MONITORING

BIO-AVAILABLE

WITH

MUSSELS IN THE

MARINE

(MYTILUS

CONTAMINANTS

E D U L I S L)

NETHERLANDS

W. CHR.

DE KOCK

TNO, P.O. Box 217, 2600 A E Delft, the Netherlands

Abstract. After discussing the usefulness of mussels (e.g. Mytilus edulis) for investigating contamination

in the marine environment, four cases are presented: (1) A survey of Cu bioavailability in and a r o u n d the Eastern Scheldt during one winter (1974 1975); (2) A surveillance with a six years interval (1974; 1980) of the bioavailability of Zn along the Dutch North Sea coast. (3) ,A monitoring p r o g r a m m e over the period 1972-1980 showing the decrease in bioavailable Hg in the water column of the Ems-Dollard estuary after decreased emissions at Delfzijl. (4) Retrospective monitoring of two P C B ' s , as a background for the effectiveness of legal restrictions to PCB use. These four cases are considered a first step in understanding the effects of pollutants in marine ecosystems. In depth physiological, histological and biochemical studies could lay the basis for improved routine biological effect monitoring.

1. Introduction

The fact that industries are interested in acquiring marine dumping licenses, and scientific evidence that certain parameters in marine exosystems deviate beyond their natural variations, call for impact monitoring and surveillance of the marine habitat as summarized in Figure 1. Water management authorities must prevent irreversible damage to vital natural functions in marine systems and, therefore, need to set limits for the quantity of specific contaminants which can be tolerated. The necessary knowledge o f doseeffect relationships can be provided by studies on the geochemical fate of contaminants and by ecotoxicological testing at different levels of biological organization. Sometimes useful data can be obtained f r o m past pollution accidents. Once a standard has been set, there is a need for monitoring, i.e. the repeated measurement of a variable so that trends are found and can be related to the standard (Holdgate, 1976). One way to monitor contaminants in the marine environment is to make use of appropriate bio-accumulating organisms, indicating the relative amounts of potentially harmful c o m p o u n d s that are bio-available. This is important, because toxic physiological effects are primarily related to the internal concentrations of the c o m p o u n d and the necessary toxicological standard, e.g. a 'no observed effect concentration' (NOEC) with a safety factor, could be derived f r o m laboratory studies, which couple concentrations in tissues to some physiological disturbance. Environmental Monitoring and Assessment 7 (1986) 209-220. 9 1986 by D. Reidel Publishing Company.

)

Fig. 1.

-

-

ib

l a b . :

by: retrospective analysis of accidents

-

by: prospective experiments on different levels of biological organization cell organ ~, organism population - field: ecosystem ('mesocosm') * simulation experiments * validation lab. tests

DOSE-EFFECT R E L A T I O N S H I P S

ECOTOXICOLOGICAL RESEARCH testing fate pollutants and effect on organisms

- development methods

- control by monitor&g * chemical * biological

ESTABLISHING STANDARD (flexibility necessary)

WATER MANAGEMENT aim: counteracting irreversible damage of vital natural functions

The role of ecotoxicological research and water management in solving aquatic contamination problems.

from: chemical field surveys (surveillance) relative concentration contaminants in abiotic components * surface water * suspended particles * sediment * interstitial water relative concentration contaminants in biotic components of ecosystem

-

from: biological field surveys (surveillance) (individuals) - mortality sublethal phenomena * growth change * reproduction change * disease * physiological or biochemical indicating parameters (populations)- species composition change * qualitative * quantitative - production change ( + or - ) (e.g. phytoplankton, zooplankton, fish)

raised by: W A R N I N G SIGNALS (retrospective)

raised by: REQUEST FOR D U M P I N G LICENSES /

C O N T A M I N A T I O N PROBLEM

non-toxicological criteria

MONITORING

BIO-AVAILABLE

MARINE

CONTAMINANTS

211

Such a 'standard' however needs reconsideration if more adequate or sensitive physiological methods become available. Moreover, a NOEC-assessment from a single-species laboratory study requires validation in suitable ecological test systems (e.g. 'mesocosms', Kuiper, 1984). Mussels are extensively applied as quantitative bioindicators of marine pollution. The filterfeeding genus Mytilus appears to fulfill the basic prerequisites of a monitoring organism, as listed by Phillips (1977, 1980), e.g. wide distribution, abundance, size, ease of collection, long life-span, euryhalinity, accumulation ability and high concentration factors for several xenobiotics. Monitoring schemes with Mytilus edulis are known internationally ('musselwatch', Goldberg et al., 1978; National Academy of Sciences, 1980). Various attempts to avoid natural variability related to factors such as age, size or vertical position on the shoreline, by translocating M. edulis to pollution gradients, have been described. Succesful efforts have been reported for instance for Hg in Scotland (Davies and Pirie, 1978), Zn and Pb in the U.K. (Simpson, 1979) Cd and PCBs in the Netherlands (de Kock, 1983) and various metals and hydrocarbons in Narragansett Bay, R.I. (Widdows et al., 1981). De Kock and Kuiper (1981) mention how temporal variation of total Hg levels in M. edulis could be reduced by replacing 'passive' monitoring with mussels sampled from the Dutch intertidal zone, by an 'active' approach. Large samples of continuously submerged mussels of a selected size class were translocated from a relatively unpolluted site to the target area and suspended from buoys in cages. This translocation technique has three advantages: (1) the resolution power between geographical locations is increased by employing statistically similar groups of animals derived from a common stock, (2) the chosen period of exposure to the polluted environment is known, and (3) the investigator can select the exposure locations independent of the natural occurence of the monitoring species (De Kock, 1983). Knowledge of contaminant equilibrium levels in the organisms concerned is required for a meaningful comparison of contaminant concentrations in biota between different locations within an observation network. The equilibrium levels characterize the average surrounding supply of a bio-available contaminant fraction at a certain location over a certain period. The location may be situated on a pollution gradient. The period is contaminantspecific and will depend on the combined rates of uptake and loss by the organisms. It should be noted that changes in ambient contaminant concentrations can result in either accumulation or elimination, defined as the net result of uptake and loss processes with time. An example of data derived from field translocation experiments with 34. edulis is shown in Figures 2A and 2B. Accumulation and elimination of the congener PCB-153 were simultaneously studied by translocating random samples of a selected length class from a relatively low-contaminated area (Eastern Scheldt) to a contaminated estuary (Western Scheldt, eastern part) and vice versa. The combined concentration data of the three accumulation series in the Western Scheldt (Figure 2A) were used in a simple mathematical model to describe accumulation and

212

W. CHR. DE KOCK 1,00

PCB - 153 pg kg -1 ash-free dry weight

500

200

100

days I

i

I

I

I

25

50

75

100

125

Fig. 2A. Mytilus edulis, L. Accumulation of congener PCB- 153 in three series of samples (90 individuals each, length class 3.0-4.5 cm), randomly composed from a single stock in the Eastern Scheldt (Kats) and translocated to the Western Scheldt (Perkpolder) on different dates (April 5, May 11, June 6, 1983). Concentrations in lag kg J ash-free dry weight.

32t~

PCB - 153 9 pg kg -1 ash-free dry weight

21,0

160

80

days

e's

Cs

,bo

,i,s

Fig. 2B. Mytilus edulis. L. Elimination of congener PCB-153 in three series of samples (75 individuals each, lenght class 3.0-4,5 cm, randomly composed from a single stock in the Western Scheldt estuary (buoy 22) and translocated to the Eastern Scheldt (Kats) on different dates (April 5, May 11, June 6, 1983). Concentrations in lag kg-~ ash-free dry weight.

MONITORING BIO-AVAILABLE MARINE CONTAMINANTS

213

elimination dynamics (De Kock and Van het Groenewoud, 1985). Assumptions were: (1) M. edulis is a one-compartment unit, (2) contaminant uptake and loss are only dependent of the ambient bio-available contaminant concentration, (3) equilibrium is reached as a consequence of simultaneously occurring reversible uptake and loss processes. With the model the equilibrium contaminant concentrations and time constants for accumulation or elimination for the Western Scheldt and the Eastern Scheldt could be estimated. Calculations showed that about 35 days were needed to approach the actual equilibrium for PCB-153 within 5%. It was thus shown feasable to estimate equilibrium levels of a bio-available contaminant fraction with an appropriate organism, notwithstanding the natural variability usually encountered in field experiments.

2. Survey, Surveillance, Monitoring The case studies presented below, illustrate the terms 'survey', 'surveillance' and 'monitoring', as used in Figure 1. Most field data have not been published before; they originate either from various reports prepared for sponsors, or from TNO data files, kept as reference material. Routine tissue sample treatment procedures have been described by de Kock (1983). 2.1. CU-SURVEY General copper pollution could be surveyed, using the translocation technique mentioned before with mussels, M. edulis, in and around the Eastern Scheldt during the winter of 1974-1975. Mussel samples were exposed for various periods to the water column at different sites (Figure 3) and recollected for heavy metal analysis in total soft tissue homogenates. Cu (among other metals) was analyzed by atomic absorption spectrometry (AAS) after wet destruction of about 2.5 g of material. Pearson's variation coefficient for the analytical programme on Cu, determined with a standard mussel homogenate, or calculated from duplicate samples, was less than 15 %. Atlantic specimens used for the survey were collected from an intertidal rocky shore site in Southern Ireland (Kilmore Quay), and are considered to represent natural background levels for Cu (de Kock and Kuiper, 1981). Table ! shows the outer and inner Eastern Scheldt as well as adjacent waters (Krammer and Volkerak) to be largely free from bio-available Cu contamination. Samples recollected in Lake Veere (saline, artificially separated from Eastern Scheldt waters in 1961) however, showed highly elevated Cu-levels near the locks at Veere, the Cu presumably originating from an industrial point source in the Walcheren Canal near Middelburg. The case was confirmed by the same procedure carried out simultaneously with the brown seaweed Fucus vesiculosus, collected in Atlantic waters (de Kock, 1975). From a toxicological laboratory study (Adema, 1972) and a field accumulation experiment (de Wolf et el., 1972) it is known that lethal Cu-levels in M. edulis tissue are to be found between 50 and 100 mg k g - l dry weight. Figure 4 shows LCsovalues

214

w . CHR. DE KOCK TABLE I

E x p o s u r e e x p e r i m e n t s with A t l a n t i c m u s s e l s a m p l e s (Mytilus edulis, 3 . 0 - 3 . 5 c m l e n g t h class, s a m p l e size 5 0 - 1 0 0 individuals). C u c o n c e n t r a t i o n s in m g k g - ~ a s h free d r y w e i g h t o f soft tissue h o m o g e n a t e s a f t e r t r a n s l o c a t i o n o f s a m p l e s f o r d i f f e r e n t p e r i o d s to e x p o s u r e sites in the E a s t e r n Scheldt a r e a . E x p o s u r e period V o l k e r a k

Krammer

E a s t e r n Scheldt

E a s t e r n Scheldt

L a k e Veere

(inner p a r t )

(outer part)

(Veere locks)

7.5 _

7.5 6.8

-

(days) 0 5 19

7.5 -

7.5 -

-

-

21 32

7.6

7.6 9.8

-

-

42 63

-

_

7.4

-

72 76

8.9

8.6

10.6-15.3

7.2-7.5

8.3

7.5 12.6 _ _ -

-

4 1 . 5

8.2 -

8.4

-

6.0-7.1

-

not a v a i l a b l e

Local populations

M. edulis

or,hS,

[fresh)

C a n a / ~ LakeVeere-/ (Saline) Fig. 3.

~ --

WesternSche/d;

E x p o s u r e sites E a s t e r n Scheldt a r e a . C u - s u r v e y 1974-1975, using Mytilus edulis

o f ambient Cu, dosed a s C u C l 2 , for different exposure times in these experiments, along with the accumulated levels in total soft tissues of still surviving mussels. The internal Cu-concentration found in Lake Veere (41.5 mg k g - l after 63 days exposure, Table I), while approaching the range of lethal effects, suggests the possible existence of a sublethal toxic situation. This case demonstrates that an

215

M O N I T O R I N G BIO-AVA1LABLE MARINE C O N T A M I N A N T S

Cu Cu-concentrations in total soft tissue LCSO in tJg I - I , homogenates at mortality in m9 kg-! ambient water Ix) dry weight (/.all}

3ooJ

200200~/F/e x

1oo. 7oo

.

~

i ~ ~ ,b

x

.

.

.

.

7 - -

.w

k

9

"

3b

x

,;o

days

s*o

Fig. 4. Cu-toxicity for M y t i l u s edulis, expressed as LC50 values (• in Ilg 1 - ~ambient water) at various exposure times. Hatched area: accompanying Cu-concentrations in total soft tissue homogenates at

mortality (in mg kg-1 dry weight).

integration of field survey data and dose-controlled experiments is necessary to estimate environmental risk of contaminants. 2.2. ZN-SURVEILLANCE In the summer of 1974 samples o f M. edulis collected f r o m a fouling stock on buoy T O 53~ '' N, 04~ '' E in the North Sea were used in exposure experiments with cages suspended f r o m buoys in coastal waters near IJmuiden and Petten. Similar caged samples attached to buoy T O itself served as an open sea reference. The procedure was repeated in the summer of 1980 with mussels of the same lenght class (4.0-5.0 cm). Recollection of samples t o o k place at various intervals in both years. The open sea reference exposure lasted about 250 days in 1974, the exposure period at the other locations did not exceed 30 days (1974) or 60 days (1980) respectively. Zn concentrations were measured by AAS after wet sample destruction, with a variation coefficient of ca. 5~ as determined with a standard mussel tissue h o m o genate. Although in comparison to 1974 relatively low levels of Zn were found in 1980, a gradient of bio-available Zn concentrations extending north of IJmuiden with decreasing values along the coast is still present (Figure 5). Contaminants along the Dutch shore are subject to a north-going residual current with a mean velocity of approximately 5 cm s e c - 1 (Otto, 1964; van Bennekom et aL, 1974). Additional data collected at more southern locations (de Kock and Marquenie, 1981) supported the

216

W. CHR. DE KOCK

North Sea ~ ~?3 ~/ Zn ~g g-I ~ .... osh-free dry weight Peften~ " ~ 506

F @/Ymulden

x Ymutden

1974

406 xpeffen

-

-

1980 i

306

206

/ ~ ~

-

50- km

Ymulden TO x

100

days

3'0

~o

~o

i~o

I~O

1~o

2~o

2~o

Fig. 5. Zn-concentrations (Ixg g-~ ash free dry weight) in M y t i l u s e d u l i s samples (4.0-5.0 cm lenght class) exposed at locations near IJmuiden (Baloeran buoy) Petten (Pettemer buoy) and in the open North Sea (buoy TO). Periods: 1974 (x, to=May 6), 1980 (O, to=June 4).

suggestion that the observed Zn gradient could possibly be related to industrial contamination o f the IJmuiden area. The case was not pursued. 2.3. HG-MONITOR1NG The Ems estuary became contaminated with mercury discharged by industrial sources near Delfzijl in the period 1960-1975. Action by water management authorities resulted in a reduction o f the estimated annual mercury input in the area from about 5 tons before 1975 to 0.6 tons in 1976 and 0.006 tons in 1977 and 1978 (Essink, 1980). The reduction o f the emissions was followed with monitoring Hg-concentrations in mussels, making use o f destructive neutron activation analysis. Figure 6 shows general results o f a biomonitoring programme carried out with M. edulis. Initially mussels were collected in the intertidal zone (1972-1974). In the years thereafter (1974-1980) monitoring was continued with translocated North Sea samples. The latter data reflect mean equilibrium levels after at least 30 days o f sublittoral exposure. The difference between mean total Hg concentrations measured in intertidal local populations o f M. edulis and those measured in the exposure experiments is probably related to age differences o f the animals. The effectiveness o f the reduction o f emissions is demonstrated by the gradual decline o f bio-available Hg in the water column after 1975. Hg-concentrations in the mussels t o o k about 4 years to drop to more natural levels. This monitoring exercise

MONITORING

total

BIO-AVAILABLE

MARINE

Hg

~ g g t ash

~

A

19'71 ';2

EMS - DOLL A R D

2

I 9

217

free dry weight ~)

xI

CONTAMINANTS

DELFZIdL X ~

8

'73 "7'4 '7'5 7'6 '7'7 '7'8 '7'9 '80 yeer

Fig. 6. Eros estuary, 1971-1980. Range of total mercury levels in Mytilus edulis tissue. (A) mean of range, local population. (B) mc,m of range, translocated samples in exposure experiments. (1), (2), and (3) sampling stations

however, did not reveal the deposition of mercury in estuarine sediments of the area. Sound ecological management will also require quantitative knowledge of Hg-immobilization in deeper layers, as well as data on local mercury mobility resulting from various dynamic processes such as evaporation, methylation and bioturbation. Monitoring studies with deposit feeding benthic macro-evertebrates (e.g. bivalves such as Macoma balthica) could provide essential information to supplement water column studies (de Kock and Marquenie, 1982). 2.4. PCB-MON1TORING Monitoring can also cover the retrospective analysis of contaminants in stored samples, provided that the analytical data match the requirement of detecting temporal changes in bioavailable contaminant load as a result of past management. This is opportune when new equipment or improved analytical methods become available or new contaminants become of interest. Figure 7 gives an example of such retrospective monitoring. In view of their toxicity and persistence the use of PCBs in open systems in the Netherlands has been drastically reduced on a voluntary base since 1972. The EEC guideline in 1976 was followed up by legal prohibition in 1979 of PCB-use in open systems. Deepfrozen soft tissue homogenates o f 45 samples stored at - 20 ~ during the period 1971-1982 were available for improved gaschromatographic analysis in 1983. Fig. 7 compares average yearly concentrations of two PCB-congeners, PCB-52

218 200

W. CHR. DE KOCK IJg kg -1

a s h - f r e e dry we/ghf

I

16(

PCB - 52

I

PCB - 180

I f I I

12(

970

74 78 South Holland

82

70

74 78 North Holland

82

I 70

74 78 years 82 Western Wadden sea

Fig. 7. Retrospective analysis of congeners PCB-52 and PCB-180 in Mytilus edulis tissue. Mean yearly concentrations (pg k g - l ash-free dry weight) for the South Holland coast, North Holland coast and Western W a d d e n Sea.

(quadrochlorinated, 2,5-2',5') and PCB-180 (heptachlorinated, 2,3,4,5-2',4',5') in tissues of M. edulis collected f r o m buoys in three different areas: (1) South Holland coastal waters just outside the Rhine-Meuse estuary, ( 2 ) N o r t h Holland coastal waters and (3) Western Wadden Sea. Spatially there is a decrease o f absolute PCB-52 concentrations going north from South Holland waters to the Wadden Sea, This is not the case for PCB-180. The lower chlorinated congeners decrease in the direction of the Wadden Sea, possibly because of better microbial or photochemical degradation in the sea. In time, however, no decline is observed in PCB-levels over the whole period since 1971, notwithstandig official attempts to reduce emissions. The study was carried out with samples of mussels f r o m natural fouling communities on buoys. Future exercises of this kind could, however, benefit f r o m combining proper storage techniques with the advantages of exposure experiments mentioned in Section 1.

3. Concluding Remarks These four case studies on contamination of marine waters of the Netherlands with bio-available copper, zinc, mercury and P C B ' s represent practical illustrations of the survey (Cu), surveillance (Zn) and monitoring (Hg, PCB's) concepts. The survey brought to light the existence of an undesirable toxic Cu pollution situation in Lake Veere. The surveillance was conducted as a repeated survey but did not have a checking function; it indicated the presence of a gradient with elevated Zn concentrations in coastal waters north of IJmuiden. The Hg-monitoring was

M O N I T O R I N G BI O- AVAI LABLE MARINE C O N T A M I N A N T S

219

carried out to check the effectiveness of pollution abatement measures by water management authorities and showed for the water column the time dimensions involved. Retrospective monitoring of P C B ' s was also carried out in relation to legal restrictions to the use of P C B ' s . These four cases of distributional patterns of bioavailable contaminants in field situations, are only a first step in understanding the effects of anthropogenic waste in marine ecosystems. More in depth studies of the effects of elevated contaminant concentrations in biotic components of the ecosystem on key biological factors like growth and reproduction are needed. Aspects such as energy budget disorders, histopathological conditions, lysosomal destabilization, enzyme induction mechanisms or various biochemical measurements as stress-indicating mechanisms in M . edulis and other c o m m o n macro-evertebrate species (e.g. Bayne et al., 1979, 1985) m a y be amenable to routine biological effect monitoring in the near future. References Adema, D. M. M., de Swaaf-Mooy, S. I., and Bais, P.: 1972, 'Laboratory Investigations Concerning the Influence of Copper on Mussels (Mytilus edulis)' TNO-nieuws 27, 482-486. Bayne, B. L., Moore, M. N., Widdows, J., Livingstone, D. R. and Salkeld, P.: 1979, 'Measurement of the Responses of Individuals to Environmental Stress and Pollution: Studies with Bivalve Molluscs', Phil. Trans. R. Soc. Lond. B 286, 563-581. Bayne, B. L., Brown, D. A., Burns, K., Dixon, D. R., Ivanovici, A., Livingstone, D. R., Lowe, D. M., Moore, M. N., Stebbing, A. R. D., and Widdows, J.: 1985, The Effects o f Stress and Pollution on Marine Animals, Preager Publ. New York, 375 pp. Bennekom, A. J. van, Krijgsman-Van Hartingsveld, E., van der Veer, G. C. M., and van Voorste, H. F. J.: 1974, 'The Seasonal cycles of Reactive Silicate and Suspended Diatoms in the Dutch Wadden Sea', Neth. J. Sea Res. 8 (2-3), 174-207. Davies, I. M. and Pirie, J. M.: 1978, 'The Mussel Mytilus edulis as a Bioassay Organism for Mercury in Sea Water', Mar. Pollut. Bull. 9, 128-132. Essink, K.: 1980, 'Mercury Pollution in the Ems Estuary', Helogltinder wiss. Meeresunters. 33, 111-121. Goldberg, E. D., Bowen, V. T., Farrington, J. W., Harvey, G., Martin, J. H., Parker, P. L., Risebrough, R. W., Robertson, W., Schneider, E., and Gamble, E.: 1978, 'The Mussel Watch', Environ. Conserv. 5, 101-125. Holdgate, M. W.: 1976, 'Closing Summary', in Jenifer M. Baker (ed.), Marine Ecology and Oil Pollution, Applied Science Publ. Ltd., England, pp. 525-535. Kock, W. Chr. de: 1975, 'Milieutoxicologische waarnemingen over de verontreiniging van het Oosterschelde-areaal met zware metalen', Report MT-TNO, CL 75/83, Delft, The Netherlands. Kock, W. Chr. de and Kuiper, J.: 1981, 'Possibilities for Marine Pollution Research at the Ecosystem Level', Chemosphere 10, 575-603. Kock, W. Chr. de and Marquenie, J. M.: 1981, 'De experimentele toepassing van de mossel Mytilus edulis L. bij het meten van zware metalen en organische microverontreinigingen in Nederlandse kustwateren', Report MT-TNO MD-N&E 81/2, Delft, The Netherlands. Kock, W. Chr. de and Marquenie, J. M.: 1982, 'The Effect of Discharges of Certain Metals and Organochlorine Compounds such as PCBs and Pesticides on Marine Ecosystems', Commission of the European Communities, EEG 4/81/81, Final report (TNO Rep. CL 82/97) 178 pp. Kock, W. Chr. de: 1983, 'Accumulation of Cadmium and Polychlorinated Biphenyls by Mytilus edulis Transplanted from Pristine Water into Pollution Gradients', Can. J. Fish. Aquat. Sci. 40, (Suppl. 2), 282-294. Kock, W. Chr. de and van het Groenewoud: 1985, 'Modelling Bioaccumulation and Elimination Dynamics of some Xenobiotic Pollutants (Cd, Hg, PCB, HCB) Based on 'in situ' Observations with Mytilus edulis', Report MT-TNO R85/048, Delft, The Netherlands.

220

w. CHR. DE KOCK

Kuiper, J.: 1984, 'Marine Ecotoxicological Tests: Multispecies and Model Ecosystem Experiments', in G. Persoone, E. Jaspers, and C. Claus (eds.), Ecotoxicological Testing for the Marine Environment. State Univ. Ghent and Inst. Mar. Scient. Res., Bredene, Belgium. Vol. 1, pp. 527-588, National Academy of Sciences: 1980, The International Mussel Watch, National Academy of Sciences, Washington D.C., 284 pp. Otto, L.: 1964, 'Results of Current Observations at the Netherlands Lightvessels over the Period 1910-1939. Part I. Tidal Analysis and the Mean Residual Currents', Meded. Verh. K. Ned. Met. Inst. 85, 1-56. Phillips, D. H. J.: 1977, 'The Use of Biological Indicator Organisms to Monitor Trace Metal Pollution in Marine and Estuarine Environments - a review', Environ. Pollut. 13, 281-317. Phillips, D. H. J.: 1980, 'Quantitative Aquatic Biological Indicators', (Pollution Monitoring Series), 488 pp. Applied Science Publishers Ltd. London. Simpson, R. D.: 1979, 'Uptake and Loss of Zinc and Lead by Mussels (Mytilus edulis) and Relationships with Body Weight and Reproductive Cycle', Mar. Pollut. Bull. 10, 74--78. Widdows, J., Phelps, D. K. and Galloway, W.: 1981, 'Measurement of Physiological Condition of Mussels Transplanted Along a Pollution Gradient in Narrangansett Bay', Mar. Environ Res. 4, 181-194. Wolf, P. de, Kock, W. Chr. de, and Stare, A.: 1972, 'Field Experiments on the Influence of Copper and Mercury in a Natural Musselbed', TNO nieuws 27, 497-504.