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Sublethal effects of treated liquid effluent from a petroleum refinery. V. Reproduction of Daphnia pulex and overall evaluation
Aquatic Toxicology, 4 (1983) 327-339
327
Elsevier
SUBLETHAL EFFECTS OF TREATED LIQUID EFFLUENT FROM A PETROLEUM REFINERY. V. REPRODUCTION OF DAPHNIA PULEX AND OVERALL EVALUATION
G.F. W E S T L A K E l, J.B. S P R A G U E 2'* and D.W. R O W E 3
i Environmental Protection Service, Environment Canada, 45 A lderney Drive, Dartmouth, Nova Scotia, Canada B2 Y 2N6, 2 Department o f Zoology, College o f Biological Science, University o f Guelph, Guelph, Ontario, Canada, N1G 2W1 and
J Chief Biologist, Erie County Public Health Division, Buffalo, NY, U.S.A. (Received 22 September 1980; accepted after revision 16 August 1983)
The 48-h LC30 o f treated refinery effluent for 2-day-old Daphnia pulex was 76070 effluent. The 14-day LCso was 6.407o effluent and this was a threshold value for mortality. For reproductive failure, the 14-day ECho was 3.1070 effluent, and the EC5 of 0.5207o effluent was considered to approximate the threshold of sublethal effect. Daphnia reproduction was the most sensitive response in a series o f studies that included fish growth, reproduction, locomotion, and respiration. Results are considered representative for a well-treated effluent from a petroleum refinery. The 48-h lethal test with D. pulex would be a useful tool for monitoring or assessing such effluents, since it is simple, small-scale, quick, and about 2.6 times as sensitive as a lethal test with trout. Key words: oil refinery; effluent; bioassay; chronic toxicity; reproduction; monitoring test; Daphnia
pulex
INTRODUCTION
This is the fifth and final paper reporting experiments on sublethal toxicity of a refinery effluent. Despite the worldwide occurrence of oil refineries, and much concern about regulating their discharges into surface waters, there had been almost no research on sublethal effects of a real refinery effluent on aquatic organisms. Without this information it is uncertain whether a given set of regulatory standards will actually achieve 'safe' concentrations in the environment. The overall objective of our work was, accordingly, to estimate 'no-effect' concentrations for various * To w h o m correspondence should be addressed. 0166-445X/83/$03.00 © Elsevier Science Publishers B.V.
328
sublethal responses of aquatic organisms. The preceding projects used fish as test animals (Rowe et al., 1983a, b; Westlake et al., 1983a, b), but invertebrates and particularly crustaceans are more sensitive than fish to a number of pollutants ( N A S / N A E , 1974). Accordingly, a species of water-flea (Daphnia pulex de Geer) was selected to complete the spectrum of testing. The specific objective was to measure for a sensitive invertebrate, acute lethality and chronic effects on reproduction, caused by an oil refinery effluent which met Canadian regulations. A secondary objective was to assess testing with Daphnia as a rapid method of screening toxicity of effluents. MATERIALS AND METHODS
General procedures including effluent characteristics have been described (Rowe et al., 1983a). In the present tests, some effluent samples showed elevated concentrations of certain components, compared to samples used in the other projects. Ammonia averaged 20 mg/1 compared to a regulatory limit of 12.5 (Canada, 1974), and oil-plus-grease had a high average o f 69 mg/l, compared to a regulatory limit of 10 and an average of 20 in all sublethal experiments. Other chemical characteristics were within government limits or well below them (see Table I, Rowe et al., 1983a). Among the nine samples used in Daphnia tests, the third one was very high in ammonia (85 mg/l as N) and cyanide (770/~g/l). In pre-experimental screening for toxicity to rainbow trout, this same third sample caused complete mortality of trout during a full-strength exposure of 24 h, but no mortality at 50°70 concentration. This sample was diluted by one-half before being used sublethally with Daphnia, since the objective was to test an effluent meeting regulations for non-lethality to fish. Combined results for all 9 lethal screening tests showed 19070 overall mortality of trout during a 3-day exposure to full-strength effluent. Seven of the 9 samples were considered to have passed a government test for non-lethality to trout. D. pulex were obtained from a clone maintained by A.L. Buikema, Jr. at Virginia Polytechnic Institute and State University, and maintained in aquaria. Gravid adults were transferred to mason jars to provide young Daphnia that had been released for 2 days or less. These were randomly introduced into test solutions by wide-mouthed pipette, to start a given exposure. No ephippial eggs were seen, indicating satisfactory conditions. A slurry of trout chow and a culture of the alga Ankistrodesmus falcatus were used as food. Both were added to stock aquaria, which also had a growth of resident algae. The same apparatus and procedure were used for acute and chronic exposures. A series of identical chambers, each a Rubbermaid plastic 'drawer organizer' 22 x 7 x 5 cm deep, contained 300 ml o f test water. Within each chamber, six cells were suspended in line, each o f them 2.5 x 5 × 3.8 cm, containing 20 ml of the abovementioned test water (Fig. 1). Each cell was a section of a 'natural' polyethylene icecube tray, with fine nylon net replacing the bottom. One Daphnia was placed in each
329
\ Fig. 1. Drawing of three chambers, with six cells in each, as used in experiments with Daphnia.
cell, thus there were six individuals at one concentration in any given chamber. A glass tube released a slow succession o f air bubbles halfway along each chamber. The top was covered with a plastic sheet to decrease evaporation. Effluent was 1.8-5.8 days old during the experiment and test water was renewed twice-daily at 1000 and 2100 h. Six hundred ml of the desired concentration of effluent was made up in a beaker; to this was added 1 ml of the slurry of trout chow and 1 ml o f the algal culture. Aliquots of 200 ml were added to the chamber through a funnel (Fig. 1). A constricted outlet caused the water level to rise, then drain to normal level. This was done three times, and trials with a salt solution showed that 84°7o of test water in the cells was replaced. Photoperiod was a 16-h day with dimming, and illumination at the chambers was about 1200 lux. Average of 439 temperatures in acute and chronic tests was 24.0°C (so = 1.40). Averages in individual tests varied from 23.1 ° to 25.5°C, but temperatures were very similar at different concentrations within each acute test. In chronic tests, 10°70 effluent averaged 0.8°C higher than the average, and 5°7o effluent was 1. I ° C lower, apparently because of the randomized position along the laboratory bench. Average p H was 7.04 (SD = 0.15), and averages for various concentrations did not vary more than 0.03 units f r o m that value. Dissolved oxygen was 76°7o saturation or greater. Nine acute tests of lethality used 3-day exposures, and two chronic tests had durations of 14 days. Daily inspection of the Daphnia was done with a dissecting
330
microscope. Floating animals were classified as alive, since they often recovered and swam normally, and some released young after floating for several days. Missing individuals were classified as dead if not found subsequently. In chronic tests, Daphnia were also characterized as to presence of eggs, carrying of young, and release of young. Young were removed from the cell. L a b o r a t o r y water was essentially that of Lake Ontario with total hardness of 130 m g / l (further description in Rowe et al., 1983a). As it came f r o m the tap, it proved unsatisfactory for rearing Daphnia, probably because traces of chlorine or its compounds passed the carbon filter. Water entering the filter contained 0.4 mg/1 chlorine (amperometric) and water leaving had 0.00 to 0.03 mg/1, whereas 0.014 m g / l has been shown to cause mortality to a species of Daphnia (Arthur, 1971-1972, in Brungs, 1973). This problem was remedied for the final four acute tests and the second chronic test by holding dilution water for several days in an aerated well-lit tank after seeding with algal culture. Detailed examination of Daphnia responses (Sprague et al., 1978) showed that the toxic element in raw water did not have a detectable influence on results because any concentration of effluent down to the lowest used (1.25°70) was apparently sufficient to detoxify the chlorine. Because of this, no adjustment was made in the 3-day lethal tests for control mortality (22070 overall). Response of control organisms in the second chronic exposure was used to correct experimental response in both chronic tests, using A b b o t t ' s formula (Tattersfield and Morris, 1924). Computerized probit analysis was used to determine median lethal concentration (LCso), median effective concentration for reproductive failure (ECso), and the associated probit lines. RESULTS W I T H DISCUSSION
A general picture of acute lethality to Daphnia is given by the combined results for all nine batches of raw effluent (Fig. 2). The 48-h LCso was 75.6070 effluent (95o7o confidence limits (CL) -- 53.6-107o70, slope of probit line (S) = 1.76). Thus Daphnia were more sensitive than trout which suffered a combined mortality of only 19%, for the same nine batches at full strength. An exposure of 48 h seemed suitable for routine testing with Daphnia. The 72-h LCso was somewhat lower as expected, at 46.5°70 effluent (CL = 3 4 . 9 - 6 1 . 9 % , S = 1.79). Slopes of the probit lines were almost identical for 48- and 72-h exposures, as indicated by Fig. 2 and the values given above. Exposure for 24 h gave less satisfactory data. Most bioassays did not achieve 50°7o mortality in that time, and in fact the 24-h LCs0 could not be estimated. The slope of the probit line was appreciably lower indicating more variability within tests. The variability in the 24-h test could reflect increased sensitivity at time of molting; some individuals would have molted during 24 h while others would not have done so. The 48-h exposure would have allowed most individuals to molt during a test (Lee and Buikema, 1979). Results can be grouped by toxicity of the batches as shown by mortality of trout
331 98
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CONCENTRATION OF EFFLUENT, PER CENT
Fig. 2. Acute mortality of D. pulex in treated oil refinery effluent. Combined data from nine bioassays with different batches of effluent. Results are shown for three exposure times on the same groups of animals, 54 at each concentration. The 950/0 confidence limits are shown for two LCso values.
TABLE I Median lethal concentrations for D. pulex exposed to nine batches of treated oil refinery effluent. Results grouped according to the toxicity of the batches to trout in full-strength effluent. N u m b e r of batches
2 4 2 1
Mortality of trout in 72 h
LCso values for Daphnia (07o effluent)
(070)
24 h
48 h
72 h
0 10 20-30 100 (LCs0 = 71)
> 100 > 100 > 100 45.7
> 100 92.3 58.6 27.8
82.9 49.2 47.4 12.5
(Table I). Daphnia LCso values were obtained at 48 h for batches that killed only 1 0 - 3 0 % of trout. For the most toxic batch, the 72-h LCso for trout was 2.6 times higher than the 48-h LCso for Daphnia. (For trout, the 72-h LCso would be almost the same as the more standard 96-h value, judging by comparisons of 30 lethal tests with this effluent (Sprague et al., 1978).) Results given above for lethality to Daphnia are based on the raw effluent, useful for comparison with the trout tests. For comparison with the chronic test, lethality data for Daphnia were adjusted for the dilution of one batch to half-strength. Doing this, the combined 48-h LCso for Daphnia, of the 7 batches used in the chronic test, would be 76.3% (CL = 51.7-113°/0, S = 1.81). This is almost identical to the LCso for combined data on all nine batches of raw effluent.
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Fig. 3. Lethality and reproductive impairment in D. pulex exposed for 14 days to treated oil refinery effluent. Results are corrected for control performance. Based on 12 individuals per concentration.
For the two chronic exposures, combined results showed a 14-day LCso of 6.4°7o effluent (CL = 3.6-11.6%, S = 1.89) (Fig. 3). This was the corrected value for 33.3°7o control mortality in the second exposure. The 10-day LCso was the same, and a toxicity curve indicated that 6.4°7o was a threshold for lethality. Ten days is a rather long time for reaching a lethal threshold, since it is about one-quarter of the animal's lifetime. The 14-day ECso for reproductive failure was 3.1°70 (CL = 1.7-5.6%, S = 2.13) (Fig. 3). This ECso incorporates correction for 33.3o7o reproductive failure in the control of the second exposure. The 14-day ECso signifies the effective concentration that prevented half o f the Daphnia f r o m producing freeswimming young. This was a reasonable evaluation of interference with reproduction, since only one Daphnia out of 60 in the chronic experiments formed eggs without releasing young. Temperature differences within the chronic tests may have slightly increased the variability of results. Buikema et al. (1976b) have shown that in the 20-25°C range, each increase of 1°C increased acute lethality of a simulated refinery effluent to D. pulex by about 17.4°70. Assuming this relationship held in our 14-day lethal tests, applying corrections for higher temperature in 10o70 effluent and lower ones in 5°70 would merely draw the two points closer to the probit line in Fig. 3, without appreciably changing its position or slope.
"Safe" concentration Estimation of a harmless concentration requires some arbitrary judgement. It might be considered that 5°70 impairment of reproduction would be a negligible effect and therefore relatively 'safe'. The value for reproductive failure a m o n g 5o70 of
333 the animals, the ECs, m a y be read f r o m Fig. 3 as 0.52°7o effluent. This is a low concentration, however it might be considered a realistic one to protect sensitive invertebrates in an ecosystem. The 'safe'-to-lethal ratio could be calculated as: EC5 for reproductive failure 48-h LCso
-
0.52 - 0.0068. 76.3
The value 0.0068 could be used as an application factor, to predict 'safe' levels o f a refinery effluent for Daphnia, having determined the 48-h LCso. This application factor is somewhat below the usual range of 0.1 to 0.01 (Sprague, 1971). Daphnia are probably a m o n g the most sensitive of aquatic animals to refinery effluent. Buikema et al. (1976a) found D. pulex and D. magna were one or two orders of magnitude more sensitive than 13 other invertebrates and 3 fish, tested against a simulated refinery effluent. Their final tests showed that D. pulex was 52 times more sensitive than trout (Buikema et al., 1976b), much greater than the factor of 2.6 which we found. Buikema and co-workers also obtained 48-h LCso values that were much different f r o m our value of 76°7o; they report 7o7o and 3.3°7o in the two reports mentioned above, and 3o70 in another publication (Geiger et al., 1980). The differences are surprising since their simulated effluent bad chemical characteristics very similar to the limits in Canadian effluent regulations. The explanation for both differences apparently involves species sensitivity to the type of oil used in the simulated effluent of Buikema and colleagues. This was Number 2 fuel oil, a relatively toxic petrochemical, and it was the m a j o r contributor of toxicity in their tests. When deleted f r o m the effluent, 48-h LCs0 values were 96 and 52o70, bracketing almost exactly our LCso of 76%. This agreement might also seem unusual since our effluents had high average values for oil-plus-grease and a m m o n i a (7 times and 1.6 times the Canadian limits). Apparently the toxicity of the whole effluent is predicted rather poorly f r o m a summation of the individual toxicities of components. This was strikingly shown by Buikema et al. (1976b) when effluents without a m m o n i a , or without phenol, had greater toxicity than the complete simulated effluent. This indicated antagonism between some components. GENERAL DISCUSSION OF FINDINGS WITH THIS EFFLUENT The first objective of this research was to establish the concentrations of a welltreated refinery waste that would be sublethally 'safe' for aquatic life. We studied a spectrum o f responses in five experiments (this paper and Rowe et al., 1983a, b; Westlake et al., 1983a, b) and the approximate thresholds of effect are summarized in Table II. The first two effects listed in Table II could clearly be meaningful for existence o f the fish in nature; the thresholds are of a similar magnitude, 5-9°70 effluent. Avoidance reactions were apparently not a problem with this effluent, and other locomotor and respiratory changes were statistically significant only at high concentrations.
334 TABLE II Estimated thresholds of sublethal effect on aquatic organisms exposed to treated effluent from a petroleum refinery. Experiment
Threshold (% effluent)
Growth of trout Life-cycle exposure of flagfish Avoidance by trout Locomotory changes in trout Gill irrigation rate, trout 'Coughing' by trout Chronic lethality to Daphnia 5070 inhibition of reproduction in Daphnia
About 5.6 (limits 3.1, 10) About 9 No effect to at least 30 About 30 (borderline statistical significance) Between 50 and 100 Between 25 and 50 6.4 0.52
Responses of D. pulex appear to be the most sensitive. Half the Daphnia showed chronic mortality at 6.4°70 effluent and failure to reproduce at 3.1 °70. The criterion of 5°70 inhibition of reproduction may be a justifiable choice as the most sensitive threshold documented in this series. Our work indicates that concentrations of this kind of well-treated refinery effluent should be 0.5°70 or less to protect freshwater communities. Lacking direct experiments on marine organisms, it might be predicted that a similar concentration would be protective of marine communities (USEPA, 1978; Klapow and Lewis, 1979). Achieving dilution to 0.5°70 would depend very much on the specific characteristics at a refinery site. Many such installations are, indeed, on the coast or other large bodies of water with high dilution. However, studies of other kinds o f effluents indicate that without facilities for diffusing the discharge, concentrations may exceed 0.5°70 in a narrow plume extending several kilometres, for example 1.7 km in Lake Superior (Minns, 1977) or 5 km in Canadian Atlantic estuaries (Whitney and Wilson, 1968). Plumes of high concentration can be greatly reduced in extent by designing outfall lines for rapid initial dilution and dispersal (Metcalf and Eddy, 1972). Such outfall design would be desirable for refineries in addition to avoidance of locations with restricted circulation or flow. Given those points, we conclude that treatment of refinery effluent to satisfy requirements such as the present Canadian ones for chemical limits and non-lethality to fish (Canada, 1974) would generally be protective of aquatic ecosystems.
Comparison with other work There is little published work on sublethal effects of refinery effluents, even if research with simulated effluents is included. Indeed this virtual absence of information was the stimulus for our research. One fragmentary report states that photosynthesis of an alga was affected by 2°70 effluent and probably also by 1°70, but not by 0.5°70 effluent (North et al., 1972). This report, for another type of organism, shows
335 a comparable but slightly less sensitive response than we concluded for Daphnia. By contrast, periphyton photosynthesis was apparently stimulated in artificial streams, even when full-strength simulated refinery effluent was present (Honig and Buikema, 1980). A shift f r o m normal species of algae was noted, however, when a 25070 concentration was present in the streams. There has been a fair amount of work on acute lethality o f whole effluents, which is of less direct interest in relation to the present study. Most of it confirms our finding that adequate treatment of a refinery effluent renders it non-lethal to fish. For example, Pessah et al. (1973) found that two Canadian refineries with good treatment produced effluents that were essentially non-lethal to rainbow trout. Two others with inadequate treatment, and one with treatment but a stripping problem, were lethal, with LCso values of 56°70, about 88070, and 21°70. They also found that a simulated waste with the maximal concentrations of parameters according to the Canadian regulations (Canada, 1974) did not cause lethality. Doubling these concentrations caused only about 20070 mortality. Similarly, G r a h a m and Dorris (1968) found no mortality of fish for two refineries with biological treatment, and LCso values of a b o u t 2007o effluent for two refineries without such treatment. G r a h a m and Dorris also did longer lethal tests of 32 days. For the more toxic effluents, concentrations which did not kill in 4 days did not prove lethal in 32 days either. Oddly enough, two effluents of low toxicity which caused no lethality in fullstrength exposure for 4 days required four-fold dilution to allow survival for 32 days. These experiments do not deal with sublethal effects by any means, but come closer to the topic than any other experiments with fish. Brown trout (Salmo trutta) and rainbow trout were the fourth and fifth most sensitive of 57 species of fish tested for 96-h LCso values in whole refinery effluent (Irwin, 1965). Gizzard shad (Dorosoma cepedianum) were most sensitive and guppies (Lebistes reticulatus) least sensitive, with a five-fold difference in tolerance. The sensitivity of rainbow trout would suggest that they were a good choice for our sublethal experiments.
Screening tests A secondary objective of our work was to look for a convenient method of biologically assessing the toxicity of refinery effluents. From our spectrum o f tests, we agree with Buikema et al. (1976a) that a 48-h lethal test with D. pulex has m a n y advantages, and we would list the following ones. (a) For monitoring, it is small-scale, simple, and inexpensive. (b) It requires only small volumes o f effluent, a useful feature for shipping from remote areas and for evaluating pilot-plant effluents. (c) It is twice as fast as a standard lethal test with fish. (d) It is approximately 2.6 times as sensitive as lethality to trout. This could mean
336 a greater yield o f information in cases when an effluent was non-lethal to the fish it could be just below the lethal level or far below it. (e) It predicts the 'safe' concentration of a refinery effluent for Daphnia, which appear to be a m o n g the most sensitive of aquatic organisms to this waste. The 'safe' level could be estimated by using the application factor of 0.0068 on the LC50, as determined by our work. (f) Using a clone of Daphnia should improve the reproducibility of tests at different times and places. Daphnia could be used in a simple pass/fail test. For example the stipulation that effluent at 40% concentration should kill not more than half a group of D. pulex in 48 h could provide an alternative to the present Canadian regulatory test with fish (Canada, 1974). A 7-day test with D. pulex may give a good assessment of chronic effects. Geiger et al. (1980) show that the body-length of pre-adult Daphnia, after seven days of exposure to simulated refinery effluent, is a good predictor of later success in reproduction. Short tests such as this or the 48-h LCs0 would be advantageous. There is a considerable art in carrying out a full reproductive test; Nebeker (1982) reports that three out of six laboratories did not successfully complete 21-day chronic experiments with D. magna. An even more rapid technique based on enzyme inhibition is currently being evaluated by Rutherford et al. (1979). For simulated refinery effluent, they found the test sensitive and in reasonable agreement with Daphnia and fish tests. The cough response of a suitable fish could be useful for on-line monitoring of a given effluent stream. As described in Westlake et al. (1983b), such a system could be computerized to trigger an alarm at times o f increased stress on the fish. This would require, however, fairly sophisticated equipment, programming, and personnel. -
Characteristics o f this effluent The effluent used in this research proved fairly consistent. During two years, 92 batches were tested for lethality to rainbow trout, with an overall mortality rate of 23%0 in full-strength effluent. O f the 92 batches, 71 were considered to have passed the Canadian test for non-lethality to trout. Most of the failures came during an unusual period of changeover in refinery processes when treatment was disrupted, and at a time when we were conducting preliminary Or trial runs. For the 68 batches actually used in sublethal experiments, full-strength effluent caused an overall mortality of 14% of the fish, and 60 o f the 68 batches were considered to have passed the regulatory test. Chemical characteristics of the effluent have been summarized (Rowe et al., 1983a). In general, the 68 batches used sublethally had phenol and sulphide levels which were one or two orders of magnitude lower than the Canadian limits of 1.0
337
and 0.3 mg/l (Canada, 1974), and the similar limits proposed in 1973 for U.S.A. (USEPA, 1973). The 68 batches averaged 17 mg/l of ammonia as N, somewhat higher than the Canadian limit of 12.5, and were about twice the limits of 10 mg/1 and 25 mg/l for oil-plus-grease and residue. From the above, our experiments are based on an effluent that came close to meeting accepted standards. However, it was not an unduly innocuous one, and our results would seem generally applicable for a well-treated petroleum refinery effluent. We found little or no indication that the degree of sublethal toxicity could be predicted from physico-chemical characteristics of the effluent. There were no obvious correlations of chemical 'strength' with biological effect, within experiments on trout growth, respiration, locomotion, or for Daphnia reproduction. We did find increased mortality in the screening tests with trout, for certain batches o f effluent which were high in one or more of phenol, ammonia, cyanide, or oil-plus-grease. However, since we did not control the effluent characteristics it could not be ascertained whether mortality was due to these components or other unmeasured toxicants which increased at the same times. C6t6 (1976) has summarized considerable work which tested individual components of refinery effluent for their toxicity. In general, this other work also showed rather poor prediction of the total toxicity of a simulated effluent, on the basis o f its components. A review by Baker (1979) concludes that it 'appears to be impossible to generalise about why refinery effluents are toxic'. In any case the topic of component toxicity is of less interest for the purpose of our work - to study a whole effluent. There is relatively little sublethal research on such effluents, compared to that on pure compounds. Especially in view of the difficulties of predicting toxic effects of mixtures, it is important to have available some findings on the real effluents which aquatic organisms face in the environment. Although it might appear that work on a whole effluent, with its day-today variability, would lead to an imprecise piece of research, this has not been our experience. Effects during the various time periods of the experiment seemed relatively similar and consistent. For example, two growth experiments on trout, in separate years, led to very similar conclusions on the threshold of damage. ACKNOWLEDGEMENTS
This project was funded and supported by the Petroleum Association for Conservation of the Canadian Environment (PACE) and by the Petroleum and Industrial Organic Chemicals Division, Water Pollution Control Directorate, Environmental Protection Service, of the Canadian Department of Environment. It was carried out in laboratories supplied at the Canada Centre for Inland Waters, Burlington, Ontario. We thank William Yasui for his technical work on the Daphnia experiment. From the petroleum industry we thank Evan C. Birchard, Peter Budzick, and Waiter Krawciw of PACE, and Norman A. Barron of BP Trafalgar refinery. Numerous people from Environment Canada gave advice, facilities, and provided
338 a massive program of chemical analyses. We thank Douglas Anderson, James P. B r u c e , V i c t o r C a i r n s , K. C o n n , O . E l Kei, R o n a l d G . G i l l e s p i e , P e t e r V. H o d s o n , Michael
LeBlanc,
A.R.
Lefeuvre,
D. Marsh,
Serge Metikosh,
R o s s E. Mills,
William A. Neff, Edward Pessah, F.J. Philbert, J.D. Salloum, N.W. Schmidtke, A.D. Stevenson, D.P. Sturtevant, Michael Thorpe, and W.J. Traversy. REFERENCES Arthur, J.W., 1971-1972. Progress report. U.S.E.P.A., National Water Quality Laboratory, Duluth, Minnesota (1971-1972) (cited through Brungs 1973). Baker, J.M., 1979. Ecological impact of refinery effluents in the marine and estuarine environment. Part III. In: The environmental impact of refinery effluents. Rept. No. 5/79, CONCAWE, The Hague, pp. 1-77.
Brungs, W.A., 1973. Effects of residual chlorine on aquatic life. J. Water Pollut. Control Fed. 45, 2180-2193. Buikema, Jr., A.L., D.R. Lee and J. Cairns, Jr., 1976a. A screening bioassay using Daphniapulex for refinery wastes discharged into freshwater. American Soc. Testing and Materials, J. Test. Mat. 4, 119-125. Buikema, Jr., A.L., D.R. Lee and J. Cairns, Jr., 1976b. Development of a simple freshwater invertebrate bioassay method for evaluating petroleum refinery effluents: a status report on Phase II. Unpubl. Rept., Department of Biology and Center for Environmental Studies, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 26 pp. Canada, 1974. Petroleum refinery effluent regulations and guidelines. Canada Department of Environment, Water Pollution Control Directorate, Rept. No. EPS 1-WP-74-1, 59 pp. Geiger, J.G., A.L. Buikema, Jr. and J. Cairns, Jr., 1980. A tentative seven-day test for predicting effects of stress on populations of Daphniapulex. In: Aquatic toxicology, edited by J.G. Eaton, P.R. Parrish and A.C. Hendricks, American Society for'Testing and Materials, Spec. Techn. Publ. 707, pp. 13-26. Graham, R.J. and T.C. Dorris, 1968. Long-term toxicity bioassay of oil refinery effluents. Water Res. 2, 643-663. Honig, R.A. and A.L. Buikema, Jr., 1980. Evaluating promulgated refinery effluent standards using artificial streams. Arch. Environ. Contam. Toxicol. 9, 607-618. Irwin, W.H., 1965. Fifty-seven species of fish in oil-refinery waste bioassay. Trans. Thirtieth North Am. Wildl. Nat. Resour. Conf. 30, 89-99. Klapow, L.A. and R.H. Lewis, 1979. Analysis of toxicity data for California marine water quality standards. J. Water Pollut. Control Fed. 51, 2054-2070. Lee, D.P. and A.L. Buikema, Jr., 1979. Molt-related sensitivity of Daphniapulex in toxicity testing. J. Fish. Res. Board Can. 36, 1129-1133. Metcalf and Eddy, Inc., 1972. Wastewater engineering. Collection treatment disposal. McGraw-Hill Book Co., New York, 781 pp. Minns, C.K., 1977. Analysis of a pulp and paper mill effluent plume. J. Fish. Res. Board Can. 34, 776-783. NAS/NAE, 1974. Water quality criteria 1972 (U.S. National Academy of Sciences/National Academy of Engineering). U.S. Environmental Protection Agency, Ecological Res. Series EPA-R3-73-033,594 PP. Nebeker, A.V., 1982. Evaluation of Daphnia magna renewal life-cycle test method with silver and Endosulfan. Water Res. 16, 739-744. North, W.J., G.C. Stephens and N.B. North, 1972. Marine algae and their relation to pollution problems. In: Marine pollution and sea life, edited by M. Ruivo, F.A.O. of U.N., Fishing News (Books) Ltd., London, pp. 330-340.
339 Pessah, E., J.S. Loch and J.C. MacLeod, 1973. Preliminary report on the acute toxicity of Canadian petroleum refinery effluents to fish. Fish. Res. Board Can. Tech. Rept. No. 408, 43 pp. Rowe, D.W., J.B. Sprague and T.A. Heming, 1983a. Sublethal effects of treated liquid effluent from a petroleum refinery. I. Chronic toxicity to flagfish. Aquat. Toxicol. 3, 149-159. Rowe, D.W., J.B. Sprague, T.A. Heming and I.T. Brown, 1983b. Sublethal effects of treated liquid effluent from a petroleum refinery. II. Growth of rainbow trout. Aquat. Toxicol. 3, 161-169. Rutherford, C.L., A.L. Buikema, Jr., D.R. Armant and J. Cairns, Jr., 1979. A simple method for screening petroleum effluents by in vitro enzyme inhibition. Bull. Environ. Contam. Toxicol. 21, 79-84. Sprague, J.B., 1971. Measurement of pollutant toxicity to fish. III. Sublethal effects and 'safe' concentrations. Water Res. 5, 254-266. Sprague, J.B., D.W. Rowe, G.F. Westlake, T.A. Heming and I.T. Brown, 1978. Sublethal effects of treated liquid effluent from a petroleum refinery on freshwater organisms. Canada Dept. of Fisheries and Environment, Environmental Protection Service, and Petroleum Association for Conservation of the Canadian Environment, Ottawa, 297 pp. Tattersfield, F. and H.M. Morris, 1924. An apparatus for testing the toxic values of contact insecticides under controlled conditions. Bull. Entomol. Res. 14, 223-233. USEPA, 1973. Petroleum refining point source category. Proposed effluent limitation guidelines and new source standards (U.S. Environmental Protection Agency). Fed. Register 30, 34542-34558. USEPA, 1978. Water quality criteria. U.S. Federal Register, Vol. 43, No. 97 (U.S. Environmental Protection Agency), pp. 21506-21518. Westlake, G.F., J.B. Sprague, R.J. Hines and I.T. Brown, 1983a. Sublethal effects of treated liquid effluent from a petroleum refinery. III. Avoidance and other locomotory responses of rainbow trout. Aquat. Toxicol. 4, 235-245. Westlake, G.F., J.B. Sprague and 1.T. Brown, 1983b. Sublethal effects of treated liquid effluent from a petroleum refinery. IV. Respiratory movements and coughing of rainbow trout. Aquat. Toxicol. 4, 317-325. Whitney, W.H. and G.G. Wilson, 1968. Effluent distribution studies on the Miramichi and Restigouche estuaries. Pulp Pap. Mag. Can. 69, 79-85.
327
Elsevier
SUBLETHAL EFFECTS OF TREATED LIQUID EFFLUENT FROM A PETROLEUM REFINERY. V. REPRODUCTION OF DAPHNIA PULEX AND OVERALL EVALUATION
G.F. W E S T L A K E l, J.B. S P R A G U E 2'* and D.W. R O W E 3
i Environmental Protection Service, Environment Canada, 45 A lderney Drive, Dartmouth, Nova Scotia, Canada B2 Y 2N6, 2 Department o f Zoology, College o f Biological Science, University o f Guelph, Guelph, Ontario, Canada, N1G 2W1 and
J Chief Biologist, Erie County Public Health Division, Buffalo, NY, U.S.A. (Received 22 September 1980; accepted after revision 16 August 1983)
The 48-h LC30 o f treated refinery effluent for 2-day-old Daphnia pulex was 76070 effluent. The 14-day LCso was 6.407o effluent and this was a threshold value for mortality. For reproductive failure, the 14-day ECho was 3.1070 effluent, and the EC5 of 0.5207o effluent was considered to approximate the threshold of sublethal effect. Daphnia reproduction was the most sensitive response in a series o f studies that included fish growth, reproduction, locomotion, and respiration. Results are considered representative for a well-treated effluent from a petroleum refinery. The 48-h lethal test with D. pulex would be a useful tool for monitoring or assessing such effluents, since it is simple, small-scale, quick, and about 2.6 times as sensitive as a lethal test with trout. Key words: oil refinery; effluent; bioassay; chronic toxicity; reproduction; monitoring test; Daphnia
pulex
INTRODUCTION
This is the fifth and final paper reporting experiments on sublethal toxicity of a refinery effluent. Despite the worldwide occurrence of oil refineries, and much concern about regulating their discharges into surface waters, there had been almost no research on sublethal effects of a real refinery effluent on aquatic organisms. Without this information it is uncertain whether a given set of regulatory standards will actually achieve 'safe' concentrations in the environment. The overall objective of our work was, accordingly, to estimate 'no-effect' concentrations for various * To w h o m correspondence should be addressed. 0166-445X/83/$03.00 © Elsevier Science Publishers B.V.
328
sublethal responses of aquatic organisms. The preceding projects used fish as test animals (Rowe et al., 1983a, b; Westlake et al., 1983a, b), but invertebrates and particularly crustaceans are more sensitive than fish to a number of pollutants ( N A S / N A E , 1974). Accordingly, a species of water-flea (Daphnia pulex de Geer) was selected to complete the spectrum of testing. The specific objective was to measure for a sensitive invertebrate, acute lethality and chronic effects on reproduction, caused by an oil refinery effluent which met Canadian regulations. A secondary objective was to assess testing with Daphnia as a rapid method of screening toxicity of effluents. MATERIALS AND METHODS
General procedures including effluent characteristics have been described (Rowe et al., 1983a). In the present tests, some effluent samples showed elevated concentrations of certain components, compared to samples used in the other projects. Ammonia averaged 20 mg/1 compared to a regulatory limit of 12.5 (Canada, 1974), and oil-plus-grease had a high average o f 69 mg/l, compared to a regulatory limit of 10 and an average of 20 in all sublethal experiments. Other chemical characteristics were within government limits or well below them (see Table I, Rowe et al., 1983a). Among the nine samples used in Daphnia tests, the third one was very high in ammonia (85 mg/l as N) and cyanide (770/~g/l). In pre-experimental screening for toxicity to rainbow trout, this same third sample caused complete mortality of trout during a full-strength exposure of 24 h, but no mortality at 50°70 concentration. This sample was diluted by one-half before being used sublethally with Daphnia, since the objective was to test an effluent meeting regulations for non-lethality to fish. Combined results for all 9 lethal screening tests showed 19070 overall mortality of trout during a 3-day exposure to full-strength effluent. Seven of the 9 samples were considered to have passed a government test for non-lethality to trout. D. pulex were obtained from a clone maintained by A.L. Buikema, Jr. at Virginia Polytechnic Institute and State University, and maintained in aquaria. Gravid adults were transferred to mason jars to provide young Daphnia that had been released for 2 days or less. These were randomly introduced into test solutions by wide-mouthed pipette, to start a given exposure. No ephippial eggs were seen, indicating satisfactory conditions. A slurry of trout chow and a culture of the alga Ankistrodesmus falcatus were used as food. Both were added to stock aquaria, which also had a growth of resident algae. The same apparatus and procedure were used for acute and chronic exposures. A series of identical chambers, each a Rubbermaid plastic 'drawer organizer' 22 x 7 x 5 cm deep, contained 300 ml o f test water. Within each chamber, six cells were suspended in line, each o f them 2.5 x 5 × 3.8 cm, containing 20 ml of the abovementioned test water (Fig. 1). Each cell was a section of a 'natural' polyethylene icecube tray, with fine nylon net replacing the bottom. One Daphnia was placed in each
329
\ Fig. 1. Drawing of three chambers, with six cells in each, as used in experiments with Daphnia.
cell, thus there were six individuals at one concentration in any given chamber. A glass tube released a slow succession o f air bubbles halfway along each chamber. The top was covered with a plastic sheet to decrease evaporation. Effluent was 1.8-5.8 days old during the experiment and test water was renewed twice-daily at 1000 and 2100 h. Six hundred ml of the desired concentration of effluent was made up in a beaker; to this was added 1 ml of the slurry of trout chow and 1 ml o f the algal culture. Aliquots of 200 ml were added to the chamber through a funnel (Fig. 1). A constricted outlet caused the water level to rise, then drain to normal level. This was done three times, and trials with a salt solution showed that 84°7o of test water in the cells was replaced. Photoperiod was a 16-h day with dimming, and illumination at the chambers was about 1200 lux. Average of 439 temperatures in acute and chronic tests was 24.0°C (so = 1.40). Averages in individual tests varied from 23.1 ° to 25.5°C, but temperatures were very similar at different concentrations within each acute test. In chronic tests, 10°70 effluent averaged 0.8°C higher than the average, and 5°7o effluent was 1. I ° C lower, apparently because of the randomized position along the laboratory bench. Average p H was 7.04 (SD = 0.15), and averages for various concentrations did not vary more than 0.03 units f r o m that value. Dissolved oxygen was 76°7o saturation or greater. Nine acute tests of lethality used 3-day exposures, and two chronic tests had durations of 14 days. Daily inspection of the Daphnia was done with a dissecting
330
microscope. Floating animals were classified as alive, since they often recovered and swam normally, and some released young after floating for several days. Missing individuals were classified as dead if not found subsequently. In chronic tests, Daphnia were also characterized as to presence of eggs, carrying of young, and release of young. Young were removed from the cell. L a b o r a t o r y water was essentially that of Lake Ontario with total hardness of 130 m g / l (further description in Rowe et al., 1983a). As it came f r o m the tap, it proved unsatisfactory for rearing Daphnia, probably because traces of chlorine or its compounds passed the carbon filter. Water entering the filter contained 0.4 mg/1 chlorine (amperometric) and water leaving had 0.00 to 0.03 mg/1, whereas 0.014 m g / l has been shown to cause mortality to a species of Daphnia (Arthur, 1971-1972, in Brungs, 1973). This problem was remedied for the final four acute tests and the second chronic test by holding dilution water for several days in an aerated well-lit tank after seeding with algal culture. Detailed examination of Daphnia responses (Sprague et al., 1978) showed that the toxic element in raw water did not have a detectable influence on results because any concentration of effluent down to the lowest used (1.25°70) was apparently sufficient to detoxify the chlorine. Because of this, no adjustment was made in the 3-day lethal tests for control mortality (22070 overall). Response of control organisms in the second chronic exposure was used to correct experimental response in both chronic tests, using A b b o t t ' s formula (Tattersfield and Morris, 1924). Computerized probit analysis was used to determine median lethal concentration (LCso), median effective concentration for reproductive failure (ECso), and the associated probit lines. RESULTS W I T H DISCUSSION
A general picture of acute lethality to Daphnia is given by the combined results for all nine batches of raw effluent (Fig. 2). The 48-h LCso was 75.6070 effluent (95o7o confidence limits (CL) -- 53.6-107o70, slope of probit line (S) = 1.76). Thus Daphnia were more sensitive than trout which suffered a combined mortality of only 19%, for the same nine batches at full strength. An exposure of 48 h seemed suitable for routine testing with Daphnia. The 72-h LCso was somewhat lower as expected, at 46.5°70 effluent (CL = 3 4 . 9 - 6 1 . 9 % , S = 1.79). Slopes of the probit lines were almost identical for 48- and 72-h exposures, as indicated by Fig. 2 and the values given above. Exposure for 24 h gave less satisfactory data. Most bioassays did not achieve 50°7o mortality in that time, and in fact the 24-h LCs0 could not be estimated. The slope of the probit line was appreciably lower indicating more variability within tests. The variability in the 24-h test could reflect increased sensitivity at time of molting; some individuals would have molted during 24 h while others would not have done so. The 48-h exposure would have allowed most individuals to molt during a test (Lee and Buikema, 1979). Results can be grouped by toxicity of the batches as shown by mortality of trout
331 98
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,
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CONCENTRATION OF EFFLUENT, PER CENT
Fig. 2. Acute mortality of D. pulex in treated oil refinery effluent. Combined data from nine bioassays with different batches of effluent. Results are shown for three exposure times on the same groups of animals, 54 at each concentration. The 950/0 confidence limits are shown for two LCso values.
TABLE I Median lethal concentrations for D. pulex exposed to nine batches of treated oil refinery effluent. Results grouped according to the toxicity of the batches to trout in full-strength effluent. N u m b e r of batches
2 4 2 1
Mortality of trout in 72 h
LCso values for Daphnia (07o effluent)
(070)
24 h
48 h
72 h
0 10 20-30 100 (LCs0 = 71)
> 100 > 100 > 100 45.7
> 100 92.3 58.6 27.8
82.9 49.2 47.4 12.5
(Table I). Daphnia LCso values were obtained at 48 h for batches that killed only 1 0 - 3 0 % of trout. For the most toxic batch, the 72-h LCso for trout was 2.6 times higher than the 48-h LCso for Daphnia. (For trout, the 72-h LCso would be almost the same as the more standard 96-h value, judging by comparisons of 30 lethal tests with this effluent (Sprague et al., 1978).) Results given above for lethality to Daphnia are based on the raw effluent, useful for comparison with the trout tests. For comparison with the chronic test, lethality data for Daphnia were adjusted for the dilution of one batch to half-strength. Doing this, the combined 48-h LCso for Daphnia, of the 7 batches used in the chronic test, would be 76.3% (CL = 51.7-113°/0, S = 1.81). This is almost identical to the LCso for combined data on all nine batches of raw effluent.
332
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Fig. 3. Lethality and reproductive impairment in D. pulex exposed for 14 days to treated oil refinery effluent. Results are corrected for control performance. Based on 12 individuals per concentration.
For the two chronic exposures, combined results showed a 14-day LCso of 6.4°7o effluent (CL = 3.6-11.6%, S = 1.89) (Fig. 3). This was the corrected value for 33.3°7o control mortality in the second exposure. The 10-day LCso was the same, and a toxicity curve indicated that 6.4°7o was a threshold for lethality. Ten days is a rather long time for reaching a lethal threshold, since it is about one-quarter of the animal's lifetime. The 14-day ECso for reproductive failure was 3.1°70 (CL = 1.7-5.6%, S = 2.13) (Fig. 3). This ECso incorporates correction for 33.3o7o reproductive failure in the control of the second exposure. The 14-day ECso signifies the effective concentration that prevented half o f the Daphnia f r o m producing freeswimming young. This was a reasonable evaluation of interference with reproduction, since only one Daphnia out of 60 in the chronic experiments formed eggs without releasing young. Temperature differences within the chronic tests may have slightly increased the variability of results. Buikema et al. (1976b) have shown that in the 20-25°C range, each increase of 1°C increased acute lethality of a simulated refinery effluent to D. pulex by about 17.4°70. Assuming this relationship held in our 14-day lethal tests, applying corrections for higher temperature in 10o70 effluent and lower ones in 5°70 would merely draw the two points closer to the probit line in Fig. 3, without appreciably changing its position or slope.
"Safe" concentration Estimation of a harmless concentration requires some arbitrary judgement. It might be considered that 5°70 impairment of reproduction would be a negligible effect and therefore relatively 'safe'. The value for reproductive failure a m o n g 5o70 of
333 the animals, the ECs, m a y be read f r o m Fig. 3 as 0.52°7o effluent. This is a low concentration, however it might be considered a realistic one to protect sensitive invertebrates in an ecosystem. The 'safe'-to-lethal ratio could be calculated as: EC5 for reproductive failure 48-h LCso
-
0.52 - 0.0068. 76.3
The value 0.0068 could be used as an application factor, to predict 'safe' levels o f a refinery effluent for Daphnia, having determined the 48-h LCso. This application factor is somewhat below the usual range of 0.1 to 0.01 (Sprague, 1971). Daphnia are probably a m o n g the most sensitive of aquatic animals to refinery effluent. Buikema et al. (1976a) found D. pulex and D. magna were one or two orders of magnitude more sensitive than 13 other invertebrates and 3 fish, tested against a simulated refinery effluent. Their final tests showed that D. pulex was 52 times more sensitive than trout (Buikema et al., 1976b), much greater than the factor of 2.6 which we found. Buikema and co-workers also obtained 48-h LCso values that were much different f r o m our value of 76°7o; they report 7o7o and 3.3°7o in the two reports mentioned above, and 3o70 in another publication (Geiger et al., 1980). The differences are surprising since their simulated effluent bad chemical characteristics very similar to the limits in Canadian effluent regulations. The explanation for both differences apparently involves species sensitivity to the type of oil used in the simulated effluent of Buikema and colleagues. This was Number 2 fuel oil, a relatively toxic petrochemical, and it was the m a j o r contributor of toxicity in their tests. When deleted f r o m the effluent, 48-h LCs0 values were 96 and 52o70, bracketing almost exactly our LCso of 76%. This agreement might also seem unusual since our effluents had high average values for oil-plus-grease and a m m o n i a (7 times and 1.6 times the Canadian limits). Apparently the toxicity of the whole effluent is predicted rather poorly f r o m a summation of the individual toxicities of components. This was strikingly shown by Buikema et al. (1976b) when effluents without a m m o n i a , or without phenol, had greater toxicity than the complete simulated effluent. This indicated antagonism between some components. GENERAL DISCUSSION OF FINDINGS WITH THIS EFFLUENT The first objective of this research was to establish the concentrations of a welltreated refinery waste that would be sublethally 'safe' for aquatic life. We studied a spectrum o f responses in five experiments (this paper and Rowe et al., 1983a, b; Westlake et al., 1983a, b) and the approximate thresholds of effect are summarized in Table II. The first two effects listed in Table II could clearly be meaningful for existence o f the fish in nature; the thresholds are of a similar magnitude, 5-9°70 effluent. Avoidance reactions were apparently not a problem with this effluent, and other locomotor and respiratory changes were statistically significant only at high concentrations.
334 TABLE II Estimated thresholds of sublethal effect on aquatic organisms exposed to treated effluent from a petroleum refinery. Experiment
Threshold (% effluent)
Growth of trout Life-cycle exposure of flagfish Avoidance by trout Locomotory changes in trout Gill irrigation rate, trout 'Coughing' by trout Chronic lethality to Daphnia 5070 inhibition of reproduction in Daphnia
About 5.6 (limits 3.1, 10) About 9 No effect to at least 30 About 30 (borderline statistical significance) Between 50 and 100 Between 25 and 50 6.4 0.52
Responses of D. pulex appear to be the most sensitive. Half the Daphnia showed chronic mortality at 6.4°70 effluent and failure to reproduce at 3.1 °70. The criterion of 5°70 inhibition of reproduction may be a justifiable choice as the most sensitive threshold documented in this series. Our work indicates that concentrations of this kind of well-treated refinery effluent should be 0.5°70 or less to protect freshwater communities. Lacking direct experiments on marine organisms, it might be predicted that a similar concentration would be protective of marine communities (USEPA, 1978; Klapow and Lewis, 1979). Achieving dilution to 0.5°70 would depend very much on the specific characteristics at a refinery site. Many such installations are, indeed, on the coast or other large bodies of water with high dilution. However, studies of other kinds o f effluents indicate that without facilities for diffusing the discharge, concentrations may exceed 0.5°70 in a narrow plume extending several kilometres, for example 1.7 km in Lake Superior (Minns, 1977) or 5 km in Canadian Atlantic estuaries (Whitney and Wilson, 1968). Plumes of high concentration can be greatly reduced in extent by designing outfall lines for rapid initial dilution and dispersal (Metcalf and Eddy, 1972). Such outfall design would be desirable for refineries in addition to avoidance of locations with restricted circulation or flow. Given those points, we conclude that treatment of refinery effluent to satisfy requirements such as the present Canadian ones for chemical limits and non-lethality to fish (Canada, 1974) would generally be protective of aquatic ecosystems.
Comparison with other work There is little published work on sublethal effects of refinery effluents, even if research with simulated effluents is included. Indeed this virtual absence of information was the stimulus for our research. One fragmentary report states that photosynthesis of an alga was affected by 2°70 effluent and probably also by 1°70, but not by 0.5°70 effluent (North et al., 1972). This report, for another type of organism, shows
335 a comparable but slightly less sensitive response than we concluded for Daphnia. By contrast, periphyton photosynthesis was apparently stimulated in artificial streams, even when full-strength simulated refinery effluent was present (Honig and Buikema, 1980). A shift f r o m normal species of algae was noted, however, when a 25070 concentration was present in the streams. There has been a fair amount of work on acute lethality o f whole effluents, which is of less direct interest in relation to the present study. Most of it confirms our finding that adequate treatment of a refinery effluent renders it non-lethal to fish. For example, Pessah et al. (1973) found that two Canadian refineries with good treatment produced effluents that were essentially non-lethal to rainbow trout. Two others with inadequate treatment, and one with treatment but a stripping problem, were lethal, with LCso values of 56°70, about 88070, and 21°70. They also found that a simulated waste with the maximal concentrations of parameters according to the Canadian regulations (Canada, 1974) did not cause lethality. Doubling these concentrations caused only about 20070 mortality. Similarly, G r a h a m and Dorris (1968) found no mortality of fish for two refineries with biological treatment, and LCso values of a b o u t 2007o effluent for two refineries without such treatment. G r a h a m and Dorris also did longer lethal tests of 32 days. For the more toxic effluents, concentrations which did not kill in 4 days did not prove lethal in 32 days either. Oddly enough, two effluents of low toxicity which caused no lethality in fullstrength exposure for 4 days required four-fold dilution to allow survival for 32 days. These experiments do not deal with sublethal effects by any means, but come closer to the topic than any other experiments with fish. Brown trout (Salmo trutta) and rainbow trout were the fourth and fifth most sensitive of 57 species of fish tested for 96-h LCso values in whole refinery effluent (Irwin, 1965). Gizzard shad (Dorosoma cepedianum) were most sensitive and guppies (Lebistes reticulatus) least sensitive, with a five-fold difference in tolerance. The sensitivity of rainbow trout would suggest that they were a good choice for our sublethal experiments.
Screening tests A secondary objective of our work was to look for a convenient method of biologically assessing the toxicity of refinery effluents. From our spectrum o f tests, we agree with Buikema et al. (1976a) that a 48-h lethal test with D. pulex has m a n y advantages, and we would list the following ones. (a) For monitoring, it is small-scale, simple, and inexpensive. (b) It requires only small volumes o f effluent, a useful feature for shipping from remote areas and for evaluating pilot-plant effluents. (c) It is twice as fast as a standard lethal test with fish. (d) It is approximately 2.6 times as sensitive as lethality to trout. This could mean
336 a greater yield o f information in cases when an effluent was non-lethal to the fish it could be just below the lethal level or far below it. (e) It predicts the 'safe' concentration of a refinery effluent for Daphnia, which appear to be a m o n g the most sensitive of aquatic organisms to this waste. The 'safe' level could be estimated by using the application factor of 0.0068 on the LC50, as determined by our work. (f) Using a clone of Daphnia should improve the reproducibility of tests at different times and places. Daphnia could be used in a simple pass/fail test. For example the stipulation that effluent at 40% concentration should kill not more than half a group of D. pulex in 48 h could provide an alternative to the present Canadian regulatory test with fish (Canada, 1974). A 7-day test with D. pulex may give a good assessment of chronic effects. Geiger et al. (1980) show that the body-length of pre-adult Daphnia, after seven days of exposure to simulated refinery effluent, is a good predictor of later success in reproduction. Short tests such as this or the 48-h LCs0 would be advantageous. There is a considerable art in carrying out a full reproductive test; Nebeker (1982) reports that three out of six laboratories did not successfully complete 21-day chronic experiments with D. magna. An even more rapid technique based on enzyme inhibition is currently being evaluated by Rutherford et al. (1979). For simulated refinery effluent, they found the test sensitive and in reasonable agreement with Daphnia and fish tests. The cough response of a suitable fish could be useful for on-line monitoring of a given effluent stream. As described in Westlake et al. (1983b), such a system could be computerized to trigger an alarm at times o f increased stress on the fish. This would require, however, fairly sophisticated equipment, programming, and personnel. -
Characteristics o f this effluent The effluent used in this research proved fairly consistent. During two years, 92 batches were tested for lethality to rainbow trout, with an overall mortality rate of 23%0 in full-strength effluent. O f the 92 batches, 71 were considered to have passed the Canadian test for non-lethality to trout. Most of the failures came during an unusual period of changeover in refinery processes when treatment was disrupted, and at a time when we were conducting preliminary Or trial runs. For the 68 batches actually used in sublethal experiments, full-strength effluent caused an overall mortality of 14% of the fish, and 60 o f the 68 batches were considered to have passed the regulatory test. Chemical characteristics of the effluent have been summarized (Rowe et al., 1983a). In general, the 68 batches used sublethally had phenol and sulphide levels which were one or two orders of magnitude lower than the Canadian limits of 1.0
337
and 0.3 mg/l (Canada, 1974), and the similar limits proposed in 1973 for U.S.A. (USEPA, 1973). The 68 batches averaged 17 mg/l of ammonia as N, somewhat higher than the Canadian limit of 12.5, and were about twice the limits of 10 mg/1 and 25 mg/l for oil-plus-grease and residue. From the above, our experiments are based on an effluent that came close to meeting accepted standards. However, it was not an unduly innocuous one, and our results would seem generally applicable for a well-treated petroleum refinery effluent. We found little or no indication that the degree of sublethal toxicity could be predicted from physico-chemical characteristics of the effluent. There were no obvious correlations of chemical 'strength' with biological effect, within experiments on trout growth, respiration, locomotion, or for Daphnia reproduction. We did find increased mortality in the screening tests with trout, for certain batches o f effluent which were high in one or more of phenol, ammonia, cyanide, or oil-plus-grease. However, since we did not control the effluent characteristics it could not be ascertained whether mortality was due to these components or other unmeasured toxicants which increased at the same times. C6t6 (1976) has summarized considerable work which tested individual components of refinery effluent for their toxicity. In general, this other work also showed rather poor prediction of the total toxicity of a simulated effluent, on the basis o f its components. A review by Baker (1979) concludes that it 'appears to be impossible to generalise about why refinery effluents are toxic'. In any case the topic of component toxicity is of less interest for the purpose of our work - to study a whole effluent. There is relatively little sublethal research on such effluents, compared to that on pure compounds. Especially in view of the difficulties of predicting toxic effects of mixtures, it is important to have available some findings on the real effluents which aquatic organisms face in the environment. Although it might appear that work on a whole effluent, with its day-today variability, would lead to an imprecise piece of research, this has not been our experience. Effects during the various time periods of the experiment seemed relatively similar and consistent. For example, two growth experiments on trout, in separate years, led to very similar conclusions on the threshold of damage. ACKNOWLEDGEMENTS
This project was funded and supported by the Petroleum Association for Conservation of the Canadian Environment (PACE) and by the Petroleum and Industrial Organic Chemicals Division, Water Pollution Control Directorate, Environmental Protection Service, of the Canadian Department of Environment. It was carried out in laboratories supplied at the Canada Centre for Inland Waters, Burlington, Ontario. We thank William Yasui for his technical work on the Daphnia experiment. From the petroleum industry we thank Evan C. Birchard, Peter Budzick, and Waiter Krawciw of PACE, and Norman A. Barron of BP Trafalgar refinery. Numerous people from Environment Canada gave advice, facilities, and provided
338 a massive program of chemical analyses. We thank Douglas Anderson, James P. B r u c e , V i c t o r C a i r n s , K. C o n n , O . E l Kei, R o n a l d G . G i l l e s p i e , P e t e r V. H o d s o n , Michael
LeBlanc,
A.R.
Lefeuvre,
D. Marsh,
Serge Metikosh,
R o s s E. Mills,
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339 Pessah, E., J.S. Loch and J.C. MacLeod, 1973. Preliminary report on the acute toxicity of Canadian petroleum refinery effluents to fish. Fish. Res. Board Can. Tech. Rept. No. 408, 43 pp. Rowe, D.W., J.B. Sprague and T.A. Heming, 1983a. Sublethal effects of treated liquid effluent from a petroleum refinery. I. Chronic toxicity to flagfish. Aquat. Toxicol. 3, 149-159. Rowe, D.W., J.B. Sprague, T.A. Heming and I.T. Brown, 1983b. Sublethal effects of treated liquid effluent from a petroleum refinery. II. Growth of rainbow trout. Aquat. Toxicol. 3, 161-169. Rutherford, C.L., A.L. Buikema, Jr., D.R. Armant and J. Cairns, Jr., 1979. A simple method for screening petroleum effluents by in vitro enzyme inhibition. Bull. Environ. Contam. Toxicol. 21, 79-84. Sprague, J.B., 1971. Measurement of pollutant toxicity to fish. III. Sublethal effects and 'safe' concentrations. Water Res. 5, 254-266. Sprague, J.B., D.W. Rowe, G.F. Westlake, T.A. Heming and I.T. Brown, 1978. Sublethal effects of treated liquid effluent from a petroleum refinery on freshwater organisms. Canada Dept. of Fisheries and Environment, Environmental Protection Service, and Petroleum Association for Conservation of the Canadian Environment, Ottawa, 297 pp. Tattersfield, F. and H.M. Morris, 1924. An apparatus for testing the toxic values of contact insecticides under controlled conditions. Bull. Entomol. Res. 14, 223-233. USEPA, 1973. Petroleum refining point source category. Proposed effluent limitation guidelines and new source standards (U.S. Environmental Protection Agency). Fed. Register 30, 34542-34558. USEPA, 1978. Water quality criteria. U.S. Federal Register, Vol. 43, No. 97 (U.S. Environmental Protection Agency), pp. 21506-21518. Westlake, G.F., J.B. Sprague, R.J. Hines and I.T. Brown, 1983a. Sublethal effects of treated liquid effluent from a petroleum refinery. III. Avoidance and other locomotory responses of rainbow trout. Aquat. Toxicol. 4, 235-245. Westlake, G.F., J.B. Sprague and 1.T. Brown, 1983b. Sublethal effects of treated liquid effluent from a petroleum refinery. IV. Respiratory movements and coughing of rainbow trout. Aquat. Toxicol. 4, 317-325. Whitney, W.H. and G.G. Wilson, 1968. Effluent distribution studies on the Miramichi and Restigouche estuaries. Pulp Pap. Mag. Can. 69, 79-85.