Sensitivity differences between eggs and larvae of walleye pollock (Theragra chalcogramma) to hydrocarbons

Marine Environmental Research 26 (1988) 285-297

Sensitivity Differences Between Eggs and Larvae of Walleye Pollock (Theragra chalcogramma) to Hydrocarbons Mark G. Carls & Stanley D. Rice Northwest and Alaska Fisheries Center Auke Bay Laboratory, National Marine Fisheries Service, NOAA, PO Box 210155, Auke Bay, Alaska 99821, USA (Received 12 April 1988; revised version received 1 June 1988; accepted 3 November 1988)

ABSTRACT Exposure of embryonic and larval walleye pollock (Theragra chalcogramma) to the water-soluble fractions ( W S F ) of Cook Inlet crude oil during embryonic development caused mortality and a variety of morphological abnormalities. Median WSF concentrations ( l'8ppm) which caused mortality after hatch did not differ significantly from those causing morphological abnormalities (1.6 ppm ). Pollock larvae exposed to WSF did not develop abnormalities, but swimming and survival were significantly affected within 4 h of exposure. The LCsofor larvae droppedfrom l'9 ppm on day 4 to 0.9ppm on day 10. The tissue of developing pollock larvae bioaccumulated significantly more dissolved toluene and naphthalene than those of embryos. Consequently, when exposed to equal concentrations of WSF, larval tissues are exposed to much higher hydrocarbon concentrations than embryonic tissues. On the basis of internal tissue concentrations, larvae were less sensitive than eggs, but the opposite conclusion is reached if external ( W S F ) concentrations are usedfor estimation of sensitivity.

INTRODUCTION

Walleye pollock (Theragra

chalcogramma), an a b u n d a n t species in the N o r t h Pacific Ocean, comprised 20-50% o f the standing stock o f demersal 285 © 1989 u s Government

286

Mark G. Carls, Stanley D. Rice

fish in the Bering Sea at the time of this study (Smith, 1981). Walleye pollock represent up to 75% (by weight) of the total annual fisheries products from the Bering Sea (Dagg et al., 1984); in 1985 the commercial catch (22 959 t) was valued at $2.1 million (Anon., 1985). Because the population of walleye pollock is large, it forms an important part of the food web. Walleye pollock prey on zooplankton and fish, and are a major prey species for other fish, pinnipeds, cetaceans, and birds (Smith, 1981). Walleye pollock spawn pelagic eggs in nearshore waters, and therefore the early life stages may be exposed to the water-soluble fraction (WSF) of oil spilled during offshore oil exploration, production, or transportation. Larvae are usually the most sensivite stage to WSF in the life history of fish (Moore & Dwyer, 1974; Cads, 1987). Pelagic fish eggs and larvae, such as walleye pollock, are generally very sensitive to the WSF of dissolved oil (Rice et al., 1979). The epipelagic habitat of planktonic eggs is also unusually susceptible to spill impacts because freshly spilled oil is usually most concentrated in the surface layers (Kuhnhold, 1970) where many icthyoplankton occur. Embryonict sensitivity to external stressors, such as WSF, may be greatest very early in development when damage to a few precursor cells will result in extensive damage as the embryo develops (Rosenthal & Alderdice, 1976; Longwell, 1977; Sharp et al., 1979). However, studies often indicate fish eggs are more resistant than larvae to dissolved petroleum hydrocarbons (Kuhnhold, 1970; Struhsaker et al., 1974; Rice et al., 1975; Middaugh & Dean, 1977; Moles et al., 1979; Rice et al., 1987). This discrepancy may occur because of differences in the bioaccumulation of hydrocarbons by eggs and larvae: embryonic tissues may accumulate substantially less hydrocarbon than tissues when exposed to an equal WSF concentration. The objective of this study was to measure and compare the sensitivities of walleye pollock eggs and larvae exposed to the WSF of Cook Inlet crude oil, and the bioaccumulation of hydrocarbons by them. Four criteria were used to determine sensitivity to oil: medial lethal concentrations (LCso), median concentrations causing abnormalities (ABso), hatching success, and swimming inhibition. METHODS Adult walleye pollock were collected 23 March, 1982 near Juneau, Alska, by trawling. Eggs and milt were stripped from one male and one female fish, mixed in a beaker without water, then transferred to 15-cm-dia. x 20-cm-tall t In this paper, the term 'embryonic'refers to the period betweenfertilizationand hatching, and the term 'larval' refers to the period betweenhatching and yolk resorption.

Walleye pollock eggs and larvae sensitivity to hydrocarbons

287

cylindrical glass jars filled with either 2-5 liters of filtered seawater (controls and delayed treatment groups), or seawater containing the WSF of Cook Inlet crude oil. After a 2-h water hardening period, all eggs were transferred to seawater or seawater plus WSF. Water temperatures were constant at 3.9 + 0.1°C; day length was 12.5 h preceded and followed by 15 min of dim, indirect light. Tanks were cleaned by siphon as necessary to remove debris and bacteria. Larvae were not fed during the tests. All bioassays were static, designed to mimic a single event oil spill. The WSF of Cook Inlet crude oil was produced by dripping seawater through a continuously replenished 40-cm emulsified layer of oil; resulting WSF was collected below the layer after dispersed oil floated out (Moles et al., 1985). WSF concentrations were monitored daily by removing 100 ml samples and extracting them with 20 ml of dichloromethane in two 10-ml steps with 30 and 15 min separation times. Extracted hydrocarbons were analyzed with fluorescence spectroscopy (290nm excitation, 340nm emission peak) (Gordon & Keizer, 1974) calibrated with glass capillary column gas chromatography (GC). The rates of loss of monoaromatic and diaromatic hydrocarbons from solution were analyzed directly by GC using the same extraction procedures (Rice et aL, 1987). Aromatic hydrocarbons with more than two rings were not detectable in the WSF; therefore, concentrations are reported as the sum of mono- and diaromatic hydrocarbons (see Table 1). Five treatment groups were tested to determine sensitivity differences between developmental stages: 0-2 h (fertilization through water hardening), 0-21 days (fertilization through hatch), 1-21 days (morula through hatch [added the following year on May 4 using another male and female to compare the ABs0 with the LCso]), 7-21 days (11-14 somite stage through hatch), and 2 4 4 4 days (newly hatched larvae through yolk absorption). Larvae that hatched from eggs exposed to WSF were placed in clean seawater and observed through complete yolk absorption (about 19 days). There were six WSF doses per experimental group (including controls) with three replicate exposures per concentration. Sample sizes ranged from 49 to 101 eggs per replicate, and from 38 to 43 larvae per replicate. The maximum biomass did not exceed 0-1 g/liter. Initial WSF concentrations ranged from 0 ppm (controls) to 4 ppm (Table 2). Hydrocarbon uptake rates were determined using 3H toluene (1.425 ppm) plus 14C naphthalene (0.075 ppm) dissolved in seawater. Conditions were static. Uptake exposure duration ranged from 10 rain to 3 h. Eggs fertilized 48 h prior to exposure (6 replicates with c. 30 eggs per replicate) were exposed at 3-0 + 0-5°C (specific activities were 452.6 mCi/mmole for naphthalene and 24.9 mCi/mmole for toluene). Larvae were exposed 3 days after hatch (7 replicates with c. 20 larvae per replicate) at 4.0 + 0.5°C (specific activities were 397"0 mCi/mmole for naphthalene and 27.9 mCi/mmole for toluene).

288

Mark G. Carls, Stanley D. Rice

TABLE 1 Distribution of Individual Aromatic Hydrocarbons in Water-soluble Fraction (WSF) of Cook Inlet Crude Oil, during an 18-day Static Exposure. Concentrations are Means of the Same Three Replicates Measured on Three Occasions by Gas Chromatography

Compound

0 days

Benzene Toluene Ethylbenzene m- and p-xylene o-xylene Mesitylene Naphthalene 2-methylnaphthalene 1-methylnaphthalene 2,6-DMN a 1,4 DMN a 1,2 DMN a Total mononuclear aromatics Total dinuclear aromatics Total mono- and dinuclear aromatics Per cent total hydrocarbons remaining

4 days

~m

%

~m

1.424 0.845 0.007 0"125 0.077 0.004 0"292 0.000 0-008 0"000 0.004 0-006 2.482 0"310

51.0 30-3 0-2 4-5 2.8 0"1 10"5 0.0 0-3 0-0 0"1 0-2 88-9 11-t

2.792

100.0

0.808 0.432 0-000 0.060 0.041 0.000 0.243 0.000 0-007 0-000 0.003 0-004 1.341 0.257

18 days %

50.6 27.0 0.0 3.7 2.6 0"0 15.2 0.0 0.4 0"0 0'2 0"3 83.9 16.1

1.598 100.0

100.0

~m

%

0.057 0.000 0.000 0.000 0-000 0.000 0"153 0.000 0.000 0.000 0.002 0.004 0.057 0.159

26.4 0-0 0-0 0-0 0.0 0'0 70.8 0.0 0.0 0"0 0"9 1.9 26.4 73'6

0.216

100.0

57'2

7.7

a DMN = dimethylnaphthalene.

TABLE 2 Initial WSF Concentrations in ppm for Each Treatment Group

Concen tration

Treatment 0-2h

0-21 days mean

Control Low Low Low Low Maximum

0-0 0-4 0-6 1'5 2"2 3"0

0-0 0-2, 0-6, 1"6, 2-3 3-6,

SE

0-00 0"08 0-09

1-21 days

7-21 days

Larvae

mean

SE

mean

SE

mean

SE

0'0 0.4, 0'8, 1'5, 2"2, 2'8,

0'01 0"02 0"01 0"12 0.10

0"0 0-3, 0-7, 1"8, 2-8, 3.1,

0"02 0"10 0"20 0-04 0"13

0-0 0-6, 1"3, 2"1, 3"2, 4.0,

0.04 0-10 0-04 0-28 0-29

Walleye pollock eggs and larvae sensitivity to hydrocarbons

289

Replicate samples (2-3 at each observation time) were rinsed with seawater, counted, and oxidized i to 1-5 min in an ash-free burning cup. Radioactivity was measured by liquid scintillation and corrected with an external standard (Gharrett & Rice, 1987). Data were transformed to tissue concentration (ppm) and bioaccumulation (concentration of each toxicant in tissues divided by concentration of toxicant in exposure water). Depuration of hydrocarbons by eggs and larvae was determined by exposing them to 3H toluene plus X*C naphthalene for 3 h (as above), and then transferring them to clean seawater. Eggs (9 replicates with c. 30 eggs per replicate) and larvae (9 replicates with c. 20 larvae per replicate) were monitored for 4 days after exposure. Samples were analyzed by liquid scintillation as described previously. All animals were observed without disturbance, except that newly hatched larvae were observed microscopically during transfer to clean water. (All larvae which hatched during egg tests were transferred to clean seawater within 12 h of hatch.) Biological parameters quantified (by count) were percent eggs fertile, hatch, abnormal, and dead. Larvae were also scored for swimming ability (swimmers, inhibited swimming, or nonswimming). Median concentrations causing death (LC50), swimming inhibition (ECs0), or abnormalities (ABso) were determined with logit analysis (Berkson, 1957) or with Spearman-Karber analysis (Hamilton et al., 1977) when necessary. Correction for control response (Abbott, 1925) was applied when needed. Internal (tissue) LCsos were calculated from external WSF LCsos multiplied by either pure compound bioaccumulation factors or factors weighted by the observed naphthalene to toluene uptake ratio. ANOVA procedures were followed with Tukey's (1949) a posteriori multiple comparison test at the 95% confidence level to test differences between groups, or Scheffe's (1953) test to compare several groups simultaneously. Reported values are averages of replicate tests. Sample error is reported as standard error (SE) or as + 95% confidence, and r is the sample correlation coefficient.

RESULTS Test conditions

Monoaromatic hydrocarbons were predominant in the static WSF tests initially (89%), but declined significantly more rapidly than diaromatic concentrations (P < 0.001), and after 18 days comprised only 26% of the remaining hydrocarbons (Table 1). The rate of total aromatic hycrocarbon

Mark G. Carls, Stanley D. Rice

290

loss from solution was not linear ( P = b~/t + a, where P - - p e r cent remaining and t = time in days); approximately half of the hydrocarbon was lost in the first 10 days. Hydrocarbon concentrations reported in this paper are based on initial values. Biological relationships between walleye pollock developmental stage and hydrocarbon exposure were not changed when calculations were based on mean WSF concentrations.

Egg exposures Pollock eggs were significantly affected by exposure to WSF in all treatments except the briefest exposure (i.e. 0-2h: fertilization through water hardening). Effects on eggs in the 0-2 h treatment were generally negligible with egg mortality increasing slightly (by 4%) with WSF concentration (0.01 < p < 0.025, r = 0"7). Egg survival and hatching success were slightly reduced (maximum reduction was 7% -I- 5% at 3-6 ppm), and correlated moderately with dose (r = -0"3 to -0"6). Survival and hatching hatching success were similar for the 0-21 day and 7-21 day treatment groups. At hatching, embryos exposed to WSF had a variety of morphological abnormalities which correlated strongly with dose (r = 0-9 [logit transformation]). Up to 100% of the exposed embryos were affected in the 0-21 day and 7-21 day treatments (Fig. 1). Median concentrations causing abnormalities (AB~o) at hatch were 2.1 ÷ 0.4 ppm and 2-1 _ 0.6 ppm for the 0-21 day and 7-21 day treatments, respectively. Embryonic abnormalities included formation of membranous vesicles, deformations of yolk, eye, brain, intestine, jaw, and pericardial sac. After hatch, abnormalities 100

-~

80-

.E 70

/

/

/

/

,/

/

,-

60

e'~

0-21

'~

50

® P

40

a.

30

7-21 d

,/

2o

10

~

0

O-2h ,

0

, 1

,

~

,

2

<~, 3

W S F Concentration ( p p m )

Fig. I.

Percentage of embryonic abnormalities at time of hatch plotted against dose. Vertical bars are _ 1 SE.

Walleye pollock eggs and larvae sensitivity to hydrocarbons

291

generally became more pronounced as developing structures failed to form properly. Mortality after hatch (measured only in the 1-21 day treatment) was strongly dose dependent and correlated with embryonic abnormalities (r = 0"9). The post-hatch LCso stabilized at 1.8 + 0.6 ppm, and did not differ significantly (0.10 < p < 0.25) from the AI35o (1.6 _ 0.2ppm). (The ABso concentration determined in this treatment differs from those reported above, possibly because of slight differences in methodology or genetic composition of the exposed eggs.) Larval exposures Walleye pollock larvae were quickly and adversely affected by the WSF, but exposures did not cause morphological abnormalities. Swimming ability was affected most rapidly, and necrosis preceded death (Fig. 2). After 4 h of exposure, in the upper 4 doses ( > 2.1 ppm) larval swimming was significantly inhibited, and 22% were necrotic or dead (versus 5% of controls). Mortality at 3.2 ppm (29%) was significantly greater than control mortality (4%) after 5 days, and in all doses (> 0.6 ppm) within 13 days. (Control mortality was < 10% for the first 14 days.) LCso dropped from 1"9 + 0"9 ppm on day 4 and stabilized at 0"9 + 0.5 ppm on day 10 (Fig~ 2). Hydrocarbon uptake and depuration Pollock eggs bioaccumulated significantly (P<0.001) less radio labeled toluene and naphthalene (7-8 times) than larvae (Table 3). Eggs equilibrated 10 ~"~ ) " -

[]

8

"Q

[]

Mortality

<3--© .ocrosi,

[]

",

<~

~

Swimming

4

0

,

0

,

2

,

,

4

,

6

8

10

12

14

16

18

DAYS

Fig. 2. Larvalresponse to WSF exposure measured by mortality (LCso), necrosis (ECso), and swimmiiaginhibition. Data were calculated with Iogit techniques: points greater than 4 ppm are extrapolations.

292

Mark G. Carls, Stanley D. Rice TABLE 3

Maximum Bioaccumulation of Radioisotopes by Eggs and Larvae Exposed to Dissolved Hydrocarbons Bioaccumulation

Eggs Larvae

Ratio

Toluene ~, SE

Naphthalene Y~ SE

Naphthalene/ toluene

1'2, 0"05 8.3, 0-06

6"1, 0"05 51.3, 2.20

5'0 6.2

7.0

8.4

Ratio of Larvae/eggs

with external hydrocarbon concentrations within 3 h; larvae reached equilibrium somewhat more slowly (Fig. 3). Naphthalene was bioaccumulated 5-6 times more than toluene (Table 3). Declines in dissolved hydrocarbon concentrations (4%) during the test were not significant. Walleye pollock eggs and larvae depurated hydrocarbons rapidly in clean seawater (59-83% was lost from the tissues in first 8h), but complete depuration took approximately 2 to 4 days (Fig. 3). Comparison of eggs and larvae

Based on external (WSF) hydrocarbon concentrations, walleye pollock larvae were significantly more sensitive than developing embryos: mean Uptake 12•

Depuration ~

! ,

'0"

~

6

I,-

4

"

4

I

LIJ. . . . ]

~ 2 !

0.5

1,0

1.5

Hours

Fig. 3.

2.0

2.5

0

10

20

30

40

50

Hours

Uptake and depuration of radiolabled toluene (all) and naphthalene (14C) by pollock eggs and larvae. Vertical bars are _+1 SE.

Walleye pollock eggs and larvae sensitivity to hydrocarbons

293

TABLE 4 Comparison of Egg and Larval LCs0 s, Based Either on Hydrocarbon Concentrations in Water or in Tissue. Tabulated Values are Means (ppm) with Standard Errors: n = 3 for Each Treatment Mean. Differences were Tested with ANOVA, using Square Root Transformations to Control Variance, Followed by Scheffe's a posteriori Multiple Comparison Test at the 96% Confidence Level Measurement

Eggs 0-21 days

In water (ppm) In tissue (ppm): weightedc based on naph based on tol

mean

SE

2.4

0.10 b

12-8 0"56 14.8 0.64 3"0 0"13

1-21 days mean

Larvae 7-21 days

All

22-41 days

SE

mean

SE

mean

SE

mean

SE

1.8

0-13

2.4

0-17b

2.2

0.19

0.9

0.06a

9"6 11"1 2"2

0'70 0-81 0.16

1 2 . 5 0"90 14"5 1.04 2-9 0"21

11'6 13.4 2-7

0 - 6 3 4 1 - 3 5.10a 0'73 47'0 3-24a 0"15 7'6 0-52a

Larval treatment(s) significantly different from egg treatments (p < 0"05). b Calculated from the ABso by dividing by the AB50/LCs0 ratio determined in the 1-21 day treatment (0.885, SE = 0-077). c Based on weighted mean toluene and naphthalene uptakes. Weighting factor was naphthalene/toluene ratio. a

LCsos based on external concentrations were 0-9 +_0.1 ppm for larvae, and 2 . 2 _ 0.2ppm for eggs (Table 4). However, embryonic tissues were significantly more sensitive when measured by internal tissue concentrations because eggs bioaccumulated much less hydrocarbon than the larvae: LC s os based on weighted internal concentrations were 41 _+ 22 ppm for larvae and 12 +_ 1 ppm for eggs (Table 4). Results were consistent when the ABso was assumed equal to the LCso in tests that did not measure post-hatch mortality. DISCUSSION Larvae responded to the WSF of Cook Inlet crude oil much differently than embryos. Embryos developed severe abnormalities, but larvae did not. Larvae were rapidly killed by the WSF, but exposed embryos generally did not die until after hatch. Walleye pollock larvae accumulated much more hydrocarbon than the eggs did. This result parallels results obtained with a similar species: Atlantic cod (Gadus morhua) larvae exposed for either 0.1 or 1.0 days accumulated slightly more naphthalene, and much more phenanthrene, benzo[a]pyrene, and 2,4,5,2',4',5'-hexachlorobiphenyl, than eggs (Solbakken et al., 1984).

294

Mark G. Carls, Stanley D. Rice

A minimum exposure time is apparently necessary to adversely affect development. Exposure to WSF for 2 h during water hardening had a negligible effect, even though the tissues within these eggs were exposed to hydrocarbons. Fertilization was not affected, but in our tests, was probably completed within a few seconds after exposure began. Linden (1978) observed that fertilization of Baltic herring (Clupea harengus) was not affected by exposure to WSF. Tissue abnormalities were the most prominent effect of exposure of pollock eggs to WSF hydrocarbons. Abnormal larvae hatched from exposed eggs have a reduced survival potential because establishment of feeding and predatory avoidance are critical to early survival (Rosenthal & Alderdice, 1976). For example, Aronovich et al. (1975) demonstrated that significantly fewer Arctic cod (Boreogadus saida) larvae with yolk sac deformities feed than healthy controls. Larvae with twisted spinal columns (an abnormality very frequent in our study) could only swim in circles, and therefore had obviously reduced prey capture and predator escape abilities. Sensitivity of pollock eggs to WSFs did not change significantly with age. This contrasts with results of some other investigations. For example, lipidrich eggs can sequester hydrocarbons, so they are not immediately available to the developing embryo and consequently sensitivity to hydrocarbons tends to increase with yolk absorption and embryonic age (Kuhnhold, 1970; Ernst & Neff, 1977; Moles et al., 1979; Eldridge et al., 1982). On the other hand, permeability of the egg membranes may decrease over time, and therefore embryonic sensitivity can decrease with egg age (Sharp et al., 1979). A discrepancy between stage of development and sensitivity to hydrocarbons is found in the literature. Many researchers have observed that, based on external (WSF) hydrocarbon concentrations, larvae and young fry are more sensitive than eggs (Kuhnhold, 1970; Struhsaker et al., 1974; Moles et al., 1979). This contradicts other research which indicates that sensitivity may be greatest early in development when damage to a few precursor cells will result in more extensive damage (Rosenthal & Alderdice, 1976; Longwell, 1977). Our study explains this discrepancy by demonstrating that under equal external concentrations, embryonic tissues are exposed to much lower internal hydrocarbon concentrations than larval tissues, and therefore embryonic tissues are more sensitive. Embryonic tissues are probably more sensitive because the rapid cellular differentiation and rapidly changing topological, structural, and functional relationships between cells and cell systems are more easily disturbed by external influences than larval tissues. Indeed, exposure to WSF caused deformation of embryonic tissue but not of larval tissue. Others have also noted this lack of larval deformation (Struhsaker et al., 1974). Developing embryonic tissues are more sensitive than larval tissues to

Walleye pollock eggs and larvae sensitivity to hydrocarbons

295

internal hydrocarbon contamination, but larvae may be more susceptible to brief exposures. Eggs were exposed 5 days before any effects were discernible, but larval swimming was affected within 4 h (WSF > 2 ppm) and > 8% had already died. Young larvae are poor swimmers and therefore have little or no more ability to avoid contaminated areas than eggs. However, larvae may be more capable of recovery than eggs (Struhsaker et al., 1974) because larval tissue structures are not permanently disrupted like embryonic tissues. Large, single event oil spill disasters can release sufficient quantities of hydrocarbons which may remain in the environment long enough to cause abnormalities and death in pelagic fish eggs. For example, oil slicks from the Argo Merchant spill persisted on Nantucket Shoals for approximately 1 month (Longwell, 1977). One month after a 2000 ton spill of Iranian crude oil, hydrocarbon concentrations (0-2-0.3 ppm) were detected in seawater about 30km from the spill site (Grahl-Nielsen, 1978). Concentrations of hydrocarbons in seawater can reach concentrations high enough to impede the swimming of larval pollock and cause mortality: after the Amoco Cadiz spill, water entering the Aber Wrac'h estuary contained more than 1.0 ppm hydrocarbons, and hydrocarbon levels within the estuary averaged 0.5 ppm (Calder & Boehm, 1981). Spills in prime spawning areas, such as Nantucket Shoals, at the time of spawning would probably have disproportionately large influences on spawning populations, although impacts would be reduced on species (such as walleye pollock) which spawn over wide geographic areas.

ACKNOWLEDGEMENTS We thank Chris Brodersen and Jessie Gharrett for conducting the radioisotope experiments presented in this paper, and Jeff Short for the analyses by gas chromatography.

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Rice, S. D., Moles, D. A., Taylor, T. L. & Karinen, J. F. (1979). Sensitivity of 39 Alaskan marine species to Cook Inlet crude oil and No. 2 fuel oil. In Proceedings of APL EPA, and USCG, 1979 Oil Spill Conference (Prevention, Behavior, Control, Cleanup). Washington, DC, API. pp. 549-54. Rice, S. D., Babcock, M. M., Brodersen, C. C., Carls, M. G., Gharrett, J. A., Korn, S., Moles, D. A. & Short, J. W. (1987). Lethal and sublethal effects of the watersoluble fraction of Cook Inlet crude oil on Pacific herring (Clupea harengus pallasi) reproduction. NOAA Tech. Memo., NMFS F/NWC-111.63 pp. Rosenthal, H. & Alderdice, D. F. (1976). Sublethal effects of environmental stressors, natural and pollutional, on marine fish eggs and larvae. J. Fish. Res. Board Can., 33, 2047-65. Scheffe, H. (1953). A method for judging all contrasts in the analysis of variance. Biometrika, 40, 87-104. Sharp, J. R., Fucik, K. W. & Neff, J. M. (1979). Physiological bases of differential sensitivity of fish embryonic stages to oil pollution. In Marine Pollution: Functional Responses. ed. W. B. Vernberg, A. Calabrese, F. P. Thurberg & F. J. Vernberg. Academic Press, New York, pp. 85-108. Smith, G. B. (1981). The biology of walleye pollock. In The Eastern Bering Sea Shelf." Oceanography and Resources. ed. D. W. Hood & J. A. Calder. University of Washington Press, Seattle, 527-51. Solbakken, J. E., Tilseth, S. & Palmork, K. H. (1984). Uptake and elimination of aromatic hydrocarbons and a chlorinated biphenyl in eggs and larvae of cod Gadus morhua. Mar. Ecol. Prog. Ser., 16, 297-301. Struhsaker, J. W., Eldridge, M. B. & Echeverria, T. (1974). Effects of benzene (a water-soluble component of crude oil) on eggs and larvae of Pacific herring and northern anchovy. In Pollution and Physiology of Marine Organisms. ed. F. J. Vernberg & W. B. Vernberg. Academic Press, New York, pp. 253-84. Tukey, J. W. (1949). One degree of freedom for no-additivity. Biometrics, 5, 23242.