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Effects of oil exposure on pink salmon, Oncorhynchus gorbuscha, alevins in a simulated intertidal environment
14arine Environmental Research 21 (1987) 49-58
Effects of Oil Exposure on Pink Salmon, Oncorhynchus gorbuscha, Alevins in a Simulated Intertidal Environment A d a m Moles, Malin M. Babcock & Stanley D. Rice Northwest and Alaska Fisheries Center Auke Bay Laboratory, National Marine Fisheries Service, NOAA, P.O. Box 210155. Auke Bay, AK 99821, USA (Received 6 June 1986, accepted 9 June 1986)
ABSTRACT Pink salmon, Oncorhynchus gorbuscha, alevins (5 and 60 days after hatching) were continuously or intermittently exposed for 30 days to the water-soluble fraction ( W S F ) o f Cook Inlet crude oil in fresh water or in a simulated freshwater-seawater cycle. Alevins exposed to 0"7-2.4 mg/liter W S F in the simulated tidal cycle were more sensitive to oil, had reduced yolk reserves, and accumulated more hydrocarbons than did alevins exposed to the same concentrations in fresh water. Alevins in fresh water were more sensitive to continuous than to intermittent exposures. In all exposures, 60day alevins were more severely affected than were 5-day alevins.
INTRODUCTION Pacific salmon, Oncorhynchusspp., after spending i-6 years in the North Pacific Ocean, return to natal streams to spawn in fresh water. However, pink salmon, O. gorbuscha, spawn in both the intertidal and freshwater portions of many streams in Alaska. ~Intertidal' in this study denotes the lower areas of streams that regularly experience seawater intrusion during high tides. Some pink salmon spawn intertidally because steep gradients or waterfalls at the high tide level block their migration, whereas others do so when no barriers exist. Intertidal spawning has also been reported in British Columbia (Hunter, 1959). In Prince William Sound, Alaska, up to 75% of a pink salmon run may spawn intertidally (Noerenberg, 1963; Helle, 1970). In the intertidal area, survival of eggs to pre-emergent fry is greater upstream, 49 Marine Environ. Res. 0141-1136/87/$03.50 ~ Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain
50
Adam 3[oles. Malin M. Babcock, Stanley D. Rice
where there is less seawater intrusion, and progressively decreases with reduced elevation (Helle et al., 1964). Although the intertidal reaches of streams may provide additional spawning habitat and, in harsh winters, a more stable environment than upstream waters provide, this habitat is vulnerable to both freshwater- and seawater-borne oil. Salmon co% and alevins tolerate short-term exposures to crude oil (Moles et al., 1979) but slowly accumulate high concentrations of hydrocarbons. As yolk is absorbed, toxicity and rate of uptake of oil increase (Korn & Rice, 1981). Alevins may therefore be sensitive to longterm exposures to low concentrations of oil. Salmonids are also more sensitive to oil in seawater than in fresh water (Moles et al., 1979: Thomas & Rice, 1981; Stickle et al., 1982). The salinity of intertidal spawning gravels depends on the stream characteristics and the salinity of the overlying waters during tidal intrusion. Intertidal salinity can be as high as 30-7%o, similar to that of adjacent coastal waters (Hanavan & Skud, 1954). We hypothesized that the periodic influx of seawater into a stream polluted by oil would increase the toxicity of hydrocarbons, even if alevins were exposed to only tide-borne oil, as in an oil spill in the nearshore marine environment. The objectives of our study were to determine the differences in survival, developmental index, and hydrocarbon uptake of pink salmon alevins (5 or 60 days after hatching) exposed continuously or intermittently to the watersoluble fraction (WSF) of Cook Inlet crude oil in fresh water or in a simulated freshwater-seawater tidal cycle. Intermittent exposures to WSF, lasting 3 h of every 12, coincided with the seawater phase of the tidal cycle. Of these four types of oil exposure, three are realistic: oil pollution may either originate in the marine environment and affect pink salmon alevins in intertidal areas during high tide, or originate from an upstream source and affect alevins developing in either fresh water or intertidal areas. The fourth condition used in our study (intermittent exposure of oil in fresh water) is unlikely and presented for comparison purposes.
MATERIALS AND METHODS Pink salmon eggs from the Auke Creek hatchery in southeastern Alaska were incubated in Heath trays until ready to be used in the tests. Two age groups of alevins were exposed for 30 clays to WSF of Cook Inlet crude oil. The first group was exposed 5 clays after hatching, and the second group, 60 days. The older alevins completed yolk absorption by the end of the tests and were ready to begin feeding. Exposure containers were 4-liter glass jars, wrapped in black tape to
Effects of oil exposure on pink salmon alevins
51
exclude light and with a 2-cm layer of pea-sized gravel to cover the bottom. Toxicant was delivered (400 ml/min) to the bottom of the jars through longstemmed glass funnels held in place by perforated Plexiglas lids through which excess toxicant overflowed. Each jar contained 150 alevins, and one jar was used per exposure condition and concentration. Different concentrations of WSF were produced and delivered to the exposure jars by a continuous-flow apparatus (Moles et al., 1985). In the apparatus, water dripped through a continuously replenished, ! 5-cm layer of oil atop a 2-m column of water to produce a WSF stock solution. Two WSF apparatuses operated simultaneously, one for fresh water and the other for seawater. The WSF stock solution from each apparatus entered a manifold that, at the desired flow rate, delivered it to an exposure jar, while another manifold delivered dilution water. Thus, each jar received toxicant and dilution water. Controls received only dilution water. Jars were moved, as needed, from one exposure system to the other. Concentrations of WSF in the test solutions were monitored daily and adjusted as needed. A 750ml test solution was extracted with 50 ml dichloromethane in two sequential 25 ml extractions with 5 min separation times. Extracted samples were analyzed by gas chromatography (Hewlett Packard 5880A). Concentrations are reported as a sum of eight selected mono- and di-aromatic hydrocarbons. These aromatics comprise 90-95% of the total aromatic hydrocarbons present in WSF of Cook Inlet crude oil and are the primary contributors to the toxicity of WSF (Rice et al., 1977). The average concentrations and proportions of individual aromatic hydrocarbons found in a typical WSF (at 248mg/liter) are presented in Table 1. Concentrations of toxicants in both freshwater and tidal exposures averaged 0 (control), 0-7, 1-5, and 2.4 mg/liter aromatic hydrocarbons and were stable with less than 3% dose deviation over the 30-day tests. Developing alevins were bathed in seawater twice daily to simulate a tidal cycle (Fig, 1). Selected jars were moved to the seawater manifold at 9.00 and 21.00 hours (it took 10rain for salinity to increase from 0%0 to 29.5%0). The containers were returned to the freshwater manitbld at 12.00 and 24.00 hours. This 3-h seawater cycle, which simulated salinities in an intertidal area of a stream, is the maximum period of seawater exposure that does not adversely affect survival (Helle et al., 1964). Also examined were differences between continuous and intermittent exposures to WSF. Some alevins were continuously exposed to 0.7, 15, and 24 rag/liter aromatic hydrocarbons in fresh water or in the tidal cycle, while others were exposed intermittently (3 h out of every 12 h), coinciding with the seawater phase of the tidal cycle. Survival was assessed daily during each test, and 10 alevins were periodically removed from each jar for analysis of tissue hydrocarbons.
52
Adam .~foles, Malin M. Babcock, Stanley D. Rice
TABLE 1 Average Concentrations of Selected Mono- and Diaromatic Hydrocarbons in 2.48 rag liter of the Watersoluble Fraction (WSF) of Cook Inlet Crude Oil. These compounds comprise 90-95% of the total aromatic hydrocarbons in WSF Component
Concentration (rag. liter )
Proportion (%)
Benzene Toluene o-Xylene m- and p-Xylene Ethylbenzene Naphthalene l-Methylnaphthalene 2-Methylnaphthalene
1.07 083 0.12 0.26 0-10 005 0-02 0-03
43.0 33-0 5'0 10.0 4.0 20 10 I-0
Sum
2-48
Whole body tissues were analyzed for the presence of 14 aromatic hydrocarbons, from o-xylene to dibenzothiophene, by gas chromatography and confirmed by mass spectrometry. Concentrations were reported as the sum of these compounds. After 30 days of exposure, 30 alevins were removed from each jar and preserved in 5% buffered formalin for 60 days. Weights and lengths were measured, and Barns' k o values (an index of fry development) calculated using the following formula (Barns, 1970):
10 (weight, in milligrams) 1/3 ko =
l e n g t h , in m i l l i m e t e r s
Higher kD values denote more girth and weight relative to length and
ES"WATER F'NTE M,TTENT O'L I O
,
I
/I
~;~.'~1 ;~.~i
I'~,r;%1 I ~:~!~
UOUSO,L
INTERMITTENT OIL TIDAL CYCLE CONTINUOUS OIL ~ I .
l
SEAWATER OIL WSF Fig. I.
0
3
6
;
1'2 115 HOURS
Experimenta! conditions.
118
211 24
Effects of oil exposure on pink salmon alevins
53
indicate an earlier development stage. As alevins develop and absorb yolk, the k~ values decrease. How completely yolk is absorbed by the time fry emerge from the streambed directly affects their survival. The optimum kz~ for emerging fry is 1-97 (Bailey & Taylor, 1974). Emerging fry with a large amount of yolk (as reflected in a k D > 1.97) are relatively poor swimmers and are therefore more vulnerable to predators. Conversely, fry that absorb all their yolk (k o < 1.97) before emerging are emaciated, weakened, and vulnerable to predators (Barns, 1970). Differences in kt) values between experimental groups were evaluated by using a two-way analysis of variance on the means.
RESULTS AND DISCUSSION Effects of tidal exposure
Alevins in the simulated tidal cycle were more sensitive to oil, had reduced yolk reserves, and accumulated more hydrocarbons than did alevins in fresh water. All alevins continuously exposed to 2.4rag/liter aromatic hydrocarbons died during the 30-day test: they survived only 6-7 days in the simulated tidal cycle and 7-16 days in fresh water (Table 2). Concentrations of 1"5 mg/liter total aromatic hydrocarbons did not kill any alevins, regardless of experimental conditions. Alevins exposed to _< 1'5 rag/liter in the tidal cycle had significantly lower kD's (P < 0-05, Fig. 2: Table 3) than did either controls or alevins exposed to the same TABLE 2
Survival (Days) of 5- and 60-day Pink Salmon Alevins Exposed to 2-4 rag/liter of the Water-soluble Fraction (WSF) of Cook Inlet Crude Oil before 100% Mortality Occurred. Alevins exposed to all other concentrations survived. N = 150 for each treatment Test condition
Continuous WSF: Tidal cycle Fresh water Intermittent WSF: Tidal cycle Fresh water
Surcical oj alecins 5-day
60-day
6 16
7 7
9 No deaths
10 No deaths
Adam 3ioles. Malin ,W. Babcock, Stanley D. Rice
54
140,
C
O
N
T
I
N
U
O
U
~
30
20
'S
~------© im r,, < u ©
---
0
OIL/TIDAL
~INTERMITTENT
"C..: INTERMITTENT OIL/FRESHWATER I
I
I
I
I
c~.o >,-r
~
60-DAY ALEVINS
30
/
OIL/FRESHWATER-/
/ ,NTER,,TTET , " \
10 /
/ 0
OIL/TIDAL / ~ 7 ,"
/
..... ¢---:=:---'LL___ / 5
[ 10
/ %~
~NTERMITTENT ~'.
v-- OIL/FRESHWATER
_ ~ _ ~ -
I l 1S 20 DAYS EXPOSED
.____._ - - . . I 25
30
Fig. 2. Mean Bams" k n values for 5-day and 60-day pink salmon alevins after 30-day exposures to the water-soluble fraction of C o o k Inlet crude oil. F W = freshwaterexposures: T C = e x p o s u r c s in a simulated tidal cycle, N = 3 0 . Control values were at 0 m g l i t e r . Confidence intervals ranged from 0.020 to 0'035.
concentrations in flesh water. As concentrations of WSF increased, the k o values decreased in both the freshwater and tidal groups. At the end of 30 days of exposure, control 60-day alevins in fresh water had a k D of 2.07, and those in the tidal cycle, 1-97 (Fig. 2). Controls held in fresh water had excess yolk reserves, whereas exposure to either oil or seawater speeded up development. Alevins exposed to oil in seawater had yolk reserves below optimum and therefore would have emerged from the gravel early or in poor condition. Alevins continuously exposed to the WSF in the tidal cycle
Effects o f oil exposure on pink salmon alevins
55
TABLE 3
Two-way ANOVA of Mean Barns k o Values for Pink Salmon Alevins Exposed to the Water-soluble Fraction of Cook Inlet Crude Oil for 30 Days
Source
df
SS
),IS
F
Total A (Concentration) B (Tidal) AB error
5-day alevins 11 0-01897 2 0"00382 t 0-00963 2 0.000 12 6 0"00540
0"001 9l 0"00963 0.00006 0-00090
2-12037 I0.703 70 * 0-06481
Total A (Concentration) B(Tidal) AB error
60-day alevins 11 0-026 60 2 0.01040 1 0-01203 2 0.00187 6 0-00230
0-00520 0.01203 0-00093 0.00038
13565 22 ** 31.39130"* 2-43478
* P < 0 " 0 5 ; * * P<0"01.
had two to six times higher tissue concentrations of aromatic hydrocarbons than did those continuously exposed in fresh water (Fig. 3). Alevin survival and growth were affected more by seawater-borne oil than oil in fresh water. This increased sensitivity to WSFs in seawater, which has been previously demonstrated with older juvenile salmon {Moles et al., 1979; Stickle et al., 1982), did not appear to be associated with a failure to osmoregulate (Stickle et al., 1982). Hydrocarbon uptake in alevins in the tidal cycle was much greater than in the alevins in fresh water and would appear to be the direct cause for the decreased survival and growth of alevins in seawater, even though the alevins were in seawater only 25% of the time. The increased accumulation of hydrocarbons in seawaterexposed alevins could be caused by increased drinking of seawater for osmoregulatory control. Thomas and Rice {1986) observed substantial retention of aromatic hydrocarbons and decreased metabolism of aromatic hydrocarbons in Dolly Varden char in seawater compared to char similarly exposed in fresh water. Seawater may have caused a decrease in metabolism and excretion of aromatic hydrocarbons, which led to greater hydrocarbon accumulation in alevins in seawater than in fresh water. Effects of age of alevins
In all exposures, 60-day alevins were more affected than 5-day alevins. During continuous exposures to 2-4 mg/liter, both age groups had similar
56
Adam Moles, Malin M. Babcock, Stanley D. Rice
2.20
S-DAY ALEVINS INTERMITTENT
2. 15 ~~"~'~"rW
FW CONTINUOUS
2,10
2.05 ~ E R M I T T E N T
2.00 l
~2-'° I
I ,o-oAY ALEV,NS
|
FW CONT,NOOOS
2.00
1.90
1.85
I
1
0.7
1
1.5 WSF (mg/I iter} Fig. 3. Tissuehydrocarbons present in pink salmon alevins during exposure to I-5 rag/liter of the water-soluble fraction of Cook Inlet crude oil. N= 10. Control tissue had < 3/~g/g aromatic hydrocarbons. survival in the tidal cycle. In fresh water, however, 5-day alevins survived longer than 60-day alevins (Table 2), probably because differing amounts of yolk influenced the rates of uptake and depuration. The ko values for 5-day alevins were generally higher during intermittent than continuous exposures, and they were similar for 60-day alevins in both types of exposures (Fig. 2). As the concentration of the WSF increased, the k o values were significantly lower (P<0-001) for 60-day than for 5-day alevins (Fig. 2). The 60-day alevins had lower k o values because they were growing faster and had less yolk than did 5-day alevins. In general, 5- and 60-day alevins accumulated similar concentrations of aromatic hydrocarbons, but the pattern of accumulation was different. The
Effects of oil exposure on pink salmon alevins
57
yolk-rich, 5-day alevins accumulated aromatic hydrocarbons throughout the study and, after 30 days, had higher concentrations of hydrocarbons than did the 60-day alevins. Maximum concentrations of hydrocarbons in tissues of 60-day alevins occurred after 13 days of exposure, then decreased by 50 to 95%, depending on the treatment, by the end of the test. The older alevins had consumed the majority of" their yolk by the end of the 30-day exposure (90-day-old alevins); there was very little of the lipid-rich yolk to trap and retain the absorbed aromatic hydrocarbons. Yolk has a high affinity for lipophilic hydrocarbons and retains them in semi-isolation from developing tissues where the hydrocarbons would be metabolized (Korn & Rice, 1981). This pattern of uptake and depuration is similar to that found in different ages of coho salmon, Oncorhynchus kisutch, alevins exposed to hydrocarbons in fresh water (Korn & Rice, 1981).
CONCLUSION Concentrations of aromatic hydrocarbons in seawater after short-term oil pollution can be higher than 0"5 ppm in areas where turbulence disperses the oil into the water column. For example, during the A m o c o Cadiz spill, Calder & Boehm (1981) found hydrocarbon concentrations that exceeded 1 ppm in the water entering the estuary and 0 5 p p m in the rest of the estuary. Admittedly our experimental concentrations are above those likely to occur in most spills; however, the combination of turbulent wave action--characteristic of the intertidal zone--and the chronic input of oil from coated beaches could cause concentrations within the range of those used in this study. It is here, in the intertidal zone rather than in fresh water, that pink salmon alevins, particularly those near emergence, would be adversely affected by exposure to oil.
ACKNOWLEDGEMENTS The tissues were analyzed by gas chromatography by the National Analytical Facility, Northwest and Alaska Fisheries Center, Seattle, Washington.
REFERENCES Bailey, J. E. & Taylor, S. G. (1974). Salmon fry production in a gravel incubator hatchery, Auke Creek, Alaska, 1971-72. US Dep. Commer., NOAA Tech. Memo. N M F S ABFL-3, p. 13.
58
Adam Moles, Malin M. Babcock, Stanley D. Rice
Barns, R. A. (I970). Evaluation of a revised hatchery method tested on pink and chum salmon fry. J. Fish. Res. Board Can., 27, 1429-52. Calder, J. A. & Boehm, P. D. (1981). The chemistry of Amoco Cadiz oil in the Aber Wrac'h. In: Amoco Cadiz, fates and effects of the oil spill, Proceedings of the International Symposium, Brest, France, November 19-22, 1979, 149-58. Hanavan, M. G. & Skud, B. E. (1954). Intertidal spawning of pink salmon. Fish. Bull., 95, 56, 167-85. Helle, J. H. (1970). Biological characteristics of intertidal and fresh-water spawning pink salmon at Olsen Creek, Prince William Sound, Alaska. 1962-63. US Fish. Wildl. Serv., Spec. Sci. Rep.--Fish., No. 602, p. 19. Helle, J. H., Williamson, R. S. & Bailey, J. E. (1964). Intertidal ecology and life history of pink salmon at Olsen Creek, Prince William Sound, Alaska. US Fish. WTldl. Serv., Spec. Sci. Rep.--Fish., No. 483, p. 26. Hunter, J. G. (1959). Survival and production of pink and chum salmon in a coastal stream. J. Fish. Res. Board Can., 16, 835-86. Korn, S. & Rice. S. (1981). Sensitivity to, and accumulation and depuration of, aromatic petroleum components by early life stages of coho salmon (Oncorhynchus kisutch), Rapp. P.-v. Reun. Cons. Int. Explor. Mer., 178, 87-92. Moles, A., Rice, S. D, & Andrews, S. (1985). Continuous-flow devices for exposing marine organisms to the water-soluble fraction of crude oil and its components. Can. Tech. Rep. Fish. Aquat. Sci., 1368, 53-61. Moles, A., Rice, S. D. & Korn, S. (1979). Sensitivity of Alaskan freshwater and anadromous fishes to Prudhoe Bay crude oil and benzene. Trans. Am. Fish. Soc., 108, 408-14. Noerenberg, W. A. (1963). Salmon forecast studies on 1963 runs in Prince William Sound. Alaska Dep. Fish Game Info. Leaflet 21, Juneau, Alaska, p. 27. Rice, S. D., Short, J. W. & Karinen, J. F. (1977). Comparative oil toxicity and comparative animal sensitivity. In: Fate and effects of petroleum hydrocarbons in marine ecosystems and organisms. (Wolfe, Douglas A. (Ed.)), Pergamon Press, New York, 78-94. Stickle, W. B., Sabourin, T. D. & Rice, S. D. (1982). Sensitivity and osmoregulation ofcoho salmon, Oncorhvnchus kisutch, exposed to toluene and naphthalene at different salinities. In: Physiological mechanisms of marine pollutant toxicity. (Vernberg, W. B., Calabrese, A., Thurberg, F.P. & Vernberg, F.J. (Eds)), Academic Press, New York, 331-48. Thomas, R. E, & Rice, S. D. (1981). Excretion of aromatic hydrocarbons and their metabolites by freshwater and seawater Dolly Varden char. In: Biological monitoring of marine pollutants. (Vernberg, F. J., Calabrese, A., Thurberg, F. P. & Vernberg, W. B. (Eds), Academic Press, New York, 425-48. Thomas, R. E. & Rice, S. D. (1986). The effects of salinity on uptake and metabolism of toluene and naphthalene by Dolly Varden, Sah:elinus mabna. Marine Ent'iron. Res., 18, 203-14.
Effects of Oil Exposure on Pink Salmon, Oncorhynchus gorbuscha, Alevins in a Simulated Intertidal Environment A d a m Moles, Malin M. Babcock & Stanley D. Rice Northwest and Alaska Fisheries Center Auke Bay Laboratory, National Marine Fisheries Service, NOAA, P.O. Box 210155. Auke Bay, AK 99821, USA (Received 6 June 1986, accepted 9 June 1986)
ABSTRACT Pink salmon, Oncorhynchus gorbuscha, alevins (5 and 60 days after hatching) were continuously or intermittently exposed for 30 days to the water-soluble fraction ( W S F ) o f Cook Inlet crude oil in fresh water or in a simulated freshwater-seawater cycle. Alevins exposed to 0"7-2.4 mg/liter W S F in the simulated tidal cycle were more sensitive to oil, had reduced yolk reserves, and accumulated more hydrocarbons than did alevins exposed to the same concentrations in fresh water. Alevins in fresh water were more sensitive to continuous than to intermittent exposures. In all exposures, 60day alevins were more severely affected than were 5-day alevins.
INTRODUCTION Pacific salmon, Oncorhynchusspp., after spending i-6 years in the North Pacific Ocean, return to natal streams to spawn in fresh water. However, pink salmon, O. gorbuscha, spawn in both the intertidal and freshwater portions of many streams in Alaska. ~Intertidal' in this study denotes the lower areas of streams that regularly experience seawater intrusion during high tides. Some pink salmon spawn intertidally because steep gradients or waterfalls at the high tide level block their migration, whereas others do so when no barriers exist. Intertidal spawning has also been reported in British Columbia (Hunter, 1959). In Prince William Sound, Alaska, up to 75% of a pink salmon run may spawn intertidally (Noerenberg, 1963; Helle, 1970). In the intertidal area, survival of eggs to pre-emergent fry is greater upstream, 49 Marine Environ. Res. 0141-1136/87/$03.50 ~ Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain
50
Adam 3[oles. Malin M. Babcock, Stanley D. Rice
where there is less seawater intrusion, and progressively decreases with reduced elevation (Helle et al., 1964). Although the intertidal reaches of streams may provide additional spawning habitat and, in harsh winters, a more stable environment than upstream waters provide, this habitat is vulnerable to both freshwater- and seawater-borne oil. Salmon co% and alevins tolerate short-term exposures to crude oil (Moles et al., 1979) but slowly accumulate high concentrations of hydrocarbons. As yolk is absorbed, toxicity and rate of uptake of oil increase (Korn & Rice, 1981). Alevins may therefore be sensitive to longterm exposures to low concentrations of oil. Salmonids are also more sensitive to oil in seawater than in fresh water (Moles et al., 1979: Thomas & Rice, 1981; Stickle et al., 1982). The salinity of intertidal spawning gravels depends on the stream characteristics and the salinity of the overlying waters during tidal intrusion. Intertidal salinity can be as high as 30-7%o, similar to that of adjacent coastal waters (Hanavan & Skud, 1954). We hypothesized that the periodic influx of seawater into a stream polluted by oil would increase the toxicity of hydrocarbons, even if alevins were exposed to only tide-borne oil, as in an oil spill in the nearshore marine environment. The objectives of our study were to determine the differences in survival, developmental index, and hydrocarbon uptake of pink salmon alevins (5 or 60 days after hatching) exposed continuously or intermittently to the watersoluble fraction (WSF) of Cook Inlet crude oil in fresh water or in a simulated freshwater-seawater tidal cycle. Intermittent exposures to WSF, lasting 3 h of every 12, coincided with the seawater phase of the tidal cycle. Of these four types of oil exposure, three are realistic: oil pollution may either originate in the marine environment and affect pink salmon alevins in intertidal areas during high tide, or originate from an upstream source and affect alevins developing in either fresh water or intertidal areas. The fourth condition used in our study (intermittent exposure of oil in fresh water) is unlikely and presented for comparison purposes.
MATERIALS AND METHODS Pink salmon eggs from the Auke Creek hatchery in southeastern Alaska were incubated in Heath trays until ready to be used in the tests. Two age groups of alevins were exposed for 30 clays to WSF of Cook Inlet crude oil. The first group was exposed 5 clays after hatching, and the second group, 60 days. The older alevins completed yolk absorption by the end of the tests and were ready to begin feeding. Exposure containers were 4-liter glass jars, wrapped in black tape to
Effects of oil exposure on pink salmon alevins
51
exclude light and with a 2-cm layer of pea-sized gravel to cover the bottom. Toxicant was delivered (400 ml/min) to the bottom of the jars through longstemmed glass funnels held in place by perforated Plexiglas lids through which excess toxicant overflowed. Each jar contained 150 alevins, and one jar was used per exposure condition and concentration. Different concentrations of WSF were produced and delivered to the exposure jars by a continuous-flow apparatus (Moles et al., 1985). In the apparatus, water dripped through a continuously replenished, ! 5-cm layer of oil atop a 2-m column of water to produce a WSF stock solution. Two WSF apparatuses operated simultaneously, one for fresh water and the other for seawater. The WSF stock solution from each apparatus entered a manifold that, at the desired flow rate, delivered it to an exposure jar, while another manifold delivered dilution water. Thus, each jar received toxicant and dilution water. Controls received only dilution water. Jars were moved, as needed, from one exposure system to the other. Concentrations of WSF in the test solutions were monitored daily and adjusted as needed. A 750ml test solution was extracted with 50 ml dichloromethane in two sequential 25 ml extractions with 5 min separation times. Extracted samples were analyzed by gas chromatography (Hewlett Packard 5880A). Concentrations are reported as a sum of eight selected mono- and di-aromatic hydrocarbons. These aromatics comprise 90-95% of the total aromatic hydrocarbons present in WSF of Cook Inlet crude oil and are the primary contributors to the toxicity of WSF (Rice et al., 1977). The average concentrations and proportions of individual aromatic hydrocarbons found in a typical WSF (at 248mg/liter) are presented in Table 1. Concentrations of toxicants in both freshwater and tidal exposures averaged 0 (control), 0-7, 1-5, and 2.4 mg/liter aromatic hydrocarbons and were stable with less than 3% dose deviation over the 30-day tests. Developing alevins were bathed in seawater twice daily to simulate a tidal cycle (Fig, 1). Selected jars were moved to the seawater manifold at 9.00 and 21.00 hours (it took 10rain for salinity to increase from 0%0 to 29.5%0). The containers were returned to the freshwater manitbld at 12.00 and 24.00 hours. This 3-h seawater cycle, which simulated salinities in an intertidal area of a stream, is the maximum period of seawater exposure that does not adversely affect survival (Helle et al., 1964). Also examined were differences between continuous and intermittent exposures to WSF. Some alevins were continuously exposed to 0.7, 15, and 24 rag/liter aromatic hydrocarbons in fresh water or in the tidal cycle, while others were exposed intermittently (3 h out of every 12 h), coinciding with the seawater phase of the tidal cycle. Survival was assessed daily during each test, and 10 alevins were periodically removed from each jar for analysis of tissue hydrocarbons.
52
Adam .~foles, Malin M. Babcock, Stanley D. Rice
TABLE 1 Average Concentrations of Selected Mono- and Diaromatic Hydrocarbons in 2.48 rag liter of the Watersoluble Fraction (WSF) of Cook Inlet Crude Oil. These compounds comprise 90-95% of the total aromatic hydrocarbons in WSF Component
Concentration (rag. liter )
Proportion (%)
Benzene Toluene o-Xylene m- and p-Xylene Ethylbenzene Naphthalene l-Methylnaphthalene 2-Methylnaphthalene
1.07 083 0.12 0.26 0-10 005 0-02 0-03
43.0 33-0 5'0 10.0 4.0 20 10 I-0
Sum
2-48
Whole body tissues were analyzed for the presence of 14 aromatic hydrocarbons, from o-xylene to dibenzothiophene, by gas chromatography and confirmed by mass spectrometry. Concentrations were reported as the sum of these compounds. After 30 days of exposure, 30 alevins were removed from each jar and preserved in 5% buffered formalin for 60 days. Weights and lengths were measured, and Barns' k o values (an index of fry development) calculated using the following formula (Barns, 1970):
10 (weight, in milligrams) 1/3 ko =
l e n g t h , in m i l l i m e t e r s
Higher kD values denote more girth and weight relative to length and
ES"WATER F'NTE M,TTENT O'L I O
,
I
/I
~;~.'~1 ;~.~i
I'~,r;%1 I ~:~!~
UOUSO,L
INTERMITTENT OIL TIDAL CYCLE CONTINUOUS OIL ~ I .
l
SEAWATER OIL WSF Fig. I.
0
3
6
;
1'2 115 HOURS
Experimenta! conditions.
118
211 24
Effects of oil exposure on pink salmon alevins
53
indicate an earlier development stage. As alevins develop and absorb yolk, the k~ values decrease. How completely yolk is absorbed by the time fry emerge from the streambed directly affects their survival. The optimum kz~ for emerging fry is 1-97 (Bailey & Taylor, 1974). Emerging fry with a large amount of yolk (as reflected in a k D > 1.97) are relatively poor swimmers and are therefore more vulnerable to predators. Conversely, fry that absorb all their yolk (k o < 1.97) before emerging are emaciated, weakened, and vulnerable to predators (Barns, 1970). Differences in kt) values between experimental groups were evaluated by using a two-way analysis of variance on the means.
RESULTS AND DISCUSSION Effects of tidal exposure
Alevins in the simulated tidal cycle were more sensitive to oil, had reduced yolk reserves, and accumulated more hydrocarbons than did alevins in fresh water. All alevins continuously exposed to 2.4rag/liter aromatic hydrocarbons died during the 30-day test: they survived only 6-7 days in the simulated tidal cycle and 7-16 days in fresh water (Table 2). Concentrations of 1"5 mg/liter total aromatic hydrocarbons did not kill any alevins, regardless of experimental conditions. Alevins exposed to _< 1'5 rag/liter in the tidal cycle had significantly lower kD's (P < 0-05, Fig. 2: Table 3) than did either controls or alevins exposed to the same TABLE 2
Survival (Days) of 5- and 60-day Pink Salmon Alevins Exposed to 2-4 rag/liter of the Water-soluble Fraction (WSF) of Cook Inlet Crude Oil before 100% Mortality Occurred. Alevins exposed to all other concentrations survived. N = 150 for each treatment Test condition
Continuous WSF: Tidal cycle Fresh water Intermittent WSF: Tidal cycle Fresh water
Surcical oj alecins 5-day
60-day
6 16
7 7
9 No deaths
10 No deaths
Adam 3ioles. Malin ,W. Babcock, Stanley D. Rice
54
140,
C
O
N
T
I
N
U
O
U
~
30
20
'S
~------© im r,, < u ©
---
0
OIL/TIDAL
~INTERMITTENT
"C..: INTERMITTENT OIL/FRESHWATER I
I
I
I
I
c~.o >,-r
~
60-DAY ALEVINS
30
/
OIL/FRESHWATER-/
/ ,NTER,,TTET , " \
10 /
/ 0
OIL/TIDAL / ~ 7 ,"
/
..... ¢---:=:---'LL___ / 5
[ 10
/ %~
~NTERMITTENT ~'.
v-- OIL/FRESHWATER
_ ~ _ ~ -
I l 1S 20 DAYS EXPOSED
.____._ - - . . I 25
30
Fig. 2. Mean Bams" k n values for 5-day and 60-day pink salmon alevins after 30-day exposures to the water-soluble fraction of C o o k Inlet crude oil. F W = freshwaterexposures: T C = e x p o s u r c s in a simulated tidal cycle, N = 3 0 . Control values were at 0 m g l i t e r . Confidence intervals ranged from 0.020 to 0'035.
concentrations in flesh water. As concentrations of WSF increased, the k o values decreased in both the freshwater and tidal groups. At the end of 30 days of exposure, control 60-day alevins in fresh water had a k D of 2.07, and those in the tidal cycle, 1-97 (Fig. 2). Controls held in fresh water had excess yolk reserves, whereas exposure to either oil or seawater speeded up development. Alevins exposed to oil in seawater had yolk reserves below optimum and therefore would have emerged from the gravel early or in poor condition. Alevins continuously exposed to the WSF in the tidal cycle
Effects o f oil exposure on pink salmon alevins
55
TABLE 3
Two-way ANOVA of Mean Barns k o Values for Pink Salmon Alevins Exposed to the Water-soluble Fraction of Cook Inlet Crude Oil for 30 Days
Source
df
SS
),IS
F
Total A (Concentration) B (Tidal) AB error
5-day alevins 11 0-01897 2 0"00382 t 0-00963 2 0.000 12 6 0"00540
0"001 9l 0"00963 0.00006 0-00090
2-12037 I0.703 70 * 0-06481
Total A (Concentration) B(Tidal) AB error
60-day alevins 11 0-026 60 2 0.01040 1 0-01203 2 0.00187 6 0-00230
0-00520 0.01203 0-00093 0.00038
13565 22 ** 31.39130"* 2-43478
* P < 0 " 0 5 ; * * P<0"01.
had two to six times higher tissue concentrations of aromatic hydrocarbons than did those continuously exposed in fresh water (Fig. 3). Alevin survival and growth were affected more by seawater-borne oil than oil in fresh water. This increased sensitivity to WSFs in seawater, which has been previously demonstrated with older juvenile salmon {Moles et al., 1979; Stickle et al., 1982), did not appear to be associated with a failure to osmoregulate (Stickle et al., 1982). Hydrocarbon uptake in alevins in the tidal cycle was much greater than in the alevins in fresh water and would appear to be the direct cause for the decreased survival and growth of alevins in seawater, even though the alevins were in seawater only 25% of the time. The increased accumulation of hydrocarbons in seawaterexposed alevins could be caused by increased drinking of seawater for osmoregulatory control. Thomas and Rice {1986) observed substantial retention of aromatic hydrocarbons and decreased metabolism of aromatic hydrocarbons in Dolly Varden char in seawater compared to char similarly exposed in fresh water. Seawater may have caused a decrease in metabolism and excretion of aromatic hydrocarbons, which led to greater hydrocarbon accumulation in alevins in seawater than in fresh water. Effects of age of alevins
In all exposures, 60-day alevins were more affected than 5-day alevins. During continuous exposures to 2-4 mg/liter, both age groups had similar
56
Adam Moles, Malin M. Babcock, Stanley D. Rice
2.20
S-DAY ALEVINS INTERMITTENT
2. 15 ~~"~'~"rW
FW CONTINUOUS
2,10
2.05 ~ E R M I T T E N T
2.00 l
~2-'° I
I ,o-oAY ALEV,NS
|
FW CONT,NOOOS
2.00
1.90
1.85
I
1
0.7
1
1.5 WSF (mg/I iter} Fig. 3. Tissuehydrocarbons present in pink salmon alevins during exposure to I-5 rag/liter of the water-soluble fraction of Cook Inlet crude oil. N= 10. Control tissue had < 3/~g/g aromatic hydrocarbons. survival in the tidal cycle. In fresh water, however, 5-day alevins survived longer than 60-day alevins (Table 2), probably because differing amounts of yolk influenced the rates of uptake and depuration. The ko values for 5-day alevins were generally higher during intermittent than continuous exposures, and they were similar for 60-day alevins in both types of exposures (Fig. 2). As the concentration of the WSF increased, the k o values were significantly lower (P<0-001) for 60-day than for 5-day alevins (Fig. 2). The 60-day alevins had lower k o values because they were growing faster and had less yolk than did 5-day alevins. In general, 5- and 60-day alevins accumulated similar concentrations of aromatic hydrocarbons, but the pattern of accumulation was different. The
Effects of oil exposure on pink salmon alevins
57
yolk-rich, 5-day alevins accumulated aromatic hydrocarbons throughout the study and, after 30 days, had higher concentrations of hydrocarbons than did the 60-day alevins. Maximum concentrations of hydrocarbons in tissues of 60-day alevins occurred after 13 days of exposure, then decreased by 50 to 95%, depending on the treatment, by the end of the test. The older alevins had consumed the majority of" their yolk by the end of the 30-day exposure (90-day-old alevins); there was very little of the lipid-rich yolk to trap and retain the absorbed aromatic hydrocarbons. Yolk has a high affinity for lipophilic hydrocarbons and retains them in semi-isolation from developing tissues where the hydrocarbons would be metabolized (Korn & Rice, 1981). This pattern of uptake and depuration is similar to that found in different ages of coho salmon, Oncorhynchus kisutch, alevins exposed to hydrocarbons in fresh water (Korn & Rice, 1981).
CONCLUSION Concentrations of aromatic hydrocarbons in seawater after short-term oil pollution can be higher than 0"5 ppm in areas where turbulence disperses the oil into the water column. For example, during the A m o c o Cadiz spill, Calder & Boehm (1981) found hydrocarbon concentrations that exceeded 1 ppm in the water entering the estuary and 0 5 p p m in the rest of the estuary. Admittedly our experimental concentrations are above those likely to occur in most spills; however, the combination of turbulent wave action--characteristic of the intertidal zone--and the chronic input of oil from coated beaches could cause concentrations within the range of those used in this study. It is here, in the intertidal zone rather than in fresh water, that pink salmon alevins, particularly those near emergence, would be adversely affected by exposure to oil.
ACKNOWLEDGEMENTS The tissues were analyzed by gas chromatography by the National Analytical Facility, Northwest and Alaska Fisheries Center, Seattle, Washington.
REFERENCES Bailey, J. E. & Taylor, S. G. (1974). Salmon fry production in a gravel incubator hatchery, Auke Creek, Alaska, 1971-72. US Dep. Commer., NOAA Tech. Memo. N M F S ABFL-3, p. 13.
58
Adam Moles, Malin M. Babcock, Stanley D. Rice
Barns, R. A. (I970). Evaluation of a revised hatchery method tested on pink and chum salmon fry. J. Fish. Res. Board Can., 27, 1429-52. Calder, J. A. & Boehm, P. D. (1981). The chemistry of Amoco Cadiz oil in the Aber Wrac'h. In: Amoco Cadiz, fates and effects of the oil spill, Proceedings of the International Symposium, Brest, France, November 19-22, 1979, 149-58. Hanavan, M. G. & Skud, B. E. (1954). Intertidal spawning of pink salmon. Fish. Bull., 95, 56, 167-85. Helle, J. H. (1970). Biological characteristics of intertidal and fresh-water spawning pink salmon at Olsen Creek, Prince William Sound, Alaska. 1962-63. US Fish. Wildl. Serv., Spec. Sci. Rep.--Fish., No. 602, p. 19. Helle, J. H., Williamson, R. S. & Bailey, J. E. (1964). Intertidal ecology and life history of pink salmon at Olsen Creek, Prince William Sound, Alaska. US Fish. WTldl. Serv., Spec. Sci. Rep.--Fish., No. 483, p. 26. Hunter, J. G. (1959). Survival and production of pink and chum salmon in a coastal stream. J. Fish. Res. Board Can., 16, 835-86. Korn, S. & Rice. S. (1981). Sensitivity to, and accumulation and depuration of, aromatic petroleum components by early life stages of coho salmon (Oncorhynchus kisutch), Rapp. P.-v. Reun. Cons. Int. Explor. Mer., 178, 87-92. Moles, A., Rice, S. D, & Andrews, S. (1985). Continuous-flow devices for exposing marine organisms to the water-soluble fraction of crude oil and its components. Can. Tech. Rep. Fish. Aquat. Sci., 1368, 53-61. Moles, A., Rice, S. D. & Korn, S. (1979). Sensitivity of Alaskan freshwater and anadromous fishes to Prudhoe Bay crude oil and benzene. Trans. Am. Fish. Soc., 108, 408-14. Noerenberg, W. A. (1963). Salmon forecast studies on 1963 runs in Prince William Sound. Alaska Dep. Fish Game Info. Leaflet 21, Juneau, Alaska, p. 27. Rice, S. D., Short, J. W. & Karinen, J. F. (1977). Comparative oil toxicity and comparative animal sensitivity. In: Fate and effects of petroleum hydrocarbons in marine ecosystems and organisms. (Wolfe, Douglas A. (Ed.)), Pergamon Press, New York, 78-94. Stickle, W. B., Sabourin, T. D. & Rice, S. D. (1982). Sensitivity and osmoregulation ofcoho salmon, Oncorhvnchus kisutch, exposed to toluene and naphthalene at different salinities. In: Physiological mechanisms of marine pollutant toxicity. (Vernberg, W. B., Calabrese, A., Thurberg, F.P. & Vernberg, F.J. (Eds)), Academic Press, New York, 331-48. Thomas, R. E, & Rice, S. D. (1981). Excretion of aromatic hydrocarbons and their metabolites by freshwater and seawater Dolly Varden char. In: Biological monitoring of marine pollutants. (Vernberg, F. J., Calabrese, A., Thurberg, F. P. & Vernberg, W. B. (Eds), Academic Press, New York, 425-48. Thomas, R. E. & Rice, S. D. (1986). The effects of salinity on uptake and metabolism of toluene and naphthalene by Dolly Varden, Sah:elinus mabna. Marine Ent'iron. Res., 18, 203-14.