Heavy metals in marine nematodes: Uptake, tissue distribution and loss of copper and zinc

Volume 1 4 / N u m b e r 7 / J u l y 1983

consequence o f a differential absorption or a selective trapping of the two kinds o f bacteria by the bivalves according to the dimensions of the microorganisms. The streptococci are arranged in chains, the shape o f which is greater than that of the bacilli rods. A second interpretation comes from a better adaptation of the faecal streptococci to the hostile conditions encountered in the digestive tract of the molluscs. This hypothesis fits with the results o f Prieur (1981 ) who showed that the bacterial population of the digestive tract of the mussels differs from the flora of the surrounding water. This selective effect may be linked to the presence o f specific digestive enzyme systems, This effect would amplify the relative resistance of the streptococci to various factors o f the environment (Geldreich & Kenner, 1969). As far as the variability o f the results is concerned, the counts performed on mussels are more consistent than those performed directly on water. This is particularly true for the streptococci. However, it has to be pointed out that samples o f the sea water concerned contained fairly low levels of contamination and thus there is considerable r o o m for variability in the results. This emphasizes the usefulness o f the bacterial enumeration obtained from filtering shellfish in mildly contaminated waters. The comparison between the level o f bacteria in mussels and in the surrounding water shows that the contamination of mussels partially reflects that o f the water at the same time. Different results would perhaps have been found if the analysis has been based on shellfish meat after the intervalvar water had been withdrawn. In conclusion, despite the limitation brought about by more laborious sampling and analyses, the use of shellfish as

concentrating material in the enumeration of bacterial pollutants appears to improve the results both in accuracy and consistency. This work is part of a general study of the bay of Concarneau, France, and has been supported by the Minist6re de I'Equipement. Babinback, J. A., G r a i k o s k i , J. F., Dudley, S. & Nitrowski, M. F. (1977). Distribution of fecal coliforms on b o t t o m sediments from New York Bight. Mar. Pollut. Bull., 8, 150-153. Breittmayer, J. P. (1978). Fiabilit6 des r6sultats. Rev. int. Oceanogr. reed., 50, 6 5 - 6 8 . Breittmayer, J. P. & Gauthier, M. J. (1979). D b n o m b r e m e n t des

bact6ries en milieu marin. Facteurs de variation et d'incertitude. Ann. Microbiol. (Inst. Pasteur), 130, 245-256. Bonde, G. J. (1977). Bacterial indication of water pollution. In Advances in Aquatic Microbiology. Academic Press, London. Cabelli, V. J. & Hefferman, W. P. (1970). Accumulation of E. coli by the mother quahaug. Appl. MicrobioL, 19, 239-244. Geldreich, E. E. (1967). Fecal coliform concept in steam pollution. Water and Sewage Works, R 98-R 100. Geldreich, E. E. & Kenner, B. A. (1969). Concepts of fecal streptococci in stream pollution, J. WaterPollut. ControlFed., 41, R 336-R 352. Hendricks, C. W. (1971). Increased recovery rate of Salmonellae from stream bottom sediments versus surface waters. Appl. Microbiol., 21, 379-380. Jorgensen, C. B. (1960). Efficiency of particle retention and rate of water transport in undisturbed lamellibranchs. J. Cons. perm. int. Explor. Mer., 26, 96-116. Majori, L., Campello, C. & Crevatin, E. (1977). Salmonella pollution of the Gulf of Trieste. Rev. int. Oceanogr. reed., 47, 181-191. Pellegrino, C., Carli, A., Cevasco, M. P & Scasso, M. 1. (1977). The Mytilus galloprovincialis Lamarck as an accumulator and indicator of telluric microorganisms in the sea waler. Rev. int. Oceanogr. reed.. 47, 155-160. Prieur, D. (1981). Nouvelles donn6es sur les relations entre bacteries et bivalves marins. Haliotis, 11,251-260. Rittenberg, S. C., Mittwer, T. & Ivler, D. (1958). Coliform bacteria in sediments around three marine sewage outfalls. Limnol. Oceanogr.. 3, 101-108.

MarinePollutionBulletin, Vol. 14, No. 7, pp. 263-268, 1983 Printed in Great Britain

0025-326X83 $3.00 + 0.00 © 1983Pergamon Pre~sI ld

Heavy Metals in Marine Nematodes: Uptake, Tissue Distribution and Loss of Copper and Zinc RICHARD HOWELL Zoology Department, The University, Newcastle upon Tyne, N E 1 7R U, U K

The uptake and loss of copper and zinc was studied in two species of marine nematode from a non-polluted site and in one species from a polluted site. Copper and zinc concentrations were measured during and after accumulation from seawater in six different tissues of the nematodes. Most of the metal taken up was found to be associated with the cuticle and with a group of tissues including the hypodermis. The importance of the different tissues in heavy metal metabolism by the nematodes is discussed together with the possible routes of uptake of the metals into the animals. The absence o f experimental studies on heavy metals in fleeliving marine nematodes prompted a general investigation on this ubiquitous and important group o f organisms (Howell, 1982 a). Two species were found to be suitable for

this investigation, from the point o f view o f size, distribution and availability. E n o p l u s brevis (Bastian) is a euryhaline species inhabiting intertidal sediment and Enoplus c o m m u n i s (Bastian) may be found living commensally in the holdfasts o f kelp (Laminaria hyperborea) or in the byssus threads of seed mussels (Mytilus edulis). Nematodes were collected from two sites on the north-east coast o f England (Fig. 1) and the characteristics o f the uptake and loss o f copper and zinc determined.

Methods Budle Bay is an unpolluted site approximately 80 km north o f Newcastle upon Type and contains large numbers of both species. The Blyth estuary is known to be signifi263

Marine Pollution Bulletin

Berwick upon

regions. The pharynx (up to the oesophago-intestinal junction) and the tail were removed first. Manipulation of the remaining 'tube' resulted in the separation of the gut tube, the cuticular tube and the remaining tissues. This latter group, comprising the hypodermis and muscle layers H o l y Island together with the reproductive structures, could not easily be UDLE BAY separated from each other and are termed 'other tissues' in the following paragraphs. The eggs of the females were successfully recovered and these formed the sixth type of tissue. The different regions were collected and pooled into groups of three to facilitate weighing. Thus there were three NORTH SEA replicates for each time interval. Tissues were first dried to constant weight on small pieces of filter paper over phosphorous pentoxide at room temperature. The desiccated pieces of filter paper and tissues were digested in a small volume of Aristar nitric acid in sealed vials at 200°C I ~ " " ~ B LY T H E S T U A R Y and the digests analysed using flameless atomic absorption Blyth spectrophotometry. The mean copper and zinc concentrations of the filters were determined by the same method and ..0~ the results of these analyses are shown in Table 1.

l

..-:

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Newcastle upon

Tyne

TABLE 1 The means and standard errors of the concentrations of copper and zinc in filter papers. Values in ~ugg 1 (ppm/.

I

20 km

I

Fig. 1 North-east coast of Britain showing the sites of study.

cantly polluted by a number of heavy metals (Cooper & Harris, 1974) and to contain large populations of nematodes (Capstick, 1959). Specimens ofE. brevis were collected by sieving the upper 2 cm of sediment through a nylon mesh (620 ~m) using clean filtered seawater and picking the nematodes off the mesh using a mounted needle and hand lens. E. communis was collected from among seed mussels also using a mounted needle and lens. Ingested sediment, which may interfere with heavy metal analyses (Flegal & Martin, 1977), was removed as described by Howell (1982b). Copper sulphate and zinc sulphate solutions in seawater were made up to a concentration of 1000/ag ml-~ (ppm). The seawater was collected from near Budle Bay and was filtered prior to use through a 0.45 ~m filter. Dilutions of the stock solutions provided experimental solutions of a concentration of 0.01 ppm. Animals were exposed to the experimental solutions in Petri dishes which were placed in a constant temperature room at 10°C and left unaerated for the duration of the experiment. Solutions were changed every 24 h. An artificial substrate of Ballotini glass beads was added to each dish. E. brevis from Budle Bay and the Blyth and E. communis from Budle Bay were used throughout the following experiments. Groups of animals were exposed to each metal in seawater solution. Samples of nine individuals were taken at time intervals of 0, 1,3, 6 and 12 days. After 12 days the animals remaining in each dish were transferred to clean seawater and further groups of nine individuals were taken at 13, 20, 30 and 40 days from the start of the experiment. After removal from the exposure medium the animals were rinsed several times in clean seawater and then dissected. It was possible to dissect the animals into six 264

Metal

Mean concentration (ppm)

Standard error

9.20 46.60

± 0.06 +4.60

Copper Zinc

Results Figures 2 and 3 show the uptake and loss of copper and zinc by the tissues of the three groups of animals. The uptake of metals was also expressed as an 'accumulation factor': TABLE 2 The accumulation factors (A.F.s) for copper and zinc in the tissues of E. brevis from Budle Bay and the Blyth and E. communis from Budle Bay exposed to 0.01 ~g m l - 1 (ppm) solutions of the metals in seawater. The A.F.s in the whole animals were weighted for the proportion of the whole worm made up by each type of tissue. Species/site

Tissue

Copper

Zinc

Cuticle Gut Eggs Tail Pharynx 'Other tissues'

2.6 1.7 2.0 2.3 6.3 7.5 4.21

2.0 1.0 2.8 1.7 6.0 6.5 3.55

E. brevis Budle Bay

Cuticle Gut Eggs Tail Pharynx 'Other tissues'

5.0 1.4 1.8 2.5 3.4 2.2 2.75

1.4 1.0 2.1 1.2 1.9 1.7 1.44

Cuticle Gut Eggs Tail Pharynx 'Other tissues'

10.6 9.0 4.5 9.1 9.2 4.7 8.37

13.0 4.0 4.0 3.9 5.0 4.7 6.25

A.F. in whole animal

E. brevis Blyth

A,F. in whole animal

E. communis Budle Bay

A.F. in whole animal

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Fig. 2 The uptake and loss of copper by the cuticle, gut, eggs, tail, pharynx and 'other tissues' of the two species of nematodes. © E. brevis from Budle Bay. • E. brevis from the Blyth. r! E. communis from Budle Bay. t Death point. Values in ~g g I (ppm). Bars indicate standard errors.

Accumulation Factor (A.F.) - Tissueconcentrationafter12daw Tissue concentration at day 0

The A.F.s for different tissues are shown in Table 2. A similar pattern of uptake and loss (strictly net uptake and loss) of copper was exhibited by the cuticle of all three groups of nematodes. There was a marked and rapid uptake of copper from solution which reached a plateau after approximately 3 days. Placing animals into clean seawater resulted in the immediate loss of copper, reducing the concentration associated with the cuticle by over 50°70. During the next 4 weeks the concentration was reduced still further but at a much slower rate and even after this period

there was a statistically significant (p< 0.02) amount of copper associated with the cuticle. The tail and pharynx showed similar characteristics to the cuticle in all three groups. This was probably due to the cuticular fraction of these tissues. Copper uptake by the gut ofE. brevis from both sites was relatively small and there was no convincing evidence that metal loss occurred during the period of depuration. In contrast, there was a marked uptake of copper by the gut of E. communis, achieving a final A.F. of over 9. An interspecific difference was also observed in the case of the 'ot her tissues'. In E. brevis from both sites, the 'other tissues' 265

Marine Pollution Bulletin Uptake

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Fig. 3 The uptake and loss of zinc by the cuticle, gut, eggs, tail, pharynx and 'other tissues' of the two species of nematodes. 0 E. brevis from Budle Bay. • E. brevis from the Blyth. F3 E. communis from Budle Bay. Values in pg g- t (ppm). Bars indicate standard errors. showed a steady accumulation of copper throughout the exposure period with no observable loss during depuration. In E. communis, however, a noticeable loss of metal from the 'other tissues' did occur during the period of exposure to clean seawater. The uptake of zinc by the cuticle of E. brevis from both sites was much less marked than for copper and the final concentrations achieved were noticeably lower. In the case ofE. communis, however, the uptake of zinc by the cuticle reached an A.F. of 13, the highest value noted throughout the experiments. As with copper, there was a rapid loss of a large percentage of this accumulated zinc during the period of depuration. There was no evidence for zinc uptake by the gut of E. 266

brevis from either site, while there was a significant net movement of the metal into the gut of E. c o m m u n i s during the experiment. Zinc uptake and loss by the tail and pharynx of E. c o m m u n i s appeared to reflect that shown by the cuticle, resulting in considerable metal accumulation in these tissues after 12 days. The tail and pharynx in E. brevis from both sites showed relatively small changes in zinc concentration. The highest zinc concentration was found in the 'other tissues' ofE. c o m m u n i s after 12 days exposure to the metal in seawater. Much of this accumulated zinc was lost during the period of depuration, in contrast to the results for E. brevis from both sites. The initial tissue distribution of copper differed both

Volume 14/Number 7/July 1983

ofE. communisaccumulates high concentrations during the 12 days' exposure. The difference in copper concentrations between the cuticle and the gut ofE. brevis from each site is perhaps indicative of the form and bioavailability of the metal at each site and hence its route of uptake. The results of total sediment analyses showed that the copper concentration at the Blyth was over six times that at Budle Bay (R. Howell, unpublished data). Despite this, the cuticular concentration of copper in Budle Bay animals is significantly (p <0.01) greater than in Blyth animals. It is suggested that this is indicative of the chemical form of the metal at the two sites. At Budle Bay the sediment contains relatively little organic matter (B. Morris, pers. comm.) and a significant amount of the copper will be present in ionic form. River Blyth sediment contains larger quantities of organic matter in various forms (sewage, coal particles; Buchanan & Longbottom, 1970) and it is likely that almost all the copper, being strongly electronegative, will be securely bound or complexed. Thus, copper ions are available in Budle Bay sediment for uptake by adsorption to the cuticle but not in Blyth sediment and this may account for the difference in the cuticular concentrations of copper initially observed in E. brevis from the two sites. Since the total copper concentration in Budle Bay sediment is low, the uptake of metal by the gut is not very great. At the Blyth, however, digestive processes in the gut lumen result in the absorption of significant amounts of copper, resulting in the high concentrations observed in the gut of Blyth animals. The 'other tissues' of Blyth animals also have high concentrations of copper and it may be that the metal is transported to these tissues after uptake by the gut. In addition, the gut Discussion may be the ultimate target of translocated metals, since it has The rapid uptake and loss of both copper and zinc by the been suggested by Jennings & Colam (1970) that the gut of cuticle of all three groups suggests that the primary event is free-living nematodes acts as a 'kidney of accumulation'. surface adsorption. There are several features in the struc- However, metals may also reach the 'other tissues' after ture and chemistry of the nematode cuticle which support absorption through the cuticle since exposure to copper and this hypothesis. The external cortical layer of the cuticle is zinc in seawater solution results in significant increases in the known to consist of nematode collagen and to contain concentrations of these metals in the 'other tissues' of E. disulphide and sulphydryl groups (Lee, 1966; Bird, 1971), brevis from both sites. This occurs despite the fact that E. both of which provide binding sites for heavy metals. The brevis tends not to imbibe seawater (personal observation) possession of these chemical groups leads to a net negative and so the most likely route for uptake from seawater charge on the cuticular surface and it has been demonstrated solution is via the cuticle. There are several structures in the pharyngeal region that the magnitude of this negative charge increases towards which may be important with regard to metal uptake and the tail of the animals (Zuckerman et al., 1975; Tattar et al., 1977). This property could account for some of the binding loss. The most prominent of these is the large glandular organ known variously as the excretory gland or the observed by the tail section. Unlike the shell of molluscs or the carapace of crusta- ventral gland, whose function is uncertain but which is ceans, the nematode cuticle is known to be highly metaboli- thought to be at least partly concerned with the moulting caUy active throughout life (Fujimoto, 1967; Samoiloff, process. The ventral gland of both species is known to 1973). Such activity would almost certainly lead to the secrete an acid mucopolysaccharide (King, 1977) which incorporation of heavy metal ions into the structure of the may have an important influence on heavy metal uptake and loss by the nematodes (Howell, 1982c). There are cuticle. Due to differences in experimental techniques (exposure various other smaller glandular structures in the pharynx, times and exposure concentration) it was not possible to together with pigment granules (L. Smith, pers. comm.) compare the A.F.s obtained for the nematodes with those and the respiratory pigment haemoglobin (Ellenby & obtained for other species reported in the literature. The Smith, 1966), all of which could affect heavy metal uptake relatively large A.F.s shown by some groups are probably and loss by this region. The role of the 'other tissues' is difficult to assess. This due to the small size of the nematodes, resulting in a large surface area to volume ratio, which will greatly influence the group included the hypodermis which is known to be highly metabolically active and as well as secreting the cuticle is rates of uptake of the metals. The low A.F.s indicate almost no net accumulation of thought to be the site of many physiological processes in the metals by the gut ofE. brevis from either site whereas the gut body (Crofton, 1968). As such it represents a highly specific

between species and between E. brevis from different sites. In E. brevis from Budle Bay, by far the greatest copper concentration was associated with the cuticle, with relatively lower concentrations being measured in the remaining five regions. In E. brevis from the Blyth, however, high copper levels were measured in the gut and 'other tissues' as well as the cuticle, with the 'other tissues' showing the highest concentration. After 12 days exposure to copper, the highest copper concentrations were associated with the cuticle in all groups of animals. Some indication of the overall permeability of the three groups of nematodes to copper can be gained from a consideration of Table 2. The sum of the A.F.s for the different tissues indicate that E. communis is far more permeable to copper than E. brevis and that there is also a noticeable difference between E. brevis from Budle Bay and the Blyth. The mechanism by which E. brevis from the Blyth restricts the net uptake of copper is not known. The intial distribution of zinc in E. brevis from both sites was quite different to that for copper. The gut showed the highest concentration in each case with smaller concentrations being measured in the cuticle. There were much lower concentrations in the tissues ofE. communis initially. Following exposure to zinc all groups showed very high concentrations in the 'other tissues' with lower, but increased concentrations in the cuticle. The A.F.s for zinc uptake by the 'other tissues' were generally lower than for copper and in fact there was no net accumulation at all in E. brevis from the Blyth.

267

Marine Pollution Bulletin target for toxicants of the heavy metal type, which are k n o w n to interfere with a wide variety of enzymes and cellular systems. A n i m p o r t a n t factor may be the position of the hypodermis, lying just beneath and contiguous with the cuticle. Uptake of metals, particularly copper, by cuticular adsorption a n d absorption (Bremner, 1961) would almost certainly result in a concomitant increase in the concentrations in the hypodermis. It is interesting to note that there was a time lag of 1-3 days before the copper concentration in the 'other tissues' of E. brevis began to rise. This may indicate that, particularly in Blyth animals, there is a translocation of the metal to the 'other tissues' after uptake at some other site. Since the animals were exposed to copper in seawater solution, the most obvious site of uptake is the cuticle. Since E. brevis tends not to imbibe seawater alone (personal observation), uptake via the gut would be much reduced. The means by which accumulated metals are b o u n d by the tissues of the nematodes has subsequently been investigated using radiotracers and electrophoresis (Howell & Smith, in preparation). The importance of various proteins, particularly components of the cuticle, has been demonstrated and in c o n j u n c t i o n with these studies a series of investigations using the techniques of electron microscopy and electron microprobe analysis has been undertaken. The results of this work will be reported in a later c o m m u n i c a tion.

This work was carried out in partial fulfilment of the degree of Ph.D (Howell, 1982a), under the supervision of Mr L. Smith. Grateful acknowledgement is made to N.E.R.C. for financial support and to Dr B. E. Brown for criticismof the manuscript.

Bird, A. F. (1971). The structure of Nematodes. AcademicPress, London. Bremner, K. C. (1961). The copper status of some helminth parasites with particular reference to host-helminth relationshipsin the gastrointestinal tracts of cattle. A ust. J. agric. Res., 12, 1188-l 199. Buchanan, J. B. & Longbottom, M. R. (1970). The determination of organic matter in marine muds: the effect of the presence of coal and the routine determination of protein. J. exp. mar. Biol. Ecol., 5, 158-169. Capstick, C. K. 0959). The distribution of free-living nematodes in relation to salinity in the middle and upper reaches of the River Blyth estuary. J. Anim. Ecol., 28, 189-210. Cooper, B. S. & Harris, R. C. (1974). Heavy metals in organic phases of river and estuarine sediment. Mar. Pollut. Bull., 5, 24-26. Crofton, H. D. (1968). Nematodes. Hutchinson, London. Ellenby, C. & Smith, L. (1966). Haemoglobin in Merrnis subnigrescens (Cobb), Enoplus brevis (Bastian) and E. comrnunis (Bastian). Cornp. Biochem. Physiol., 19, 871-877. Flegal, A. R. & Martin, J. H. (1977). Contamination of biological samples by ingestedsediment. Mar. Pollut. Bull., 8, 90-91. Fujimoto, D. (1967). Biosynthesisof collagenhydroxyprolinein Ascaris. Biochim. biophys. Acta, 140, 148-154. Howell, R. (1982a). The incidenceof heavy metal pollutants in Enoplus brevis (Bastian) and Enoplus cornrnunis (Bastian) with some observations on toxicity, accumulation, depuration and metal binding by proteins. Ph.D. thesis, Universityof Newcastle upon Tyne. Howell, R. (1982b). Levels of heavy metals in two species of marine nematodes. Mar. Pollut. Bull., 13,396-398. Howell, R. (1982c). The secretion of mucus by marine nematodes (Enoplus spp.). A possiblemechanisminfluencingthe uptake and loss of heavy metal pollutants. Nernatologica, 28, 110-114. Jennings, J. B. & Colam, J. B. (1970). Gut structure, digestive physiology and food storage in Pontonema vulgar& (Nematoda: Enoplida). J. ZooL (Lond.), 161, 211-221. King, T. P. (1977). Organelle associations in some nematode cells. Ph.D. thesis, Universityof Newcastle upon Tyne. Lee, D. L. (1966). The structure and composition of the helminth cuticle. Adv. Parasitol., 4, 187-254. Samoiloff, M. R. (1973). Nematode morphogenesis: Pattern of transfer of protein to the cuticle of adult Panagrellus silusiae (Cephalobidae). Nernatologica, 19, 15-18. Tattar, T. A., Stack, J. P. & Zuckerman, B. M. (1977). Apparent non destructive penetration of Caenorhabditis elegans by micro electrodes. Nernatologica, 23,267-269. Zuckerman, B. M., Himmelhoch, S. & Kisiel, M. J. (1975L Membrane surface charge changes with age in the nematode Caenorhabditis briggsae. 10th Int. Conf. Gerontology, Jerusalem.

Marine Pollution Bulletin, Vol. 14, No. 7. pp. 268-271, 1983

0025 326X/83 $3.00 ~ 0,00 © 1983 Pergamon Press l t d .

Printed in Great Britain

Oil Pollution on the Egyptian Red Sea Coast RIFAAT G. M. H A N N A Institute o f O c e a n o g r a p h y & Fisheries, R e d Sea Branch, Cairo, E g y p t

A simple weighing method using carbon tetrachloride as solvent was used to survey the dissolved hydrocarbon and tarball levels in the area of the Egyptian Red Sea coast. The area of investigation covers the coastal area from RasGharib down to the port of Qosier during the period from September 1979 to February 1981. This survey confirms the presence of extensive oil pollution in the area, especially at Ras-Gharib and Ras-Shukhair. Minimum concentrations were obtained at Ghardaqa where no dissolved hydrocarbons were detected (i.e. less than 10 ~g I-q in 1979-1980 but an increase to 20 ~g I-~ in 1981 was recorded. The high levels of pollution recorded in the southern part of the Gulf of Suez are related to the presence of the offshore oil fields, most of which are located at Ras-Gharib. 268

A b o u t 6 m tons of petroleum hydrocarbons are estimated to be released annually into the marine environment through m a n ' s various activities (NAS, 1975). Certain fractions of petroleum hydrocarbons are quite toxic to marine organisms, and some crude oils are more toxic than others, because of differences in basic composition (Ktihnhold, 1972). Oil has a immediate ecological impact on the marine environment, but the m a i n problems associated with oil spills have usually been related to amenities. Also commercial a n d recreational fishing can be impeded and shellfish beds may be covered by tarry oil residues. Extensive and serious oil pollution was observed in the coastal area of the Red Sea. This has been reported in a report by I M C O (The Intergovernmental Maritime Consultative Organization, 1971). The levels of oil pollution to