Toxicity of polynuclear aromatic hydrocarbons to the polychaete Neanthes arenaceodentata

Marine Pollution Bulletm Blohm, H W & Becker, E I (1952) Allophanates Chem R e v , 51, 471-504 Cassmgne, M A , Lacoste, A M & Neuzd, M E (1976) Recherches sur le catabohsme des acldes phosphomques blodegradatlon de la hason C-P par Pseudomonas aerugmosa C R Acad Set Parts, s e r w D , 282, 1637 Cooney, J J & Summers, R J (1976) Hydrocarbon using microorgamsms m three fresh-water ecosystems In Proc o f the Thtrd Int Btodegr Symposmm Sess I, XIII, XXV, XVII, XIX J M Sharpie and A M Kaplan (eds), held at the Umverslty of Rhode Island, Kingston, Rhode Island, U S A pp 141-155 London Appl So Pubhshers Ltd Floodgate, G D (1976) Oil blodegradatlon m the oceans In Proc o f the ThtrdInt Btodegr Sympostum Sess I, XIII, XXV, XVII, XIX J M Sharpie and A M Kaplan (eds), held at the University of Rhode Island, Kingston, Rhode Island, U S A pp 87-91 London Appl Sci Publishers Ltd Gsbbs, C F (1975) Quantitative studies on marine blodegradation of od I Nutrient limitation at 14°C Proc R Soc Lond B , 188, 191 Kosolapoff, G M (1945) Isomenzatlon of Alkylphosphltes lIl The synthesis of n-Alkylphosphonic aods J A m chem Soc, 67, 1180-1182

Nleuwenhuyzen, W Van (1976) Leothm production and properties J A m OtlChem Soc, 53,425-427 Ohvleri, R , Bacchm, P , Roberuello, A , Oddo, N , Degen, L & Tonolo, A (1976) Microbial degradation of oll spills enhanced by a slow-release fertdlzer Appl envtron Mwrobtol, 31(5), 629-634 Petit, J Le & N'Guyen, H (1976) Besoms en phosphore des bactenes metabohsant les hydrocarbures en mer Can J Mwrobtol, 22, 1364-1373 Scocca, P M (1976) Utilization of lecithin J A m OdChem Soc, 53,428-429 Suzuki, T , Komatsu, K & Tazimura, K (1973) Thmlayer chromatography of amino aod hydantoms J Chromat, 80, 199-204 Walker, J D & Colwell, R R (1976) Petroleum degradation by estuarlne mlcroorgamsms In Proc o f the Third Int Btodegr Syrnposmm Sess I, XIII, XXV, XVII, XIX J M Sharpie and A M Kaplan (eds), held at the University of Rhode Island, Kingston, Rhode Island, U S A pp 197-204 London Appl Sci Publishers Ltd Werner, A E A & Gray, J (1947) Scwnt Proc R Dubl Soc, 24, 209 Zeleznick, L D , Myers, T C & Titchner, E B (1963) Growth of E coh on methyl and ethyl phosphonic acids Btochlm btophys Acta, 78, 546-547

MarmePolluttonBulletm, Vol 9, pp 220-223 © PergamonPressLtd 1978 Printedin GreatBritain

0025-326X/78/080141220 $02 00/0

Toxicity of Polynuclear Aromatic Hydrocarbons to the P olychaete Neanthes arenaceodentata S S R O S S I * a n d J M NEFF Department o f Btology, Texas A & M Umverslty, College Statton, T X 77843, U S A *Present address Marme Btology Research Dtvtston, Scripps Instttutton of Oceanography, La Jolla, CA 92093, U S A

The toxicity of ten polynuclear aromatic hydrocarbons (PNAs) to N e a n t h e s a r e n a c e o d e n t a t a was studied in replicate 96 h bionssays with specific hydrocarbons in solution. Relative solubilities for each PNA were derived in short-term equilibration experiments. Disappearance of representative di-, tri, tetra- and pentacyclic-aromatic hydrocarbons from test solutions was also characterized by ultraviolet spectrophotometry. PNA toxicity and residence time in test solutions were directly related to molecular weight (MW), while relative solubility is inversely related to MW. 96 h T L m values for the diaromatic PNAs naphthalene, 2,6-dimethylnaphthalene, and 2,3,6-trimethylnaphthalene were 3.8, 2.6 and 2.0 ppm, respectivdy. Phenanthrene, fluorene and 1-methylphenanthrene (triaromatics) exhibited 96 h T L m values of 0.6, 1.0 and 0.3 ppm, respectively. None of the tetra- and pentacyclicaromatics (chrysene, 3,4-benzo(a)pyrene, 1,2,5,6-dibenzanthracene) were toxic up to the highest concentration tested (1 ppm), excepting fluoranthene, with a 96 h T L m of 0.5 ppm. PNA toxicity appears to be related to both solubility and residence lime in test solutions. Much recent attention has focused on the occurrence and fate of polynuclear aromatic hydrocarbons (PNAs) in the marine environment (Blumer, 1976, Hltes, 1976, Pancirov & Brown, 1977) Several studtes have dealt with 220

uptake and release of specific PNAs by marine organisms, and have concerned themselves primarily with the highly carcinogenic 3,4-benzo(a)pyrene (Neff & Anderson, 1975; Dunn & Stlch, 1976, Lee et al, 1972, 1976) In the marine environment, higher molecular weight aromatic hydrocarbons (e g chrysene, perylene, pyrene) are often associated with organically-rich sediments (Alzenshtat, 1973, Borles et al, 1976) Very little is known about the toxicity of PNAs to marine organisms These observations stimulated the following experiments with several specific PNAs, and the sediment-dwelling marine worm Neanthes arenaceo-

dentata Materials and Methods Bioassays were performed with immature young adult polychaetes from a laboratory population originally cultured by D J Relsh, Cahfornta State University, Long Beach (Retsh & Rlchards, 1966) Each bioassay involved the use of 6 or 7 PNA concentrations in addition to a control group (concentrations of 5, 2 5, l, 0 5, 0 1, 0 05, 0 01 and 0 ppm were generally employed) Erlenmeyer flasks (125 ml) containing 50 ml unaerated PNA-test solution served as bIoassay chambers Hydrocarbons were Introduced to seawater by addition of

Volume 9/Number 8/August 1978 TABLE 1

Relatwe solubfllt~esof selected polynuclear aromatic hydrocarbons m 32%0 S seawater at 22°C Equdlbratmn period, 24 h Mean values ± S D for three determmatmns are hsted PNA N DMN TMN FLR PHE MPH FLA CHR B(a)P DBA

Relatwe solubdlty ~ g g-I seawater)

(naphthalene) (&methylnaphthalene) (tnmethylnaphthalene) (fluorene) (phenanthrene) (methylphenanthrene) (fluoranthene) (chrysene) (benzo(a)pyrene) (dlbenzanthracene)

20 + 2 2 4+0 5 1 7+0 6 0 8 +0 2 0 6+0 1 0 3 +0 1 0 01 +0 06 0 05, 0 001 0 010, 0 005 0 010, 0 005

techniques described above 'Equilibrated' solutions were p r o d u c e d b y stirring a n excess o f P N A in 0 . 4 5 / t m M i l h p o r e - f l l t e r e d s e a w a t e r ( u n d e r N2) f o r 24 h at r o o m temperature T r i p l e f i l t r a t i o n t h r o u g h glass w o o l c o l u m n s (lightly p a c k e d - 1 × 10 cm) p r i o r to h e x a n e extraction eliminated interference from suspended PNA crystals It is r e c o g n i z e d t h a t the s h o r t st~rring times e m p l o y e d here p r o d u c e (only) a n o p e r a t i o n a l d e f i n i t i o n o f solubility, since s o m e P N A s m a y require m o r e t h a n 24 h to b e c o m e t r u l y e q u l h b r a t e d with a q u e o u s s o l u t i o n s at r o o m t e m p e r a t u r e ( E g a n h o u s e & C a l d e r , 1976)

Results TABLE 2

Toxicity of selected polvnuclear aromatic hydrocarbons (PNAs) for Nenathes arenaceodentata 96 h TLm values, 95 % confidence hmlts, and corresponding slope functmns were calculated by the method of Lltchfleld & Wdcoxon (1949) TLravalues are expressed as concentratmn (ppm) PNA mltmlly present m test solutmn as determined by UV analysis Values for chrysene, benzo(a)pyrene, and dlbenzanthracene are not presented since these hydrocarbons were not lethal at the highest concentration tested (1 ppm) PNA

96 h TLrn

Confidence interval

Slope

N DMN TMN FLR PHE MPH FLA CHR B(a)P DBA

38 26 20 !0 06 03 05 ----

41-35 29-23 24-1 6 1 3-0 7 0 8-0 4 0 4-0 2 0 7-0 3 ----

15 14 12 14 l7 11 l 2 ----

appropriate volumes of stock PNA solutions dissolved in U V g r a d e a c e t o n e T h e highest c o n c e n t r a t i o n o f a c e t o n e c a r r i e r e m p l o y e d was 1 ml 1-1 a c e t o n e in seaw a t e r Ten a n i m a l s were used p e r c o n c e n t r a t i o n a n d held w i t h o u t f o o d m i n d i v i d u a l flasks S u r v i v a l was m o n i t o r e d o n d a y s l , 2 a n d 4 (96 h) E a c h b i o a s s a y was r e p e a t e d once, a n d all were p e r f o r m e d at r o o m t e m p e r a ture (22 + 2 ° C ) with a r t i f i c i a l s e a w a t e r ( I n s t a n t O c e a n , A q u a r i u m S y s t e m s , I n c ) a d j u s t e d t o a s a h n i t y o f 32%o A l l P N A s h a d a p u r i t y g r e a t e r t h a n 9 8 % , a n d were o b t a i n e d f r o m the f o l l o w i n g ( U S A ) sources n a p h thalene, 2 , 6 - d l m e t h y l n a p h t h a l e n e , 2 , 3 , 6 - t r i m e t h y l n a p h thalene and phenanthrene - Chemical Samples C o m p a n y , 1 - m e t h y l p h e n a n t h r e n e , f l u o r e n e , chrysene, fluoranthene and 1,2,5,6-dlbenzanthracene- Aldrich Chemical Company, Inc; 3,4-benzo(a)pyrene-Pflatz a n d Bauer, I n c R e p l i c a t e flasks w i t h o u t a n i m a l s were set u p to m o n i t o r decreases in P N A c o n c e n t r a t i o n d u r i n g test p e r i o d s P N A c o n c e n t r a t i o n s were d e t e r m i n e d b y ultraviolet s p e c t r o p h o t o m e t r y , f o l l o w i n g e x t r a c t i o n f r o m s e a w a t e r with 5 ml o f U V g r a d e n - h e x a n e F o l l o w i n g s e p a r a t i o n , the c o n c e n t r a t i o n o f P N A in h e x a n e extracts o f test s o l u t i o n s was d e r i v e d b y c o m p a r i n g the heights o f U V a b s o r p t i o n m a x i m a o f the u n k n o w n s with t h o s e o f k n o w n P N A s t a n d a r d s ( G i g e r & B l u m e r , 1974) R e l a t i v e solubilities were d e r i v e d b y c o m p a r i n g P N A c o n c e n t r a t i o n s In h e x a n e extracts o f ' e q u i l i b r a t e d ' P N A - s e a w a t e r s o l u t i o n s with k n o w n s t a n d a r d s , using

Relative solubilities in seawater for ten P N A s are listed in T a b l e 1 S o l u b i l i t y was closely r e l a t e d to m o l e c u l a r v o l u m e a n d m o l e c u l a r weight ( M W ) , s o l u b i l i t y v a r i e d inversely with M W T h e lighter dlaromatic hydrocarbons naphthalene, dimethylnapht h a l e n e a n d t r l m e t h y l n a p h t h a l e n e ( M W - 1 3 8 , 156 a n d 170, respectively) e x h i b i t e d s e a w a t e r solubilities in the low p p m r a n g e T h e t r l a r o m a t i c c o m p o u n d s p h e n anthrene, fluorene, and methylphenanthrene (MW178, 166 a n d 192 respectively) h a d solubilities a p p r o x i m a t e l y a n o r d e r o f m a g n i t u d e b e l o w t h o s e o f the d l a r o m a t l c s , i e slightly b e l o w 1 p p m H i g h m o l e c u l a r weight h y d r o c a r b o n s such as chrysene, b e n z o ( a ) p y r e n e a n d d l b e n z a n t h r a c e n e were m i n i m a l l y s o l u b l e in seawater, e x h i b i t i n g v a r i a b l e solublhtles in the low p p b r a n g e T h e t h r e s h o l d o f d e t e c t i o n f o r the U V s p e c t r o p h o t o m e t r i c p r o c e d u r e s e m p l o y e d here was 1 p p b It seems likely t h a t the latter three P N A s r e q u i r e m o r e t h a n 24 h to e q u i l i b r a t e with seawater, a n d this c o u l d a c c o u n t for v a r i a t i o n o b s e r v e d m their relative solubilities Results o f b l o a s s a y s with N e a n t h e s arenaceodentata a n d specific P N A s a r e p r e s e n t e d in T a b l e 2 O n l y 96 h TLm values are given since m o r t a l i t i e s g r e a t e r t h a n 5 0 % were n o t o b s e r v e d a f t e r 24 a n d 48 h e x p o s u r e P N A toxicity was a l s o closely r e l a t e d to M W , 1 e a t r e n d t o w a r d s i n c r e a s i n g toxicity with increasing M W was o b s e r v e d m the P N A series n a p h t h a l e n e t h r o u g h f l u o r a n t h e n e 1 - m e t h y l p h e n a n t h r e n e was the m o s t toxic P N A tested Its toxicity was a p p r o x i m a t e l y a n o r d e r o f m a g n i t u d e g r e a t e r t h a n t h o s e o f the d i a r o m a t l c n a p h t h a l e n e s P N A s with very low s e a w a t e r solublhtles p r o v e d least toxic, to the extent t h a t 5 0 % m o r t a h t y was n o t o b s e r v e d in s o l u t i o n s c o n t a i n i n g chrysene, b e n z o ( a ) pyrene, o r & b e n z a n t h r a c e n e at ( n o m i n a l ) c o n c e n t r a tions u p to 1 p p m Results o f analyses o f P N A c o n c e n t r a t i o n s p r e s e n t in e x p o s u r e m e d i a d u r i n g the b m a s s a y p e r i o d are s u m m a r i z e d in F i g 1 C o n c e n t r a t i o n s o f r e p r e s e n t a t i v e dl-, trl-, tetra- a n d p e n t a - a r o m a t l c h y d r o c a r b o n s a r e p l o t t e d vs time Initial c o n c e n t r a t i o n s o f the f o u r P N A s p r e s e n t e d m F i g 1 were i n t e n d e d t o be n a p h t h a l e n e 2 5 ppm, phenanthrene-0 5 ppm, fluoranthene-0 1 p p m , a n d b e n z o ( a ) p y r e n e - 0 05 p p m A c t u a l initial c o n c e n t r a t i o n s , as d e t e r m i n e d b y UV s p e c t r o p h o t o m e t r y , d e v i a t e d m i m m a l l y f r o m these n o m i n a l values A s expected, significant decreases in P N A c o n c e n t r a t i o n o c c u r r e d d u r i n g the test p e r i o d , p r i m a r i l y d u r i n g the first 48 h O b s e r v e d decreases were p r e s u m a b l y d u e to 221

Marine Pollution Bulletin 5

o~ O6

/

006

o

.o

"~ +c

o ,,.) "

004 002

.............I.......---:i :

OOI 0 008 0 006

.....

ttt

t

Benzo ( a ) p y r e n e

0004 0 002 0 001

214

48

Time,

96

hr

Fig 1 Concentration of specific polynuclear aromatic hydrocarbons m bloassay test solutions, as determined by ultraviolet spectrophotometry Symbols represent mean values for n = 10 samples, :t: standard deviation (verttcal bars)

volatlhzation (naphthalene) and/or photo-oxidation of hydrocarbons In test solutions (Acheson et al, 1976) Additional experiments with air-free containers and antibiotic seawater solutions indicated little or no detectable loss of PNA due to adsorption onto glass and/or microbial degradation Residence times of PNAs in test solutions appeared to be directly related to MW, 1 e decreases in naphthalene concentration were relatively greater than those of the higher MW hydrocarbons fluoranthene and benzo(a)pyrene

Discussion L~ttle m known about the solublhty of PNAs in seawater Schwarz & Wasik (1976) report maximum solubilities in fresh water at 25°C of naphthalene, fluoranthene and benzo(a)pyrene of 22, 0 236 and 0.004 ppm, respectively These values are simdar to those reported here, differences accounted for by the lower solubility of aromatic hydrocarbons in seawater than in fresh water (Eganhouse & Calder, 1976) McAuhffe (1966) showed that for a homologous series of monocyclic aromatic hydrocarbons, the logarithm of the solublhty m water is a linear function of the hydrocarbon molar volume The relationship between solubdlty and molar volume becomes more complex for PNAs For instance, lsopropylbenzene, with a molar volume of 139 5 ml mol -t at 20°C, has an aqueous solubility of 50 ppm (Sutton & Calder, 1975), while naphthalene, with a molar volume of 133 2 ml mol -~ at 25°C, has an aqueous solubdity of only 22 ppm, somewhat lower than would be predicted from a strict linear relationship between log solubihty and molar volume As shown by McAuhffe (1966) each homologous series of hydrocarbons has a shghtly different solubihty/molar volume relationship Scant information is available about the toxicity to marme ammals of aromatic hydrocarbons with molecular weights exceeding those of naphthalene Dlaromatlc hydrocarbons are toxic to shrimp (Penaeus aztecus and Palaemonetes pugto) at concentrations around 1 ppm, which corresponds well with findings 222

presented here (Anderson et al, 1974) Trlaromatic PNAs were quite toxic, their toxicity for N areanaceodentata was an order of magnitude greater than that of the naphthalenes The higher MW PNAs, chrysene, benzo(a)pyrene and dibenzanthracene exhibited little acute toxicity This finding may have been related to the low relative solubility of these compounds (see Table 1) Higher MW hydrocarbons may not have formed true solutions with seawater, thereby affecting their mobility across biological membranes, and hence modifying their apparent toxicity It seems likely that these hydrocarbons could have formed biologically macttve micelles with dissolved organic matter in test solutions, in the manner suggested by Beohm & Qulnn (1976) Analyses of concentrations within test organisms at selected times throughout bloassays would have shed much light on the biological avallablhty of specific PNAs in test solutions These data support the hypothesis that alkylatlon (--methylation) can substantially modify the chemical and biological characteristics of aromatic hydrocarbons (Anderson et al, 1974) For example, in addmon to bemg less soluble than their parent compounds (Table 1), &- and trimethylnaphthalene, and 1-methylphenanthrene proved significantly more toxic than naphthalene and phenanthrene, respectively These findings are of s~gmficance when one considers the relative abundance of alkyl-homologues (over parent hydrocarbons) of PNA constituents of commonly transported oils (Youngblood & Blumer, 1975) and recent marine sediments (Hltes, 1976) Concentrations of specific PNAs in marine sediments can exceed those which have proven toxic to the mfaunal representative studied hete (Armstrong, pers c o m m , Niaussat et al, 1975, Youngblood & Blumer, 1975) However, great caution should be taken before the environmental relevance of the laboratory findings reported here is fully assessed Factors such as synergistic effects of additional pollutants, as well as the role of sediment-hydrocarbon binding, could greatly modify the biological actwlty (availability) of PNAs under natural condmons The authors gratefully acknowledge the support of the American Petroleum Institute under contract No OS20C

Acheson, M A , Harrison, R M , Perry, R &Welhngs, R A (1976) Factors affecting the extraction and analysis of polynuclear hydrocarbons m water W a t e r R e s , l O , 2 0 7 - 2 1 2 Alzenshtat, Z (1973) Perylene and its geochemical significance Geochtm cosmochtm Acta, 37,559-567 Anderson, J W , Neff, J M , Cox, B A , Tatem, H E & Hlghtower, G M (1974) The effects o f o d on estuarine animals toxicity, uptake and depuratton, respiration Pollution and Physiology o f Marme Orgamsms, pp 285-410 Adademlc Press, New York Blumer, M (1976) Polycychc aromatic compounds in nature Scwnt A m , 234, 35 - 4 5 Boehm, P D & Quinn, J B (1976) The effect of dissolved orgamc matter m seawater on the uptake of mixed individual hydrocarbons and Number 2 Fuel Oil by a marine falter-feeding bivalve (Mercenarta mercenarta) Est cstl mar Scl, 4 , 9 3 - 1 0 5 Bories, G , Tulhez, J , Pelter, J C & FleckInger, R (1976) Evolution de la teneur en hydrocarbures ahpatiques et naphtenlques, ansi que an 3 4-benzopyrene, de Moules prelevees dans une zone cotiere pollue par une nappe du (fuel) C r hebd S~anc Acad Sct , Parts, 282, 1641-1655 Dunn B P &Such, H F (1976) Release of the carcinogen benzo(a) pyrene from environmentally contaminated mussels Bull environ contam Toxtcol, 15,389-401

Volume 9/Number 8/August 1978 Eganhouse, R P & Calder, J A (1976) The solubdlty of medmm molecular weight aromanc hydrocarbons and the effects of hydrocarbon co-solutes and sahmty Geochtm cosmochtm Acta, 40, 555 -561 Giger, W & Blumer, M (1974) Polycychc aromanc hydrocarbons m the enwronment ~solanon and charactenzatton by chromatography, visible, ultravtolet and mass spectrometry Analyt Chem, 46, 1663-1671 Hltes, R A (1976) Sources ofpolycychc aromanc hydrocarbons m the aquatic enwronment Proc Symp Sources, Effects and Sinks o f Hydrocarbons m Aquatw Envtron, A I B S , Arhngton, VA, pp 325 -332 Lee, R F , Sauerheber, R &Benson, A A (1972) U ptake, metabohsm and discharge of polycvchc hydrocarbons by marine fish Mar Btol, 17,210-208 Lee, R F , Ryan, C & Neuhauser, M L (1976) Fate of petroleum hydrocarbons taken up from food and water by the blue crab, Calhnectessaptdus Mar Btol, 37, 363-370 Litchfield, J T & Wflcoxon, F (1949) A simphfied method for evaluating dose effectwe experiments J Pharmac exp Ther, 96, 99-113 McAuhffe, C (1966) Solubility in water of paraffin, cycloparaffin,

Sub-lethal Effects of Pollution T h e R o y a l S o c i e t y held a 2 - d a y d i s c u s s i o n m e e t i n g in L o n d o n o n 2 4 - 2 5 M a y o n ' T h e a s s e s s m e n t o f sublethal effects o f p o l l u t a n t s in the sea' It revealed s o m e i m p o r t a n t d~chotomies o f a p p r o a c h a n d o u g h t to provoke a good deal of thought and reappraisal when the p a p e r s a n d discussions are p u b l i s h e d later this y e a r in the P r o c e e d i n g s o f the R o y a l S o c i e t y P a p e r s b y D r A R D S t e b b l n g , o n the use o f responses o f h y d r o i d clones as tests for low c o n c e n t r a tions o f p o l l u t a n t s a n d Dr. B r i a n B a y n e , o n sensitive tests f o r the h e a l t h o f M y t d u s e p i t o m i z e d the experim e n t a l , a n d b y n o w very s o p h i s t i c a t e d a p p r o a c h to toxicity testing a n d the d e t e c t i o n o f a n i m p a c t o f m i n u t e c o n c e n t r a t i o n s o f p o l l u t a n t s on, as n e a r as we are likely to get, ' s t a n d a r d ' o r g a n i s m s D r E J P e r k i n s , D r J D B u r t o n a n d D r G W B r y a n gave a series o f c a u t i o n s a b o u t the need for this sort o f s o p h i s t i c a t i o n B u r t o n o n speciatxon o f m e t a l s , for e x a m p l e , d r o p p e d a p r e t t y b r o a d hint t h a t in m u c h l a b o r a t o r y w o r k o n m e t a l s a n d m o s t field studies o n m e t a l p o l l u t i o n , we h a v e no i d e a o f the f o r m in w h i c h the m e t a l is a v a i l a b l e to the a n i m a l o r p l a n t , a m a t t e r which is a l m o s t c e r t a i n l y o f s u p r e m e i m p o r t a n c e , B r y a n s h o w e d t h a t we need to be m u c h less glib a b o u t b i o a c c u m u l a t i o n , a n d P e r k i n s t h a t ' l o n g - t e r m ' toxicity tests at best, u s u a l l y r e p r e s e n t such a small f r a c t i o n o f the l i f e - s p a n o f the o r g a n i s m t h a t a g o o d d e a l o f thought needs to be given to the usefulness o f struggling to e x t e n d t h e m f r o m 5°70 to 6°70 o f the l i f e - s p a n o f a n a n i m a l t h a t lives 9 o r 10 years T h e r e IS o b v i o u s l y s c o p e f o r a c o n s i d e r a b l e refinem e n t o f l a b o r a t o r y testing a n d e x p e r i m e n t a t i o n , m u c h o f w h i c h n o w a p p e a r s to be o f q u e s t i o n a b l e v a l i d i t y even In its o w n right W h a t is m o r e i m p o r t a n t is h o w the results o f r e s p o n s e s o f o r g a n i s m s to p o l l u t a n t s c a n be t r a n s l a t e d i n t o r e s p o n s e s o f m a r i n e e c o s y s t e m s . T h e d e v e l o p m e n t o f ' b i g b a g ' e x p e r i m e n t s In large enclos-

olefm, acetylene, cycloolefin, and aromanc hydrocarbons J Phys Chem 70, 1267-1275 Neff, J M & Anderson, J W (1975) Accumulanon, release and distribution of benzo(a)pyrene-[C TM] m the clam Rangta cuneata Proc J Conf Prev Cont Od Poll Amencan Petroleum Insntute, Washington, DC, pp 469-472 Nmussat, P M , Tncket, J , Heros, M , Luong, N G T & Ehrhardt, J P (1975) Mlse en ewdence de benzo-3,4-pyrene dans l'eau et les sediments organlques de mares saumatres d'atolls polyneslens Edute de certams facteurs bmnques et abloneues assocles C r hebd S~anc Acad Sct, Parts, 281, 1031-1034 Panorov, R J & Brown, R A (1977) Polynuclear aromanc hydrocarbons m marine nssues Envtron Sct Technol, 11,989-992 Schwarz, F P & Waslk, S P (1976) Fluorescence measurements of benzene, naphthalene, anthracene, pyrene, fluoranthene, and benzo(a)pyrene m water Analyt Chem, 48, 524-528 Sutton, C & Calder, J A (1975) Solubdlty of alkylbenzenes m distilled water and seawater at 25 0°C J chem Engng Data, 20, 320-327 Youngblood, W W & Blumer, M (1975) Polycychc aromanc hydrocarbons m the environment homologous series m sods and recent marine sediments Geochtm cosmochtm Acta, 39, 1303-1314

ures was an a t t e m p t to m o v e i n t o larger scale, b u t still in s o m e degree c o n t r o l l e d e x p e r i m e n t a t i o n D r J M Davis a n d D r J G G a m b l e reviewed p r o g r e s s In this field and Dr J H Steele gave the results o f the Scottish big b a g experiments in e c o s y s t e m simulation U n f o r t u n a t e l y , large enclosures d o n o t a p p e a r to p r o v i d e the p o w e r f u l t o o l t h a t they p r o m i s e d o n l y a c o u p l e o f y e a r s a g o T h e r e a r e p r o b l e m s in i n t e r p r e t ing the results o b t a i n e d f r o m them, w h i c h m a y be i n h e r e n t In the w h o l e c o n c e p t , a n d Steele c o n c l u d e d t h a t they have p r o b a b l y t a u g h t us m o r e a b o u t general e c o l o g i c a l i n t e r a c t i o n s t h a n a b o u t subtle l o n g - t e r m effects o f p o l l u t a n t s T h e d i f f i c u l t y o f assessing the i m p l i c a t i o n s o f small o r large scale e x p e r i m e n t s o n the subtle effects o f p o l l u t a n t s was u n d e r l i n e d in v a r i o u s ways in p a p e r s b y Dr M Waldichuk, Professor J S Gray, Dr D H C u s h i n g a n d M r A l a n P r e s t o n E a r l i e r in the meeting, one o r two s p e a k e r s h a d e m p h a s i z e d the g r e a t significance o f p o l l u t a n t s which depress the p r o d u c t i o n o f a species, b u t in fact C u s h i n g s h o w e d the wide a n d erratic n a t u r a l f l u c t u a t i o n In r e c r u i t m e n t to fish stocks a n d o t h e r m a r i n e o r g a n i s m s which d w a r f s a n y I m p a c t the s u b t l e effects n o t e d in the l a b o r a t o r y m i g h t have G r a y p o i n t e d to the g r e a t genetic h e t e r o g e n e i t y we n o w k n o w exists in a n u m b e r o f m a r i n e o r g a n i s m s such that, p a r t i c u l a r l y a m o n g o p p o r t u n i s t i c species, resistant f o r m s a r e selected on the s p o t to c o p e with local e n v i r o n m e n t a l c o n d i t i o n s (This places a big q u e s t i o n m a r k over the usefulness o f a search for genetically homogeneous 'standard' laboratory animals ) P r e s t o n ' s I m p o r t a n t p a p e r on the strategy f o r estabhshing s t a n d a r d s f o r p o l l u t i o n c o n t r o l e v i d e n t l y reflects the official U K a t t i t u d e a n d is s h a r p l y c o n t r a s t e d with the N o r t h - A m e r i c a n E P A a n d E P S a p p r o a c h e s T h e latter were e x a m p l l f I e d in Dr. R E i s l e r ' s p a p e r on b e h a v i o u r a l responses o f m a r i n e a n i m a l s to p o l l u t a n t s It is scarcely a p a r o d y o f his a r g u m e n t to say t h a t a test o r g a n i s m w o u l d be m a i n t a i n e d in a c o n t i n u o u s flow o f f a c t o r y effluent, s u r r o u n d e d b y a j u n g l e o f electronics, a n d each t i m e it b l i n k e d o u t o f t u r n the f a c t o r y w o u l d be a u t o m a t m a l l y s w i t c h e d o f f I n c o n t r a s t to this 223