“Survival in air” of the blue mussel Mytilus edulis L. as a sensitive response to pollution-induced environmental stress

.I. Exp. Mar. Biol. Ecol., 170 (1993) 179-195 0 1993 Elsevier Science Publishers B.V. All rights reserved

JEMBE

179 0022-0981/93/$06.00

01993

“Survival in air” of the blue mussel ~~~~2~~e&is L. as a sensitive response to pollution-induced environments stress Richard H. M. Eertman a, Arco J. Wagenvoort”, Herman Hummel” and Aad C. Smaalb “lvetherlands Institute of Ecology, Centre for Estuarine and Coastal Ecology, Yerseke. The Netherlands: ‘R~kswaterstaat, Tidal Waters Division, ~idde~burg, The Netheriands (Received

14 December

1992; revision received

15 March

1993; accepted

26 March

1993)

Abstract: Mussels, Mytihu edulis, were exposed for periods of 6 weeks at various locations in Dutch coastal waters during 1989 and 1990. “Survival in air” showed to be a sensitive response parameter for indicating pollution induced environmental stress in transplanted mussels sampled from eight field sites. Increased tissue contaminant levels, especially PCBs and PAHs, correlated with a reduced survival time during aerial exposure. Three weeks exposure of mussels in the laboratory to 1 pg.l-’ PCBs affected the aerial survival time negatively. Laboratory experiments did not indicate that lowered salinity influences the “Survival in air” response after sufficient acclimation (15 days), facilitating the use of this response parameter in both marine and estuarine waters. Key words: Aerial exposure;

Monitoring;

Mortality;

PCB; Salinity stress

INTRODUCTION

The blue mussel Mj&z~ e&&s L. is commonly used as indicator species of environmental pollution in routine monitoring programmes in coastal waters (satin et al., 1984; Borchardt, 1988; Widdows & Johnson, 1988; Nelson, 1990; Smaal et al., 1991). Through its sessile and filter-feeding behaviour the blue mussel accumulates contaminants, such as trace metals, polychlorinated biphenyls and polycyclic aromatic hydrocarbons, which reflect the contaminant levels at the exposure locations. In search of sensitive biological effect parameters that can serve as early warning indicators of pollution induced environmental stress in coastal waters, “Survival in air” was developed as a stress response that utilises the natural ability of mussels to survive periods of aerial exposure. Intertidal mussels will be exposed to air during periods of low tide. When mussels are exposed to air they will close their valves and are unable to feed. The mussels may maintain a small valve gap through which oxygen can diffuse into the mantle cavity. The rate of oxygen consumption under these circumstances may vary according to ration, acclimatisation, season and temperature (Shick et al., 1988). In intertidal musCorrespondence Coastal Ecology,

address: Vierstraat

R.H.M. Eertman, Netherlands Institute 28, 4401 EA Yerseke, The Netherlands.

of Ecology,

Centre for Estuarine

aned

R.H.M.EERTMANETAL.

180

sels oxygen consumption tion, whereas suddenly

subtidally

may account acclimatised

for up to 40% of total metabolic mussels

may not consume

heat dissipa-

oxygen at all when

exposed to air. In the latter group of mussels the tissues will progressively

deprived of oxygen, the mussels aerobic to anaerobic metabolism.

be

will lower their energy demand and will switch from Under anaerobic conditions carbohydrates, glycogen

in particular, become the only or main source of energy (de Zwaan and Wijsman, 1976). The fermentation of glycogen under anaerobic conditions is considerably less efficient in comparison to the aerobic oxidation of glycogen. However, instead of using the universal Embden-Mayerhof-Parnas pathway, in which glycogen is fermented into lactate, mussels are capable of using a more efficient glycogen fermentation pathway leading to the formation of succinate and propionate, more than doubling the anaerobic ATP yield (Zandee et al., 1986). M. edulis is able to survive aerial exposure for many days, but under sustained aerial exposure the mussels will eventually die. Mussels subjected to toxic substances or natural stress in general have an increased metabolic rate (de Zwaan & de Kock, 1988) and are therefore expected to have a reduced aerial survival time in comparison to mussels from less contaminated locations. The “Survival in air” response was first experimentally tested in mussels by Veldhuizen-Tsoerkan et al. (1991). Short term laboratory exposure to cadmium and long term exposure to PCBs resulted in a significantly reduced tolerance to aerial exposure. The first application of the “Survival in air” response of mussels following field exposure produced significant inverse correlations between tissue contaminant concentrations and tolerance to aerial exposure (Smaal et al., 1991) confirming the potential of this parameter as stress indicator in coastal waters. In this paper the results of “Survival in air” experiments are presented that were performed on mussels in 1989 and 1990 following 6 weeks exposition periods at locations in the Dutch coastal waters with a varying degree of contamination. Some of these locations are situated in the Westerschelde estuary where a decreasing salinity gradient is present. Salinity is a naturally occurring stressor which may affect the “Survival in air” response. To assess the effect of salinity on “Survival in air”, mussels were exposed in the laboratory to unpolluted seawater with a lowered salinity. Natural studied validate cability mental

mussel populations from the Oosterschelde and Westerschelde estuaries were to observe seasonal variations in “Survival in air” response. Furthermore, to field responses, mussels were exposed in the laboratory to PCBs. The appliof the “Survival in air” as a sensitive response to pollution induced environstress will be discussed.

MATERIALSAND

METHODS

Animals

The mussels that were used for the field and laboratory obtained from a single, subtidally acclimatised, reference

exposure experiments were population in the relatively

“SURVIVAL

IN AIR” OF THE BLUE MYTILUS

EDULIS

L.

181

unpolluted Oosterschelde estuary in the south-western part of the Netherlands. Following transport to the laboratory, located near the entrance of Oosterschelde estuary (see Fig. I), the experimental animals were selected randomly from the 45 to 50 mm length class and cleaned of epibionts. Prior to the start of the exposure experiments the mussels were left in running Oosterschelde seawater for 24 h. All laboratory exposure experiments were conducted under semi-field conditions, indicating the use of how-through systems with a continuous flow of Oosterschelde seawater of ambient temperature and salinity, and natural suspended particles. Field exposure

Mussels were exposed for periods of 6 weeks during summer (July-August) and autumn (October-November) of 1989 and 1990 and winter (Feb~~-Mach) of 1990. Figure I shows the locations of the eight exposure sites: [ 1] and [2]: North Sea (NS), [ 31: Oosterschelde (OS), [4], [ 51 and [6 1: Westerschelde estuary (WS-west, centre and east), a busy shipping route to Antwerp harbour, [ 71: entrance to the Haringvliet (HV), a basin which receives water from the rivers Rhine and Meuse, and [8]: a station in the northerly coastal current (NAM). The field exposure in the winter of 1990 was limited to the Oosterschelde and Westerschelde exposure sites, due to stormy weather on the North Sea.

1m

Sea

Fig. 1. Map of the study area in the south-western part of the Netherlands. For locations terials and Methods, Field exposure. L = location of laboratory.

see under Ma-

1x2

R. H. M. EERTMAN

ET AL.

For each exposure site 170 mussels were placed in polye~ylene baskets, which were mounted in a stainless steel frame. At each exposure site a frame was connected to a buoy. At the end of an exposure period the mussels from each exposure site were collected, wrapped in disposable nappies and stored in freezepack cooled polystyrene boxes. The temperature of the mussel ~omp~tment varied between 4 and 6 “C. The boxes were transported to the laboratory, where they arrived at intervals but within 24 h after collection. To standardise for the various transportation times, the boxes were left untouched until the standard 24-h transport/storage period had elapsed. At the completion of the storage period the boxes were opened and the mussels were transferred to tanks with running natural seawater, where they were allowed to recover for a period of 24 h. Laborutoiy exposure

Mussels were exposed in the laboratory for 15 days to three saline environments: 3.5, 28 and 23x,. The exposure media were composed of a mixture of natural seawater, filtered seawater and distilled water to obtain the desired salinities, and maintaining equal algal diets and concentrations of suspended particles. For each expe~mental salinity 150 mussels were exposed in a 20 1 flow-through system, in which the seawater was completely renewed every 4 h. To prevent nutritional depletion in the exposure systems due to the filtration activity of 150 mussels in 20 1 seawater, the algal diet was increased by adding 60000 cellsm-‘*h-l of the diatom P~~eodact~IM~tr~corn~~~ to each of the exposure systems. Exposure to PCBs

Mussels were exposed to PCBs under semi-field conditions in 80 1 fibreglass tanks with a continuous supply of fresh seawater (20 l*h-“). One ml of a stock-solution of PCBs (technical mixture Clophen A50, Bayer, Leverkusen; 500 mg*l’), using DMSO as organic solvent, was added to every 24 1 of a culture of P. tricornutum (DMSO concentration in algal suspension: 0.004% v/v). Follo~ng sufficient mixing, spiked algae were added continuously (1 1-h-l) to the experimental tank to obtain a nominal concentration of 1 pg PCBs*l-‘. A control group of mussels was exposed to natural seawater with added DMSO solvent. To guarantee a sufficient algal diet additional, untreated, algae were added to the experimental and control tanks. Survival in air

At the start of each test 30 mussels were collected at random from a group that was exposed in the held or an experimental group and placed on a tray. The mussels were damp dried with paper tissue and the tray was placed in a constant temperature room of 18 “C and 83% relative humidity. In 1989 the temperature in the constant temper-

“SURVIVAL

IN AIR” OF THE BLUE ~~ILUS

EDVLIS

L.

183

ature room was slightly lower (16-17 “C). The response in survival time is affected by the temperature at which the animals are exposed. Lower temperatures result in higher LT,, values (Veldhuizen-Tsoerkan et al., 1991). From 1990 onwards, the temperature and relative humidity in the constant temperature room were recorded continuously. Daily recordings were made of the number of animals alive, also registering the time of inspection. Animals were considered dead when shell-gape occurred and an external stimulus (prodding tissues with a probe and/or squeezing of the valves) did not generate any response. Occasionally dead animals did not show she&gape, but distributed a specific smell. Dead animals were removed immediately. Gbcogen content

The glycogen content of mussels was determined using the method described by Bergmeyer (1984).

The developmental stages of the gonads of mussels were determined using the Gonad-Index of Seed (1975). The Gonad-Index ranges from 0, resting stage, to 5, spawning. Tissue contaminant concentrations The mussels were frozen in dry ice immediately after sampling and, in the case of field exposures, transported to the laboratory. The soft body tissue of 100 mussels was dissected, homogenised and divided into four equal portions of which one was freezedried prior to chemical analyses. Chemical analyses were performed in duplicate and the variation coefficient was less than 10%. Analyses of trace metals were performed by AAS, following digestion with concentrated nitric acid. PCBs and PAHs were extracted from the samples by hexane/acetone (1:3) for 4 h. The extracts were subjected to a clean-up procedure using a column with Florisil and elution with hexane/acetone (1:3). For the estimation of PCBs, the extract was further purified on a dried (180 “C) SiO, column by elution with hexane. PCBs were measured with a Hewlett Packard (HP) 5880 gaschromatograph fitted with two capillary columns and two ECD detectors. PAHs were further isolated on a dried (180 ’ C) SiO, column by elution with hexane/diethylether and the extract was brought quantitatively on a column with 1 g AlO, (dried at 180 “C). PAHs were separated by a Vydac 201 PB-5 (250 x 4.4 mm) reverse-phase column and a methanol gradient, and analysed by a HP 1090 liquid chromatograph with two HP 1046A fluorescence detectors.

184

R.H.M.EERTMAN ETAL.

As the survival time of animals does not conform to a normal distribution, the non-parametric Kaplan-Meier test was used to estimate the survival curves of the various groups of mussels (Kaplan and Meier, 1958). A confidence limit of 95% was used to test the significance of differences between groups. LT,, values were estimated using the trimmed Spearman-Karber method (LX= 10%) (Hamilton et al., 1977). Correlation coefficients between tissue contaminant levels and the “Survival in air” response were tested statistically, using the t-test for significance of the product-moment correlation coeficient (Sokal and Rohlf, 1981). The presence of correlations between the various tissue contaminants was tested with the Pearson correlation, using Bonferroni adjusted probabilities (Wilkinson, 1990).

RESULTS

Figure 2 shows the tissue concentrations of various contaminants at the end of the five exposure experiments. The lowest tissue concentrations of PCBs were found in mussels exposed at the two off-shore North Sea locations. The highest concentrations were observed in mussels exposed in the Westerschelde, where an increasing gradient was present from west to east. Mussels exposed in the Oosterschelde had moderately elevated tissue PCB concentrations in comparison to mussels exposed at the North Sea exposure sites. At the locations HV and NAM intermediate PCB concentrations were observed, comparable to the levels found in the western part of the Westerschelde. The distribution in tissue concentrations of PAHs was comparable to that of PCBs. However, mussels exposed at the stations HV and NAM had tissue concentrations of PAHs that were lower in comparison to the Westerschelde and similar to those in the Oosterschelde. The spatial distribution patterns in the tissue concentrations of Cd and Cu were less pronounced in comparison to those for the organic contaminants. Elevated levels of Cd and Cu could be observed only in mussels exposed in the Westerschelde. However, the tissue concentrations of Cu were elevated in only two of the five series. When examining the temporal distribution in tissue contaminant levels, it appears that mussels accumulate higher concentrations of PAHs in autumn in comparison to summer, but the highest PAH concentrations were found in winter. In the Westerschelde mussels accumulated lower levels of PCBs in 1990 in comparison to 1989. At the end of each exposure period, the “Survival in air” response of the exposed mussels was determined. The results of these experiments are summarised in Table I. Mussels exposed in the Westerschelde estuary have a reduced survival time in air in comparison to mussels from the Oosterschelde. A declining survival time was observed from west to east. Mussels exposed at the two off-shore North Sea locations, with the lowest tissue contaminant levels, had a “Survival in air” response which was usually similar to or higher than that of Oosterschelde mussels. Table II shows that, with

ws-w

ws-0

WSe

HV

NAM

q

0 NSl

NS-2

OS

NS-1

NS-2

OS

WS-c

m Summer'89

Exposure sites

WSw

m

WSe

Autumn’89

HV

NAM

Winter+$XJ

q

NS-1

OS

Summer ‘90

NS-2

a

sites

WSe

Autumn 90

lfixpsure

WSw

WSe

HV

NAM

NAM

i

_

_.

Fig. 2. Contaminant levels in M. erlulis following 6 weeks exposition in the Dutch coastal water. PCB concentrations are the sum of PCBs 18, 28, 31, 44, 52, 101, 118, 138. 153. 170, 180 and 187. PAH concentrations are the sum of Fen. Ant, Flu. Pyr. BaA. Chr, BeP, BbF, BkF, BaP, BghiP, DBahA and Inp.

”

0

2

4

6

8

10

12

14

WSc

WS-w wsc

16

OS

ttM0

NS2

0.8

1.6

Cd concentration (x&kg Dw)

Cu concentration @g/kg Dw)

NSl

(x&g DW)

PAH concentration (rig/g DW)

0

PCB ~n~ntration

5

186

R. H. M. EERTMAN

ET AL.

TABLE I “Survival in air” response by M. edulis following 6 weeks field exposure. For abbreviations see under Ma(OS) animals terials and Methods, Field exposure. * Response differs significantly from Oosterschelde (p < 0.05). a: Mussels were lost during exposure period, b: Mussels from these locations could not be used for “Survival in air” response as transport from exposure site to laboratory lasted 40 h, exceeding the maximum 24 hour standard period, nd.: not determined. LT 50 (days)

Locations Summer NS-1 NS-2 OS ws-w ws-c WS-e HV NAM

‘89

7.0 6.6 7.5 6.8 6.1* 5.4* 5.0* 5.3*

Autumn

Winter ‘90

‘89

Summer

n.d. n.d. 4.6 4.0* 3.7% 3.1* n.d. n.d.

7.8* 8.5* 6.3 5.1* 4.8* 4.0* 4.6* 6.5

‘90

4.0* 3.4 3.3 3.: 3.0* 2.9* 2.3*

Autumn

‘90

4.2 b 3.9 3.1 3.5* 3.1* 3.5* b

exception of the summer measurements, significant correlations exist between “Survival in air” response and tissue contaminant concentrations. However, the Pearson correlation, using Bonferroni adjusted probabilities, demonstrated that the tissue concentrations of PCBs, PAHs and Cd are correlated significantly. During the autumn and winter measurements significant correlations were always found for the organic compounds. In summer significant correlations could not be demonstrated. As the “Survival in air” response was studied during field exposure experiments in different seasons, the possibility of seasonal influences on the biological response had to be investigated. Therefore the “Survival in air” response of reference mussels from the Oosterschelde was studied at regular intervals during 1991. Figure 3 shows that the LT,, values decline in winter, reaching the lowest values in March, coinciding with the spawning season. LT,, values increase gradually during summer, reaching the highest

TABLE II Correlation

coefficients between tissue concentrations of contaminants “Survival in air” response. Significant correlations:

Contaminants

Z PCBs Z PAHs Cd cu

Correlation

following 6 weeks field exposure

and

*p
coefficients

(r)

Summer ‘89 (n = 8)

Autumn ‘89 (vi = 8)

Winter ‘90 (n = 4)

Summer ‘90 (n = 7)

Autumn ‘90 (n = 6)

0.562 0.406 0.32 1 0.124

0.928** 0.879** 0.618 0.564

0.995*x 0.964* 0.990** 0.975*

0.554 0.384 0.098 0.091

0.927* 0.860* 0.795 0.943**

“SURVIVAL

LT 50 (days) 2-P

IN AIR” OF THE BLUE ~~~~~~

Gb-gen@e’gDw)

EDULIS

187

L.

Glycosyl @mole/g DW)

80

60

i

“;a=_,,,,, 8.

JFMAMJJAi-

Gonad Index

T(OC) 2.5,

lime (months) Fig. 3. Seasonal variations during 1991 in (A) “Survival in air” response, (B) glycogen content, (C) gonadal development and (D) seawater temperature of mussels from the relatively unpolluted Oosterschelde.

values towards the end of the year. The glycogen content of the mussels was lowest during March and April, followed by a sharp increase during May and June. A gradual decline in glycogen content was observed towards the end of the year. The increase in LT,, values and glycogen content in spring and the moment of spawning started when the seawater temperature reached 10 “C. During a study of natural mussel populations in the Westerschelde estuary, with monthly sampling for one year, a comparison could be made between “Survival in air” response and the body glycogen content of mussels inhabiting areas with different contaminant levels. Figure 4 shows that, despite corresponding glycogen concentrations, mussels from the eastern, most polluted, region of the Westerschelde had a lower “Survival in air” response in comparison to mussels from the western region of the Westerschelde. In the Westerschelde estuary a salinity gradient prevails, ranging from 32s0 in the western region to z 12x0 in the eastern region. As the salinity at the most eastern exposure site (WS-e) ranges from 14.5 &0.7x, in winter to 23.3 rf 1.2g0 in summer (1989-1990 values), a possible effect of lowered salinity on the “Survival in air”

R. H. M. EERTMAN

188

ET AL.

LT 50 (days)

20

40

60

80

loo

120

140

GLYCOGEN (mg/g DW) Fig. 4. Fig. 4. Glycogen content versus “Survival in air” response of mussels from the western and eastern region of the Westerschelde estuary. Correlation coefficients for both curves are 0.7.

response had to be investigated. Mussels were exposed for 15 days in the laboratory under semi-field conditions to seawater media with lowered salinities. Other variables, such as the availability of food and the concentration of suspended particles, were kept identical in all three experimental situations. The results of the “Survival in air” experiments are shown in Table III. After 1 and 4 days adaptation to 23& or 4 days adaptation to 28x0 salinity the survival time in air of the mussels was significantly reduced in comparison to control mussels (35x,). A similar reduction was not observed after 7 and 15 days adaptation. Mussels adapted for 15 days to 23x0 had a significantly increased survival time in air in comparison to the other groups.

TABLE III “Survival in air” response by M. edulis following laboratory exposure to seawater with lowered salinity. * Response is significantly lower in comparison to response of control mussels (35%&p < 0.05). # Response is significantly higher in comparison to response of control mussels (p < 0.05). Exposure

start 1 day 4 days 7 days 15 days

time

LT,a (days) 35%0

28%0

23%0

3.1 2.8 3.2 3.1 3.2

3.1 2.8 2.4* 3.4 3.2

3.1 2.5* 2.v 3.2 4.0 #

189

“SURVIVAL IN AIR’ OF THE BLUE MYTILUS EDULIS L. In the field mussels establish

the toxicity

exposure

experiments,

are exposed to a mixture of contaminants, of individual mussels

contaminants.

were exposed

To validate

in the laboratory

making it difficult to the results under

of the field

semi-field

con-

ditions to 1 pg.ll’ PCBs. Two weeks exposure to 1 pg.ll’ PCBs did not reduce the tolerance to aerial exposure significantly, but mussels exposed for 3 and 4 weeks had a significantly reduced survival time in air compared to control mussels (Fig. 5). Control mussels had LT,, values of 3.9 and 4.1 days after 3 and 4 weeks exposition, respectively, while the LT,, values of the mussels exposed to PCBs had declined to 3.1 and 3.4 days, respectively. During the exposure period the mussels had accumulated 494 and 656 pg PCBs.kg-’ dry weight after 3 and 4 weeks exposure respectively. DISCUSSION Most adult, subtidally acclimatised, mussels tightly close their valves when acutely exposed to air, cease to consume oxygen and are predominantly anoxic during aerial reexposure (Shick et al., 1988). In these animals “Survival in air” is predominantly lated to anaerobic metabolic processes. Under anaerobic conditions glycogen is used as the only substrate for ATP generation, with the exception of the initial hours of anoxia where aspartate is the major substrate (Zandee et al., 1986). The results of the study into the seasonal effects on the “Survival in air” response indicated that the survival time in air of reference mussels from the relatively unpolluted Oosterschelde appears to be influenced by the glycogen content of the mussels, the stage of gonadal development and the seawater temperature. The process of gonadal development,

0 Days of aerial exposure

1

2 3 4 5 6 Days of aerial exposure

7

8

Fig. 5. Anoxic survival curves of mussels following (A) 3 weeks and (B) 4 weeks exposition to 1 pg.1 ~’ PCBs under semi-field conditions. Mussels exposed to PCBs had a significantly reduced survival time: (A) pc 0.01, (B) p
R. H. M. EERTMAN

190

which is activated metabolic

by the seawater

energy production

lowest body glycogen

content

temperature

(Bayne, coincide

ET AL.

(Seed,

1975), requires

up to 94% of

1976a). The lowest survival time in air and the with the spawning

period.

The observed

gly-

cogen cycle corresponds with earlier observations by de Zwaan and Zandee (1972) and Hummel et al. (1988). De Zwaan and Wijsman (1976) estimated that mussels consume ~37 pmol ATP.g-‘.WW during the initial 48 h of anoxia, of which 21 pmol were linked to glycogen fermentation (z 168 pmol.ATP.g-‘.DW, assuming a WW:DW ratio of 8). During prolonged anoxia energy demand is met by the sole fermentation of glycogen into propionate with a yield of 6.4 mol ATP per mol glycosyl (de Zwaan, 1983). Moreover, the ATP utilisation rate reduces during prolonged anoxia (Ebberink et al., 1979). Consequently, during the “Survival in air” test mussels ferment less than 26 pmol of glycosyl.g-‘.DW. The glycogen content of the mussels varied from 125 to 500 pmol glycosyl*g-r*DW. Therefore, it seems unlikely that the “Survival in air” response is influenced directly by the glycogen content of mussels, as the glycogen content does not show to be the limiting factor. This conclusion is confirmed by the results of the study of natural mussel populations in the Westerschelde. Mussels from the eastern, most polluted, region of the Westerschelde estuary showed a reduced tolerance to aerial exposure in comparison to mussels from the western region, despite corresponding glycogen contents. The glycogen content of mussels can therefore not be responsible for the reduced survival time in air in contaminated organisms. In early spring, during the final stages of gametogenesis, the high energy requirement of mussels seems to be mainly responsible for the low tolerance to aerial exposure. LT,, values immediately increase following the spawning period. The “Survival in air” response may also be influenced negatively by a temperature shock. Following transfer of the mussels from the field locations to the laboratory, the mussels were allowed to recover in running seawater of ambient temperature at the end of the storage period. Subsequently, the mussels were exposed to air in a constant temperature room of 18 “C, introducing a temperature shock which may range from = + 14 “C in winter to -2 “C in summer. Figure 3 shows that from April to August increasing LT,, values coincide with a decreasing AT. However, a gradual increase of AT, as experienced from September to December, did not result in decreased LTSo values. Nevertheless it is feasible that a large temperature shock in combination with the process of gametogenesis, a period during which mussels are most vulnerable to stress, may have a negative effect on the “Survival in air” response. The influence of the seasonal variation in tissue contaminant levels on the annual cycle in “Survival in air” response appears to be relevant only when relatively large and consistent fluctuations in contaminant concentrations occur during the year. The seasonal cycle in LT,, values shown in Fig. 3A was measured in mussels from the Oosterschelde with relatively low tissue contaminant concentrations. Although the mussels used for the determination of the seasonal variation in tolerance to aerial exposure were not the same as the ones used in the field study, the tissue contaminant levels are comparable to the values measured at the Oosterschelde exposure site during the field

“SURVIVAL IN AIR” OF THE BLUE MI’fI,5US

study. The low LT,, values in winter-early contaminant location.

levels (especially

However,

PAHs

spring coincide

and Cu) in mussels

despite higher tissue contaminant

to summer, similar LT,, ences in tissue cont~in~t

191

EDULIS L.

with the highest seasonal from this relatively

levels in autumn

clean

in comparison

values were measured in both periods. The seasonal differlevels in mussels from the Oosterschelde seem to be too

small to have a significant effect on the seasonal pattern in “Survival in air” response. Field exposure experiments have demonstrated that “Survival in air” is a useful early warning biological response parameter for indicating pollution induced stress in mussels. Especially in winter and autumn significant correlations could be demonstrated between “Survival in air” response and tissue cont~inant levels. However, in summer significant correlations could not be demonstrated (Table II). The relatively high seawater temperatures in summer may have a negative effect on the “Survival in air” response. When the water temperature varies between 5 and 20 “C the physiological response of mussels is relatively independent of temperature (Bayne, 1976b). At 20 “C the mechanism of metabolic compensation starts to decline. During the summer experiments the maximum water temperature at the exposure sites approached the upper limit of this temperature range (1989: 19.9 t 0.3 ‘C; 1990: 18.3 it 0.4 ’ C). Furthermore, in summer the tissue concentrations of PAHs were 35-60% lower in ~omp~ison to autumn, possibly caused by a higher biotransformation rate of compounds due to higher water temperatures. A reduced concentration range in summer may explain the lower correlation coefficients for PAHs, but similar seasonal discrepancies were not observed for the other contaminants. Close examination of the “Survival in air” responses of mussels in summer and the accompanying tissue contaminant concentralions revealed that mussels exposed at stations HV and NAM, exposure sites under the influence of the Haringvliet basin, had lower LT,, values than might be expected with reference to their tissue contaminant levels. If the results from these two stations are excluded from the data set, for statistical purposes only, the correlation coefficients between individual contaminants and the “Survival in air” response improve considerably (Table IV). Exclusion of the stations HV and NAM from the data set does not only improve the correlations of the summer measurements, resulting in significant correlations for PCBs and Cd in the summer of 1989, but also further improves the already significant

correlations

of the autumn

measurements.

It seems that other fac-

tors must have a negative effect on the “Survival in air” response at these stations, particularly in summer. The variation in the concentrations of the most obvious natural variables, availability of food, expressed as the concentration of chlorophyll-~, and the concentration of suspended particulates in the seawater at the exposure sites is shown in Fig. 6. Although seasonal and spatial variation occurs, significant correlations between each of the parameters and the “Survival in air” response could not be demonstrated (Table V). As both stations HV and NAM are influenced by the Haringvliet basin, which in itself is a recipient of water from the rivers Rhine and Meuse, it seems possible that mussels at these stations are afkcted by contaminants which are transported to the Haringvliet basin by the rivers Rhine and Meuse, especially in

R. H, M. EERTMAN

192

ET AL.

TABLE IV Correlation coefficients between tissue concentrations of contaminants following 6 weeks field exposure and “Survival in air” response, excluding the results from stations HV and NAM. Significant correlations: *p
Contaminants

C PCBs C PAHs Cd CU

coefficients

(r)

Summer ‘89 (n = 6)

Autumn ‘89 (n = 6)

Winter ‘90 (n=4)

Summer ‘90 (n = 5)

Autumn ‘90 (n = 5)

0.850’ 0.759 0.897* 0.460

0.959** 0.949** 0.783 0.755

0.995** 0.964s 0.990** 0.975*

0.691 0.704 0.588 0.465

0.972** 0.977** 0.930* 0.975**

summer, but which are not chemically analyzed in the mussel monitoring programme. Although the correlation coefficients improve considerably in the summer measurements when stations HV and NAM are excluded, the r-values remain lower than the winter and autumn values. The results of the salinity experiment showed that the survival time in air may be affected negatively after short-term exposure to lowered salinities of 28 or 23x,, but a significantly decreased survival time was not observed after 7 days and 15 days exposure. Although the salinity at the most eastern exposure site in the Westerschelde estuary fluctuated around 2 1.9 + 1.4x,, during the summer and autumn exposure periods, lower values down to 14x,, may be experienced during the winter period. At this latter salinity the survival time in air may be affected negatively (Weber et al., 1992). The results of the laboratory experiment do not give any indication that the “Survival in air” response is affected negatively after long-term adaptation to salinities as low as 23~~. Veldhuizen-Tsoerkan et al. (1991) demonstrated toxic effects of PCBs on the “Survival in air” response after long term (3 months) semi-field exposure of mussels to 1 pg*l-r PCBs. The results of the present study show that the survival time in air of

TABLEV Correlation coefficients between the average concentrations chlorophyll-a (Chl-a) and suspended particles (S.P.) in seawater at the exposure sites and the “Survival in air” response. No significant correlations, for all r-values, p> 0.05. Variables

Chl-a S.P.

Correlation

coefficients

Summer ‘89 (n=8)

Autumn ‘89 (,z = 8)

Winter ‘90 (?I = 4)

0.514 0.257

0.556 0.775

0.543 0.572

(r) Summer (n=7) 0.221 0.297

‘90

Autumn ‘90 (n = 6) 0.536 0.580

“SURVIVAL

Chlorophyll-a

IN AIR” OF THE BLUE MYTILUS

EDULIS

L.

193

@g/L)

ISI

NS-1

NS-2

Suspended particulates

NS-1

NS-2

OS

WS-tV

WS-c

WS-e

HV

NAM

ws-w

ws-c

WS-e

HV

NAM

(ma)

OS

Bipmure sites I Summer ‘89 q Autumn ‘89 D Winter ‘90 IZISummer ‘90 q Autumn ‘91) Fig. 6. Average concentrations

of chlorophyll-a and suspended particulates field exposure experiments.

at the exposure

sites during the

mussels is si~i~c~tly reduced after short term (3 to 4 weeks) exposure to 1 pg*l-’ PCBs, although no sign&ant change in response occurred between 3 and 4 weeks exposure. The accumulated PCB levels after 3 weeks exposure are comparable to the highest values found in the eastern part of the Westerschelde. This would suggest that tissue PCB concentrations as found at the eastern most polluted location in the Westerschelde could have a significant negative effect on the tolerance to aerial exposure of

EERTMANETA~,

R.H.M.

194

mussels, but lower PCB concentrations, as observed at the other exposure locations, would not have a significant effect on the “Survival in air” response. Possible synergistic effects with other toxicants can of course not be excluded. Considering the tissue contaminant concentrations in mussels at various locations around the world (Picard-Berube et al., 1983; Farrington et al., 1983; Hamilton, 1988; Widdows & Johnson, 1988 and Claisse & Simon, 1991), the contaminant levels in mussels exposed in the Dutch coastal waters belong to the lower ranges of measured values. This shows that “Survival in air” is a sensitive response parameter, which, thanks to its methodological simplicity, has the potential of becoming a universally applicable stress parameter in marine and estuarine waters.

ACKNOWLEDGEMENTS

The authors wish to thank Mr F. Smedes for the chemical analyses, Mr R. Duin and MS B. Kater for statistical advise,and Drs W. Zurburg and A. de Zwaan for critically reading the m~uscript. This study was performed under contract of the project BEON*EFFECT of the Tidal Waters Division, Ministry of Transport and Public Works.

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