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Observations on the interaction of zinc and cadmium uptake rates in crustaceans (amphipods and crabs) from coastal sites in UK and France differentially enriched with trace metals
Aquatic Toxicology 50 (2000) 189 – 204 www.elsevier.com/locate/aquatox
Observations on the interaction of zinc and cadmium uptake rates in crustaceans (amphipods and crabs) from coastal sites in UK and France differentially enriched with trace metals P.S. Rainbow a,*,1, C. Amiard-Triquet b, J.C. Amiard b, B.D. Smith a, W.J. Langston c a b
Department of Zoology, The Natural History Museum, Cromwell Road, London SW 7 5BD, UK Ser6ice d’Ecotoxicologie, CNRS-GDR 1117, ISOMer, Faculte´ de Pharmacie, 1 Rue Gaston Veil, 44035 Nantes Cedex 01, France c Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB, UK
Received 18 May 1999; received in revised form 3 November 1999; accepted 24 November 1999
Abstract This paper presents results on the possible interaction of zinc and cadmium uptake rates in crustaceans. Zn and Cd uptake rates were measured in amphipods (Orchestia gammarellus) and crabs (Carcinus maenas and Pachygrapsus marmoratus) from five coastal sites in Britain and France subjected to different degrees of trace metal enrichment. The presence or absence of 100 mg l − 1 of one metal (1.53 mM l − 1 Zn, 0.89 mM l − 1 Cd) had an inconsistent effect on the rate of uptake of the other metal by O. gammarellus. The presence or absence of 50 mg l − 1 of either zinc (0.76 mM l − 1) or cadmium (0.45 mM l − 1) had no effect on the rate of uptake of the other metal by C. maenas (from Millport, Scotland). Zinc and cadmium uptake rates were correlated in individual amphipods and crabs of both species from the five sites. These correlations indicate that zinc and cadmium might share common routes of uptake from solution by crustaceans, but the metals do not consistently interact competitively or synergistically at the exposure concentrations investigated. Regression coefficients of the relationship between zinc and cadmium uptake rates in amphipods and crabs showed occasional, but inconsistent, differences between sites and over time. All three crustaceans take up zinc from solution at a higher rate than cadmium for the same total dissolved metal molar concentration, but at a lower rate than cadmium per free metal ion molar concentration. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Trace metals; Uptake rates; Amphipods; Crabs; Orchestia; Carcinus; Pachygrapsus
* Corresponding author. Tel.: + 44-207-9425275; fax: + 44-207-9425054. E-mail address: [email protected] (P.S. Rainbow). 1 Previous address: School of Biological Sciences, Queen Mary and Westfield College, London, E1 4NS, UK. 0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 9 9 ) 0 0 1 0 3 - 4
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1. Introduction Due to their similar chemistries, zinc and cadmium generally occur together in metal inputs from different sources, particularly in mining effluents which are responsible for metal contamination in the metal-rich sites studied in the present paper (Restronguet Creek, Dulas Bay, Gironde Estuary). Moreover, as a result of their chemical affinities, these trace metals may share uptake pathways into aquatic invertebrates (Rainbow, 1997a,b). Most of the literature dealing with the fate and effects of metals in the aquatic environment report data obtained from single metal exposures, whereas, in the field, organisms are generally exposed to mixtures of contaminants (see Amiard-Triquet and Amiard, 1998). The interaction of one trace metal on the uptake or accumulation of another by aquatic invertebrates can result from competition for absorption sites at the level of biological membranes (Simkiss and Taylor, 1989), and/or from competition for metabolic sites within the cell (Mason and Jenkins, 1995). Interactive effects vary from synergistic (where the concurrent presence of one trace metal enhances the bioaccumulation of another), to antagonistic (where the concurrent presence of another metal decreases the bioaccumulation of the first). Many studies dealing with Cd – Zn interactions have used bivalves as experimental models (see Amiard-Triquet and Amiard, 1998). On consideration of these data, the biological role of a trace metal — whether it is essential like zinc or nonessential like cadmium — seems to be a feature that can influence the nature of any interactive effect. In many of these molluscan species, exposure to the non-essential metal Cd has no effect on Zn accumulation, whereas exposure to the essential metal Zn often has an antagonistic effect on Cd accumulation (Amiard-Triquet and Amiard, 1998). There are fewer studies available on Cd – Zn interactions in crustaceans (Ray et al., 1980; Ahsanullah et al., 1981; Devineau and Amiard-Triquet, 1985; Chou et al., 1987; Nugegoda and Rainbow, 1995; Bat et al., 1998). The opportunity is taken here to make a selective in-depth analysis of Cd – Zn competition, us-
ing data recently published in an extensive study of trace metal uptake rates in amphipods and crabs from a range of coastal sites differentially enriched with trace metals (Rainbow et al., 1999). This latter paper addresses (primarily) whether crustaceans from metal-rich sites are under selective pressure to reduce trace metal uptake rates, and whether the presence or absence of a dispersive larval phase affects such selective pressure. In the event we found that the exposure of the crustaceans to raised trace metal availabilities at these sites had not been sufficient to select for a reduction in dissolved trace metal uptake rates, even in the case of the in situ populations of amphipods lacking a dispersive larval stage. In the study we exposed amphipods (Orchestia gammarellus) and crabs (Carcinus maenas and Pachygrapsus marmoratus) to dissolved zinc or cadmium in the laboratory, either singly or in combination. These data can be analysed to test for the effect of the presence or absence of one metal on the rate of uptake of the other, and to seek correlations between the rates of uptake of each metal in individual crustaceans. We present the results of these analyses here. The crustaceans were collected from up to five sites (Rainbow et al., 1999). Three were considered as metal-rich — Restronguet Creek, Cornwall and Dulas Bay, Anglesey in Britain and the Gironde Estuary in France, and two were control (reference) sites — Millport in the Firth of Clyde, Scotland and Talmont St. Hilaire, Vende´e near Les Sables d’Olonne on the Atlantic coast of France (Fig. 1). Restronguet Creek contains extraordinarily high levels of As, Cd, Cu, Fe, Mn and Zn (Bryan and Gibbs, 1983; Bryan et al., 1987). Dulas Bay has very high levels of Cu, Fe, Mn and Zn (Foster, 1976; Foster et al., 1978; Boult et al., 1994). The Gironde estuary contains elevated availabilities of Cd, Cu and Zn (RNO, 1995).
2. Materials and methods Samples from metal-exposed populations of O. gammarellus and C. maenas were collected intertidally in the UK from Restronguet Creek
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(50°12%N; 05°03%W) and from Dulas Bay (53°22%N; 04°17%W). Control amphipods and crabs from the shore (55°44%N; 04°54%W) and immediate sublittoral near Millport (Isle of Cumbrae, Firth of Clyde) were obtained from the University Marine Biological Station, Millport.
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Contaminated O. gammarellus, C. maenas and P. marmoratus were also collected from the south shore of the Gironde Estuary — O. gammarellus at Le Phare de Richard (45°22%N; 00°55%W) and the crabs at Le Verdon–La Chambrette (45°32%N; 01°03%W), near La Pointe de Grave. The control
Fig. 1. Collection sites.
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site for French populations of all three crustaceans was the shore at Talmont St. Hilaire, Vende´e (46°24%N; 01°33%W). Dates of collection are detailed in Tables 1 and 2, and the sites are shown in Fig. 1. The study requires a measure of the rate of uptake of metal into the body of the crustacean concerned. Uptake refers to the flux of all metal entering the body of the crustacean (in these experiments, always from solution and always of radioactively labelled metal). Accumulation, on the other hand, is equivalent to net uptake or net flux; that is absolute uptake minus excretion. If there is no excretion of metal in the time period of the experiment, then accumulation is directly equivalent to uptake. The exposure concentrations of 50 and 100 mg l − 1 zinc and cadmium were chosen in order to ensure measurable uptake and accumulation in the time scale of the experiments. The zinc concentration is within the range found in some metal-contaminated estuaries, including Restronguet Creek (100 – 2000 mg Zn per litre, Bryan and Gibbs, 1983) and Dulas Bay (170 – 5650 mg Zn per litre in the stream Afon Goch at the head of the estuary, Foster et al., 1978; Boult et al., 1994). The cadmium concentration is less environmentally realistic but needs to be of the same order as that of zinc for comparative purposes, particularly when investigating the possibility of interaction. Details of acclimation, laboratory handling and experiments are provided by Rainbow et al. (1999).
2.1. Amphipod uptake experiments Experiments were carried out at 10°C in fully aerated artificial seawater (TMN-Tropic Marin Neu, Tropicarium Buchshlag, Dreieich, Germany) at 33%, to ensure reproducibility of physicochemical conditions that affect trace metal uptake rates (Rainbow, 1995, 1997a,b). Groups of ten amphipods, of both sexes and of similar size (usually \10 mg dry wt.) and moult stage (intermoult), were held individually in acid-washed per-
forated plastic containers (Toby ‘Teaboys’, Aldridge Plastics, Aldridge, UK) in replicate 1 l acid-washed plastic tanks. Amphipods were exposed to one or both simultaneously of the trace metals Zn and Cd, using the radioisotopes 65Zn and 109Cd (NEN Life Science Products, Boston, MA) added to stock solutions of the respective metal chloride (Analar grade, BDH) to give experimental exposures of 100 mg l − 1 (1.53 mM Zn per litre, 0.89 mM Cd per litre) with 185 kBq l − 1 (5 mCi l − 1) tracer in TMN. Amphipods were counted live at daily intervals for 4 days, giving a measure of ‘new’ labelled metal accumulated. Accumulation was linear and best-fit linear regression lines were fitted to data for individual amphipods for days 1–4, the zero point being excluded to allow for adsorption of labelled metal onto the exoskeleton. Regression coefficients (ng g − 1 per day) represent the metal uptake rates of individual amphipods. Details of the five amphipod uptake experiments are given in Table 1. There is no excretion of radiolabelled metal accumulated from solution by O. gammarellus over the short time period used in the experiments, and so the rate of accumulation of labelled metal in this short term is indeed a direct measure of the rate of metal uptake from solution (Rainbow and White, 1989; Weeks and Rainbow, 1991; Rainbow et al., 1993; Rainbow and Kwan, 1995). For example, Weeks and Rainbow (1991) exposed O. gammarellus to a series of radiolabelled Zn concentrations for 21 days at 10°C, and showed that, at all exposures, the labelled Zn concentration newly accumulated by the amphipods matched the increases in total Zn concentration. Thus, all Zn taken up from solution in this period [in contrast to Zn assimilated from food (Weeks and Rainbow, 1991, 1994)] is retained as accumulated Zn without excretion, a conclusion supported by the confirmation of the absence of labelled Zn in the urine of amphipods exposed to dissolved Zn (Weeks and Rainbow, 1991). Rates of uptake of Zn and Cd from solution by O. gammarellus can therefore be measured using sequential live counting of amphipods exposed to radioactively labelled metal tracers.
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Table 1 O. gammarellus a Mean Zinc uptake rate Experiment 1 [20/5/96] (a) Zn only (c) Zn+Cd Experiment 2 [10/6/96] (a) Zn only (c) Zn+Cd Experiment 3 [5/8/96] (a) Zn only (c) Zn+Cd Experiment 4 [14/7/97] (a) Zn only
(c) Zn+Cd
Experiment 5 [29/10/97] (c) Zn+Cd
Cadmium uptake rate Experiment 1 (20/5/96) (b) Cd only (c) Cd+Zn Experiment 2 [10/6/96] (b) Cd only (c) Cd+Zn Experiment 3 [5/8/96] (b) Cd only (c) Cd+Zn Experiment 4 [14/7/97] (b) Cd only
S.D.
n
ANOVAb
Restronguet Creek (16/5/96) Millport (15/5/96) Restronguet Creek (16/5/96) Millport (15/5/96)
6.73 12.2 7.39 7.06
3.17 8.10 4.30 2.98
9 9 9 8
A
Dulas Bay (5/6/96) Millport (15/5/96) Dulas Bay (5/6/96) Millport (15/5/96)
6.21 11.4 38.0 42.3
1.74 4.71 28.2 10.1
6 8 6 7
Gironde (29/7/96) Talmont (31/7/96) Gironde (29/7/96) Talmont (31/7/96)
31.7 24.4 7.48 10.1
31.3 9.89 3.30 2.60
9 9 10 10
Gironde (23/6/97) Talmont (4/7/97) Millport (27/6/97) Gironde (23/6/97) Talmont (4/7/97) Millport (27/6/97)
10.8 10.6 23.3 9.24 8.50 17.8
5.55 6.62 15.5 5.21 5.24 16.6
7 5 8 7 8 8
Gironde (23/6/97) Gironde (15/10/97) Dulas Bay (23/9/97) Millport (27/6/97)
9.13 17.3 7.75 9.80
2.32 13.9 3.44 6.64
10 8 9 10
Restronguet Creek (16/5/96) Millport (15/5/96) Restronguet Creek (16/5/96) Millport (15/5/96)
0.93 2.18 1.25 1.24
0.48 1.45 0.61 0.59
7 10 9 10
Dulas Bay (5/6/96) Millport (15/5/96) Dulas Bay (5/6/96) Millport (15/5/96)
2.38 4.59 6.81 5.02
0.94 3.73 3.48 2.93
10 9 7 10
A
Gironde (29/7/96) Talmont (31/7/96) Gironde (29/7/96) Talmont (31/7/96)
3.36 3.37 1.91 2.58
1.16 0.94 0.75 0.75
10 10 10 9
A
Gironde (23/6/97) Talmont (4/7/97) Millport (27/6/97)
3.67 3.26 5.37
1.80 1.30 2.42
8 6 8
A A A A A B B A A B B A A A A A A
A A A A
A B A
A B A A A A
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Table 1 (Continued) Mean (c) Cd+Zn
Experiment 5 [29/10/97] (c) Zn+Cd
S.D.
n
Gironde (23/6/97) Talmont (4/7/97) Millport (27/6/97)
4.15 3.04 3.57
3.29 1.22 2.62
8 8 8
Gironde (23/6/97) Gironde (15/10/97) Dulas Bay (23/9/97) Millport (27/6/97)
4.80 8.18 2.07 5.78
1.64 4.72 0.52 1.70
10 8 9 10
ANOVAb A A A
a Mean uptake rates (nM g−1 per day) with standard deviation (S.D.) of labelled Zn or Cd in amphipods from five sites (date of collection) exposed to labelled (a) 1.53 mM l−1 Zn, (b) 0.89 mM l−1 Cd, or (c) 1.53 mM l−1 Zn and 0.89 mM l−1 Cd together at 10°C [date of experiment]. b ANOVA: metal uptake rates of amphipods from the same site subjected to metal exposure in the same experiment in the presence or absence of the other metal do not differ significantly (P\0.05) if they share the same letter in a column.
2.2. Crab uptake experiments Measures of rates of short-term whole body net accumulation of radiolabelled Zn and Cd can also be considered to provide measures of absolute rates of Zn and Cd uptake from solution by crabs (Rainbow et al., 1999). Chan and Rainbow (1993a,b) have shown that labelled Zn taken up from solution (as opposed to food) by C. maenas is accumulated without excretion within the time scales and at the exposure concentrations used here. All radiolabelled Zn taken up from solution by crabs exposed to radiolabelled dissolved Zn concentrations up to 100 mg l − 1, was added sequentially to the Zn concentration already present in the crabs (Chan and Rainbow, 1993a), and this accumulated Zn is not excreted – only at exceptionally high Zn exposure concentrations is excretion observed (Chan and Rainbow, 1993b). Measures of rates of whole body net accumulation of radiolabelled Zn therefore do provide measures of absolute rates of Zn uptake from solution. Published literature on Cd accumulation by crustaceans (Jennings and Rainbow, 1979; Bjerregaard, 1982; Rainbow, 1985, 1988, 1998; Rainbow and White, 1989) indicates that the rate of accumulation of Cd by the crab will similarly reflect the rate of Cd uptake. Other relative measures of the uptake rates of Zn and Cd from solution by C. maenas, are provided by features of
the accumulation kinetics of either metal in the haemolymph of the crab (Martin and Rainbow, 1998; Rainbow et al., 1999).
2.2.1. Uptake into blood Following Martin and Rainbow (1998), up to ten C. maenas (\ 44 mm carapace width) were exposed in each experiment to (a) 50 mg l − 1 (0.76 mM l − 1) Zn in TMN, (b) 50 mg l − 1 (0.45 mM l − 1) Cd in TMN, or (c) 50 mg l − 1 (0.76 mM l − 1) Zn and 50 mg l − 1 (0.45 mM l − 1) Cd in TMN simultaneously, at 10°C for 4 days in individual acidwashed plastic containers (Rainbow et al., 1999). Zn and Cd solutions were labelled with 185 kBq l − 1 (5 mCi l − 1) 65Zn and 109Cd as appropriate. Experimental details are provided in Table 2. Haemolymph samples were taken each day and counted for labelled metal concentration. The labelled Zn concentration in the haemolymph continues to increase over the exposure period. The rate of this increase (the regression coefficient of the best-fit line, nM ml − 1 per day) is directly proportional to the concentration of available Zn in the exposure solution, and is a relative measure of the crab’s uptake rate of dissolved metal (Martin and Rainbow, 1998; Rainbow et al., 1999). The labelled Cd concentration in the haemolymph, on the other hand, rapidly reaches an equilibrium as its rate of removal from the haemolymph matches its rate of uptake into the
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Table 2 C. maenas a
Zinc uptake rate Experiment 1 [20/5/96] (a) Zn only (c) Zn+Cd Experiment 2 [10/6/96] (c) Zn+Cd Experiment 3 [5/8/96] (c) Zn+Cd Experiment 4 [14/7/97] (c) Zn+Cd
Cadmium uptake rate Experiment 1 (20/5/96) (b) Cd only (c) Cd+Zn Experiment 2 [10/6/96] (c) Cd+Zn Experiment 3 [5/8/96] (c) Cd+Zn Experiment 4 [14/7/97] (c) Cd+Zn
Mean
S.D.
n
ANOVAb
Millport (15/5/96) Millport (15/5/96) Restronguet Creek (16/5/96)
0.88 0.69 1.05
0.65 0.30 0.67
6 9 8
A A
Dulas Bay (5/6/96) Millport (15/5/96)
3.58 0.75
1.11 0.30
8 9
Gironde (29/7/96) Talmont (31/7/96)
1.24 1.59
1.10 0.82
6 8
Restronguet Creek (25/6/97) Millport (27/6/97) Gironde (23/6/97) Talmont (4/7/97)
0.78 1.15 1.39 3.10
0.38 0.54 1.22 2.49
6 7 6 6
Millport (15/5/96) Millport (15/5/96) Restronguet Creek (16/5/96)
0.157 0.160 0.167
0.081 0.089 0.134
7 9 8
Dulas Bay (5/6/96) Millport (15/5/96)
0.677 0.138
0.117 0.058
8 9
Gironde (29/7/96) Talmont (31/7/96)
0.153 0.233
0.111 0.176
6 7
Restronguet Creek (25/6/97) Millport (27/6/97) Gironde (23/6/97) Talmont (4/7/97)
0.176 0.271 0.254 0.404
0.133 0.131 0.166 0.197
6 7 6 6
A A
a Mean relative uptake rates with standard deviations (S.D.) of labelled Zn (nM ml−1 per day in haemolymph) and Cd (nM ml−1 in haemolymph) of crabs from five sites (date of collection) exposed to labelled (a) 0.76 mM l−1 Zn; (b) 0.45 mM l−1 Cd, or (c) 0.76 mM l−1 Zn and 0.45 mM l−1 Cd together at 10°C [date of experiment]. b ANOVA: relative metal uptake rates of crabs subjected to metal exposure in the presence or absence of the other metal in the same experiment do not differ significantly (P\0.05) if they share the same letter.
haemolymph under constant exposure (Martin and Rainbow, 1998). The equilibrium concentration of Cd in the haemolymph (nM ml − 1) increases with increased concentration of available Cd in solution and is a relative measure of the rate of uptake of Cd from solution by the whole crab (Martin and Rainbow, 1998; Rainbow et al., 1999).
2.2.2. Whole crab accumulation P. marmoratus and smaller specimens of C. maenas (B44 mm carapace width) were used in experiments in which the accumulated concentrations of labelled Zn and Cd were measured at intervals in the whole crabs. The rates of (net) accumulation of Zn and Cd during these shortterm experiments are considered to be direct mea-
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sures of the absolute rates of uptake of Zn and Cd from solution by the whole crabs (Rainbow et al., 1999). Collection and experimental details are provided for C. maenas in Table 2. P. marmoratus were collected on the same day as C. maenas from the same site (Table 2). In 1996 C. maenas from Restronguet Creek, Dulas Bay and Millport, and P. marmoratus from the Gironde and Talmont St. Hilaire, were exposed for 11 days at 10°C to 50 mg l − 1 (0.76 mM l − 1) Zn and 50 mg l − 1 (0.45 mM l − 1) Cd (labelled with 185 kBq l − 1 65Zn and 185 kBq l − 1 109Cd) together in TMN. Up to five C. maenas and four P. marmoratus were sampled on days 1, 2, 4 and 7, and all remaining crabs on day 11, prior to counting for labelled Zn and Cd contents. In 1997 C. maenas from Restronguet Creek, Millport, Gironde and Talmont, and P. marmoratus from Gironde were similarly exposed for 21 days. Crabs were sampled on days 7, 14 and 21, before being prepared and counted as above (Rainbow et al., 1999). Data for any crab that moulted during the experiments have been ignored.
2.3. Statistical analysis All statistical analyses, including regression analysis, ANOVA and ANCOVA, were carried out using STATISTICA (Statsoft), preliminary tests for normality of distributions of data and equality of variances having shown that it was valid to employ parametric statistics. Where a priori ANOVA or ANCOVA revealed statistically significant (PB0.05) differences among groups, a posteriori testing was used to identify which groups differed from each other. 3. Results
3.1. Amphipod uptake rates Table 1 gives details of the zinc and cadmium uptake rates of O. gammarellus exposed to 100 mg l − 1 of one metal (1.53 mM l − 1 Zn, 0.89 mM l − 1 Cd) in the presence or absence of 100 mg l − 1 of the other metal, and the results of ANOVA comparisons of these uptake rates as appropriate.
3.1.1. Zinc uptake The presence of a high dissolved cadmium concentration (0.89 mM Cd per litre) had an inconsistent effect on the zinc uptake rate (Table 1). In one case out of three, the uptake rate of zinc by Millport amphipods increased in the presence of high cadmium, as did the zinc uptake rate of Dulas Bay amphipods in the same experiment (experiment 2). In experiment 3, on the other hand, the zinc uptake rates of amphipods from Gironde and Talmont decreased significantly in the presence of raised cadmium concentrations. There was no such significant effect, however, in the case of the same amphipods in experiment 4. It is impossible therefore to draw a consistent general conclusion on the effect of raised cadmium availability on the zinc uptake rate of amphipods exposed to both metals at 100 mg l − 1. 3.1.2. Cadmium uptake The presence of a high zinc concentration (1.53 mM Zn per litre) similarly had an inconsistent effect on the cadmium uptake rates of the amphipods. The cadmium uptake rate of Dulas Bay amphipods in June 1996 (experiment 2) was increased significantly in the presence of high zinc, but that of Gironde amphipods was decreased significantly in May 1996 (experiment 3) and unaffected in July 1997 (experiment 4) (Table 1). The cadmium uptake rates of Millport, Restronguet Creek and Talmont amphipods were unaffected by the presence of high zinc (Table 1). 3.1.2.1. Correlation of zinc and cadmium uptake rates in indi6idual amphipods. It is possible to use these data to address the question whether zinc and cadmium uptake rates of individual amphipods are correlated — i.e. do individual amphipods show characteristically high or low uptake rates for both metals? Data were analysed for amphipods in all joint zinc/cadmium experiments (Table 1), firstly for all the amphipods from each site in each experiment, secondly for all the amphipods from each site combined across all experiments, and then for the whole data set (Fig. 2). Uptake rates for the two metals were correlated in the amphipods from each site in most of the separate experiments (regression lines not
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shown), in all the combined data sets for each site (single regression lines, Fig. 2), and in the whole data set (Fig. 2f). It can be concluded therefore that the uptake rates of zinc and cadmium in individual O. gammarellus are correlated. Data are also available to check whether the correlation varies between sites or over time. Comparisons were made by ANCOVA between regression coefficients of the best-fit regression lines of Zn uptake rate (x) against Cd uptake rate (y) as in Fig. 2. No consistent pattern emerged. A comparison of regression coefficients for amphipods from each site (using all data combined between experiments) showed no significant difference between sites with the exception of Gironde amphipods, but a comparison experiment by experiment indicated Dulas Bay as the only site differing from the others. Relationships
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for amphipods within one site did not differ between experiments, except in the one case of Millport amphipods in experiment 5.
3.2. Crab uptake rates 3.2.1. As measured from blood parameters Table 2 presents data on the relative uptake rates of zinc and cadmium by C. maenas derived from patterns of short term accumulation of the metals in the blood of individual crabs exposed to 50 mg l − 1 of the metals (0.76 mM l − 1 Zn, 0.44 mM l − 1 Cd), either singly or simultaneously. As explained in Section 2, the slope of the best-fit regression line (nM Zn ml − 1 per day) is a relative measure of the uptake rate of zinc by the crabs, whereas in the case of cadmium it is the steadystate equilibrium concentration of cadmium in the
Fig. 2. O. gammarellus. Correlation of rates of uptake (nM g − 1 per day) of labelled zinc and cadmium in individual amphipods exposed simultaneously to labelled 1.53 mM l − 1 Zn and 0.89 mM l − 1 Cd at 10°C in experiments 1 – 4 of Table 1. The different symbols in (a – e) indicate amphipods from the same site of collection but in different experiments (see Table 1). The different symbols in (f) each represent all amphipods from one site combined between experiments. * PB0.05, ** PB 0.01, *** P B0.001.
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Fig. 3. C. maenas. Correlation of relative rates of uptake of labelled zinc (nM ml − 1 per day) and cadmium (nM ml − 1) (see text for explanation) in individual crabs exposed simultaneously to labelled 0.76 mM l − 1 Zn and 0.45 mM l − 1 Cd at 10°C in experiments 1–4 of Table 2. The different symbols in (a–e) indicate crabs from the same site of collection but in different experiments (see Table 2); the different symbols in (f) each represent all crabs from one site combined between experiments.
blood (nM Cd per millilitre) that is the relative measure of the rate of cadmium uptake by the crabs. Table 2 summarises the data obtained, with the results of ANOVA comparisons of uptake rates of one metal in the presence or absence of the other. The presence of 0.44 mM Cd per litre had no significant effect on the rate of zinc uptake by Millport crabs, nor did the presence of 0.76 mM Zn per litre on the rate of uptake of cadmium (Table 2).
3.2.1.1. Correlation of zinc and cadmium uptake rates in indi6idual crabs. Relative uptake rates of zinc and cadmium are known for individual crabs in the double metal exposure experiments (experi-
ments 2–4, Table 2). Uptake rates for the two metals were correlated in the crabs from each site in most of the separate experiments (not shown), in all data sets combined for each site (Fig. 3), and in the whole combined data set for all crabs from all sites (Fig. 3f). As for the amphipods therefore, the uptake rate of zinc in individual C. maenas is correlated with the rate of cadmium uptake. As in the case of the amphipods, the data can also be used to check whether the nature of the correlation between Zn and Cd varies between sites or over time. Comparison of regression coefficients for all crabs from each site (all data combined between experiments) showed no differences between sites except for Talmont crabs, but
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comparison experiment by experiment showed no intersite differences at all. There were no differences in regression coefficients for crabs from the same site between experiments.
3.2.2. As measured by whole crab accumulation rates 3.2.2.1. Correlation of zinc and cadmium accumulation in indi6idual crabs. The whole crab accumulation data can be used to test whether the concentrations of labelled zinc and cadmium accumulated by individual crabs are correlated when crabs are exposed to labelled zinc and cad-
199
mium simultaneously. Zinc and cadmium concentrations for all individual crabs from each site were correlated significantly (PB 0.01) in most experiments for both C. maenas and P. marmoratus (data not presented separately for each experiment). The trace metal concentrations were also significantly correlated when all the data for each species were combined across experiments and years (Fig. 4). Thus, crabs with a high or low rate of accumulation of zinc tend to have a respectively high or low rate of cadmium accumulation. The whole crab accumulation data (Fig. 4) can also be used to check whether the correlation between Zn and Cd varies intraspecifically between sites or between species. ANCOVA comparisons of the relevant regression coefficients showed Millport C. maenas to differ significantly from Restronguet Creek and Dulas Bay crabs (no difference) in 1996, and from Restronguet Creek, Gironde and Talmont crabs (no difference) in 1997. For P. marmoratus there was no significant difference between regression coefficients for Gironde and Talmont in 1996, although there was for Gironde P. marmoratus between 1996 and 1997. Interspecifically, C. maenas (whole data set) had a regression coefficient significantly different from that of P. marmoratus. At Gironde in 1997, however, the regression coefficients for the two species did not differ significantly. No consistent pattern has emerged.
4. Discussion
Fig. 4. Correlations of accumulated concentrations of labelled zinc and cadmium by individual whole crabs [(a) C. maenas; (b) P. marmoratus] exposed simultaneously to 0.76 mM l − 1 Zn and 0.45 mM l − 1 Cd at 10°C for either up to 11 days (1996) or up to 21 days (1997). Different symbols each represent crabs from one site combined between experiments and years.
Experimental studies of metal–metal interactions in crustaceans are inconsistent or even contradictory. In the amphipod Corophium 6olutator, zinc addition induced a depletion of cadmium toxicity and accumulation (Bat et al., 1998). In the decapod Callianassa australiensis, exposure to a mixture of Zn and Cd increased the bioaccumulation of both elements (Ahsanullah et al., 1981). In the lobster Homarus americanus (another decapod), Cd added to food enhanced Zn accumulation in the hepatopancreas but not in the tail muscle (Chou et al., 1987). In the caridean decapod Pandalus montagui, the response was variable according to organ or dose; in the presence of Zn,
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particularly at the lower concentration tested (1.07 mM l − 1), Cd concentration increased in the hepatopancreas, decreased in the carcass and was unaffected in the other tissues (Ray et al., 1980). In another caridean decapod Palaemon serratus, the presence of cadmium reduced the accumulation of zinc by larvae (Devineau and Amiard-Triquet, 1985). Thus, interactions of metals are variable, and may change from antagonism to synergism with change of external concentrations, length of exposure, species studied, and tissues or organs examined (Amiard-Triquet and Amiard, 1998). Another factor that may have an influence is the tolerance of a population, within a species, to one or other of the metals concerned (AmiardTriquet and Amiard, 1998). Brown (1978), for example, described two metal-tolerant populations of the freshwater isopod crustacean Asellus meridianus, where the metal accumulation patterns (and mechanisms of tolerance) differed between the two populations, with consequences on the nature of the interaction of the metals concerned. In one population resistant to Pb, lead tolerance was reflected in reduced accumulation of both Pb and Cu; in the other population, resistant to both Pb and Cu, there was no reduced accumulation of either metal, and both metals competed for sites of detoxified storage (cuprosomes) in the hepatopancreatic caeca (Brown, 1978; Amiard-Triquet and Amiard, 1998). The studies mentioned above have generally been based upon comparative assessments of bioaccumulation, and therefore involve the possible competition of dissolved cadmium and zinc for both uptake and storage sites in the crustaceans. The present study is not strictly comparable since, in two of the three sets of experiments, it takes into account only the first step of bioaccumulation, that of uptake. In the amphipod O. gammarellus, the presence of 0.89 mM l − 1 cadmium variably increased, decreased or had no significant effect upon the uptake of zinc from 1.53 mM Zn per litre, as did the reverse scenario of zinc on cadmium uptake. In the case of C. maenas neither metal at 50 mg l − 1 (0.76 mM Zn per litre, 0.45 mM Cd per litre) affected the rate of uptake of the other. When P. elegans were ex-
posed to zinc and cadmium together, both at 20 mg l − 1 (0.31 mM Zn per litre, 0.18 mM Cd per litre), the rate of zinc uptake decreased and the rate of cadmium uptake increased in comparison with the uptake rates of each metal in single metal exposures at the same concentration (Nugegoda and Rainbow, 1995). Thus, even when interactive effects on influx are examined independently of interactions within the cell, the possible competition of dissolved Cd and Zn for uptake sites still appears inconsistent and contradictory. In both the amphipods and the crabs in this study, there was a significant correlation between zinc and cadmium uptake rates of individuals, suggesting that these two trace metals may share one or more routes of uptake from solution (Rainbow, 1995, 1997b). The same correlation has been observed in P. elegans (Nugegoda and Rainbow, 1995). Recent findings also indicate that cadmium and zinc may use the same uptake mechanism in the gills of fish, apparently by both sharing the route of cadmium entry (Bentley, 1992; Hogstrand and Wood, 1996; Olsson et al., 1998). Nevertheless, any potential sharing of routes of uptake of zinc and cadmium from solution, has not led to consistent synergism or antagonism in the experiments run here. It is possible that the chosen metal exposure concentrations are not high enough to cause significant interaction between the metals entering the crustaceans from solution. This is unlikely, for the experimental concentrations used exceed those of typical coastal waters and most estuaries (Bryan and Gibbs, 1983; Bryan et al., 1985; Bryan and Langston, 1992), including for example cadmium in the Gironde ( 0.3 mg Cd l − 1; Boutier et al., 1989). In the case of zinc, however, even higher dissolved concentrations can be found in some metal-contaminated estuaries, including Restronguet Creek (100–2000 mg Zn per litre, 0.25– 5.0 mg Cd per litre, Bryan and Gibbs, 1983) and Dulas Bay (170–5650 mg Zn per litre in the stream Afon Goch at the head of the estuary, Foster et al., 1978; Boult et al., 1994). Restronguet Creek has extremely high levels of zinc and cadmium relative to most coastal and estuarine waters (Bryan and Gibbs, 1983). Zinc is accumulated to atypically high concentrations in
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Table 3 Total metal molar uptake indices and metal ion molar uptake indices for zinc and cadmium (see text for explanation and units), and their ratios in the amphipod O. gammarellus, and the crabs C. maenas and P. marmoratus (data from Table 1 and Fig. 4)
Orchestia gammarellus Millport Zinc Zn/Cd separate Expt. 1 Expt. 2 Expt. 4 Zn/Cd together
Cadmium Zn/Cd separate
Zn/Cd together
Expt. Expt. Expt. Expt.
Total metal uptake index
Ratio Zn:Cd total metal uptake index
Metal ion uptake index
Ratio Zn:Cd metal ion uptake index
0.0080 0.0075 0.0152
3.33: 1.44: 2.53: 2.43: 3.29: 4.33: 2.90: 0.98: 3.03:
0.0126 0.0118 0.0240
0.19: 0.08: 0.15: 0.14: 0.19: 0.29: 0.17: 0.06: 0.18:
1 1 1 1 (mean) 1 1 1 1 1 (mean)
1 2 4 5
0.0046 0.0276 0.0116 0.0064
Expt. 1 Expt. 2 Expt. 4
0.0024 0.0052 0.0060
0.0661 0.1391 0.1627
Expt. Expt. Expt. Expt.
0.0014 0.0056 0.0040 0.0065
0.0376 0.1521 0.1082 0.1752
1 2 4 5
Carcinus Maenas Millport 1996 Dulas Bay 1996 Restronguet Creek 1996 Restronguet Creek 1997 Gironde 1997 Talmont 1997 Pachygrapsus marmoratus Gironde 1996 Talmont 1996 Gironde 1997
0.0073 0.0436 0.0184 0.0101
1 1 1 1 (mean) 1 1 1 1 1 (mean)
0.85: 1 1.14: 1 0.89: 1
0.050: 1 0.067: 1 0.052: 1
10.5: 1
0.623: 1
4.89: 1 4.70: 1
0.286: 1 0.275: 1
2.73: 1 2.34: 1 1.37: 1
0.160: 1 0.137: 1 0.080: 1
many of the indigenous aquatic fauna and flora (Bryan and Gibbs, 1983; Bryan et al., 1987), whilst Cd accumulation can be considered atypically low, relative to environmental levels. A sharing of (and potentially competition for) uptake routes from solution by zinc and cadmium would be consistent with this observation (Bryan and Gibbs, 1983). The nature of the relationship between zinc and cadmium uptake rates in amphipods and crabs showed occasional differences between sites and
over time, but no consistent pattern was discernible. Generally, for a given molar concentration, the molar uptake (crustaceans — Rainbow and White, 1990) and bioaccumulation (bivalve molluscs — Wang et al., 1996; Lee et al., 1998) of dissolved zinc by marine invertebrates is greater than that of cadmium. It is possible to recalculate the zinc and cadmium uptake rates of O. gammarellus given in Table 1 in terms of molar uptake rate (mM g − 1 per day) per molar metal exposure
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(mM l − 1) to give a (total metal) molar uptake index (l g − 1 per day). Table 3 shows this index for O. gammarellus from Millport (data from experiments detailed in Table 1). As expected, the molar uptake rate of zinc is higher than that of cadmium, whether the amphipods are exposed to zinc and cadmium separately or together (Table 3). Lee et al. (1998), however, recalculated their data to take account of the model that considers the free metal ion to be the major determinant of the bioavailability of many trace metals including Zn and Cd (Campbell, 1995; Rainbow, 1995, 1997b). [It would be simplistic, however, to consider that the free metal ion is the only bioavailable form of the metal, given that neutral inorganic or organic complexes of a trace metal ion may diffuse directly across the cell membrane, bypassing any (more important?) membrane-embedded transport system for the free metal ion (Simkiss and Taylor, 1989; Campbell, 1995; Van Ginneken et al., 1999)]. Recalculation of the data in terms of the free metal ion concentration reverses the previous conclusion, showing the bivalves to be more permeable to Cd2 + than to Zn2 + (Lee et al., 1998). It is possible to make the same recalculation here. In the artificial seawater TMN at 33‰, the free Zn2 + ion is estimated to make up 63.5% of total dissolved Zn (Rainbow et al., 1993) and the free Cd2 + ion 3.7% of total dissolved Cd (Rainbow and Kwan, 1995). Thus, any decrease in salinity makes a proportionately greater increase in the activity of the free cadmium ion than in the activity of the free zinc ion as chloride complexation decreases (Zirino and Yamamoto, 1972; Mantoura et al., 1978; Rainbow et al., 1993; Rainbow and Kwan, 1995). By calculating the free ion concentrations at the experimental exposure concentrations, it is now possible to calculate a metal ion molar uptake index (mM g − 1per day per mM M2 + l − 1) (Table 3). As for the bivalves, the free Cd2 + ion is taken up by the amphipod at a greater rate per unit molar exposure than the free Zn2 + ion. The higher molar uptake rate for Zn, described above, is therefore largely attributable to the greater proportion of the free metal ion present. Table 3 also presents similar data for the crabs C. maenas and P. marmoratus recalculated from
the raw data (Rainbow et al., 1999) contributing to Fig. 4. As for the amphipod, in both crabs zinc has a higher total metal molar uptake index than cadmium but a lower metal ion molar uptake index (Table 3). This study set out to investigate the possible interaction of zinc and cadmium uptake rates in crustaceans. In fact the presence or absence of a raised dissolved concentration of one metal had an inconsistent effect on the rate of uptake of the other metal by the amphipod O. gammarellus, and no effect on the uptake of the other metal by the crab C. maenas. Zinc and cadmium uptake rates are correlated in individual amphipods and crabs (of two species) from all the sites examined. Thus zinc and cadmium appear to share common routes of uptake from solution by these crustaceans, but the metals do not consistently interact competitively or synergistically during uptake at the exposure concentrations investigated. It remains possible that other results might have been obtained at other exposure concentrations and combinations, and/or that other mechanisms (e.g. excretion, detoxification) are involved in the overall manifestation of competition in terms of body burdens. Acknowledgements We are very grateful for support for travel from the Central Research Fund of the University of London, and from the Alliance: Franco–British Joint Research Programme scheme of the British Council and APAPE (France) (Alliance Project PN 97.076). We also thank two referees for their constructive criticism. References Ahsanullah, M., Negilski, D.S., Mobley, M.C., 1981. Toxicity of zinc, cadmium and copper to the shrimp Callianassa australiensis. III. Accumulation of metals. Mar. Biol. 64, 311 – 316. Amiard-Triquet, C., Amiard, J.-C., 1998. Influence of ecological factors on accumulation of metal mixtures. In: Langston, W.J., Bebianno, M. (Eds.), Metal Metabolism in Aquatic Environments. Chapman and Hall, London, pp. 351 – 386.
P.S. Rainbow et al. / Aquatic Toxicology 50 (2000) 189–204 Bat, L., Raffaelli, D., Marr, I.L., 1998. The accumulation of copper, zinc and cadmium by the amphipod Corophium 6olutator. J. Exp. Mar. Biol. Ecol. 223, 167–184. Bentley, P.J., 1992. Influx of zinc by channel catfish (Ictalurus punctatus): uptake from external environmental solutions. Comp. Biochem. Physiol. 101C, 215–217. Boutier, B., Chiffoleau, J.F., Jouanneau, J.M., Latouche, C., Philipps, I., 1989. La contamination de la Gironde par le cadmium: origine, extension, importance, IFREMER. Rapp. Sci. Tech. 14, 1–105. Bjerregaard, P., 1982. Accumulation of cadmium and selenium and their mutual interaction in the shore crab Carcinus maenas (L.). Aquat. Toxicol. 2, 113–125. Boult, S., Collins, D.N., White, K.N., Curtis, C.D., 1994. Metal transport in a stream polluted by acid mine drainage — the Afon Goch, Anglesey, UK. Environ. Pollut. 84, 279 – 284. Brown, B.E., 1978. Lead detoxification by a copper-tolerant isopod. Nature (Lond.) 276, 388–390. Bryan, G.W., Gibbs, P.E., 1983. Heavy metals in the Fal Estuary, Cornwall: a study of long-term contamination by mining waste and its effects on estuarine organisms. Occ. Publ. Mar. Biol. Assoc. UK 2, 1–112. Bryan, G.W., Langston, W.J., 1992. Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries: a review. Environ. Pollut. 76, 89 – 131. Bryan, G.W., Langston, W.J., Hummerstone, L.G., Burt, G.R., 1985. A guide to the assessment of heavy-metal contamination in estuaries using biological indicators. Occ. Publ. Mar. Biol. Assoc. UK 4, 1–92. Bryan, G.W., Gibbs, P.E., Hummerstone, L.G., Burt, G.R., 1987. Copper, zinc, and organotin as long-term factors governing the distribution of organisms in the Fal Estuary in Southwest England. Estuaries 10, 208–219. Campbell, P.G.C., 1995. Interaction between trace metals and aquatic organisms: a critique of the free-ion activity model. In: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Aquatic Systems. Wiley, New York, pp. 45–102. Chan, H.M., Rainbow, P.S., 1993. The accumulation of dissolved zinc by the shore crab Carcinus maenas (L.). Ophelia 38, 13 – 30. Chan, H.M., Rainbow, P.S., 1993. On the excretion of zinc by the shore crab Carcinus maenas (L.). Ophelia 38, 31–45. Chou, C.L., Uthe, J.F., Castell, J.D., Kean, J.C., 1987. Effect of dietary cadmium on growth, survival and tissue concentrations of cadmium, zinc, copper, and silver in juvenile American lobster (Homarus americanus). Can. J. Fish. Aquat. Sci. 44, 1443 –1450. Devineau, J., Amiard-Triquet, C., 1985. Patterns of bioaccumulation of an essential trace metal (zinc) and a pollutant metal (cadmium) in larvae of the prawn Palaemon serratus. Mar. Biol. 86, 139 – 143. Foster, P., 1976. Concentrations and concentration factors of heavy metals in brown algae. Environ. Pollut. 10, 45–53. Foster, P., Hunt, D.T.E., Morris, A.W., 1978. Metals in an acid mine stream and estuary. Sci. Tot. Environ. 9, 75–86.
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Hogstrand, C., Wood, C.M., 1996. The physiology and toxicology of zinc in fish. In: Taylor, E.W. (Ed.), Toxicology of Aquatic Pollution. Society for Experimental Biology Seminar Series 57. Cambridge University Press, Cambridge, pp. 61 – 84. Jennings, J.R., Rainbow, P.S., 1979. Studies on the uptake of cadmium by the crab Carcinus maenas in the laboratory. I. Accumulation from seawater and a food source. Mar. Biol. 50, 131 – 139. Lee, B.-G., Wallace, W.G., Luoma, S.N., 1998. Uptake and loss kinetics of Cd, Cr and Zn in the bivalves Potamocorbula amurensis and Macoma balthica: effects of size and salinity. Mar. Ecol. Prog. Ser. 175, 177 – 189. Mantoura, R.F.C., Dickson, A., Riley, J.P., 1978. The complexation of metals with humic materials in natural waters. Estuar. Coast Mar. Sci. 6, 387 – 408. Martin, D.J., Rainbow, P.S., 1998. The kinetics of zinc and cadmium in the haemolymph of the shore crab Carcinus maenas (L.). Aquat. Toxicol. 40, 203 – 231. Mason, A.Z., Jenkins, K.D., 1995. Metal detoxification in aquatic organisms. In: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Bioavailability in Aquatic Systems. Wiley, Chichester, pp. 479 – 608. Nugegoda, D., Rainbow, P.S., 1995. The uptake of dissolved zinc and cadmium by the decapod crustacean Palaemon elegans. Mar. Pollut. Bull. 31, 4 – 12. Olsson, P.-E., Kling, P., Hogstrand, C., 1998. Mechanisms of heavy metal accumulation and toxicity in fish. In: Langston, W.J., Bebianno, M. (Eds.), Metal Metabolism in Aquatic Environments. Chapman and Hall, London, pp. 321 – 350. Rainbow, P.S., 1985. Accumulation of Zn, Cu and Cd by crabs and barnacles. Estuar. Coastal Shelf Sci. 21, 669 – 686. Rainbow, P.S., 1988. The significance of trace metal concentrations in decapods. Symp. Zool. Soc. Lond. 59, 291 – 313. Rainbow, P.S., 1995. Physiology, physicochemistry and metal uptake — a crustacean perspective. Mar. Pollut. Bull. 31, 55 – 59. Rainbow, P.S., 1997. Trace metal accumulation in marine invertebrates: marine biology or marine chemistry? J. Mar. Biol. Assoc. UK 77, 195 – 210. Rainbow, P.S., 1997. Ecophysiology of trace metal uptake in crustaceans. Estuar. Coastal Shelf Sci. 44, 169 – 175. Rainbow, P.S., 1998. Phylogeny of trace metal accumulation in crustaceans. In: Langston, W.J., Bebianno, M. (Eds.), Metal Metabolism in Aquatic Environments. Chapman and Hall, London, pp. 285 – 319. Rainbow, P.S., Kwan, M.K.H., 1995. Physiological responses and the uptake of cadmium and zinc by the amphipod crustacean Orchestia gammarellus. Mar. Ecol. Prog. Ser. 127, 87 – 102. Rainbow, P.S., White, S.L., 1989. Comparative strategies of heavy metal accumulation by crustaceans: zinc, copper and cadmium in a decapod, an amphipod and a barnacle. Hydrobiologia 174, 245 – 262.
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Rainbow, P.S., White, S.L., 1990. Comparative accumulation of cobalt by three crustaceans: a decapod, an amphipod and a barnacle. Aquat. Toxicol. 16, 113–126. Rainbow, P.S., Malik, I., O’Brien, P.O., 1993. Physicochemical and physiological effects on the uptake of dissolved zinc and cadmium by the amphipod crustacean Orchestia gammarellus. Aquat. Toxicol. 25, 15–30. Rainbow, P.S., Amiard-Triquet, C., Amiard, J.-C., Smith, B.D., Best, S.L., Nassiri, Y., Langston, W.J., 1999. Trace metal uptake rates in crustaceans (amphipods and crabs) from coastal sites in NW Europe differentially enriched with trace metals. Mar. Ecol. Prog. Ser. 183, 189–203. Ray, S., McLeese, D.W., Waiwood, B.A., Pezzack, D., 1980. The disposition of cadmium and zinc in Pandalus montagui. Arch. Environ. Contam. Toxicol. 9, 675–681. RNO, 1995. Les contaminants dans la matie`re vivante. In: Anon. (Ed.), Surveillance du milieu marin. Min. Environ., Paris and IFREMER, Nantes, pp. 9–24. Simkiss, K., Taylor, M.G., 1989. Metal fluxes across the membranes of aquatic organisms. CRC Crit. Rev. Aquat. Sci. 1, 173 – 187.
Van Ginneken, L., Chowdhury, M.J., Blust, R., 1999. Bioavailability of cadmium and zinc to the common carp, Cyprinus carpio, in complexing environments: a test for the validity of the free ion activity model. Environ. Toxicol. Chem. 18, 2295 – 2304. Wang, W.X., Fisher, N.S., Luoma, S.N., 1996. Kinetic determinations of trace element bioaccumulation in the mussel Mytilus edulis. Mar. Ecol. Prog. Ser. 140, 91 – 113. Weeks, J.M., Rainbow, P.S., 1991. The uptake and accumulation of zinc and copper from solution by two species of talitrid amphipods (Crustacea). J. Mar. Biol. Assoc. UK 71, 811 – 826. Weeks, J.M., Rainbow, P.S., 1994. Interspecific comparisons of zinc and cadmium relative assimilation efficiencies in an ecological series of talitrid amphipods. Oecologia 97, 228 – 235. Zirino, A., Yamamoto, S., 1972. A pH-dependent model for the chemical speciation of copper, zinc, cadmium, and lead in seawater. Limnol. Oceanogr. 17, 661 – 671.
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Observations on the interaction of zinc and cadmium uptake rates in crustaceans (amphipods and crabs) from coastal sites in UK and France differentially enriched with trace metals P.S. Rainbow a,*,1, C. Amiard-Triquet b, J.C. Amiard b, B.D. Smith a, W.J. Langston c a b
Department of Zoology, The Natural History Museum, Cromwell Road, London SW 7 5BD, UK Ser6ice d’Ecotoxicologie, CNRS-GDR 1117, ISOMer, Faculte´ de Pharmacie, 1 Rue Gaston Veil, 44035 Nantes Cedex 01, France c Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB, UK
Received 18 May 1999; received in revised form 3 November 1999; accepted 24 November 1999
Abstract This paper presents results on the possible interaction of zinc and cadmium uptake rates in crustaceans. Zn and Cd uptake rates were measured in amphipods (Orchestia gammarellus) and crabs (Carcinus maenas and Pachygrapsus marmoratus) from five coastal sites in Britain and France subjected to different degrees of trace metal enrichment. The presence or absence of 100 mg l − 1 of one metal (1.53 mM l − 1 Zn, 0.89 mM l − 1 Cd) had an inconsistent effect on the rate of uptake of the other metal by O. gammarellus. The presence or absence of 50 mg l − 1 of either zinc (0.76 mM l − 1) or cadmium (0.45 mM l − 1) had no effect on the rate of uptake of the other metal by C. maenas (from Millport, Scotland). Zinc and cadmium uptake rates were correlated in individual amphipods and crabs of both species from the five sites. These correlations indicate that zinc and cadmium might share common routes of uptake from solution by crustaceans, but the metals do not consistently interact competitively or synergistically at the exposure concentrations investigated. Regression coefficients of the relationship between zinc and cadmium uptake rates in amphipods and crabs showed occasional, but inconsistent, differences between sites and over time. All three crustaceans take up zinc from solution at a higher rate than cadmium for the same total dissolved metal molar concentration, but at a lower rate than cadmium per free metal ion molar concentration. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Trace metals; Uptake rates; Amphipods; Crabs; Orchestia; Carcinus; Pachygrapsus
* Corresponding author. Tel.: + 44-207-9425275; fax: + 44-207-9425054. E-mail address: [email protected] (P.S. Rainbow). 1 Previous address: School of Biological Sciences, Queen Mary and Westfield College, London, E1 4NS, UK. 0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 9 9 ) 0 0 1 0 3 - 4
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1. Introduction Due to their similar chemistries, zinc and cadmium generally occur together in metal inputs from different sources, particularly in mining effluents which are responsible for metal contamination in the metal-rich sites studied in the present paper (Restronguet Creek, Dulas Bay, Gironde Estuary). Moreover, as a result of their chemical affinities, these trace metals may share uptake pathways into aquatic invertebrates (Rainbow, 1997a,b). Most of the literature dealing with the fate and effects of metals in the aquatic environment report data obtained from single metal exposures, whereas, in the field, organisms are generally exposed to mixtures of contaminants (see Amiard-Triquet and Amiard, 1998). The interaction of one trace metal on the uptake or accumulation of another by aquatic invertebrates can result from competition for absorption sites at the level of biological membranes (Simkiss and Taylor, 1989), and/or from competition for metabolic sites within the cell (Mason and Jenkins, 1995). Interactive effects vary from synergistic (where the concurrent presence of one trace metal enhances the bioaccumulation of another), to antagonistic (where the concurrent presence of another metal decreases the bioaccumulation of the first). Many studies dealing with Cd – Zn interactions have used bivalves as experimental models (see Amiard-Triquet and Amiard, 1998). On consideration of these data, the biological role of a trace metal — whether it is essential like zinc or nonessential like cadmium — seems to be a feature that can influence the nature of any interactive effect. In many of these molluscan species, exposure to the non-essential metal Cd has no effect on Zn accumulation, whereas exposure to the essential metal Zn often has an antagonistic effect on Cd accumulation (Amiard-Triquet and Amiard, 1998). There are fewer studies available on Cd – Zn interactions in crustaceans (Ray et al., 1980; Ahsanullah et al., 1981; Devineau and Amiard-Triquet, 1985; Chou et al., 1987; Nugegoda and Rainbow, 1995; Bat et al., 1998). The opportunity is taken here to make a selective in-depth analysis of Cd – Zn competition, us-
ing data recently published in an extensive study of trace metal uptake rates in amphipods and crabs from a range of coastal sites differentially enriched with trace metals (Rainbow et al., 1999). This latter paper addresses (primarily) whether crustaceans from metal-rich sites are under selective pressure to reduce trace metal uptake rates, and whether the presence or absence of a dispersive larval phase affects such selective pressure. In the event we found that the exposure of the crustaceans to raised trace metal availabilities at these sites had not been sufficient to select for a reduction in dissolved trace metal uptake rates, even in the case of the in situ populations of amphipods lacking a dispersive larval stage. In the study we exposed amphipods (Orchestia gammarellus) and crabs (Carcinus maenas and Pachygrapsus marmoratus) to dissolved zinc or cadmium in the laboratory, either singly or in combination. These data can be analysed to test for the effect of the presence or absence of one metal on the rate of uptake of the other, and to seek correlations between the rates of uptake of each metal in individual crustaceans. We present the results of these analyses here. The crustaceans were collected from up to five sites (Rainbow et al., 1999). Three were considered as metal-rich — Restronguet Creek, Cornwall and Dulas Bay, Anglesey in Britain and the Gironde Estuary in France, and two were control (reference) sites — Millport in the Firth of Clyde, Scotland and Talmont St. Hilaire, Vende´e near Les Sables d’Olonne on the Atlantic coast of France (Fig. 1). Restronguet Creek contains extraordinarily high levels of As, Cd, Cu, Fe, Mn and Zn (Bryan and Gibbs, 1983; Bryan et al., 1987). Dulas Bay has very high levels of Cu, Fe, Mn and Zn (Foster, 1976; Foster et al., 1978; Boult et al., 1994). The Gironde estuary contains elevated availabilities of Cd, Cu and Zn (RNO, 1995).
2. Materials and methods Samples from metal-exposed populations of O. gammarellus and C. maenas were collected intertidally in the UK from Restronguet Creek
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(50°12%N; 05°03%W) and from Dulas Bay (53°22%N; 04°17%W). Control amphipods and crabs from the shore (55°44%N; 04°54%W) and immediate sublittoral near Millport (Isle of Cumbrae, Firth of Clyde) were obtained from the University Marine Biological Station, Millport.
191
Contaminated O. gammarellus, C. maenas and P. marmoratus were also collected from the south shore of the Gironde Estuary — O. gammarellus at Le Phare de Richard (45°22%N; 00°55%W) and the crabs at Le Verdon–La Chambrette (45°32%N; 01°03%W), near La Pointe de Grave. The control
Fig. 1. Collection sites.
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site for French populations of all three crustaceans was the shore at Talmont St. Hilaire, Vende´e (46°24%N; 01°33%W). Dates of collection are detailed in Tables 1 and 2, and the sites are shown in Fig. 1. The study requires a measure of the rate of uptake of metal into the body of the crustacean concerned. Uptake refers to the flux of all metal entering the body of the crustacean (in these experiments, always from solution and always of radioactively labelled metal). Accumulation, on the other hand, is equivalent to net uptake or net flux; that is absolute uptake minus excretion. If there is no excretion of metal in the time period of the experiment, then accumulation is directly equivalent to uptake. The exposure concentrations of 50 and 100 mg l − 1 zinc and cadmium were chosen in order to ensure measurable uptake and accumulation in the time scale of the experiments. The zinc concentration is within the range found in some metal-contaminated estuaries, including Restronguet Creek (100 – 2000 mg Zn per litre, Bryan and Gibbs, 1983) and Dulas Bay (170 – 5650 mg Zn per litre in the stream Afon Goch at the head of the estuary, Foster et al., 1978; Boult et al., 1994). The cadmium concentration is less environmentally realistic but needs to be of the same order as that of zinc for comparative purposes, particularly when investigating the possibility of interaction. Details of acclimation, laboratory handling and experiments are provided by Rainbow et al. (1999).
2.1. Amphipod uptake experiments Experiments were carried out at 10°C in fully aerated artificial seawater (TMN-Tropic Marin Neu, Tropicarium Buchshlag, Dreieich, Germany) at 33%, to ensure reproducibility of physicochemical conditions that affect trace metal uptake rates (Rainbow, 1995, 1997a,b). Groups of ten amphipods, of both sexes and of similar size (usually \10 mg dry wt.) and moult stage (intermoult), were held individually in acid-washed per-
forated plastic containers (Toby ‘Teaboys’, Aldridge Plastics, Aldridge, UK) in replicate 1 l acid-washed plastic tanks. Amphipods were exposed to one or both simultaneously of the trace metals Zn and Cd, using the radioisotopes 65Zn and 109Cd (NEN Life Science Products, Boston, MA) added to stock solutions of the respective metal chloride (Analar grade, BDH) to give experimental exposures of 100 mg l − 1 (1.53 mM Zn per litre, 0.89 mM Cd per litre) with 185 kBq l − 1 (5 mCi l − 1) tracer in TMN. Amphipods were counted live at daily intervals for 4 days, giving a measure of ‘new’ labelled metal accumulated. Accumulation was linear and best-fit linear regression lines were fitted to data for individual amphipods for days 1–4, the zero point being excluded to allow for adsorption of labelled metal onto the exoskeleton. Regression coefficients (ng g − 1 per day) represent the metal uptake rates of individual amphipods. Details of the five amphipod uptake experiments are given in Table 1. There is no excretion of radiolabelled metal accumulated from solution by O. gammarellus over the short time period used in the experiments, and so the rate of accumulation of labelled metal in this short term is indeed a direct measure of the rate of metal uptake from solution (Rainbow and White, 1989; Weeks and Rainbow, 1991; Rainbow et al., 1993; Rainbow and Kwan, 1995). For example, Weeks and Rainbow (1991) exposed O. gammarellus to a series of radiolabelled Zn concentrations for 21 days at 10°C, and showed that, at all exposures, the labelled Zn concentration newly accumulated by the amphipods matched the increases in total Zn concentration. Thus, all Zn taken up from solution in this period [in contrast to Zn assimilated from food (Weeks and Rainbow, 1991, 1994)] is retained as accumulated Zn without excretion, a conclusion supported by the confirmation of the absence of labelled Zn in the urine of amphipods exposed to dissolved Zn (Weeks and Rainbow, 1991). Rates of uptake of Zn and Cd from solution by O. gammarellus can therefore be measured using sequential live counting of amphipods exposed to radioactively labelled metal tracers.
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193
Table 1 O. gammarellus a Mean Zinc uptake rate Experiment 1 [20/5/96] (a) Zn only (c) Zn+Cd Experiment 2 [10/6/96] (a) Zn only (c) Zn+Cd Experiment 3 [5/8/96] (a) Zn only (c) Zn+Cd Experiment 4 [14/7/97] (a) Zn only
(c) Zn+Cd
Experiment 5 [29/10/97] (c) Zn+Cd
Cadmium uptake rate Experiment 1 (20/5/96) (b) Cd only (c) Cd+Zn Experiment 2 [10/6/96] (b) Cd only (c) Cd+Zn Experiment 3 [5/8/96] (b) Cd only (c) Cd+Zn Experiment 4 [14/7/97] (b) Cd only
S.D.
n
ANOVAb
Restronguet Creek (16/5/96) Millport (15/5/96) Restronguet Creek (16/5/96) Millport (15/5/96)
6.73 12.2 7.39 7.06
3.17 8.10 4.30 2.98
9 9 9 8
A
Dulas Bay (5/6/96) Millport (15/5/96) Dulas Bay (5/6/96) Millport (15/5/96)
6.21 11.4 38.0 42.3
1.74 4.71 28.2 10.1
6 8 6 7
Gironde (29/7/96) Talmont (31/7/96) Gironde (29/7/96) Talmont (31/7/96)
31.7 24.4 7.48 10.1
31.3 9.89 3.30 2.60
9 9 10 10
Gironde (23/6/97) Talmont (4/7/97) Millport (27/6/97) Gironde (23/6/97) Talmont (4/7/97) Millport (27/6/97)
10.8 10.6 23.3 9.24 8.50 17.8
5.55 6.62 15.5 5.21 5.24 16.6
7 5 8 7 8 8
Gironde (23/6/97) Gironde (15/10/97) Dulas Bay (23/9/97) Millport (27/6/97)
9.13 17.3 7.75 9.80
2.32 13.9 3.44 6.64
10 8 9 10
Restronguet Creek (16/5/96) Millport (15/5/96) Restronguet Creek (16/5/96) Millport (15/5/96)
0.93 2.18 1.25 1.24
0.48 1.45 0.61 0.59
7 10 9 10
Dulas Bay (5/6/96) Millport (15/5/96) Dulas Bay (5/6/96) Millport (15/5/96)
2.38 4.59 6.81 5.02
0.94 3.73 3.48 2.93
10 9 7 10
A
Gironde (29/7/96) Talmont (31/7/96) Gironde (29/7/96) Talmont (31/7/96)
3.36 3.37 1.91 2.58
1.16 0.94 0.75 0.75
10 10 10 9
A
Gironde (23/6/97) Talmont (4/7/97) Millport (27/6/97)
3.67 3.26 5.37
1.80 1.30 2.42
8 6 8
A A A A A B B A A B B A A A A A A
A A A A
A B A
A B A A A A
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P.S. Rainbow et al. / Aquatic Toxicology 50 (2000) 189–204
Table 1 (Continued) Mean (c) Cd+Zn
Experiment 5 [29/10/97] (c) Zn+Cd
S.D.
n
Gironde (23/6/97) Talmont (4/7/97) Millport (27/6/97)
4.15 3.04 3.57
3.29 1.22 2.62
8 8 8
Gironde (23/6/97) Gironde (15/10/97) Dulas Bay (23/9/97) Millport (27/6/97)
4.80 8.18 2.07 5.78
1.64 4.72 0.52 1.70
10 8 9 10
ANOVAb A A A
a Mean uptake rates (nM g−1 per day) with standard deviation (S.D.) of labelled Zn or Cd in amphipods from five sites (date of collection) exposed to labelled (a) 1.53 mM l−1 Zn, (b) 0.89 mM l−1 Cd, or (c) 1.53 mM l−1 Zn and 0.89 mM l−1 Cd together at 10°C [date of experiment]. b ANOVA: metal uptake rates of amphipods from the same site subjected to metal exposure in the same experiment in the presence or absence of the other metal do not differ significantly (P\0.05) if they share the same letter in a column.
2.2. Crab uptake experiments Measures of rates of short-term whole body net accumulation of radiolabelled Zn and Cd can also be considered to provide measures of absolute rates of Zn and Cd uptake from solution by crabs (Rainbow et al., 1999). Chan and Rainbow (1993a,b) have shown that labelled Zn taken up from solution (as opposed to food) by C. maenas is accumulated without excretion within the time scales and at the exposure concentrations used here. All radiolabelled Zn taken up from solution by crabs exposed to radiolabelled dissolved Zn concentrations up to 100 mg l − 1, was added sequentially to the Zn concentration already present in the crabs (Chan and Rainbow, 1993a), and this accumulated Zn is not excreted – only at exceptionally high Zn exposure concentrations is excretion observed (Chan and Rainbow, 1993b). Measures of rates of whole body net accumulation of radiolabelled Zn therefore do provide measures of absolute rates of Zn uptake from solution. Published literature on Cd accumulation by crustaceans (Jennings and Rainbow, 1979; Bjerregaard, 1982; Rainbow, 1985, 1988, 1998; Rainbow and White, 1989) indicates that the rate of accumulation of Cd by the crab will similarly reflect the rate of Cd uptake. Other relative measures of the uptake rates of Zn and Cd from solution by C. maenas, are provided by features of
the accumulation kinetics of either metal in the haemolymph of the crab (Martin and Rainbow, 1998; Rainbow et al., 1999).
2.2.1. Uptake into blood Following Martin and Rainbow (1998), up to ten C. maenas (\ 44 mm carapace width) were exposed in each experiment to (a) 50 mg l − 1 (0.76 mM l − 1) Zn in TMN, (b) 50 mg l − 1 (0.45 mM l − 1) Cd in TMN, or (c) 50 mg l − 1 (0.76 mM l − 1) Zn and 50 mg l − 1 (0.45 mM l − 1) Cd in TMN simultaneously, at 10°C for 4 days in individual acidwashed plastic containers (Rainbow et al., 1999). Zn and Cd solutions were labelled with 185 kBq l − 1 (5 mCi l − 1) 65Zn and 109Cd as appropriate. Experimental details are provided in Table 2. Haemolymph samples were taken each day and counted for labelled metal concentration. The labelled Zn concentration in the haemolymph continues to increase over the exposure period. The rate of this increase (the regression coefficient of the best-fit line, nM ml − 1 per day) is directly proportional to the concentration of available Zn in the exposure solution, and is a relative measure of the crab’s uptake rate of dissolved metal (Martin and Rainbow, 1998; Rainbow et al., 1999). The labelled Cd concentration in the haemolymph, on the other hand, rapidly reaches an equilibrium as its rate of removal from the haemolymph matches its rate of uptake into the
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195
Table 2 C. maenas a
Zinc uptake rate Experiment 1 [20/5/96] (a) Zn only (c) Zn+Cd Experiment 2 [10/6/96] (c) Zn+Cd Experiment 3 [5/8/96] (c) Zn+Cd Experiment 4 [14/7/97] (c) Zn+Cd
Cadmium uptake rate Experiment 1 (20/5/96) (b) Cd only (c) Cd+Zn Experiment 2 [10/6/96] (c) Cd+Zn Experiment 3 [5/8/96] (c) Cd+Zn Experiment 4 [14/7/97] (c) Cd+Zn
Mean
S.D.
n
ANOVAb
Millport (15/5/96) Millport (15/5/96) Restronguet Creek (16/5/96)
0.88 0.69 1.05
0.65 0.30 0.67
6 9 8
A A
Dulas Bay (5/6/96) Millport (15/5/96)
3.58 0.75
1.11 0.30
8 9
Gironde (29/7/96) Talmont (31/7/96)
1.24 1.59
1.10 0.82
6 8
Restronguet Creek (25/6/97) Millport (27/6/97) Gironde (23/6/97) Talmont (4/7/97)
0.78 1.15 1.39 3.10
0.38 0.54 1.22 2.49
6 7 6 6
Millport (15/5/96) Millport (15/5/96) Restronguet Creek (16/5/96)
0.157 0.160 0.167
0.081 0.089 0.134
7 9 8
Dulas Bay (5/6/96) Millport (15/5/96)
0.677 0.138
0.117 0.058
8 9
Gironde (29/7/96) Talmont (31/7/96)
0.153 0.233
0.111 0.176
6 7
Restronguet Creek (25/6/97) Millport (27/6/97) Gironde (23/6/97) Talmont (4/7/97)
0.176 0.271 0.254 0.404
0.133 0.131 0.166 0.197
6 7 6 6
A A
a Mean relative uptake rates with standard deviations (S.D.) of labelled Zn (nM ml−1 per day in haemolymph) and Cd (nM ml−1 in haemolymph) of crabs from five sites (date of collection) exposed to labelled (a) 0.76 mM l−1 Zn; (b) 0.45 mM l−1 Cd, or (c) 0.76 mM l−1 Zn and 0.45 mM l−1 Cd together at 10°C [date of experiment]. b ANOVA: relative metal uptake rates of crabs subjected to metal exposure in the presence or absence of the other metal in the same experiment do not differ significantly (P\0.05) if they share the same letter.
haemolymph under constant exposure (Martin and Rainbow, 1998). The equilibrium concentration of Cd in the haemolymph (nM ml − 1) increases with increased concentration of available Cd in solution and is a relative measure of the rate of uptake of Cd from solution by the whole crab (Martin and Rainbow, 1998; Rainbow et al., 1999).
2.2.2. Whole crab accumulation P. marmoratus and smaller specimens of C. maenas (B44 mm carapace width) were used in experiments in which the accumulated concentrations of labelled Zn and Cd were measured at intervals in the whole crabs. The rates of (net) accumulation of Zn and Cd during these shortterm experiments are considered to be direct mea-
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sures of the absolute rates of uptake of Zn and Cd from solution by the whole crabs (Rainbow et al., 1999). Collection and experimental details are provided for C. maenas in Table 2. P. marmoratus were collected on the same day as C. maenas from the same site (Table 2). In 1996 C. maenas from Restronguet Creek, Dulas Bay and Millport, and P. marmoratus from the Gironde and Talmont St. Hilaire, were exposed for 11 days at 10°C to 50 mg l − 1 (0.76 mM l − 1) Zn and 50 mg l − 1 (0.45 mM l − 1) Cd (labelled with 185 kBq l − 1 65Zn and 185 kBq l − 1 109Cd) together in TMN. Up to five C. maenas and four P. marmoratus were sampled on days 1, 2, 4 and 7, and all remaining crabs on day 11, prior to counting for labelled Zn and Cd contents. In 1997 C. maenas from Restronguet Creek, Millport, Gironde and Talmont, and P. marmoratus from Gironde were similarly exposed for 21 days. Crabs were sampled on days 7, 14 and 21, before being prepared and counted as above (Rainbow et al., 1999). Data for any crab that moulted during the experiments have been ignored.
2.3. Statistical analysis All statistical analyses, including regression analysis, ANOVA and ANCOVA, were carried out using STATISTICA (Statsoft), preliminary tests for normality of distributions of data and equality of variances having shown that it was valid to employ parametric statistics. Where a priori ANOVA or ANCOVA revealed statistically significant (PB0.05) differences among groups, a posteriori testing was used to identify which groups differed from each other. 3. Results
3.1. Amphipod uptake rates Table 1 gives details of the zinc and cadmium uptake rates of O. gammarellus exposed to 100 mg l − 1 of one metal (1.53 mM l − 1 Zn, 0.89 mM l − 1 Cd) in the presence or absence of 100 mg l − 1 of the other metal, and the results of ANOVA comparisons of these uptake rates as appropriate.
3.1.1. Zinc uptake The presence of a high dissolved cadmium concentration (0.89 mM Cd per litre) had an inconsistent effect on the zinc uptake rate (Table 1). In one case out of three, the uptake rate of zinc by Millport amphipods increased in the presence of high cadmium, as did the zinc uptake rate of Dulas Bay amphipods in the same experiment (experiment 2). In experiment 3, on the other hand, the zinc uptake rates of amphipods from Gironde and Talmont decreased significantly in the presence of raised cadmium concentrations. There was no such significant effect, however, in the case of the same amphipods in experiment 4. It is impossible therefore to draw a consistent general conclusion on the effect of raised cadmium availability on the zinc uptake rate of amphipods exposed to both metals at 100 mg l − 1. 3.1.2. Cadmium uptake The presence of a high zinc concentration (1.53 mM Zn per litre) similarly had an inconsistent effect on the cadmium uptake rates of the amphipods. The cadmium uptake rate of Dulas Bay amphipods in June 1996 (experiment 2) was increased significantly in the presence of high zinc, but that of Gironde amphipods was decreased significantly in May 1996 (experiment 3) and unaffected in July 1997 (experiment 4) (Table 1). The cadmium uptake rates of Millport, Restronguet Creek and Talmont amphipods were unaffected by the presence of high zinc (Table 1). 3.1.2.1. Correlation of zinc and cadmium uptake rates in indi6idual amphipods. It is possible to use these data to address the question whether zinc and cadmium uptake rates of individual amphipods are correlated — i.e. do individual amphipods show characteristically high or low uptake rates for both metals? Data were analysed for amphipods in all joint zinc/cadmium experiments (Table 1), firstly for all the amphipods from each site in each experiment, secondly for all the amphipods from each site combined across all experiments, and then for the whole data set (Fig. 2). Uptake rates for the two metals were correlated in the amphipods from each site in most of the separate experiments (regression lines not
P.S. Rainbow et al. / Aquatic Toxicology 50 (2000) 189–204
shown), in all the combined data sets for each site (single regression lines, Fig. 2), and in the whole data set (Fig. 2f). It can be concluded therefore that the uptake rates of zinc and cadmium in individual O. gammarellus are correlated. Data are also available to check whether the correlation varies between sites or over time. Comparisons were made by ANCOVA between regression coefficients of the best-fit regression lines of Zn uptake rate (x) against Cd uptake rate (y) as in Fig. 2. No consistent pattern emerged. A comparison of regression coefficients for amphipods from each site (using all data combined between experiments) showed no significant difference between sites with the exception of Gironde amphipods, but a comparison experiment by experiment indicated Dulas Bay as the only site differing from the others. Relationships
197
for amphipods within one site did not differ between experiments, except in the one case of Millport amphipods in experiment 5.
3.2. Crab uptake rates 3.2.1. As measured from blood parameters Table 2 presents data on the relative uptake rates of zinc and cadmium by C. maenas derived from patterns of short term accumulation of the metals in the blood of individual crabs exposed to 50 mg l − 1 of the metals (0.76 mM l − 1 Zn, 0.44 mM l − 1 Cd), either singly or simultaneously. As explained in Section 2, the slope of the best-fit regression line (nM Zn ml − 1 per day) is a relative measure of the uptake rate of zinc by the crabs, whereas in the case of cadmium it is the steadystate equilibrium concentration of cadmium in the
Fig. 2. O. gammarellus. Correlation of rates of uptake (nM g − 1 per day) of labelled zinc and cadmium in individual amphipods exposed simultaneously to labelled 1.53 mM l − 1 Zn and 0.89 mM l − 1 Cd at 10°C in experiments 1 – 4 of Table 1. The different symbols in (a – e) indicate amphipods from the same site of collection but in different experiments (see Table 1). The different symbols in (f) each represent all amphipods from one site combined between experiments. * PB0.05, ** PB 0.01, *** P B0.001.
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Fig. 3. C. maenas. Correlation of relative rates of uptake of labelled zinc (nM ml − 1 per day) and cadmium (nM ml − 1) (see text for explanation) in individual crabs exposed simultaneously to labelled 0.76 mM l − 1 Zn and 0.45 mM l − 1 Cd at 10°C in experiments 1–4 of Table 2. The different symbols in (a–e) indicate crabs from the same site of collection but in different experiments (see Table 2); the different symbols in (f) each represent all crabs from one site combined between experiments.
blood (nM Cd per millilitre) that is the relative measure of the rate of cadmium uptake by the crabs. Table 2 summarises the data obtained, with the results of ANOVA comparisons of uptake rates of one metal in the presence or absence of the other. The presence of 0.44 mM Cd per litre had no significant effect on the rate of zinc uptake by Millport crabs, nor did the presence of 0.76 mM Zn per litre on the rate of uptake of cadmium (Table 2).
3.2.1.1. Correlation of zinc and cadmium uptake rates in indi6idual crabs. Relative uptake rates of zinc and cadmium are known for individual crabs in the double metal exposure experiments (experi-
ments 2–4, Table 2). Uptake rates for the two metals were correlated in the crabs from each site in most of the separate experiments (not shown), in all data sets combined for each site (Fig. 3), and in the whole combined data set for all crabs from all sites (Fig. 3f). As for the amphipods therefore, the uptake rate of zinc in individual C. maenas is correlated with the rate of cadmium uptake. As in the case of the amphipods, the data can also be used to check whether the nature of the correlation between Zn and Cd varies between sites or over time. Comparison of regression coefficients for all crabs from each site (all data combined between experiments) showed no differences between sites except for Talmont crabs, but
P.S. Rainbow et al. / Aquatic Toxicology 50 (2000) 189–204
comparison experiment by experiment showed no intersite differences at all. There were no differences in regression coefficients for crabs from the same site between experiments.
3.2.2. As measured by whole crab accumulation rates 3.2.2.1. Correlation of zinc and cadmium accumulation in indi6idual crabs. The whole crab accumulation data can be used to test whether the concentrations of labelled zinc and cadmium accumulated by individual crabs are correlated when crabs are exposed to labelled zinc and cad-
199
mium simultaneously. Zinc and cadmium concentrations for all individual crabs from each site were correlated significantly (PB 0.01) in most experiments for both C. maenas and P. marmoratus (data not presented separately for each experiment). The trace metal concentrations were also significantly correlated when all the data for each species were combined across experiments and years (Fig. 4). Thus, crabs with a high or low rate of accumulation of zinc tend to have a respectively high or low rate of cadmium accumulation. The whole crab accumulation data (Fig. 4) can also be used to check whether the correlation between Zn and Cd varies intraspecifically between sites or between species. ANCOVA comparisons of the relevant regression coefficients showed Millport C. maenas to differ significantly from Restronguet Creek and Dulas Bay crabs (no difference) in 1996, and from Restronguet Creek, Gironde and Talmont crabs (no difference) in 1997. For P. marmoratus there was no significant difference between regression coefficients for Gironde and Talmont in 1996, although there was for Gironde P. marmoratus between 1996 and 1997. Interspecifically, C. maenas (whole data set) had a regression coefficient significantly different from that of P. marmoratus. At Gironde in 1997, however, the regression coefficients for the two species did not differ significantly. No consistent pattern has emerged.
4. Discussion
Fig. 4. Correlations of accumulated concentrations of labelled zinc and cadmium by individual whole crabs [(a) C. maenas; (b) P. marmoratus] exposed simultaneously to 0.76 mM l − 1 Zn and 0.45 mM l − 1 Cd at 10°C for either up to 11 days (1996) or up to 21 days (1997). Different symbols each represent crabs from one site combined between experiments and years.
Experimental studies of metal–metal interactions in crustaceans are inconsistent or even contradictory. In the amphipod Corophium 6olutator, zinc addition induced a depletion of cadmium toxicity and accumulation (Bat et al., 1998). In the decapod Callianassa australiensis, exposure to a mixture of Zn and Cd increased the bioaccumulation of both elements (Ahsanullah et al., 1981). In the lobster Homarus americanus (another decapod), Cd added to food enhanced Zn accumulation in the hepatopancreas but not in the tail muscle (Chou et al., 1987). In the caridean decapod Pandalus montagui, the response was variable according to organ or dose; in the presence of Zn,
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particularly at the lower concentration tested (1.07 mM l − 1), Cd concentration increased in the hepatopancreas, decreased in the carcass and was unaffected in the other tissues (Ray et al., 1980). In another caridean decapod Palaemon serratus, the presence of cadmium reduced the accumulation of zinc by larvae (Devineau and Amiard-Triquet, 1985). Thus, interactions of metals are variable, and may change from antagonism to synergism with change of external concentrations, length of exposure, species studied, and tissues or organs examined (Amiard-Triquet and Amiard, 1998). Another factor that may have an influence is the tolerance of a population, within a species, to one or other of the metals concerned (AmiardTriquet and Amiard, 1998). Brown (1978), for example, described two metal-tolerant populations of the freshwater isopod crustacean Asellus meridianus, where the metal accumulation patterns (and mechanisms of tolerance) differed between the two populations, with consequences on the nature of the interaction of the metals concerned. In one population resistant to Pb, lead tolerance was reflected in reduced accumulation of both Pb and Cu; in the other population, resistant to both Pb and Cu, there was no reduced accumulation of either metal, and both metals competed for sites of detoxified storage (cuprosomes) in the hepatopancreatic caeca (Brown, 1978; Amiard-Triquet and Amiard, 1998). The studies mentioned above have generally been based upon comparative assessments of bioaccumulation, and therefore involve the possible competition of dissolved cadmium and zinc for both uptake and storage sites in the crustaceans. The present study is not strictly comparable since, in two of the three sets of experiments, it takes into account only the first step of bioaccumulation, that of uptake. In the amphipod O. gammarellus, the presence of 0.89 mM l − 1 cadmium variably increased, decreased or had no significant effect upon the uptake of zinc from 1.53 mM Zn per litre, as did the reverse scenario of zinc on cadmium uptake. In the case of C. maenas neither metal at 50 mg l − 1 (0.76 mM Zn per litre, 0.45 mM Cd per litre) affected the rate of uptake of the other. When P. elegans were ex-
posed to zinc and cadmium together, both at 20 mg l − 1 (0.31 mM Zn per litre, 0.18 mM Cd per litre), the rate of zinc uptake decreased and the rate of cadmium uptake increased in comparison with the uptake rates of each metal in single metal exposures at the same concentration (Nugegoda and Rainbow, 1995). Thus, even when interactive effects on influx are examined independently of interactions within the cell, the possible competition of dissolved Cd and Zn for uptake sites still appears inconsistent and contradictory. In both the amphipods and the crabs in this study, there was a significant correlation between zinc and cadmium uptake rates of individuals, suggesting that these two trace metals may share one or more routes of uptake from solution (Rainbow, 1995, 1997b). The same correlation has been observed in P. elegans (Nugegoda and Rainbow, 1995). Recent findings also indicate that cadmium and zinc may use the same uptake mechanism in the gills of fish, apparently by both sharing the route of cadmium entry (Bentley, 1992; Hogstrand and Wood, 1996; Olsson et al., 1998). Nevertheless, any potential sharing of routes of uptake of zinc and cadmium from solution, has not led to consistent synergism or antagonism in the experiments run here. It is possible that the chosen metal exposure concentrations are not high enough to cause significant interaction between the metals entering the crustaceans from solution. This is unlikely, for the experimental concentrations used exceed those of typical coastal waters and most estuaries (Bryan and Gibbs, 1983; Bryan et al., 1985; Bryan and Langston, 1992), including for example cadmium in the Gironde ( 0.3 mg Cd l − 1; Boutier et al., 1989). In the case of zinc, however, even higher dissolved concentrations can be found in some metal-contaminated estuaries, including Restronguet Creek (100–2000 mg Zn per litre, 0.25– 5.0 mg Cd per litre, Bryan and Gibbs, 1983) and Dulas Bay (170–5650 mg Zn per litre in the stream Afon Goch at the head of the estuary, Foster et al., 1978; Boult et al., 1994). Restronguet Creek has extremely high levels of zinc and cadmium relative to most coastal and estuarine waters (Bryan and Gibbs, 1983). Zinc is accumulated to atypically high concentrations in
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201
Table 3 Total metal molar uptake indices and metal ion molar uptake indices for zinc and cadmium (see text for explanation and units), and their ratios in the amphipod O. gammarellus, and the crabs C. maenas and P. marmoratus (data from Table 1 and Fig. 4)
Orchestia gammarellus Millport Zinc Zn/Cd separate Expt. 1 Expt. 2 Expt. 4 Zn/Cd together
Cadmium Zn/Cd separate
Zn/Cd together
Expt. Expt. Expt. Expt.
Total metal uptake index
Ratio Zn:Cd total metal uptake index
Metal ion uptake index
Ratio Zn:Cd metal ion uptake index
0.0080 0.0075 0.0152
3.33: 1.44: 2.53: 2.43: 3.29: 4.33: 2.90: 0.98: 3.03:
0.0126 0.0118 0.0240
0.19: 0.08: 0.15: 0.14: 0.19: 0.29: 0.17: 0.06: 0.18:
1 1 1 1 (mean) 1 1 1 1 1 (mean)
1 2 4 5
0.0046 0.0276 0.0116 0.0064
Expt. 1 Expt. 2 Expt. 4
0.0024 0.0052 0.0060
0.0661 0.1391 0.1627
Expt. Expt. Expt. Expt.
0.0014 0.0056 0.0040 0.0065
0.0376 0.1521 0.1082 0.1752
1 2 4 5
Carcinus Maenas Millport 1996 Dulas Bay 1996 Restronguet Creek 1996 Restronguet Creek 1997 Gironde 1997 Talmont 1997 Pachygrapsus marmoratus Gironde 1996 Talmont 1996 Gironde 1997
0.0073 0.0436 0.0184 0.0101
1 1 1 1 (mean) 1 1 1 1 1 (mean)
0.85: 1 1.14: 1 0.89: 1
0.050: 1 0.067: 1 0.052: 1
10.5: 1
0.623: 1
4.89: 1 4.70: 1
0.286: 1 0.275: 1
2.73: 1 2.34: 1 1.37: 1
0.160: 1 0.137: 1 0.080: 1
many of the indigenous aquatic fauna and flora (Bryan and Gibbs, 1983; Bryan et al., 1987), whilst Cd accumulation can be considered atypically low, relative to environmental levels. A sharing of (and potentially competition for) uptake routes from solution by zinc and cadmium would be consistent with this observation (Bryan and Gibbs, 1983). The nature of the relationship between zinc and cadmium uptake rates in amphipods and crabs showed occasional differences between sites and
over time, but no consistent pattern was discernible. Generally, for a given molar concentration, the molar uptake (crustaceans — Rainbow and White, 1990) and bioaccumulation (bivalve molluscs — Wang et al., 1996; Lee et al., 1998) of dissolved zinc by marine invertebrates is greater than that of cadmium. It is possible to recalculate the zinc and cadmium uptake rates of O. gammarellus given in Table 1 in terms of molar uptake rate (mM g − 1 per day) per molar metal exposure
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(mM l − 1) to give a (total metal) molar uptake index (l g − 1 per day). Table 3 shows this index for O. gammarellus from Millport (data from experiments detailed in Table 1). As expected, the molar uptake rate of zinc is higher than that of cadmium, whether the amphipods are exposed to zinc and cadmium separately or together (Table 3). Lee et al. (1998), however, recalculated their data to take account of the model that considers the free metal ion to be the major determinant of the bioavailability of many trace metals including Zn and Cd (Campbell, 1995; Rainbow, 1995, 1997b). [It would be simplistic, however, to consider that the free metal ion is the only bioavailable form of the metal, given that neutral inorganic or organic complexes of a trace metal ion may diffuse directly across the cell membrane, bypassing any (more important?) membrane-embedded transport system for the free metal ion (Simkiss and Taylor, 1989; Campbell, 1995; Van Ginneken et al., 1999)]. Recalculation of the data in terms of the free metal ion concentration reverses the previous conclusion, showing the bivalves to be more permeable to Cd2 + than to Zn2 + (Lee et al., 1998). It is possible to make the same recalculation here. In the artificial seawater TMN at 33‰, the free Zn2 + ion is estimated to make up 63.5% of total dissolved Zn (Rainbow et al., 1993) and the free Cd2 + ion 3.7% of total dissolved Cd (Rainbow and Kwan, 1995). Thus, any decrease in salinity makes a proportionately greater increase in the activity of the free cadmium ion than in the activity of the free zinc ion as chloride complexation decreases (Zirino and Yamamoto, 1972; Mantoura et al., 1978; Rainbow et al., 1993; Rainbow and Kwan, 1995). By calculating the free ion concentrations at the experimental exposure concentrations, it is now possible to calculate a metal ion molar uptake index (mM g − 1per day per mM M2 + l − 1) (Table 3). As for the bivalves, the free Cd2 + ion is taken up by the amphipod at a greater rate per unit molar exposure than the free Zn2 + ion. The higher molar uptake rate for Zn, described above, is therefore largely attributable to the greater proportion of the free metal ion present. Table 3 also presents similar data for the crabs C. maenas and P. marmoratus recalculated from
the raw data (Rainbow et al., 1999) contributing to Fig. 4. As for the amphipod, in both crabs zinc has a higher total metal molar uptake index than cadmium but a lower metal ion molar uptake index (Table 3). This study set out to investigate the possible interaction of zinc and cadmium uptake rates in crustaceans. In fact the presence or absence of a raised dissolved concentration of one metal had an inconsistent effect on the rate of uptake of the other metal by the amphipod O. gammarellus, and no effect on the uptake of the other metal by the crab C. maenas. Zinc and cadmium uptake rates are correlated in individual amphipods and crabs (of two species) from all the sites examined. Thus zinc and cadmium appear to share common routes of uptake from solution by these crustaceans, but the metals do not consistently interact competitively or synergistically during uptake at the exposure concentrations investigated. It remains possible that other results might have been obtained at other exposure concentrations and combinations, and/or that other mechanisms (e.g. excretion, detoxification) are involved in the overall manifestation of competition in terms of body burdens. Acknowledgements We are very grateful for support for travel from the Central Research Fund of the University of London, and from the Alliance: Franco–British Joint Research Programme scheme of the British Council and APAPE (France) (Alliance Project PN 97.076). We also thank two referees for their constructive criticism. References Ahsanullah, M., Negilski, D.S., Mobley, M.C., 1981. Toxicity of zinc, cadmium and copper to the shrimp Callianassa australiensis. III. Accumulation of metals. Mar. Biol. 64, 311 – 316. Amiard-Triquet, C., Amiard, J.-C., 1998. Influence of ecological factors on accumulation of metal mixtures. In: Langston, W.J., Bebianno, M. (Eds.), Metal Metabolism in Aquatic Environments. Chapman and Hall, London, pp. 351 – 386.
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