following exposure to selected environmental. S.D. BarnbeT*, M.H. Depledge. Plymouth Environmental Research Centre, Universit~~ of Plymouth, Drake.
Aquatic
Toxicology
40 (1997) 79-92
Responses of shore crabs to physiological following exposure to selected environmental S.D. BarnbeT*, M.H. Plymouth
Environmental Received
Research
22 January
Depledge
Centre, Universit~~ of Plymouth, Devon PL4 XAA. UK
1997; revised 6 March
challenges contaminants
Drake Circus. Plynouth.
1997; accepted
3 June
lYY7
Abstract
The major objective of this study was to determine whether sub-lethal effects of contaminant exposure could be detected in crabs subjected to standardised, environmentally realistic physiological challenges in the laboratory. Heart rate changes, associated with transition from rest to physical exercise, together with osmoregulatory ability were assessed and used as measures of the physiological competence of crabs following exposure to various concentrations of copper, arsenite and benzo[a]pyrene (B[a]P). Increasing concentrations of copper caused a corresponding increase in the heart rate of both resting crabs and crabs subjected to physical stress, suggesting an impairment to one or more of the processes associated with normal respiratory functioning. Furthermore, exposure of crabs to copper was clearly detrimental to osmoregulatory ability, with a concentration of 0.1 mg 1-l sufficient to cause greater reduction in haemolymph osmolality than that recorded for control animals exposed to similar low salinity conditions. The mean heart rate of crabs exposed to increasing concentrations of arsenite decreased under both rest and stress conditions. suggesting an impairment to one or more of the control processes associated with cardiac physiology. Arsenite appeared to have little impact on the osmoregulatory ability of crabs, even at a concentration of 1 mg 1-l. No significant impairment of either cardiac performance or osmoregulatory capability was observed in crabs exposed to relatively high doses of B[a]P, leading to the conclusion that acute exposure to B[a]P poses little immediate toxic threat to the physiological processes controlling heart rate and osmoregulation in adult shore crabs. 0 1997 Elsevier Science B.V. Kqwords:
rene;
Curcinus
maenas;
Osmoregulation;
Heart
rate;
Biomarker
*Corresponding
author.
Tel.: (01752) 232961:
0166-445X/97/$17.00 0 1997 Elsevier PII SO 166-445X(97)00040-4
fax: (01752) 232970
Science B.V. All rights
reserved
Copper;
Arsenite;
Benzo[a]py-
80
S.D. Bamber. M. H. DepledgelAquatic
Toxicology 40 (1997)
79-92
1. Introduction
At present, toxicological assessment of chemicals typically employs lethal dose tests which focus on mortality and therefore fail to take into account sub-lethal effects of exposure (Peakall, 1992). Furthermore, such tests are normally undertaken on animals maintained in a stable physical environment, demanding little or no response from physiological processes which may be critical to the long term survival of these animals in their natural environment (Cairns, 1986). Thus it is very difficult, in the absence of environmentally realistic physical challenges in laboratory test regimes, to accurately determine the potential impact that contaminants may have on natural populations of animals. The environmental relevance of such tests must therefore be questioned. The shore crab, Carcinus maenas, is commonly found in estuaries which are characterised by periodic fluctuations in salinity, temperature and aerial exposure. To successfully exploit this region animals must possess physiological and/or behavioural mechanisms to buffer them from such rapid environmental changes and it is the susceptibility to pollution of these processes that could prove key to their survival. In this study, shore crabs have been exposed to three contaminants commonly found in the environment, copper, arsenite and benzo[a]pyrene, with subsequent evaluation of the impact such exposures had on selected physiological processes. Variability in heart rate provides a potential measure of physiological competence in crabs (Depledge and Lundebye, 1996). Typically, heart rate is elevated in response to physical stress and this capacity for a rapid increase in respiration rate is presumably critical to the survival of crabs attempting escape from predators or physical perturbation. Depledge (1984) showed that circulatory and respiratory activity in shore crabs was disrupted when crabs were exposed to selected trace metals. In the present study, crabs were subjected to a standardised physical stress regime with heart rates measured both at rest and under physical stress in order to establish if such measurements provided sufficient sensitivity to detect exposure to low doses of contamination. Shore crabs are hyper-osmoregulators, maintaining their haemolymph osmolality above that of a dilute medium. In this way it is possible for them to tolerate a range of salinity from full seawater down to 4 ppt (Crothers, 1967) making exploitation of the rich resources of estuaries possible. Previous laboratory studies have indicated that some metals are detrimental to the effective functioning of the osmoregulatory processes (Thurberg et al., 1973; Johnson, 1988; Weeks et al., 1993). Contaminant concentrations used in these studies, however, were in excess of environmentally realistic levels. In the present study, osmoregulatory ability was assessed in crabs exposed to a range of metal concentrations, including levels more typically found in the environment. The aim of this study was to determine whether sub-lethal effects of contaminant exposure could be detected in crabs subjected to standardised, environmentally realistic physiological challenges in the laboratory.
SD.
2. Material
Bamber, M. H. DepledgelAquatic
Toxicology 40 (1997)
79-92
81
and methods
2.1. Collection and maintenance
sf crabs
Carcinus maenas were collected from the shore using a baited dropnet. Only green colour morph males with carapace widths in the range 5572 mm were selected for testing. Crabs subsequently exposed to copper and arsenite solutions were collected from the estuary of the river Yealm and crabs exposed to B[a]P collected from the estuary of the river Avon. Both rivers are located in south Devon in the UK. On return to the laboratory, crabs were maintained in an aquarium at 15 + 2°C. Crabs to be used for heart rate assessments following exposure to copper and arsenite were maintained in holding tanks of filtered seawater with a salinity of 34 ppt. Crabs to be assessed for osmoregulatory ability were immediately transferred to tanks containing water with a salinity of 30 ppt, made up with commercial sea salt (Instant Ocean). Crabs exposed to copper and arsenite were not fed prior to or during the experiments. Crabs exposed to B[a]P were initially held in filtered seawater at a salinity of 34 ppt and were fed following the regime described below. Photoperiod in the aquarium was 12:12 L:D. 2.2. Measurement
of heart rate
Heart rates of crabs were measured initially at rest and subsequently under physical stress, following a pre-defined period of exposure to contaminants. Heart rate was measured using a non-invasive infrared light system described fully in the account of Depledge and Andersen (1990). In brief, a coupled infrared transmitter and detector unit, located on the outer surface of the crabs carapace, beams infrared light onto the surface of the heart. As the conformation of the heart changes with each cardiac cycle the intensity of light reflected back to the detector fluctuates. The detected signal is fed to an analogue-to-digital converter and displayed on a computer screen. Cardiac pulses are visually identified and custom designed software used to detect and record each cardiac cycle, with a cumulative counter incremented with each complete cycle and downloaded at one minute intervals. Prior to the start of heart rate measurement, all crabs were transferred to individual 2.5 1 volume plastic tanks covered in 1ightLtight material. Water (0.5 1) taken from the holding tank was added to each test tank. Crabs were assessed in groups of eight with four tanks stacked upon four others, all on top of a rotary shaking table. Sensors were connected to the crabs which were then placed into the tanks and permitted 1 h to acclimatise. During this time the heart rate from each crab was monitored and the detection equipment tuned. Previous observations have shown that 1 h is sufficient time for heart rate to stabilise following the handling of crabs and that this stable rate is maintained for several hours thereafter if crabs are not further disturbed. The start of all experimental procedures was timed to minimise the potential influence of small changes in heart rate associated with endogenous rhythmicity. Following acclimatisation, recording of heart rate commenced. In the first hour of recording, the basal heart rate for each crab was established after
82
S. D. Bamber, M. H. DepledgelAquatic
Toxicology 40 (1997) 79-92
which stress, in the form of physical agitation, was applied for 1 h with the shaker table operating at 70 rpm. 2.3. Measurement
of haemolymph
osmolality
Following a predetermined period of exposure to the selected contaminants a haemolymph sample was taken from each crab just prior to their transfer to tanks containing water at salinity 5 + 0.5 ppt (copper and arsenite exposure) and 42 0.5 ppt (B[aJP exposure) made up with commercial sea salt. Further samples were taken at 2, 4, 8, 12, 24 and 48 hourly intervals. Approximately 50 ~1 of haemolymph was taken from each crab using a Drummond pipette to draw up the samples from a small wound made in the arthrodal membrane at the base of a walking limb. Samples were transferred to capped microcentrifuge tubes and immediately frozen in liquid nitrogen. In this manner a series of low volume samples was obtained from each single crab with a minimum of stress. Osmolality was measured using a vapour pressure osmometer (Wescor 5500) requiring a 10 ~1 volume sample for each evaluation. 2.4. Exposure
of crabs to copper and arsenite
solutions
Copper test solutions were formulated from copper chloride, CuC12, and arsenite solutions from sodium arsenite, NaAsO:! (Sigma chemicals). Copper concentrations of 0 (control), 0.1, 0.5 and 1 mg 1-l and arsenic concentrations of 0 (control), 0.01, 0.1 and 1 mg 1-l were tested. Crabs were exposed to test solutions for 40 h prior to evaluation of heart rate variability. The Salinity of water in exposure test tanks was 34 ppt (n = 8 for each test solution). Assessment of osmoregulatory ability was undertaken on a second group of crabs which were exposed to test solutions for 18 h in water with a salinity of 30 ppt prior to transfer to tanks containing contaminant free water at a salinity of 5 ppt (n = 8 for each test solution). 2.5. Exposure
of crabs to benzofalpyrene
Due to the extremely low solubility of B[a]P in water, an alternative route of exposure to crabs, via their food intake, was chosen. It is likely that this method represents a more environmentally realistic mode of exposure when compared to the artificial enhancement of the solubility of B[a]P in water using detergents. Squid mantle tissue was chosen as the carrier as it is readily consumed by crabs and, furthermore, the nature of the tissue structure ensures it does not disintegrate into the surrounding water when crabs are feeding, thus ensuring the majority of the contaminant is ingested by the crab. Squid tissue was cut and prepared into pieces weighing approximately 0.75 g. B[a]P was first dissolved in ethanol and subsequently transferred to the surface of the carrier tissue using a pipette. The treated food was then left in a fume hood for 15 min to facilitate the evaporation of
S.D. Bamber, M.H. DepledgelAquatic
Toxicology 40 (1997)
79-92
83
the ethanol, leaving the B[a]P adsorbed to the squid. As a control, further squid pieces were prepared and treated only with ethanol. Crabs collected from the shore were returned to the aquarium and maintained in holding tanks without food for a period of one week prior to the commencement of the experiment. This was done principally to ensure the crabs would readily consume the treated food. Eight crabs were given a total of 20 ng of B[a]P over a period of 7 days, in four separate feeds, each dose delivering 5 ng of the contaminant. Control treatments were fed to a further group of eight crabs at times coincident with the B[a]P exposures. Heart rate assessment and osmoregulatory ability tests were performed in sequence on the same two groups of eight crabs. Heart rate assessment was undertaken 8 days from the start of the experiment. On completion of this procedure crabs were returned to their test tanks containing seawater at a salinity of 34 ppt for a minimum of 18 h with no further food or B[a]P administered. The following day the osmoregulatory ability of the two groups of crabs was assessed employing the methodology described above.
3. Results 3.1. Crabs exposed to copper Mean heart rates of crabs monitored
over a 60 min rest period and a similar
140
controi
O.lmg/l
Copper
1q/l
0.5mg/l
Concentration
Fig. 1. Mean heart rates + SEM of Carcinus maenas exposed under physical stress (n = 8).
to copper,
both
at rest and when put
84
S. D. Bamber.
M. H. DepledgelAquatic
Toxicology
40 (1997)
79-92
period of physical stress are shown for all test groups in Fig. 1. Analysis of variance indicated a significant difference between treatments both at rest and under stress conditions (,**I’< 0.001). Least-squared difference (LSD) tests identified copper concentrations of 0.5 mg 1-l and 1 mg 1-l as producing significant increases in rate compared with control crabs, suggesting an impairment to a process, or multiple processes, associated with normal respiratory functioning. No obvious deviation in heart rate, either at rest or under stress, was evident in crabs exposed to 0.1 mg 1-l copper when compared to the controls. This indicates that, in isolation, environmentally realistic levels of copper associated with polluted sites are insufficient to cause overt interference in cardiac physiology. Copper at a concentration of 0.1 mg 1-l did, however, cause impairment to the osmoregulatory ability of crabs. Fig. 2 shows the change in haemolymph osmolality with time for all treatments with continued immersion in a low salinity medium. Analysis of variance indicted large inter-group differences (Table 1) and (LSD) tests confirmed the inability of copper exposed crabs to osmoregulate as effectively as control crabs. A relatively rapid fall in osmolality over the first 8 h was seen in control crabs, which slowed over the remainder of the test period. With increasing concentrations of copper the ability of treated crabs to maintain elevated haemolymph osmolality levels, similar to controls, was increasingly impaired. After 4 h in the low salinity medium, crabs exposed to 1 mg 1-l copper lost mobility, due to apparent muscular dysfunction, and were removed from the analysis. Mortality of crabs was zero during all analyses (including arsenite and B[a]P exposures). Crabs removed from the 1 mg 1-l copper and low salinity challenge subsequently recovered mobility and apparent health after transfer to clean, fully saline water. 1000 .,
0.1 ‘i-
mg/l
COPPtR
----__
.
1
500 0.5
4oo
A,_-_ 024
Hours
..~
.~_
17
24
from
transfer
8
Fig. 2. Comparative plot of changes in haemolymph to a range of copper concentrations and subsequently (n = 8).
mg/l
COPPER
~~~ -1 48
to 5ppt
water
osmolality with time (2 maintained in a medium
SEM) of crabs exposed with a salinity of 5 ppt
SD.
Bamher,
Table 1 Analysis of variance and (LSD) posed to various concentrations (n=S in all cases)
M.H. DepledgelAquatic
tests of haemolymph of copper following
Toxicology 40 (1997)
79-92
85
osmolality between control crabs and crabs extheir transfer to low salinity (5 ppt) medium
Hours after transfer to dilute medium
F-ratio
P value
Statistically significant concentration pairings
2 4 8” 12 24 48
8.16 15.64 2.98 16.91 23.14 33.82
“‘P
