The Sea-surface Microlaver of Puget Sound: Part I. Toxic Effects on F’ish Eggs and Larvae

John Hardy.“* Steven Kiesser.” Liam Antrim,a Alan Stubin.” Richard Kocanb & John Strand’

“ Battclle Marine Research Laborator). 439 \Vcst Scquim Bay Rd.

Sequim. Washington 98352. USA

h School of Fisheries, Unibersity of IVashington. Seattle. IVashington 98195. L’S;\

(Rcccived 7 January 1957: revised version received 30 March 1987:

accepted I Scptembcr 1987)

A BSTR.4 CT

The sea .surjircc i.s at2 ittlportutlt huhitar fk llw tieccdoptt~enrcrl stages (eggs rmi irmue) oftnun~~,~i.sb utd inwrrehrares: ir is a1.w N cotwentrufiotl point for

ati~lvwpogcnic c~0tilirttiitrcitit.s etrrcritlg rhe seu. S~~rtiie.s Lowe cotirhtcreti lo detcrtnitie rllc cs1ctt( lo \c~hich (lit seu .wrfke of Piigel Sound n’as toxic to Ihe euri~ ii/e hi.stor>~ stuges of Jislt. Three ltrhun ha.ss with .sll.spcc~eti cotitaniittritioti. u rwul rcftwnw t7a.k.. uttd u Cenlrul So~ind sire ww

compared. Siir~bce-ti~t~ciii~~g eggs utrti 0rgtrtli.vtt.s ( -_oonelr.ston ) lc‘ere coliecreci \c.ifh a surfiicc-.sX-ittitriitig tieikstoti ticl utiri rlieir ciensiries t~nwmwtt~ci. Sand .sole (Psettichthys melunostictus) rmhr>~o.s \c’ere exposed in the field and iuhorutor~~ 10 rile sea-.sut$rce ttlicroltr~w. TO riecriop u Lrsejirl !.eur-rod upproucli fo nioniroring .scu-arrfirce rosicir!.. lutwl riewlopttwnt ofuticho~ies. kelp bass, and sect urchins \cus ulso ecuiwreri u.s un indicu~ion of sea-nrrjhce

ttiicroiu~er ro.ricirJ.. Dwing the spawning season ( Fehrmr>, utd Murch ), urban hu?,.s it1 PLtger

Sound huci io\l,er concenlrutions oJ‘satd soie e,, vv.s undntwsrotiic organisms 011 rile seu .nrt$ce thun did Ilie rwui hcij. or Centrui Sowxi referewe sires. Conrpured 10 the refrrence sites. iuhorutorj, e.yto.wre 10 surjkce ttiicroiuj,et .sunipie.s coliecreti from w-hari ha>. sires getierail~ resulted iti more chromo.sottlul uherrurions in deceioping sole enlhr>~o.s. twhrceti hatching .siicce.s.s of .soie iurrue. atid redwed growrh in troul cell cultures. In situ hunching .~~~cce.ss of’ sole eggs HUS reduced h!. half or more it1 w+atl ba>,.s compureci lo rc>ference sires.

* Present address: General Scicncc Department. Oregon State University, Conallis, Oregon

97330. USA.

/Marine Enciron. Rex 013 I-I I36!871SO3.50 c 1987 Elscvicr Applied Science Publishers Ltd,

England. Printed in Great Britain

IYTRODC:CTION

The sea surface represents a highly productive, metabolically active interface and a vital biological habitat. Microneuston inhabiting this layer include bacteria (Carlucci it rrl., 195% tintinnids, small ciliates (Zaitsev, 1971) and microalgae (Souza-Lima, 1982: Hattori er crl., 1983; Hardy & Apts, 1984). Neustonic copepods and larger organisms feed on the high densities of such microneuston (Zaitsev, 197 1).

The sea surface also has economic \xlue because of its importance as a nursery ground for pelagic eggs and larvae. Numerous species of fish, including cod, sole, Hounder, hake. anchovy, mullet, flying fish, greenling, saury, rockfish, halibut and many others (Zaitsev, 1971; Ahlstrom & Stevens, 1975; Breiver, 198 I: Safronov, 198 1; Kendall & Clark, 1982) have surface-dwelling cob w or larval stages. Crab and lobster larvae in estuarine, coastal, and shelf areas also concentrate in the surface film during midday as a result of positive phototasis (Smyth, 1980; Jacoby, 1982; Provenzano c’t rrl.,

1983). These measurements of neustonic egg or larval abundance are based on

neuston net tows, which generally sample the upper 5- to 20-cm IGyer. However, in normal seawater salinities (greater than 30%) most pelagic fish eggs, because of their high lipid content, float directly in contact with the surface microlayer (SMIC) (upper 50 {tm) (Zaitsev, 197 1). As Zaitsev (197 1) noted, ‘... most of the eggs must rise to the surface film and remain suspended there. Although the specific weight of the egg increases during development, it remains low enough to hold the egg close to the surface film

. . . and surface enrichments of organisms were found to be stable even at sea states of 5 to 6 Beaufort.’

In Puget Sound, English sole (Parophrrss cetrtlus) and sand sole (Psctticlt~h~~s nwhostictrrs) spawn between January and April, releasing trillions of eggs that collect on the ivater surface. The embryos float (Fig. 1) until hatching occurs, generally 6 to 7 days after fertilization (Budd, 1940 and personal observation). Because of the buoyancy of their large yolk

Fig. 1. Sole eggs Roating in contact bvith the sra-surtcic~ microla!:r.

sacs, newly hatched larvae of both species often float upside-down at the surface of the water (Budd, 1940). Within a salinity range of90 to 37o/ou and a

temperature range of65 to IO’C about 90% of the fertilized egg hatch into normal live larvae. The salinity of neutral buoyancy ri?;Fts from 17.Soo0 ;tt fertilization to 30.8% at hatching (Alderdice & Forrester. 1968).

The surface microlayer (SMIC). also sert’es ~1s. ;t concentration point for many contaminants entering the sea. A large portion of these contaminants associate with suspended particles and deposit in the bottom sediments. However, contaminants that have low vvater solubility or that associate with floatable particles concentrate in the SIMIC. Concentrations of potentially toxic PAHs, PCBs, and metals. orders of magnitude greater than LS Environmental Protection Agency water quality criteria standards. ha\,e been found in the SMIC of Puget Sound (Hardy cf ~1.. 1985r. 19%). Chesapeake Bay (Hardy t’f al., 1987) and elsewhere (rev,iew in Hardy. I%?).

Fish eggs are sensiti\e to environmental toxicants ~Purdom & CVoodhead. 1973: Kligerman t’f ~71.. 1975: Longwell. 1976). Abnormalities in embryos and larvae have been found following laboratory e.xposure to petroleum hydrocarbons (.lnderson. 1977; Kern 51 Rice. 19S 1). metAs (&‘esternha,oen c’! ~1.. 19791 and pesticid<s (Dethlefzn. 19‘11. Despite the demonstrated importance of the sea surface as both ;t vital nursery area and a concentration point for contaminants. spati;tl distributions or temporal trends in surface contaminants. or their impacts on the reproduction of \ziuable marine species, remain unkrwsn.

Our studies wre intended as a tirst step to determine. at a few sites and times: (1) the densities of neuston. including fish eggs: 17) the lethal and sublethal tosic effects of the SYIIC in the laboratory and it1 .tittr: and 13) the degree of association of toxicity with contaminated urban bay areas and ivith visible sea-surface films or slicks. Because sole eggs are available for toxicity tests for onI>- about 3 months of the year. ue also undertook to develop another (surrogate) toxicit!, test that could be used routineI> throughout the t’ear for monitoring trends in SMfC toxicity.

k3ETHODS

Neuston densities

Samples were collected by neuston net tobvs and vertical, plankton net toivs (both having a 300-~m mesh) near the gencrral center of the area at two

rcfcrwce sites (Sequim Bay and Central Sound) and at two urban-bay site’s (Elliott Bay and Commencement Bay) (Fig. 7) once per month between February and May. 19X5. The neuston net. attached to a frame Lvith Iloats. had a mouth opening %-cm \\.icle and tit o tiers that simultaneously collected the O- to J-cm and the 3 to 43-cm la~,.ers of the bvater surface. Tow volumes (6 to 19 m3) bt’ere recorded by an attached How meter. Samples were preserved in 551 formalin. In each sample the number of individuals of 1 1 major taxonomic dikions (fish e ggs. copepods. barnacle larvae. tunicates. meduw, prosobranch larvae, decapod lar\xe, chaetognaths, polychaete larl ae. amphipods and miscellaneous organisms) uere counted using an invcrttxi compound plankton microscope. Densities (organisms m _ “) ivere computed from the original net tow \.olumes. Site-to-site comparisons of t:lw abundances Lt’ere made using an IBM-PC cluster analysis program ISq’stat. 1981) Lvith Euclidean distance as the measure of separation.

Ziicrolayer colitction

During 1985. sea-surface microlayer sampies were collected from 13 prcselrcted stations in Puget Sound (Fig. 3) for use in laboratory toxicity

‘3 i

tests. At each station. a sample of the

plate microlayer sampler (Harve)- & Briefly. samples of the upper 50/m dippiny of a glass plate and removed

SMlC was collected using the gl:ljs- Burzell, 1972; Hardy C[ LL 1985b). \vere collected b> repeated vertical by a silicon squeegee. Samples were

collected at the pre-determined station. regardless of the degree of visible surface slicks. The surface pressure !an indicator of the presence ofsurface- active organic films) !vas measured at a minimum of fix poinrs at each station using the spreadin g oil method (.Adam. 1937). This measurement pro\+des it semi-quantitati\-e measure (dynes cm-‘) of surface pressure. Higher surface pressure is indicative ofthe presence of surface acti\-e organic compounds and generally confirms the degree of \isible surface slick. Stations sampled included relatively uncontaminated rural reference areas that are remote from areas of documented contamination: i.e.. Sequim Ba) and Central Sound (Stations 7. Y and 9). three urban bays-Elliott Bay near

Seartie r Stations 1.2. dtxi 3 1. Commen<emsnt BAY near Tacoma (Station< t. 5. 6. 10 and 11 I, and Port Angeles Harbor istation 13). Aiso. a station ~93 lo~ar~d near Seattk’s Ll’est Point setvage treatment plant discharge diffuser 1Station 12). IFis. 2). Subsurface bulk water samples v.ere coflscred oni! at three stations IS. 11 and 13) by opening ;I ?.i-liter gla+ jug ;!t :i C!cpth oi

0.2 in. Samples i\‘ere collected four time5 during l9j5. desi~n;lted .-\

[February 12). B(March 3 to 6). C (Xlarch 10 to 121. and D 1\1~tv 31. Sampies i\<re immediatel>- stored in the dark on ice and returned to the I::borat~~r\~. Toxicit). tests ivith unfiltered samples were Initiarcd u ithin 2-l h ofsol!ection.

Laboratory toxicity to sole embryos and larvae

To.Gcity tests \\c’rc conducted using sand sale csg:‘. \i,hich occur abundantI>. on the surL~e of PuEet Sound during the kite byinter 3nd spring. Tests from March 3 to 6. 1985. lvere conducted using early-stage embryos collected by neuston net t‘rom Sequim Ba!. ,411 other tests used laboratory- fertilized eggs produced from gametes of one male and one female sand sole. In the laborator). J to 10 replicate dishes containing about 15 eggs each and either 30 ml of microlayer or bulk water sample \verc incubated ;~t 9 C for 6 days. Numbered dishes were arranged in a random, in\.estisntor-blind

manner. To avoid the etyects ofsalinity differences, the salinity ot‘each sample ii-as

adjusted to 30% using American Society for Testing and Xlateriais sea s;tlt. .A parallcl test, in which the reference Sequin1 Ba! sealvater ~v;ls diluted to 30% ivith deionized water and then reconstituted to 30’%0 \vith sea salts. demonstrated that no significant acute tosicity resulted from the A!ition of the se3 salts.

.At the end of the toxicity exposure period, dishes u’erc’ csamined and the number of normal live lar~~ae recorded. Normal live larvae were completel> free from the egg shell, shoued active motility, and appeared to ha\,e normal morphology. Data are reported as the percentage of initial eggs in each treatment that hatched into normal live larvae. Serial dilution tests were conducted by exposing embryos to microlayer diluted l\ith Sequim Bay reference seawater.

To determine the effects of freezing on toxicity. six SMIC samples from Commencetnent Bay, used in the sole tosicit): tests, \vcre frozen to -50 C. then tha\ved and the test re-run. Analysis of variance follo\ved b>- Tuke>‘s Studentized Range Test (SAS, 1985) was used to test the hypothesis that there ivere no significant differences in toxicity bettveen fresh and frozen/ thawed samples. Ten 1985 samples that showed significant toxicity. as well as a Sequim Bay reference sample, were frozen and used later for additional tosicity tests. including those on other species.

Relationship of toxicity to surface slicks

To determine the relationship, if any. betueen SMIC toxicity and the presence of surface slicks. 61 samples were coIlected and tested for toxicity during 1986 using a rotating drum sampler. The sampler was similar to that of Harvey (1966) but the drum was coated with hydrophobic teflon. Samples were from the same general sites as 1985. but were purposely collected either inside or outside areas of visible surface slick.

Duplicate SMIC samples were collected from four slick and four nonslick stations in two urban bays (Commencement Bay and Elliott Bay) and from the Central Sound (off Pt. Pully). Duplicate samples vvere collected from one slick and one nonslick station in a rural bay (Sequim Bay). In addition, subsurface bulkwater (0.5-m depth) samples were collected from Elliott Bay and Commencement Bay. Surface pressure at each station was measured by oil drop spreading (Adam, 1937). Sole embryos were exposed to these samples in the laboratory as described above. and the percentage of live- larval hatch was recorded.

Slick-to-nonslick and site-to-site comparisons of toxicity were examined using ANOVA and Tukey’s Studentized Range Test (SAS, 1985). The relationship between mean surface pressure of the surface film material at the site and toxicity to sole eggs was examined by a least squares linear regression model.

In sifu toxicity

In the laboratory exposures (above) floating eggs were placed in dishes containing collected surface microlayer (SMIC). The eggs were completely surrounded by, and all surfaces were free to accumulate contaminants from, the SMIC. In the field the 1 mm diameter eggs float in contact with, but not necessarily surrounded by, SMIC. Toxicity could, therefore, be less in sifrr than in the laboratory. To test this hypothesis toxicity tests were also conducted i/l s&r. The field exposure system consisted of duplicate polyethylene cups (150 ml) held in a 0-09-m’ Styrofoam floatation board. The bottoms of the cups were open for vvater exchange through a 0.5-mm mesh nylon screen. The apparatus was submerged below the water surface and slowly brought to the surface to trap the surface film within each dish. Exposure systems were tethered to buoys in Sequim Bay (reference) and in urban bay areas with suspected contamination. Eggs from one female sand sole were fertilized with sperm from one male. At about 2 h post fertilization. approximately 50 embryos were added to each dish. allowed to incubate for 6 days in situ, retrieved, and examined for normal live-larval hatching success.

Tosicity to fish cell cultures

Extracts of 10 microlayer samples were tested for their effects on in uirro

grovvth of rainbow trout cells. Ether estracts of the microlayer samples were evaporated to dryness and resuspended in nanograde dimethyl sulfoxide (DMSO). Rainbow trout gonad cells (RTG-7) were grown at 18’C I Wolf 8: Quimbly. 1963 C II- ). e 5 were placed into multiwell culture dishes (2-I vveilsdish and 2 cm2 surface area vvell) at a density of 20 000 viable cells per well and allowed to settle and attach for 1 S h. Crlis from five wells vvere then removed and counted on an electronic particle counter. These counts were used to establish the number of cells per culture at the beginning of the exposure period (time 0). Simultaneously, cells were exposed to microlayer extract dissolved in 0.5% DiMSO as a carrier. Following incubation in the presence of the microlayer extract for 96 h, cultures vvere washed w,ith buffered saline to remove dead cells and harvested for counting. Control cultures were exposed only to 0.5% DMSO; treatment cultures consisted of extracts of microlayer from the different sites. To determine if there was a significant e(Tect on growth or survival as a result of exposure to the microlayer extract, growth data were transformed to the arcsine of the square root of the per cent of the control and analyzed by one-way ANOVA. If a significant difference in growth was found between the treatment and the control, then Fisher’s test of least significant difference was applied (Systat. 1984).

Embryo anaphase aberrations

The incidence of chromosome abnormalities in mitotically active sand sole embryos exposed to SMIC samples was determined according to Longwell & Hughes (1980) as modified by Liguori & Landolt (1985). During the sole toxicity tests, at 35 h post fertilization one of the replicate dishes from each treatment was preserved in 4% buffered formalin and used for anaphase aberration analysis of the blastodisc stage embryos. Embryos were stained with aceto-orcein and squash preparations were examined at 400x to 1000 x magnification on a compound microscope. The per cent of abnormal anaphase figures (chromosome bridges, lagging fragments, attached fragments, and multipolar cells) was determined and the mean number of abnormal anaphase cells in the treated groups was compared to the controls (baseline) using ANOVA and Fisher’s test as described above (*Toxicity to fish cell cultures’).

On microlayer samples collected durin, * 1986, similar techniques were

employed, but frozen and thawed microlayer samples were used. Eggs were obtained from kelp bass (Parafahra.v cfurhrutus) collected off Dana Point, California. Toxicity tests were conducted using twenty-five fertilized eggs

Scu- sirrtdc-e microlu~er ot‘ Puget Soirntl: Par: I

added to each of six replicate l-liter beakers containing 150 ml ofmicrolay.er

and incubated at 17 k 0.5’C. ,4t 60 h post fertilization. all larvae in the sisth beaker from each series were preserved in J’?,d buffered formahn for enumeration of anaphase chromosome aberrations. j-elk sac epithelium was dissected free from the larvae, stained. and analyzed as above. When possible 10 larvae vvere evaluated and all cytogenetic observations were

performed using blind review.

Surrogate toxicity tests

In Pupet Sound. sole eggs are available only during the spawning season- generally late January through March. In order to develop a surrogate methodology for conducting year-round monitoring of SMIC toxicity, embryos of several other species were evaluated and compared to sole. Ten

previously frozen SMIC samples (which had been tested in March 1986 using sand sole eggs) were thawed and tested between June and August, using embryos of three other species: anchovies (Engradis tnorhs), kelp

bass (Parchhrcm c.lcrthrcrt~ls), and sea urchins (L~techims pic,tus). The samples selected for testing were from stations where the sand sole embryo tests had previously indicated a range of effects from 100 to 0% live-larval hatch (LLH). Samples vvet-e thawed and all tests were conducted in the same manner as the sole embryo tests (i.e. incubated in 30 ml of SM IC in dishes, but at the known optimum temperature for the species).

Anchovies were spawned at the National Marine Fisheries Service, La Jolla, California (Leong. 1971) and fertilized eggs were shipped to us in a temperature insulated container. Tests were begun within 10 h of fertilization. Floating embryos vvere dispensed in dishes containing SMIC samples and exposed at 16’C for 48 h at which time most of the control embryos had hatched. For kelp bass, frozen SMIC samples were shipped to the laboratory of the Southern California Water Research Project Authority, where fertilized eggs were exposed to the thawed Sh4IC samples for 4 days at 2O’C. Sea urchins were obtained from a commercial biological supply house and maintained and fertilized as described by Hinegardner (1967) with modification by Oshida (1977). They were incubated at 20-C for 48 h (normally the advanced pluteus larval stage) and then preserv,ed in 2’,“0 buffered formalin. Results were expressed as the percentage of morphologi- cally abnormal or delayed-development embryos in the treatment compared to the same percentage in the reference (Sequim Bay seawater) sample. Samples from different stations were ranked in order of toxicity as measured by each of the tests. Toxicity results of the four tests were compared using Kendall’s coefficient of concordance, W, which represents a measure of the extent of association among several sets of rankings (Zar, 1984).

‘:6 _-

RESULTS

Xeuston densities

Densities of zooneuston (not including fish eggs) in urban bays were, on average, approximately 15% to 35% as great as in the Central Sound or Sequim Bay (Fig. 3). Likewise, flatfish e,, 0~ densities Lvere much lower in the urban bays than in the reference areas of Sequim Bay and Central Sound (Fig. 3). The number of eggs in the surface layer of the urban bays during the spawning period of February and March averaged only 1% of the number at the reference sites. Flatfish eggs were found almost esclusively in the top neuston net during the peak of the spawning season. Later. in March to April, some eggs were found in the subsurface water and by mid-May the spawning season was over and eggs were absent.

Other abundant surface layer organisms included copepods. amphipods and crab larvae. Detailed organism densities have been reported previously (Hardy ef al., 1986). A few taxa were exclusively or mostly neustonic. Copepods were frequently abundant in the top-layer neuston collections,

700-

SOO-

soo-

i; E VI

2 0’ 400-

5 B f

g E-8 300-

3

8 -1 zoo- d P

lOO-

0

I ZOONEUSTON

m FISH EGGS

XNTFULSOUNO ELLIOTT B*V

Fig. 3. Mean density (February through May) of zooneuston organisms not including fish

eggs, and mean density (February and March) of neustonic eggs at four sites in Puget Sound.

Samples collected by neuston net tows from the upper 0 to 4cm surface layer.

&cl-.Wr]&Y microla~w of Pucyr Sound: Parr I ,.- _\

especially from Sequim Bay. At the Central Sound site, a Hyperiid amphipod (Phroninza sp.) occurred frequently in the neuston. Examination of live samples revealed the amphipod actually moving on top of the air-water interface. It was occasionally very abundant. reaching S98 and 137 individuals m - 3 on March 11 and Play 17. 1985. respectively. Dungeness crab (Carrcer majester) larvae were often abundant in May and June neuston samples.

Site-to-site comparison of neuston (0- to 4-cm layer) net collections by cluster analysis also indicated that the reference sites (Sequim Bay. station 8 and Central Sound, station 7) were different from the urban bay. sites in terms of both the abundance of major taxonomic groups (Fig. 41.4)) and the density of fish eggs (Fig. 4(B)). Three separate groups were identified based on the total abundance of organisms: group 1 with low densities (Stations 1. 2, 5. 6, 11; mean = 22 m- ‘), group 2 with intermediate densities (Stations 9 and 10; mean = 224m-‘), and group 3 with high densities (Stations 7 and 8: mean = 832 me3). Fish eggs were present in high densities at Stations 7 and

A

r

STATION

B

7 3 11 5 4 6 2 1 10 9

STATION

Fig. 4. Cluster analysis dendrograms of sampling station groups, (A) based on abundances

of major neuston taxa (not including fish eggs), and(B) based on neustonic egg densities (not

including other taxa groups). Stations 7 and 8 are rural reference sites: other stations are in

urban bays.

S. intermediate densities at Stations 3 and 9. and low densities or absent at

other stations.

Laboratory toxicity to sole embryos and larvae

The per cent of normal live-larval hatch (LLH) at the end of a 6-day exposure period ranged from 0 to 96 9,‘” (Table 1). Samples were analyzed by

ANOVA and ranked by a Newman-Keul’s multiple range test. Significantly toxic ( P < 0.05) samples were all from urban bay sites. These included Stations 1 and 2 (Elliott Bay). 4 and 10 (Commencement Bay), and 13 (Port Angeles Harbor). Only microlayer samples from the urban bays showed significant tosicity. No toxicity was found in bulk vv.ater samples (0.2 m depth) from either urban or rural bay stations. The greatest survival, 86% to 96%. occurred in samples from the Central Sound (Station 7) and Sequim Bay (Station 8) sites. Out of 12 microlayer samples tested from urban bays,

five showed significant toxicity (i.e. 5j% or less LLH). Station 9, the microlayer collected from a thick visible slick in rural Sequim Bay, resulted in about a 12% (non-significant) reduction in LLH. In dilution tests, percent LLH generally decreased with logarithmic increasing percentages of toxic microlayer. For example, a sample from Station 1 (May 5, 1985) yielded:

I’ = I 6. I - 6.7 In .\- (1)

vvhere Y = per cent normal live larvae. X = per cent microlayer, and LV = 20 (i.e. four concentrations x five replicate dishes); r = 0.90.

Salinities of SMIC samples were generally 27 to 30%0 (reference stations), 24 to 38%0 (Elliott Bay), and 8 to 26%0 (Commencement Bay). The salinity of the microlayer samples was adjusted to 30% prior to conducting the bioassays. There was no apparent correlation between the original sample salinity and subsequent toxicity in terms of the percentage of live larval hatch in the bioassays. Analysis of v,ariance of the 1986 data indicated that there vvere no significant differences in toxicity between samples attributable to freezing/thawing (P[F, ,3 = I.821 = 0.270). parentage (the spawning batch of eggs), or time of collection (for the three Sequim Bay sample collections)

(W2.41= 1.731 = 0.189).

Relationship of toxicity to surface slicks

*Mean LLH in nonslick samples vvas 96% to 98%, except in Commencement Bay where LLH was only 82%. In visible slick areas LLH was 94% to 96% in the reference areas (Sequim Bay and Central Sound), but was reduced in the urban bays to only 4% and 61% in Commencement Bay and Elliott Bay, respectively (Fig. 5).

239

Commencement Elliott Bay Central Sound Sequim Bay Bay

aslick 6anowslick m bulk water

Fig. 5. Per cent normal livr-larval hatch for sand soieembryosexposcd in ths laboratory to

scu-surfxc microluycr samples from slick and non-slick microktyer samples from urban sites

(Commcnccmcnt Bay. Elliott Bny) and rural sites (Ccntraf Sound and Scquim Bxy).

Analysis of variance indicated significant difference (P[F,,,, = 49.833 =

I x IO-‘) in toxicity that were due to the presence of visible slicks. Surface microlayer samples from Elliott Bay slicks and Commencement Bay slicks were all significantly (Tukey’s Studentized Range test) more toxic than slick or non-slick samples from the non-urban sites. Linear regression analysis

indicated that, in the rural sites, no significant relationship occurred between toxicity and mean surface pressure as measured by the oil drop spreading technique. However, in both the urban bays, normal LLH decreased with increasing quantities of surface active organic films according to:

Y= -365X+ 112 (2)

where Y= % normal live larvae, X = mean surface pressure (dynes cm - ‘) at the urban bay station sampled; iv = 32, I’ = 0.6505 and P = <0401.

In situ toxicity

Fertilized sole eggs, placed in exposure chambers in situ and allowed to float

in contact with the SMIC during embryogenesis, displayed marked reduction in hatching success in the urban bays compared to the reference stations (Fig. 6). In 1985 normal live larval hatch in Port Angeles Harbor and Commencement Bay was only 4% and 42%, respectively, of that at the reference site (Sequim Bay). In Commencement Bay all larvae showed

80

70

60 3

1985 1985 ’ 1986 1986

Port Angeles Commencement Bay Elliott Bay

Fig. 6. Per cent normal live larvae for scmd sole embryos exposed in sip ;tt stverul urban bay

sites compared to ;t rurill (Squim Bay) reference site (considered ;LS loo?;,).

morphological abnormalities, primarily kyphosis (bent spine). In 1986, a Commencement Bay exposure resulted in only 55% normal live larvae and two exposures in Elliott Bay, performed a ueek apart, gave 79”/ and 78% as many normal live larvae as Sequim Bay. These results compare well to those found in the laboratory tests.

Toxicity to fish cell cultures

Compared to cell growth .in the control, trout cell cultures treated with microlayer extracts generally showed less cell growth. Cell grovvth was not significantly reduced in cultures exposed to microlayer extracts from Sequim Bay or the Central Sound. in contrast, cell growth i+as signiticantly (F= S-63, P < O-001) reduced in cultures esposed to urban bay microlayer extracts (Fig. 7).

Embryo anaphase aberrations

The mean incidence of chromosome abnormalities in mitotically active fish embryos or larvae of two fish species (sand sole and kelp bass) \vas 8% in

Sequim Bay Central Sound Elliott Bay West Point CommG-nt

Fig. 7. Growth of trout cclis exposed in ritro to sea-surface microlayer samples. pooled

samples &- standard error (* = significant reduction P < 0901 compared to control cell

cultures).

Anchovy Larvae Sole Larva4 !%a Urchin Lawat Kelp Sass Larva8

Fig. 8. Percentage of normal live larvae in laboratory toxicity tests of four species exposed

to sea-surface microlayer samples collected from Puget Sound.

0 ‘j\ c-l 0 ~~~000 s -_- -

Sequim Bay (reference) and greater than 20” 0 at other sites. with percentages greater than 300/b occurring in two samples from urban bays (Table 2). Considerable variance associated with the presence of slicks occurred so that overall differences in chromosome aberrations between different sites were not statistically significant (F = 1.97. P = 0.197). Hotvever, embryos exposed to urban slick samples shovved a significantly (F= 6.16. P = 0.021) greater incidence of chromosome aberrations than those exposed to bulk water.

Surrogate toxicity tests

The toxicity of SMIC samples was similar when evaluated by either sole, anchovy, kelp bass, or sea urchin tests. Some differences in species sensitivity were found (Fig. 8); however, when the effects of samples from different sites were compared, the rank order of toxicity was not significantly different when evaluated by the four species (the coefficient of concordance, using corrections for tied ranking, was Ct’= 0.853).

DISCUSSION

Extremely high concentrations of contaminants, especially polycyclic aromatic hydrocarbons, have been found in the SMIC of Puget Sound (Hardy et al., 1986) and Chesapeake Bay (Hardy et cd., 1987). Our previous data indicate that larval hatching success decreases with increasing concentrations of polycyclic aromatic hydrocarbons and, secondarily, heavy metals in the surface microlayer (Hardy rr crf., 1986). In a subsequent report (‘Part II. Concentrations of contaminants and relation to toxicity’) we will present chemical data on the concentrations of metal and organic contaminants in the SMIC of Puget Sound and their relationship to the toxic effects described above.

Densities of neuston, including sole eggs, were lower in urban bays than at the reference sites. This could result from the effects of surface contamination. However, other causes (e.g. differences in currents, salinity, etc.) cannot be ruled out. Our sampling was biased toward a larger number of stations in urban bays; thus, in 1985, only three samples were collected outside bays (two in the Central Sound and one off the West Point Sewage waste diffuser). Thus, these results do not indicate that toxicity is restricted to the urban bays of Puget Sound. If we consider significant contamination as PAHs > 1000 ng liter- ’ and metals > 50 pg liter- ‘, i.e. toxic levels (Hardy et

al., 1986), then two of the three non bay samples were highly contaminated. In 1986, eight samples were collected at one Central Sound station at one time. They were not toxic; however, one station at one time obviously should

not be used to extrapolate to Puget Sound. In Chesapeake Bay, samples were collected at 12 widely separated stations once each Lveek over a j-week period. Using the same chemical criteria as ‘significant’ contamination. I2 of the 36 samples, includin g one from the Central Bay. Lvere highly contaminated (Hardy et 01.. 1987). Other studies indicate that surface contamination (e.g. tar balls and plastic particles) exists far oRshore (Snap et

ul., 1986) and that cytogenetic and embryologic damage to floating fish eggs may occur in offshore coastal waters with elevated contaminant con- centrations (Longwell & Hughes, 1980).

Neuston nets generally collect the upper 4- to 15cm of water surface and thus are not able to differentiate how many of the eggs or larvae are contained in only the upper millimeter of water. However, our experience indicates that, when collected eggs are placed directly into beakers or pans of seawater from the collection site, they very rapidly float to the water surface, where they concentrate in the upper 1 mm (Fig. I). We have no reason to believe that the upper 1 mm is not their natural place of occurrence throughout their 6-day period prior to hatching. Fish eggs persist at the surface even with wind and sea states of Beaufort 4 and 5 (Zaitsev, 1971; Hardy & Antrim, 1987).

Although we found that toxicity was strongly correlated with the presence of slicks this does not diminish, in our vielv, the overall spatial importance of sea surface toxicity. Visible slicks are only the extreme manifestation of the organic surface film that is present to a greater or lesser degree everywhere. The film forms from biogenic organic exudates, released by plankton in the water column, that collect at the sea surface (Baier ct (II., 1974). In areas remote from contaminant sources (e. g. Station 9) these slicks may contain little or no contamination. However. in urban areas, toxic hydrophobic contaminants concentrate in natural films (Hardy, 1983). Wind and current patterns collapse the films into thicker visible slicks. Such slicks are not restricted to urban bays, but appear to move from place to place. Contaminated su&ce films could be carried by wind and surface currents, deposit in intertidal beach areas, contaminate shellfish, and impact other species, such as herring, that deposit eggs intertidally.

We have found that, when contaminants are present. tosicity increases with the relative degree of organic surface film (surface pressure, dynescm- ‘). Thus, a film may be moderately toxic, but not visible. Ifz sirrr, embryos may be exposed intermittently to such drifting films or they may concentrate in, and be transported by. such films (personal observation and Shanks, 1983). Thus, though the denser contaminated films may not cover the entire area, their effects may be greater than their spatial coverage would suggest. Horizontally collapsed and concentrated (i.e. visible) films frequently cover 50% of a large area (personal observation). The toxicity of

that film may be 100% in laboratory bioassays. thus in the absence of convergent [wind and tide) forces the film still be spread over the entire area and a sample would produce a toxicity of 50%. Once an area has been found to contain surface contamination. daily or bveekly monitoring could be conducted simply by measuring the presence of organic films b> oil drop spreading-a rapid and inexpensive technique (Adam. 1937).

Although only five in sirll experiments ivere conducted. 1t.e be1iei.e these results confirm the trends found in the laboratory tosicity tests: namely, that hatching success of sole is reduced by more than 50% at many sites in Puget Sound. Obviously, other Factors not measured could be responsible for this effect. However, temperature and salinity values were similar and well within the range for normal development of the larvae.

Our evaluation of alternative (surrogate) toxicity tests indicates that the sea urchin embryo;larval test, which can be conveniently conducted throughout the year, would make an ideal test for monitoring SMIC toxicity. By enlargin g the comparative data base further, the sea urchin toxicity test could be used to monitor changes in sea-surface toxicit!, throughout the year.

Overall, these results raise considerable concern regarding the environ- mental quality of the sea surface as a habitat for the developmental stages of fiatfish and other species. Our results strengthen the argument that maintenance of a healthy marine environment will require development of quality standards and monitoring programs, not only for the water and the bottom sediments, but for the sea surface as well.

ACKNOWLEDGEMENTS

This research was supported by the National Oceanic and Atmospheric Administration (NOAA), Ocean Assessments Division under a Related Services Agreement with the US Department of Energy. The Battelle, Marine Research Laboratory is part of the Pacific Northwest Laboratory, which is operated for the US Department of Energy by Battelle .Memorial Institute under Contract DE-AC06-76RL0 1830. We greatly appreciate the valuable suggestions of Thomas O’Conner and Edward Long (NOAA) throughout the course of the study. The conduct of the surrogate bioassays was kindly assisted by Roger Leong(NOAA, NMFS, LaJolla) \cho provided anchovy eggs and by Jeff Cross (Southern California Water Research Project Authority) who performed the kelp bass embryo exposures. JoEllen Hose (Occidental College) evaluated anaphase chromosomal aberrations in the kelp bass larvae. We thank Gilbert Fellingham for his able assistance in the study design and statistical treatment of the data and John Skalski for the initial review of the manuscript.

247

REFERENCES

Xdam. N. K. (1937). A rapid method for determining the loitering of tension of exposed water surf;lces. M ith some observations on the surface tension of the sea and inland ivaters. Proc. Rood Sot. Lon. Ser. B.. 122. 134-9.

Ahlrtrom. E. H. & Stevens, E. I 1975). Report on neuston (surfact) coilectlons made on an extended CalCOFI cruise during May 1972. Calit Coop. Fish. Int.estig. 28, 1 Julc 1973 to 30 June 1975.

Alderdice. D.-F. & Forrester, C. R. I 1965). Some effects ofsalinitb and temperature on early development and survival of the English Sole (Parophr~.s !.tjtdu.~). J. Fish. Rex. Bd. Candy. 25. 495-52 I.

Anderson, J. W. (1977). EtTects of petroleum hydrocarbons on the grotvth of marine organisms. Rtrpp. P. I’. Rrun. Cons. /nr. E~plor. Mu. 157-65.

Baier. R. E.. Goupil, D. W., Perlmutter, S. & King, R. (1974). Dominant chemical composition of sea-surface films, natural slicks, and foams. J. & Recherche.s A tnrosplrcricllres, 8, 57 I-600.

Brewer, G. D. ( 19s I ). Abundance and vertical distribution of fish eggs and larvae in the Southern California Bight: June and October 1975. Rapp. P. V. Rem Consr. Itrt. E.vpior. .Clrr, 178, 165-7.

Budd, P. L. (1940). Development of the eggs and early larvae of six California fishes. Stule of Culjfbrniu, Dcpnrttmwt of Ntrtwd Resow-ccs. Dirisio11 0-f Fish mcl Ganw, Burcwu of Marim Fishcrks. Fish Bull. No. 55. 20 pp.

Carlucci. A. F., Craven, D. B. & Henrichs, S. M. (1985). Surface-film microhetero- trophs: Amino acid metabolism and solar radiation effects on their activities. Mar. Bid., 85. 13-22.

Dethlefsen, V. (1974). Effects of DDT and DDE on eritbryos and larvae of cod, flounder and plaice. ltzl. Council Esplor. Seu, C. M, 197-I E : 6, 1240-S.

Hardy, J. T. (1982). The sea surface microlayer: Biology, chemistry and anthropogenic enrichment. Prog. Occclnoy.. 11, 307-X.

Hardy, J. T. & Antrim, L. D. (1987). Refinement of methods for determining the toxicity of emissions from at-sea incinerators: Task 3--Evaluation of a microlayer sampler and toxicity tests at sea. Final Report. EP.4 Contract 68-03-3319, WA 36. Battelle Ocean Science and Technology Department. Duxbury. Massachusetts.

Hardy, J. T. & Apts. C. W. (1984). The sea-surface microlayer: Ph>toneuston productivity and etTects of atmospheric particulate matter. Mur. Bid., 82, 293-300.

Hardy, J. T.. Apts, C. W.. Crecelius, E. A. & Bloom, N. S. (1985~). Sea-surface microlayer metals enrichments in an urban and rural bay. E$fmr. Cwsfd Shelf Sci.. 20, 299-311.

Hardy, J. T.. Apts. C. W., Crecelius, E. A. & Fellingham, G. U’. (1985h). The sea- surface microlayer: Fate and residence time of atmospheric metals. Linml. Ocranogr.. 30, 93-101.

Hardy, J. T., Crecelius, E. A. & Kocan. R. (1986). Concentration and toxicity of sea- surface contaminants in Puget Sound. PNL-5834, Pacific Sorthwest Laboratory, Richland. Washington.

Hardy, J. T., Crecelius. E. A., Antrim, L. D., Broadhurst, V. L., Boehm, P. D. & Steinhauer, W. G. (1987). Aquatic surface contamination in Chesapeake Bay. Final Report, State of Maryland, Department of Natural Resources, Battelle Northwest, Richland, Washington, 32 pp.

Harvey. G. W’. I 1966). ~licrol;~)crcollection from thesea SurfAce: A IX\\ method and initial results. f_.i/~rjlo/. Oc~~r,lf),yr.. 1 l(4). 6OS 13.

Harley, G. LV. 2% Burzsll. L. .I. [ 1972). X jimpk microlu)er method for small samples. Lit?Jtzol. Ow~~tJo~r.. 17( I L 156-7.

Hattori. H.. Yuki, K.. Zsitsev. \I’. P. Sr Jlotodu. S. I 1983. .A pr;liminar> observation on the neuston in Surgua Bay. CII~-.I~CI... 21. I I-20.

Hinegardner. R. T. I 1967). Echinoderms. In: .\lt~r/~otl.s irl tlrcrlopt~~~v~fd hiolog~~. (Wilt. F. H. and Wesjelj. N. K. (Eds)). Se\\- York. Sew York. Thomas Y.

Crowell Cornpan)-. 139-55. Jacoby. C. A. ( 1981). Behavioral responses of the larvae of C‘lncrr ttJagi.ster DtrtJcJ to

light. pressure, and gravity. :Clllr. BdJu~ior. Plr~~siol.. 8. 267-83. Kendall. A. W.. Jr & Clark. J. (1982). k~lJt/z~.oplcJtrktotJ of If bdJ;t~gtotJ. OregotJ, trtd

~l’ort/JertJ Cd~~iwtJitJ. .-lpril-.tlu>. IYSO. NW.\FC Processed Report 82-I I. Northbvest and Alaska Fisheries Center, NMFS, NO.%& Seattle, Washington.

Kligerman, A. D.. Bloom. J. E. & Ho\vell. W. S. ( 1975). Umbra iimi: A model for the study of chromosome aberrations in fish. .Kut. Rrs., 31. 225.

Knap, A. H.. Burns. K. A.. Daivson. R., Ehrhardt. M. & Palmork. K. H. (1986). Dissolved :dispersed hydrocarbons. tarballs and the surface microlayer: Experiences from an IOC,‘UNEP workshop in Bermuda. December, 1984. Mw. PollJlr. Bull.. 7. 3 l3- 19.

Korn, S. & Rice. S. (1981). Sensitivity to and accumulation and depuration of aromatic petroleum components by earl! life stages of Coho salmon (OtJc~or/J~.tJc.lJJl.s kimtdr). Rrrpp. P. I . . RrJJtJ. Cot1.s. ItJt. E.vplor. :1/u, 178. 87-92.

Leong, R. ( 197 I). lnduccd spawning of northern anchovy, Et1yruJlli.v t~1cmlrr.v Gird. Fislr. Brrll. US. 69. 357-60.

Liguori, V. M. & Landolt, M. L. ( 1985). Anaphase aberrations: An it1 L.&W measure of genotonicity. In: Slrorl-tcrtt1 g:cvJcl t ic hiotr.v.stJ~r~.s it1 tlJe utJui.\..si.s q/‘ cmJpiv.~ cvJrirotJttJ~vJtrtl t?Ji.utJrrc.s lb’. (Waters. M. D.. Sandhu. S. S.. Lewtas. J., Cluston,

L., Strauss. G. Sr Nesnow, S. (Eds)). New York, Plenum Press. Long, E. R. (1982). An itssesjment of marine pollution in Puget Sound. .llrJr. Pollut.

Bldl., 13, 380-3. Longwell, A. C. ( 1976). Chromosome mutagenesis in developing mackerel eggs

sampled from the New York Bight. NOAA Tw/J. Mm. ERL-.\/ESA-7. US Dept. Commerce.

Longwell, A. C. & Hughes, J. B. (1980). Cytologic. cytogenetic and developmental state of Atlantic mackerel eggs from sea surface waters of the Neu, York Bight, and prospects for biological effects monitoring with ichthyoplankton. R(~pp. P. V. RcJrtJ. Cons. ItJt. E.rplor. Mrr. 179, 275-9 I.

Oshida, P. S. (1977). Toxicity of a chlorinated benzene to sea urchin embryos. in: Cmstul IVater Resrcrrch ProjLjct AtJtJ~rcrl Report. Southern California Research Project Authority, Long Beach, California. 187-91.

Provenzano, A. J., Jr, McConaugha, J. R., Philips. K. B., Johnson, D. F. & Clark, J. (1983). Vertical distribution of first stage larvae of the blue crab. Ccrll~‘tJc~ctc.s .supih.s, at the mouth of the Chesapeake Bay. E.stucJr. Coust. SiJrY Sci., 16, 489-99.

Purdom. C. W. & Woodhead, D. S. (1973). ErnlJruriotJ of grtzrtic risks o/ rrJcirotJnJetJrcr1 clrcnJicul.s. Ambio Special Report, Ro)ltl Swedish Acad. Sci. Universitetsforlaget No. 3. 27 pp.

Safronov, S. G. (1981). On neuston in Kamchatka Waters of the Sea of Okhotsk. Bid. MorJw, 3. 73-4.

SAS (1985). VMS Version of SAS Release 5.03 at Batreile Xorthkvest-PSLB VXS.

Gary. North Carolina. SAS Institute. Inc. Shanks. A. L. (1983). Surface slicks associated with tidall) forced internal iiaves ma>

transport pelagic larvae of benthic invertebrates and fishes shorward. .\ILIT. Ed. Prog. Ser., 13. 31 l-15.

Shenker. J. M. (in press). Oceanographic associations of neustonic larval and juvenile fishes and Dungeness crab mcgalope off Oregon. L...S. fix/r. &II.

Smyth. P. 0. (1980). Callinectes (Decapoda: Portunidae) larvae in the Middle Atlantic Bight, 1975-77. Fish. Bull. C*S. 78. 3-65.

Souza-Lima. Y. de ( 1982). Air-water interface: Ecological aspects of microneuston during investigations of surface films. Paper presented at the Second Symposium on Marine Microbiology, June 24, 198 I. Marseille. France, 175-8 I

Systat (1984). The ~~~stenr fbr statistics. Evanston. Illinois S\-stat. Inc.. ‘63 pp. Westernhagen, H., Dethlefsen, V. & Rosenthal. H. (1979). Combined effects ot

cadmium, copper and lead on developing herring eggs and larvae. Hrls~ol. ~t’ks. iUwre.wnt~rs., 32, 257-78.

Wolf, K. & Quimbly. A. (1962). Established euryhaline line of fish cells in vitro. Science, 135. 1005-66.

Zaitsev, Y. P. (1971). Marine neustonology (Translated from Russian.) National Marine Fisheries Service, NOAA and National Science Foundation. National Technical Information Service, Springfield, Virginia, 107 pp.

Zar, J. H. (1984). Biostutistical crnal~.si.s (2nd edn), Prentice Hall. Engleu-ood Cliffs. NJ, 718 pp.