimation period. Initial dissolved oxygen concentrations were measured using an Orion dissolved oxygen electrode (Model 97-08) attached to a specific ion meter (Orion Model 407A), then the flasks were sealed. One hour later, final dissolved oxygen concentrations were determined and the fish were measured and weighed. Differential routine oxygen consumption rates were calculated according to the equation: 0 2 (mg/kgfish h) - - A O 2 (mg/litre) x (total water volume - (weight) - (time). After initial determination of the oxygen consumption rate, fish were placed in the behaviour chamber and fed ad libitum with brine shrimp. The behaviour chamber was a modification of the counterflow system described by Dinnel et aL (1979) with replicate sides which allowed duplicate tests to be performed simultaneously (Fig. 1). Seawater (20 °C) was supplied to a central header tank which distributed the seawater to two small header tanks, one of which received only unchlorinated seawater. An NaOCI solution (5 ~ Chlorox ® in deionised water) was delivered to the test header tank at flow rates ranging from 1 to 115 ml/min. Seawater from control and test header tanks was delivered to opposite ends of the behavioural chamber at approximately 5 litres/min and flowed towards a central drain. The following morning, fish were exposed to subthreshold levels of chlorinated seawater ( < 0.16 mg/litre TRO) in the chamber for 4 to 5 h (Hose & Stoffel, 1980). The exposure regimen consisted of a stepwise increasing time-concentration gradient (Fig. 2). Four fish were placed into each replicate side of the chamber and acclimated to the water flow for 1 h before the test commenced. After acclimation, a set of control observations was recorded preceding chlorination. Time spent in each quadrant was calculated from twenty-five observations of fish positi6ns taken at 15-s intervals. After the control observation set, the hypochlorite pump was started and the chlorine residual concentration was allowed to stabilise for 30min. Preliminary measurements revealed that the desired concentration was reached within a few minutes. Three test observation sets were

244

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Fig. 1, Behaviour chamber used to study responses of marine fish to chlorinated seawater. Filtered seawater flows into the big header tank (BHT), which is then distributed to the left and right head tanks (LHT and RHT, respectively). A chlorine solution (stippled areas) is then added to the left header tank.

245

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Fig. 2. Calculation of the cumulative TRO dose sustained by fish before totally avoiding the chlorinated quadrants of the behaviour chamber. The equation Cumulative Exposure = (TRO concentration x duration of exposure x ~o of time on chlorinated side) describes the area under the time-concentration curve. Vertical arrows indicate the times at which TRO measurementswere performed and solid rectangles indicate the time intervals over which observations of fish positions were recorded. Percentages indicate time fish spent in chlorinated quadrants. Cumulative exposure: (0.0 mg/litre x 75 minx 0.59) +(0.075 mg/litre x 75min x 0.35) +(0-135 mg/litre x 75 min x 0.21) + (0.195mg/ litre x 70 min x 0.01) at the equivalent of 1-00mg/litre TRO = 4-24min. conducted at stepwise increasing hypochlorite concentrations o f approximately 0.07, 0.13 and 0.18mg/litre TRO. Four replicate test experiments were performed, yielding a total o f forty chlorine-treated fish. A group o f eight control fish was subjected to the same regimen except that no hypochlorite was introduced into the chamber. Test fish averaged 4 g wet weight and control fish, 7 g. The small weight difference between the groups did not influence results of respirometry experiments since effects of chlorination on the oxygen consumption rate were not correlated with fish size. Measurements o f T R O concentrations were obtained from seawater samples drawn from amber latex tubes extending into each of the quadrants. T R O concentrations were measured according to the procedure described by Orion (1977). The residual chlorine electrode (Model 97-70) is reportedly sensitive to 0.010mg/litre T R O with 2 ~ reproducibility between 0.2 and 2.0 mg/litre T R O and was coupled to an Orion rrticroprocessor Ionalyzer 901. T R O concentrations were averaged from readings taken in each of the quadrants before and after each stepwise increase in concentration. Although recent research has indicated the preferred unit for reporting residual chlorine concentrations to be /~M hypohalite (Wong, 1980), we have used the unit o f mg/litre seawater at 34%o salinity for ease of comparison with previously published data.

246

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Water quality analyses were performed using Orion specific ion electrodes (dissolved oxygen Model 97-08, ammonia .....Model 95-10 and pH .... Model 91-01) attached to an Orion Ionalyzer 407A. Dissolved oxygen levels were maintained near saturation. Ammonia concentrations were below 0.06ppm and pH values ranged from 7-5 to 7.9. Water temperatures were monitored throughout the experiment using a Yellow Springs Instruments telethermometer Model 47, Temperature determinations were averaged from readings taken in each of the quadrants before and alter each observation set. No significant differences in water quality were detected between the chlorinated and non-chlorinated quadrants. Mean cumulative doses of T R O sustained by each group of eight fish before totally avoiding the chlorinated quadrants of the behaviour chamber were calculated (Fig. 2). First, the time spent at various concentrations of T R O was determined. The mean cumulative T R O exposure was then calculated using the weighted average technique of Mattice & Zittel (1976) where: Cumulative Exposure = Z ( T R O concentration x duration of exposure x 0,i of time on chlorinated side). Exposure levels were standardised to the equivalent number of minutes at 1.00mg/litre TRO. This method has been supported by biological experiments conducted by Larson & Schlesinger (1978) relating mortality and the area under the time-concentration exposure curve Fish were returned to holding tanks. Since previous experiments (Buckley et al., 1976: Capuzzo, 1977) showed that animals exposed to chlorine exhibited signs of toxicity at 20 days but not at 28 days post exposure, oxygen consumption rates of C. punctipinnis were measured at I, 7 and 31 days after treatment. Fish in the highest exposure group (the equivalent of 5.23 min at 1.0 mg/litre TRO) developed a fungal infection unrelated to chlorine exposure on day 6, and experiments using this group were terminated. In all subsequent tests, control and treated fish were treated prophylactically with furacin (20 ppm) and formalin (62.5 ppb) on consecutive days at approximately 14 days post exposure. Significant differences between the oxygen consumption rates of the different groups were determined using a one-way analysis of variance (SPSS, Kim & Kohout, 1975) followed by the Student Newman-Keuls multiple range test. Differences between paired observations (i.e. initial and 7 days post exposure) were tested for significance using paired t tests. Since the fish had grown by day 31, the oxygen consumption rates measured at this time were compared with the expected oxygen consumption rates calculated from the regression equation obtained from

Fish responses to chlorine

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respirometry experiments using untreated conspecifics (n = 60, r 2 = 0 " 8 0 , p < 0.01): Routine oxygen consumption rate = ( - 12.72 x standard length) + (38.18 x temperature)+ 301-60 where routine oxygen consumption rate is in milligrams of 02 consumed per kilogram of body weight per h, standard length is in millimetres and temperature is in °C.

Survival and growth Sixty fish were weighed, anaesthetised with MS222 (tricaine methane sulphonate, Sigma Chemical Co., 1 part to 5000 parts seawater) and tagged through the epaxial musculature beneath the dorsal fin with small coloured beads attached by monofilament thread. Fish were then held for a l-week recovery period. Thirty test fish were exposed to chlorinated seawater in the behaviour chamber as described previously. Thirty control fish were placed in the behaviour chamber and subjected to the same regimen as that of the test fish, except that no hypochlorite was introduced into the chamber. Fish from control and test groups averaged 9 g. Five test and five control fish were placed together into each of six replicate 100-1itre holding tanks. Fish were fed a maintenance ration (7 ~o dry weight brine shrimp/dry weight fish) once a day and fish were reweighed on days 7 and 14 post exposure. The maintenance ration for C.punctipinnis was determined in feeding and growth experiments performed by Gardiner & Shrode (1978). Mortalities were recorded daily.

RESULTS

Oxygen consumption rates Juvenile C. punctipinnis sustained cumulative oxidant exposures ranging from the equivalent of 0-38 to 5.23 min at 1 mg/litre TRO before totally avoiding chlorinated quadrants. Initial routine oxygen consumption rates averaged 555.8 + 200.3 mg O2/kg h (X + SD). One day after exposure, the mean rate was 386.4 + 91.6mg O2/kg h, with a minimum of 144-8 mg O2/kg h. At this time, routine respiratory rates were significantly decreased from initial rate determinations (paired t test, p = 0.05). Mean decreases from 25 to 45 % of the initial rate were exhibited by the test fish (Table 1). By day 7, oxygen consumption rates of two out of three

248

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Hose. W ttunt, R J St(!Oi't

TABLE 1 Percentage Decrease in Routine Oxygen C o n s u m p t i o n Rate of Juvenile C h r o m i s puncttpinnis After Exposure to S u b t h r e s h o l d C o n c e n t r a t i o n s of Seawater 1 day P E ~

Control 0.38 min 1.20min 2-35min 5.23 min

at at at at

1.0mg/litre l-0mg/litre 1.0mg/litre 1.0 mg/litre

TRO TRO TRO TRO

- 18.9 :~ 23.9 -~ 44.63 29.1 ~ 38.73

4-S 58 4.8 5-3 1~6

7 days P E 1

--3,5 8.2 17.8 34.73

5,3 4-8 4.0 5.3

31 da~s P E ~

--13.!) -0.9 9,5 --5.7

{[-2 116 5.9 54

PE = post exposure. 2 SE = s t a n d a r d error. 3 Significantly different from initial rates at the p = 0-05 level.

groups were similar to the initial rates. Only the group with the highest exposure, the equivalent of 2.35 min at 1-0 mg/litre TRO, demonstrated a significant decrease below the pre-exposure rate. On~ month after exposure, repiration rates had returned to pre-exposure levels. At 1-day post exposure, oxygen consumption rates of control fish were significantly elevated above initial rates (paired t test, p = 0.05). Initial routine oxygen consumption rates were 260-4 +_ 42.3 mg O2/kg h (~" + SD) but increased to 305.4 +_ 28.2 mg O2/kgh following control treatment in the behaviour chamber. The mean increase of 19 ° o exhibited by control fish is in contrast to the depressed oxygen consumption rates measured in test fish. At 7 and 31 days post exposure, the respiratory rates of the control fish were similar to the pre-exposure rate. One-day post exposure, respiratory rates of the control groups were significantly different (analysis ofvariance, Student Newman-Keuls test, p = 0.05) from those of the chlorine-treated groups. Six days later, the rates of the control group and the two lowest exposure groups (the equivalent of 0.38 and 1.20min at 1.0mg/litre TRO) were not significantly different. At 31 days post exposure, the oxygen consumption rates of the control and test groups were similar. The magnitude of the decrease in the oxygen consumption rate after exposure to chlorinated seawater was directly proportional to the degree of exposure at 1 day (r = 0.62, p < 0.01) and 7 days (r = 0-70, p < 0.01) after treatment, However; the effect of chlorination on the oxygen

Fish responses to chlorine

249

consumption rate was not correlated with the size (standard length) of the fish (r < 0.54, p > 0.10).

Competition with untreated conspecifics C.punctipinnis exposed to subthreshold levels of chlorinated seawater were forced to compete with untreated fish for a limited food supply. Both lethal and sublethal (growth) effects of this competition were monitored. Survival: At 7 days post exposure, only one control fish (n = 30) had died, whereas the treated group experienced four mortalities (n---30). This difference was not statistically significant (;~2 = 1.96, p > 0-10). Both groups had four mortalities at 14 days post exposure, equalling 87 ~o survival. Growth: Seven days after exposure to chlorinated seawater, the twentysix treated fish gained an average of 0.26 __+0.08 g (,~'+ SE), whilst the twenty-nine control fish had grown 0.14+0.09g. Only the weight increase of the chlorine-treated fish was statistically significant (t = 3; 16, p < 0.005). At 14 days post exposure, treated fish had gained 0.18 - 0.13 g from their initial weights (an average loss of 0.08 g from day 7). At this time, control fish gained 0.09 __+0.10 g. Neither of these values represented a significant weight gain (t < 0.96,p > 0.10). Although test fish exhibited a statistically significant weight gain 7 days post exposure, weight changes between the control and test groups were not significantly different on either day 7 (t=0.99, p>0.10) or day 14 (t=0.57, p>0.10) post exposure.

DISCUSSION Chromis punctipinnis avoid chlorinated effluents containing > 0.20 mg/ litre TRO in the laboratory (Hose & Stoffel, 1980) and in the environment (Hose et al., in press). Under the laboratory conditions used in this study (20°C), the total avoidance threshold was 0.15 to 0.16mg/litre TRO. Previous work has shown that the avoidance threshold was reduced with higher water temperatures and increased to 0.20 mg/litre TRO when food was present in the chlorinated effluent. Avoidance of chlorinated seawater by juvenile C.punctipinnis did not preven t oxidant-induced physiological effects. Even at relatively low exposures of 0.38 to 5.23 min at 1.0 mg/litre .TRO, the routine oxygen

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/ E Hose, W. Hunt. R J. sto[]ei

consumption rates of chlorine-treated fish determined at 1 day post exposure were 25 to 45 ° 0 lower than comparable pre-exposure rates. This decrease to a minimum value of 144.8 mg Oz/kg h is expected to be easily tolerated under non-stressful conditions as the minimum routine metabolic rate necessary to support motile fish in this size range is estimated to be 34mgOz/kgh (Brett & Groves, 1979). However, under stressful conditions such as elevated temperature (often associated with chlorinated effluents), low food availability or high predation pressure, such metabolic depression could become limiting for growth and/or survival. Respiratory rates were depressed by 33 to 83 °,/,, in juvenile killilish, Fundulus heteroclitus, after exposure to 2 to 8 mg/litre applied chlorine (roughly equivalent to 0.3-1-4 mg/litre TRO) for 30 min (Capuzzo et al., 1977). Thus, compared with the killifish, ('.punctipinnis appears to be very sensitive to the toxic effects of chlorinated seawater. Results from both studies emphasise the direct relationship between the loss of respiratory function and the degree of exposure to oxidants. Further evidence that the avoidance response does not protect marine fish from the toxic effects of chlorine-produced oxidants is found in recent experiments in our laboratory (Hose et al., in press). Gill, liver, kidney and haematologic damage was noted in juvenile C. punctipmnis exposed to a subthreshold TRO dose (the equivalent of 3.0min at 1.0mg/litre) consistent with cumulative exposures described in this study. Loss of ion regulation and haemolysis occurred in sub-adult C. punctipinnis exposed to 0" 10 mg/litre TRO for 30 min, a level below the laboratory and field avoidance threshold of approximately 0.20 mg/litre T R O Although depressed respiratory function was evident in chlorinetreated C.punctipinnis for up to 1 week, complete recovery was demonstrated by 1 to 4 weeks after exposure. Following a l-week recovery period, plasma osmolarity, Ca 2 ÷, and Mg :~ of C.punctipinnis exposed to 0-10mg/litre TRO for 30min were similar to pre-treatment values (Hose et al., in press). Although significantly less oxidant damage was noted in similarly treated juvenile fish 1 week after chlorine exposure, residual changes such as blunting of secondary gill lamellae and the presence of circulating immature red blood cells were evident. The results of this study suggest that such residual pathological changes do not significantly affect oxygen consumption rates. However, analogous metabolic recovery has not been documented in other species. Capuzzo et al. (1977) found that the oxygen consumption rates of chlorine-treated killifish remained depressed even after 48 h in chlorine-free seawater.

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Similarly, respiratory rates of larval lobsters were significantly reduced for 19 days after exposure to I mg/litre of free chlorine (Capuzzo, 1977). The observed decrease in respiratory function did not impair the longterm survival of chlorine-treated C.punctipinnis. No significant differences were found in mortality on either day 7 or day 14 post exposure. Actual cumulative exposures in this experiment were quite low compared with the 96-h LCso'S of other temperate Pacific fish, approximately 0-07mg/litre TRO (Thatcher, 1978). Thus, the avoidance response of juvenile C.punctipinnis to chlorinated seawater can protect against exposure to lethal oxidant doses. Observations during experimental chlorinations further support the protective r61e of the avoidance response. No mortalities occurred during or following experimental chlorinations of the effluent of a southern California power plant (Hose et al., in press). Fish avoided the heated (mean A T = 8 °C) chlorinated effluent at plume TRO concentrations from 0.2 to 0.5 mg/litre. Giattina et al. (1981) found that most freshwater fish species studied avoided chlorine residuals at 50 ~o of the median lethal concentration. The ability of pollutant-stressed fish to successfully compete with unstressed fish for a limited resource has been employed as an indicator of overall fitness (Warren, 1971). C.punctipinnis which avoided concentrations of chlorinated seawater ( > 0.16 mg/litre TRO) successfully competed with untreated conspecifics for a restricted food supply. Although no differences in growth were measured between fish exposed to subthreshold levels of chlorinated seawater and control fish, test fish had gained weight at 7 days post exposure. This finding may reflect the depressed oxygen consumption rate since decreased oxygen consumption rates in humans due to hypothyroidism result in weight gains (Guyton, 1971). It should be noted that the slight increase in the weight of test fish 14 days post exposure was not significant. At that time period, the routine oxygen consumption rate is expected to return to pre-exposure levels. The lack of significant weight gains by control and test fish at day 14 supports the assumption that the diet (7 ~ dry weight brine shrimp/dry weight fish) was, in fact, restricted. These results are in contrast to those obtained in experiments with marine invertebrates. Capuzzo (1977) found significant decreases in the dry weight of larval lobsters up to 19 days after exposure to 1 mg/litre free chlorine for 60 min. Reduced feeding and egg production were also noted in rotifers and copepods exposed to chlorinated seawater (Capuzzo, 1979). Differences in the modes of chlorine toxicity have been discussed

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by Goldman (1979). A direct relationship between mortality and chlorine concentration was demonstrated for invertebrates while juvenile fish responded after a certain toxicant threshold was reached (Block, 1977). The relationship between this toxicant threshold and the avoidance threshold is unknown In conclusion, the avoidance response of C. punctipinnis to chlorinated seawater did not prevent temporary depression of the routine oxygen consumption rate resulting from subthreshold exposure to chlorineproduced oxidants. But the avoidance response did promote long-term survival and growth comparable with those of untreated fish. The effects of subthreshold oxidant exposure on other long-term processes such as development and reproduction should be addressed in further studies. ACKNOWLEDGEMENTS This study was supported by Southern California Edison under contract No. C06050901. We thank L. Purcell and T. King for their technical assistance and S. Warschaw and S. Gutierrez for their secretarial help in preparing this manuscript. Critical review by J. S. Stephens, Jr., M. Love and P.H. Dorn is appreciated. REFERENCES Block, R. M. (1977). Physiological responses of estuarine organisms to chlorine. Chesapeake Sci. 18, 158-60. Block, R. M., Burton, D. T., Gullans, S. R. & Richardson. L. B. (1978). Respiratory and osmoregulatory responses of white perch (Morone americana) exposed to chlorine and ozone in estuarine waters. In: Water chlorination." Em'ironmental impact and health effects, vol. 2. (Jolley, R. L., Gorchev, H. & Hamilton, D. C. (Eds)) Ann Arbor, Ann Arbor Science Pub. Inc. 351-60. Block, R. M., Helz, G. R. & Davis, W. P. (1977). The fate and effects of chlorine in coastal waters: Summary and recommendations. Chesapeake Sci., 18, 97-101. Brett, J. R. & Groves, T. D. (1979). Physiological energetics. In: Fish physiology. vol. 8. (Hoar, W.S., Randall, D.J. & Brett, J.R. (Eds)) New York, Academic Press. Buckley, J. A., Whitmore, C. M. & Matsuda, R. I. (1976). Changes in blood chemistry cell morphology in coho salmon (Oncorhynchus kisutch) following exposure to sublethal levels of total residual chlorine in municipal wastewater. J. Fish. Res. Board Can. 33, 776-82.

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Capuzzo, J. M. (1977). The effects of free chlorine and chloramine on growth and respiration rates of larval lobsters ( Homarus americanus). Water Res. 11, 1021--4. Capuzzo, J. M. (1979). The effect of temperature on the toxicity of chlorinated cooling waters to marine animals--A preliminary review. Mar. Poll. Bull. 10, 45-7. Capuzzo, J. M., Davidson, J. A., Lawrence, S. A. & Libni, M. (1977). The differential effects of free and combined chlorine on juvenile marine fish. Estuar. Coastal Mar. Sci., 5, 733-41. Dinnel, P. A., Stober, Q. J. & DiJulio, D. H. (1979). Behavioral responses of shiner perch to chlorinated primary sewage effluent. Bull. Environm. Contam. Toxicol., 22, 708-14. Gardiner, D. M. & Shrode, J. B. (1978). Laboratory analysis of food utilization and growth. In Effects of thermal effluent from Southern California Edison's Redondo Beach Steam Generating Plant on the warm temperate fish fauna of King Harbor Marina. Field and Laboratory Study Reports for Phase IlL Annual Report submitted to Southern California Edison, Research and Development Series 78-RD-47, 79-97. Giattina, J. D., Cherry, D. S., Cairns, J., Jr. & Larrick, S. R. (1981). Comparison of laboratory and field avoidance behavior of fish in heated chlorinated water. Trans. Am. Fish. Soc. 11,526--35. Goldman, J. C. (1979). Chlorine in the marine environment. Oceanus. 22(2), 37-43. Guyton, A. C. (1971). Textbook of medical physiology. Philadelphia, W. B. Saunders Co. 1032 pp. Hose, J. E. & Stoffel, R. J. (1980). Avoidance response of juvenile Chromis punctipinnis to chlorinated seawater. Bull. Environm. Contain. Toxicol. 25, 929-35. Hose, J. E., King, T. D., Zerba, K. E., Stoffel, R. J., Stephens, J. S., Jr. and Dickinson, J. A. (in press). Does avoidance of chlorinated seawater protect fish against toxicity? Laboratory and field observations. In: Water chlorination: Environmental impact and health effects, vol. 4 (Jolley, R. L. (Ed)), Ann Arbor, Ann Arbor Science Pub. Inc. Kim, J. & Kohout, F. J. (1975). Multiple regression analysis: Subprogram regression. In: SPSS: Statistical package for the social sciences. (2nd edn) (Nie, N.H., Hull, C. H., Jenkins, J. G., Steinbrenner, K. & Bent, D.H. (Eds)) Chapter 20. New York, McGraw-Hill Book Co., 320-67. Larson, G. L. & Schlesinger, D. A. (1978). Toward an understanding of the toxicity of intermittent exposures of total residual chlorine to freshwater fishes. In: Water chlorination: Environmental impact and health effects. Vol. 2. (Jolley, R. L., Gorchev, H. & Hamilton, D. H. (Eds)). Ann Arbor, Ann Arbor Science Pub. Inc., 111-22. Mattice, J. S. & Zittel, H. E. (1976). Site-specific evaluation of power plant chlorination. J. Water Poll. Control Fed. 48, 2284-308. Middaugh, D. P., Crane, A. M. & Couch, J. A. (1977). Toxicity of chlorine to juvenile spot, Leiostomus xanthurus. Water Res. 11, 1089-96.

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Orion (1977). Instruction manual--Residual chlorine electrode, mode! 97-70. Cambridge, Orion Research Inc. 19 pp. Schumacher, P. D. & Ney, J. J. (1980). Avoidance response of rainbo~ trout (Salmo gairdneri) to single-dose chlorination in a power phmt discharge canal. Water Res., 14, 651-5, Shrode, J. B. & Gerking, S. D. (1977). Effects of constant and fluctuating temperature on reproductive performance of a desert pupfish. Cyprinodon 11. neradensis, Physiol. Zool. 50, l-d0. Shrode, J. B., Zerba, K. E. & Stephens, Jr., J, S. {1982). Ecological significance of temperature tolerance and preference ot" some inshore California fishes. Trans. Am. Fish, Soc. !11, 45-51. Thatcher, T. O. (19781. The relative sensitivity of Pacific Northwest fishes and invertebrates to chlorinated sea water. In: Water chlorination: Enrironmental impact and health eJ[/eets. Vol. 2. (Jolley, R. L., Gorche,,', R. & Hamilton. D.C.), Ann Arbor, Ann Arbor Science Pub. Inc. 341 50. Warren, C. E. (19711. Biology and water pollution control Philadelphia, Pa., W.B. Saunders Co, 434 pp. Wong, G, T. F. (1980). Some problems in the determination of total residual "chlorine" in chlorinated sea-water, l,Vater Res, 14, 51-60