The beach-hopper Orchestia gammarellus (Crustacea: Amphipoda) as a biomonitor for copper and zinc: North Sea trials

The Science of the Total Environment, 106 (1991) 221-238 Elsevier Science Publishers B.V., Amsterdam

221

The beach-hopper Orchestia gammarellus (Crustacea: Amphipoda) as a biomonitor for copper and zinc: North Sea trials P.G. Moore a, P.S. Rainbow b and Elaine Hayes b aUniversity Marine Biological Station, Millport, Isle of Cumbrae, Scotland KA28 OEG, United Kingdom bCentre for Research in Aquatic Biology, School of Biological Sciences, Queen Mar)' and Westfield College, Mile End Road, London E1 4NS, United Kingdom (Received June 22nd, 1990; accepted September 27th, 1990)

ABSTRACT Collections of the beach-hopper, Orchestia gammarellus (Pallas) (Amphipoda: Talitridae), have been taken from 25 sites along the east coast of mainland Scotland, from four sites in the Orkney Is. and from single sites in Norway and Denmark. Total body burdens of the essential trace metals Cu and Zn have been analysed. Typically 'background' levels of a standard 10 mg dry wt animal were ~ 70#g g-~ Cu and ~ 170/~g g-~ Zn. Samples with significantly higher metal burdens (up to 218#g g i Cu, 340pg g ~ Zn) were associated with local sources of enrichment, due to anthropogenic inputs (antifouling paint leachates in harbours and marinas) or metal-rich mineralogy. Significant seasonal changes in Cu and Zn concentration in O. gammarellus at North Queensferry (Firth of Forth), a site with some of the highest levels of Cu and Zn recorded in this amphipod in the UK, may reflect temporal variation in contamination. At unpolluted sites, seasonal changes in Cu, and especially Zn, are generally slight. A reduction of body Cu concentration in some individuals seemed to be associated with the immediately recent loss of juveniles from the brood pouch of some pregnant females. Copper and Zn concentrations in North Sea O. gammarellus are generally elevated compared with animals from the Atlantic coast of southwest Scotland. Orchestia gammarellus provides a very convenient and sensitive biomonitoring system for these trace metals along North Sea coasts. INTRODUCTION

This paper presents the results of a study into the use of the talitrid amphipod crustacean Orchestia gammarellus (Pallas) as a biomonitor of the toxic metals copper and zinc primarily in eastern Scottish coastal waters. It follows a pilot investigation of the general suitability of talitrids as biomonitors (Rainbow et al., 1989) which considered several species from a range of sites including an extensive subset from western Scottish coastal

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waters. The opportunity has been taken here to include data sets collected in Norway and Denmark. MATERIAL AND METHODS

Material The collections of Orchestia gammarellus made during 1989 and in January 1990 are detailed in Table 1. They include a wide-ranging survey of the entire eastern seaboard of Scotland (including the Orkney Is.) and a more intensive survey of the Firth of Forth, supplemented with single samples from Norway and Denmark. The amphipods were frozen individually in sealed polythene bags within hours of collection. For pragmatic reasons, amphipods were not allowed to depurate the gut contents (see Rainbow et al., 1989). Amphipods were not segregated by sex, although records would allow it, since earlier work (Moore and Rainbow, 1987) discounted any differences in heavy metal concentrations being associated with gender in this species. On two occasions (N. Queensferry and South Alloa, both May 1989), individual females released juveniles from the brood pouch into the sealed polythene bag before freezing. On subsequent analysis these females were found to have low body concentrations of copper (not apparent in females retaining young in the brood pouch or non-brooding). Statistical analyses (see below) were therefore made of copper and zinc concentrations in these samples both including and excluding data on such spent females. Additional samples were taken at N. Queensferry primarily, but also at Skinflats (see Table 1), as part of a study of seasonal variation at a contaminated site, with comparative samples being taken at Millport to act as a control and to allow direct comparison with an earlier data set for 1987 at that site (Rainbow and Moore, 1990).

Methods Individual amphipods were thawed in pre-weighed Pyrex tubes, dried to constant weight at 60°C, digested in Aristar conc. nitric acid (BDH Ltd) at 100°C and made up to volume (2 ml) with double-distilled water. A subsample of this digest was diluted five-fold, again with double-distilled water. Digests, and/or dilutions thereof as appropriate, were analysed for copper and zinc (and cadmium for selected samples) by atomic absorption spectrophotometry (flame atomization, background correction for Zn, Cd) on an International Laboratory IL-157 atomic absorption spectrophotometer. Digest detection limits were 0.01/xg ml ~ for all three metals; equivalent in 2ml digests to

223

ORCHESTIA GAMMARELLUS: BIOMONITOR FOR COPPER AND ZINC

TABLE 1 Details of collection sites of Orchestia gammarellus Site

OS Grid ref.

Date(s) (day/month/year)

Orkney West Scapa Bay Birsay St Peter's Pool Widewall Bay

HY HY HY ND

436088 246277 550035 434915

29/3/89 29/3/89 29/3/89 29/3/89

ND 244348 ND 035152 NH 773968 NH 666687 NH 652479 NJ 145700 NJ 555666 NK 134427 NO 880867 NO 730558 NO 396264 NO 514168 NT 325946 NT 378742 NT 720773 NT 947645

25/4/89 25/4/89 25/4/89 25/4/89 21/3/89 21/3/89 21/3/89 21/3/89 22/3/89 22/3/89 17/3/89 17/3/89 17/3/89 14/3/89 14/3/89 14/3/89

NS NS NS NT NT NS NS NT

879919 913888 977857 022860 132825 961812 925841 129805

25/5/89 25/5/89 25/5/89 25/5/89 25/5/89 25/5/89 14/3/89, 25/5/89, 26/7/89 17/3/89, 25/5/89, 26/7/89 26/9/89, 20/11/89, 12/1/90

NS 173542

16/3/89, 31/5/89, 24/7/89 23/9/89, 24/11/89, 11/1/90

Mainland Scotland Lybster Helmsdale Loch Fleet Alness N. Kessock Hopeman Sandend Boddam Stonehaven Sillo Craig Wormit St Andrews West Wemyss Prestonpans Barns Ness Eyemouth Firth of Forth South Alloa Kennetpans Dunimarle Castle Torryburn Inverkeithing Kinneil Kerse Skinflats (Grangemouth) N. Queensferry Firth of Clyde Millport, Cumbrae Scandinavia Fyn Hoved, Denmark Bergen, Norway

16/7/89 15/8/89

224

P.G. MOORE ET AL.

0.47 pg g-' dry wt in the heaviest amphipod analysed (0.0427 g dry wt) and 20#g g ~ in the smallest (0.0010g dry wt). All metal concentrations are quoted in microgrammes per gramme dry weight. Analytical quality was checked against Tort-1 Lobster Hepatopancreas marine reference material (National Research Council, Canada) and was very satisfactory: means 4- l so (n = 4) were 410 4- 7.5#gCug ', 164 4- 3.7#g Zn g-~ and 25.1 4- 0.6pg Cd g-' in comparison with certified values (95% tolerance limits) of 439 4- 22 #g Cu g- I, 177 4- 10 pg Zn g- I and 26.3 4- 2.1 #g Cd g-~.

Statistical analysis Rainbow and Moore (1986) have shown that it is meaningless to compare mean metal concentrations between populations of amphipods, for allowance must be made for possible relationships between body weight and heavy metal concentration. In order to minimize such effects, only large (>2rag) individuals have been analysed. Nevertheless it was necessary to account for remaining significant relationships. Data for individual dry weights and heavy metal concentrations were transformed logarithmically (see Rainbow and Moore, 1986). The transformed data were fitted (least squares regression) to the straight line equation log y = log a + b log x (derived from the power relationship), where y is the metal concentration and x the amphipod dry weight. This equation is typically a very good model for such data (Rainbow and Moore, 1986), and the derived linear regressions can be compared by analysis of covariance (ANCOVA). ANCOVA tests for significant differences in transformed metal concentration after allowance for differences in the transformed dry weight, with the precondition that there is no significant difference between the regression coefficients (slopes) of the relationships compared. RESULTS

Copper Table 2 summarizes information on the double log regressions of copper concentration against dry weight for Orchestia gammarellus. There were several significant regressions of metal concentration against dry weight (both before and after transformation of the data into logarithms) in spite of the choice of large amphipods, so confirming the necessity to use ANCOVA for statistical comparisons. ANCOVA allows the comparison of copper concentrations in amphipods

ORCHESTIA GAMMARELLUS:BIOMONITORFOR COPPER AND ZINC

225

TABLE 2

Orchestia gammarellus (1989): t h e n u m b e r a n d d r y w e i g h t (g, m e a n a n d r a n g e ) o f a m p h i p o d s a n a l y s e d for c o p p e r c o n c e n t r a t i o n s w i t h d e t a i l s o f t h e r e l a t i o n s h i p log y = l o g a + b log x, w h e r e y is t h e m e t a l c o n c e n t r a t i o n (/~g g - ~), x is t h e d r y w e i g h t (g) a n d a a n d b (the r e g r e s s i o n coefficient) a r e c o n s t a n t s . Sig.reg. is t h e s i g n i f i c a n c e o f t h e r e g r e s s i o n ; N S n o t s i g n i f i c a n t ( P > 0.05) n

N. Queensferry (17/3/89) Sandend Sillo Craig Bergen (Norway) St Andrews Alness Wormit N. Queensferry (26/7/89) N. Kessock Stonehaven West Wemyss Lybster Boddam Skinflats (26/7]89) N. Queensferry (26/9/89) Widewall Bay Loch Fleet Hopeman N. Queensferry (20/11/89) Eyemouth St Peter's Pool Helmsdale N. Queensferry (12/1/90) West Scapa Bay Birsay Millport (24/7/89) Kennetpans N. Queensferry (25/5/89)* Dunimarle Castle Millport (16/3[89) Torryburn Skinflats (25/5/89) South Alloa* Millport (1 I/I/90) Prestonpans Millport (31/5/89) I nverkeithing Fyn Hoved (Denmark) Barns Ness Millport (24/11/89)

15 15 15 17 15 15 15 14 15 15 15 15 15 9 15 15 15 15 15 15 15 15 15 15 15 15 15 I0 15 15 15 15 9 15 14 [5 15 10 15 15

Dry weight (g)

Double log regression

Mean

Range (min. max.)

0.0125 0.0181 0.0229 0.0153 0.0155 0.0150 0.0192 0.0093 0.0176 0.0146 0.0161 0.0113 0.0219 0.0080 0.0134 0.0109 0.0149 0.0228 0.0131 0.0165 0.0137 0.0141 0.0102 0.0136 0.0177 0.0107 0.01 I0 0.0094 0.0114 0.0158 0.0114 0.0115 0.0093 0.0183 0.0145 0.0104 0.0122 0.0055 0.0154 0.0174

0.0064. 0.0079, 0.0069, 0.0036, 0.0097, 0.0010, 0.0121, 0.0059, 0.0076, 0.0100, 0.0125, 0.0064, 0.0083. 0.0055. 0.0097, 0.0057, 0.0112, 0.0084, 0.0046, 0.0094, 0.0061. 0.0072. 0.0054, 0.0081, 0.0078. 0.0084. 0.0084. 0.0066, 0.0067. 0.0098, 0.0085, 0.0070, 0.0076, 0.0106. 0.0071. 0.0067. 0.0066. 0.0042 0.0080. 0.0104.

0.0285 0.0365 0.0388 0.0323 0.0210 0.0262 0.0362 0.0123 0.0320 0.0243 0.0232 0.0265 0.0427 0.0107 0.0250 0.0176 0.0214 0.0344 0.0202 0.0245 0.0232 0.0246 0.0202 0.0191 0.0274 0.0197 0.0176 0.0142 0.0148 0.0236 0.146 0.0153 0.0127 0.0345 0.0377 0.0151 0.0220 0.0071 0.0262 0.0380

h

- 0.435 0.555 -0.559 - 0.468 -0.073 - 0.038 -0.378 -0.407 -0.435 -0.338 -0.163 -0.584 - 0.098 -0.721 0.117 -0.630 -0.032 -0.183 0.410 - 0.064 -0.366 -0.259 - 0.021 -0.374 -0.322 -0.520 - 0.600 0.649 -0.045 -0.215 0.220 0.368 -0.023 -0.157 -0.163 0.042 0.121 -0.414 - 0.529 --0.101

Sig.reg.

Log a

P< P< P < P< NS NS NS P < NS NS NS P < NS NS NS P < NS NS P < NS P < NS NS NS P < P < P < NS NS NS NS NS NS NS NS NS NS NS NS NS

1.467 1.164 1.114 1.236 2.309 2.044 1.362 1.301 1.238 1.398 1.734 0.891 1.844 0.595 2.245 0.754 1.947 1.613 1.158 1.840 1.226 1.418 1.893 1.[83 1.265 0.867 0.696 0.583 1.787 1.422 2.279 2.564 1.748 1.469 1.439 1.847 1.987 0.910 0.662 1.514

0.001 0.001 0.01 0.001

0.05

0.01

0.001

0.01 0.05

0.01 0.05 0.05

(continued)

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P.G. M O O R E ET AL.

TABLE 2 (continued)

n

Millport (23•9/89) Kinneil Kerse Skinflats (14/3/89) N. Queensferry (25/5/89) + South Alloa +

Dry weight (g)

15 15 5 15 15

Double log regression

Mean

Range (rain. max.)

0.0171 0.0120 0.0092 0.0099 0.0087

0.0067, 0.0088, 0.0064, 0.0066, 0.0011,

b

0.0249 0.0166 0.0116 0.0142 0.0127

Sig.reg.

-0.249 -0.530 1.715 - 2.270 0.010

Log a

P < 0.05 NS NS NS NS

1.195 0.615 5.025 - 2.983 1.458

+Whole sample. *Sample without spent females.

from different locations, by comparing the elevation of respective regression lines, with the precondition that regression coefficients do not differ significantly. A convenient method of presenting such comparative data is to use each regression equation to estimate the copper concentration of amphipods of standard body weight. Table 3 presents the estimated copper concentrations of talitrids of 10 mg dry weight with associated 95% confidence limits (asymmetrical about the estimate after antilogging of the transformed data) in descending order. (This order is used in Table 2.) Table 3 also provides a summary of the results of comparisons by ANCOVA, carried out on subsets of the samples. The copper concentrations of amphipods from sites sharing a common letter in the ANCOVA column do not differ significantly (P > 0.05). Table 3 illustrates several points. In a comparison of 45 graded samples, copper concentrations in Orchestia gammarellus in samples at the top and TABLE 3

Orchestia gammarellus: estimates of copper concentrations (/~g g ~) with 95% confidence limits (CL) for amphipods of 10rag dry weight, as derived from best-fit double log regressions. Amphipods from locations in the Orkneys (i), Firth of Forth, May 1989 (ii), North Queensferry, 1989 seasonal (iii), Skinflats, 1989 seasonal (iv), Millport, 1989 seasonal (v), mainland Scotland east coast March, 1989 (vi), are compared in subsets. Samples showing any c o m m o n letter in the same A N C O V A column are not significantly different in Cu concentration Site

Cu

CL

ANCOVA

conc.

(i) N. Queensferry (17/3/89) Sandend Sillo Craig Bergen (Norway)

218 188 170 148

208, 167, 130, 139,

227 211 223 159

(ii)

(iii) a

(iv)

(v)

(vi) a b b

227

ORCHESTIA GAMMARELLUS: BIOMONITOR FOR COPPER AND ZINC

TABLE 3 (continued) Site

Cu cone.

CL

ANCOVA (i)

St Andrews Alness Wormit N. Queensferry (26/7/89) N. Kessock Stonehaven West Wemyss Lybster Boddam Skinflats (26/7/89) N. Queensferry (26/9/89) Widewall Bay Loch Fleet Hopeman N. Queensferry (20/11/89) Eyemouth St Peter's Pool Helmsdale N. Queensferry (12/1/90) West Scapa Bay Birsay Millport (24/7/89) Kennetpans N. Queensferry (25/5/89)* Dunimarle Castle Millport (16/3/89) Torryburn Skinflats (25/5/89) South Alloa* Millport (11/1/90) Prestonpans Millport (31/5/89) Inverkeithing Fyn Hoved (Denmark) Barns Ness Millport (24/11/89) Millport (23/9/89) Kinneil Kerse Skinflats (14/3/89) N. Queensferry (25/5/89) + South Alloa ÷

145 132 131 130 128 119 115 114 110 109 103 103 102 95.2 94.9 92.8 90.7 86.1 86.0 85.4 81.2 80.6 78.8 76.1 75.4 70.9 69.1 67.4 62.2 60.8 58.1 58.0 55.7 54.6 52.4 52.0 49.3 47.2 39.4 36.0 27.4

108, 117, 95.9, 120, 97.0, 86.7, 93.4, 99.1, 87.5, 81.4, 90.2, 97.0, 66.5, 77.9, 84.5, 73.1, 80.5, 76.4, 73.9, 73.6, 72.4, 73.0, 70.5, 61.8, 66.8, 46.0, 60.6, 59.8, 52.7, 50.1, 36.0, 42.6, 49.5, 18.5, 39.2, 44.6, 42.7, 47.2, 17.0, 20.5, 14.1,

196 148 179 141 169 162 141 132 138 146 117 110 157 116 107 118 102 97.1 100 99.1 91.1 89.0 88.0 93.8 85.2 110 78.6 75.9 73.4 73.8 93.7 78.9 62.8 161 70.0 60.7 56.9 77.5 91.3 63.2 53.4

~Whole sample. *Sample without spent females.

(ii)

(iii)

(iv)

(v)

(vi) b,c

c,d d d d,e d,e d,e,f e,f

e,g e,h f,g,h a,b g,h d

a a,e

d,e

a,b a,b b e

b c,e

c,d,e e,e

f

228

P.G MOORE ET AL.

bottom of the list are clearly significantly different (P < 0.05), with amphipods from sites close together in the list showing no significant difference in copper concentration. Potentially, there is a large number of site groupings which do not differ significantly, but not all variations of such subgroupings are listed in Table 3 for the sake of clarity. Certain comparisons, for example, have been restricted in the table to subgroupings of locations or seasonal samples from one site. Furthermore, a significant difference in regression coefficients may prevent the use of A N C O V A for a particular relevant comparison. Apparent inconsistencies in the A N C O V A list can be explained. Copper concentrations in amphipods from sites far apart in the list may not differ significantly, although the copper concentration of amphipods from a site intermediate in the list may differ from that in one of the original pair. This situation results from differing degrees of variation at each site, indicated by wide or narrow confidence limits accordingly. It must also be remembered that differences have been accepted as significant at the 5% probability level, and such differences will occur by chance at a rate of 1 in 20. Given the large number of comparisons presented, several apparently significant differences will occur by chance. It is important therefore to be sceptical of apparently significant differences which are atypical. Inspection of copper concentration data for Orchestia gammarellus (Table 3) allows certain conclusions to be drawn. Firstly, the presence of females which have lost mature juveniles from the brood pouch in the sample from South Alloa (25/5/89) significantly lowered the copper concentration of the sample (Fs -- 22.42, df. 1,27; P < 0.001). Thus the loss of these juveniles has removed more than a proportionate amount of body copper from the mother relative to the dry weight lost. In contrast the difference in the case of the North Queensferry sample was not significant (Fs = 3.70; dr. 1,22; NS) - - a result both of the wider confidence limits, but perhaps also of the higher body concentration of copper in the North Queensferry amphipods. Seasonal changes in body copper concentrations were recognized at the three sites intensively sampled. The range of seasonal change was most marked at N. Queensferry, smaller at Skinflats (both in the Firth of Forth) and least at Millport. There was no consistent pattern among the three sites as to which time of year was associated with the highest or lowest body copper concentrations. Amphipods from the four Orkney sites (March 1989) varied in body copper concentrations, those from Widewall Bay having the highest loadings with lowest at West Scapa Bay and Birsay. In the comparison of the Firth of Forth sites (May 1989), there was a gradation in the order Kennetpans, N. Queens-

ORCHESTIA GAMMARELLUS: BIOMONITOR FOR COPPER AND ZINC

229

ferry, Dunimarle Castle, Torryburn, Skinflats, South Alloa, Inverkeithing and Kinneil Kerse. In the wider ranging comparison of east coast mainland Scotland samples (March 1989), the N. Queensferry sample was significantly highest in copper concentration, with Sandend, Sillo Craig and St Andrews also high. Bottom position was held by Skinflats (although May and July samples were significantly higher), preceded in ascending order by Barns Ness, Prestonpans, Helmsdale, Eyemouth, Hopeman and Loch Fleet. Amphipods from Bergen in Norway were relatively high in copper, those from Fyn Hoved in Denmark relatively low.

Zinc Data for zinc have been treated similarly. Table 4 summarises information on double log regressions of the zinc concentration against dry weight for Orchestia gammarellus. There are again significant regressions, confirming the need to use ANCOVA. Table 5 presents the estimated zinc concentration of talitrids of 10 mg dry weight with 95% confidence limits, as well as summaries of comparisons by ANCOVA. As Table 5 shows, there is a wide range of zinc concentrations in Orchestia gammarellus from the sites sampled. The presence in a sample of females which have just lost mature juveniles from the brood pouch has lowered the zinc concentration at South Alloa (May 1989) (Fs = 5.12; df. 1,21; P < 0.05), as it did for copper. In the case of the amphipods collected from N. Queensferry in May 1989, the sample including these females had a higher zinc concentration than that with the females excluded. It was, however, not possible to test the significance of this difference because of a significant difference in relevant regression coefficients. Seasonal changes in amphipod zinc concentrations were less pronounced than for copper. At N. Queensferry, all samples had extremely high zinc concentrations. There were no seasonal differences in zinc concentrations at Skinflats, but significant changes did occur in zinc concentration over this year in O. gammarellus at Millport. In the case of the Orkney (March 1989) amphipods, the zinc concentration of those from West Scapa Bay was significantly lower than that of any of the others. In the Firth of Forth (May 1989), amphipods from N. Queensferry had the highest zinc concentration, followed in descending order by those from South Alloa, Torryburn, Dunimarle Castle, Kennetpans, Inverkeithing, Skinflats and Kinneil Kerse. When considering samples from the east coast of mainland Scotland (March 1989), the highest zinc concentration was found in amphipods from

230

P.G. M O O R E ET AL.

TABLE 4

Orchestia gammarellus (1989): the number and dry weight (g, mean and range) of amphipods analysed for zinc concentrations with details of the relationship log y = log a + b log x, where y is the metal concentration (/~g g-~), x is the dry weight (g) and a and b (the regression coefficient) are constants. Sig.reg. is the significance of the regression; NS not significant (P > 0.05) n

N. Queensferry(26/7/89) N. Queensferry(26/9/89) N. Queensferry(17/3/89) N. Queensferry(25/5/89) ÷ N. Queensferry (20/11/89) N. Queensferry(12/l/90) Eyemouth N. Kessock Hopeman Millport (24/11/89) Bergen (Norway) South Alloa* Torryburn Millport(ll/l/90) Millport(23/9/89) DunimarleCastle St Peter's Pool Sandend LochFleet Kennetpans Inverkeithing Birsay Millport(16/3/89) Wormit Lybster Helmsdale SilloCraig West Wemyss WidewallBay Millport(24/7/89) Skinflats(25/5/89) Skinflats(14/3/89) St Andrews Boddam N. Queensferry (25/5/89)* Skinflats (26/7/89) Prestonpans

15 15 15 15 15 15 15 15 15 14 17 9 15 15 15 15 15 14 15 15 15 15 15 15 15 15 15 14 15 15 15 5 14 15 10 9 14

Dry weight (g)

Double log regression

Mean

Range

(min. max.)

0.0092 0.0134 0.0125 0.0099 0.0131 0.0102 0.0165 0.0176 0.0228 0.0178 0.0153 0.0093 0.0114 0.0183 0.0171 0.0114 0.0137 0.0181 0.0149 0.0110 0.0122 0.0177 0.0158 0.0192 0.0113 0.0141 0.0229 0.0163 0.0109 0.0107 0.0115 0.0092 0.0156 0.0219 0.0094 0.0080 0.0145

0.0059, 0.0097, 0.0064, 0.0066, 0.0046, 0.0054, 0.0094, 0.0076, 0.0084, 0.0104, 0.0036, 0.0076, 0.0085, 0.0106, 0.0067, 0.0067, 0.0061, 0.0079, 0.0112, 0.0084, 0.0066, 0.0078, 0.0098, 0.0121, 0.0064, 0.0072, 0.0069, 0.0134, 0.0057, 0.0084, 0.0070, 0.0064, 0.0097, 0.0083, 0.0066, 0.0055, 0.0071,

0.0123 0.0250 0.0285 0.0142 0.0202 0.0202 0.0245 0.0320 0.0344 0.0380 0.0323 0.0127 0.0146 0.0345 0.0249 0.0148 0.0232 0.0365 0.0214 0.0176 0.0220 0.0274 0.0236 0.0362 0.0265 0.0246 0.0388 0.0232 0.0176 0.0197 0.0153 0.0116 0.0210 0.0427 0.0142 0.0107 0.0377

b -0.053 -0.063 -0.449 -0.325 -0.306 -0.176 -0.565 -0.302 -0.324 -0.445 -0.141 -0.042 -0.074 -0.379 -0.322 0.011 -0.482 -0.277 -0.652 -0.407 -0.354 -0.268 -0.425 -0.256 -0.620 -0.505 -0.290 0.036 -0.165 -0.444 -0.017 0.346 0.043 -0.076 - 1.771 - 1.012 -0.439

Sig.reg. NS NS P<0.05 NS P < 0.01 NS NS NS P<0.05 P < 0.01 P < 0.05 NS NS P<0.01 NS NS P < 0.01 P<0.05 NS NS P<0.05 NS NS NS P<0.01 NS P<0.05 NS NS P<0.05 NS NS NS NS P < 0.05 P < 0.05 NS

Log a 2,427 2.379 1.600 1,759 1,793 2.048 1.262 1,778 1.719 1.465 2.095 2.249 2.085 1.573 1.686 2.342 1.351 1.743 0.984 1.472 1.576 1.746 1.240 1.761 1.031 2.166 1.682 2.333 1.926 1.362 2.208 2.925 2.315 2.068 - 1.324 0.161 1.286

ORCHESTIA GAMMARELLUS:

231

BIOMON[TOR FOR COPPER AND ZINC

TABLE 4 (continued)

n

Fyn Hoved (Denmark) Kinneil Kerse West Scapa Bay Stonehaven Barns Ness Millport (31/5/89) Alness South Alloa+

Dry weight (g)

10 15 15 14 15 15 15 15

Double log regression

Mean

Range

(min. max.)

0.0055 0.0120 0.0136 0.0146 0.0154 0.0104 0.0150 0.0087

0.0042, 0.0088 0.0081. 0.0100 0.0080. 0.0067. 0.0010. 0.0011.

0.0071 0.0166 0.0191 0.0243 0.0262 0.0151 0.0262 0.0127

b - 0.662 -0.101 -0.236 -0.347 -0.075 -0.184 - 0.087 - 0.026

Sig.reg.

Log a

NS NS NS NS NS NS NS NS

0.834 1.949 1.676 1.350 1.951 1.903 2.128 1.893

+Whole sample. *Sample without spent females.

N. Queensferry with high levels also present in samples from Eyemouth, N. Kessock and Hopeman. Lowest zinc concentrations were present in O. gammarellus from Alness, Barns Ness and Stonehaven. As in the case of copper, the Norwegian amphipods from Bergen had relatively high body zinc concentrations, while the Danish amphipods were relatively low in zinc.

TABLE5

Orchestia gammarellus: estimates of zinc concentrations (#g g ~) with 95% confidence limits (CL) for amphipods of 10mg dry weight, as derived from best-fit double log regressions. Amphipods from locations in the Orkneys (i), Firth of Forth, May 1989 (ii), North Queensferry 1989 seasonal (iii), Skinflats, 1989 seasonal (iv), Millport, 1989 seasonal (v), mainland Scotland east coast, March 1989 (vi) are compared in subsets. Samples showing any common letter in the same ANCOVA column are not significantly different in Zn concentration. *This N. Queensferry sample could not be compared against the others because of a significant difference in regression coefficient Site

Zn conc.

CL

ANCOVA

(i) N. N. N. N. N.

Queensferry Queensferry Queensferry Queensferry Queensferry

(26/7/89) (26/9/89) (17/3/89) (25/5/89) + (20/I 1/89)

340 320 314 256 254

308, 256, 263, 228, 235,

376 401 376 287 274

(ii)

(iii)

(iv)

(v)

(vi)

a a a a,c

b b

a,c

(continued)

232

P.G. MOORE ET AL.

TABLE 5 (continued)

Site

Zn conc.

ANCOVA

CL

(i) N. Queensferry (12/1/90) Eyemouth N. Kessock Hopeman Millport (24/11/89) Bergen (Norway) South Alloa* Torryburn Millport (11/1/90) Millport (23/9/89) Dunimarle Castle St Peter's Pool Sandend Loch Fleet Kennetpans Inverkeithing Birsay Millport (16/3/89) Wormit Lybster Helmsdale Sillo Craig West Wemyss Widewall Bay Millport (24/7/89) Skinflats (25/5/89) Skinflats (14/3/89) St Andrews Boddam N. Queensferry (25/5/89)* Skinflats (26/7/89) Prestonpans Fyn Hoved (Denmark) Kinneil Kerse West Scapa Bay Stonehaven Barns Ness Millport (31/5/89) Alness South Alloa + + Whole sample.

252 246 242 233 227 218 215 215 214 213 209 206 198 194 193 192 191 187 187 186 185 183 182 180 178 174 171 168 166 166 153 146 144 142 141 132 126 123 108 87.9

227, 17 l, 194, 184, 194, 197 191, 185, 182, 160, 164, 183, 166, 133, 167, 171, 158, 168, 149, 161, 165, 152, 126, 160, 165, 149, 77.1, 141, 135, 105, 116, 110, 82.8, 127, 121, 106, 85.0 78.2, 98.2, 42.7,

279 356 300 295 265 240 243 249 252 284 265 233 235 285 223 215 232 207 234 216 207 219 264 203 192 204 381 199 205 262 202 192 250 159 164 164 186 193 119 181

* Sample without spent females.

(ii)

(iii)

(iv)

(v)

(vi) b,d b,c,d b,d

b b,d

b,c,d d,e

e,f b,d,e b,d

e,f e,f f,g

f,g f,g C a a

e,f

f,g

*

ORCHESTIA GAMMARELLUS: BIOMON[TOR FOR COPPER AND ZINC

233

Cadmium In all cases body concentrations of cadmium were below detectable limits ( < 0.47 to < 20 Ftg Cd g-1 according to body weight). DISCUSSION

The loss of juveniles from the brood pouch of females from South Alloa seemingly resulted in a reduction of the body copper concentration. It is probable that the copper transferred is for essential purposes, including components of copper-based enzymes and the respiratory pigment haemocyanin (see White and Rainbow, 1985). The higher starting concentration of copper in the N. Queensferry amphipods, as opposed to those from South Alloa, may be a reflection of a larger detoxified store of copper in the copper-contaminated adults, e.g. as copper-rich granules in the midgut caeca (Dr J.M. Weeks, QMWC, personal communication, 1990). This store would be unaffected by transfer of essential metabolically available copper to the offspring, thereby diminishing to a non-significant level any reduction in the total body copper concentration of the mother. A similar argument may explain the same effect in the case of changes in body zinc concentration in the South Alloa sample, but not for that from N. Queensferry. In this case the presence of juveniles in the brood pouch increased the zinc concentration of the sample. As Table 5 indicates the bioavailability of zinc at N. Queensferry is extremely high. If much of this zinc is in dissolved form, it is possible that the exoskeletons of the small juveniles in the brood pouch represent a vast surface area (relative to volume) to absorb atypically high zinc loadings. These juveniles would then have had atypically high body zinc concentrations in comparison with their mothers. Rainbow and Moore (1990) have reported on seasonal changes in body copper and zinc concentrations of Orchestia gammarellus at Millport through 1987. Copper showed some variation, body concentrations being raised in March and lowered in November, but body zinc concentrations did not vary significantly. In this study higher copper concentrations were found in samples collected at Millport in July and March 1989, with lower body loadings in January, May, September and November. It is difficult to recognize any consistent pattern here. In contrast to the situation in 1987 (Rainbow and Moore, 1990), zinc concentrations in O. gammarellus in this study showed some apparent seasonal variation; the November 1989 peak value being (just) significantly different from that for May 1989, but the difference may not be real. The significant seasonal changes in both copper and zinc concentrations in O. gammarellus from N. Queensferry may well reflect real temporal changes

234

P.G. M O O R E ET AL.

in trace metal loadings of local metal-rich inputs, given the high bioavailabilities of each metal there. A similar effect was recognized by Rainbow et al. (1989) for copper concentrations in amphipods collected near whisky distillery outfalls on Islay. Seasonal changes in body copper concentrations in amphipods from Skinflats may be similarly explicable. Body zinc concentrations of O. gammarellus from Skinflats did not change seasonally. Rainbow et al. (1989) provide directly comparable data from which to assess the national relevance of both copper and zinc concentrations found in the talitrids analysed here. Overall, the east coast samples are somewhat enriched in both copper and zinc compared with the west coast of Scotland (cf. Davies and Pirie, 1980). In the case of copper, several of the concentrations reported here are in excess of the top equivalent concentration reported by Rainbow et al. (1989): viz. their 139#g Cu g-1 estimate for a 10mg dry weight amphipod from Restronguet Creek, Cornwall, a notorious copper and zinc hotspot. Dr J.M. Weeks (personal communication, 1990), however, has recorded even higher concentrations of copper (estimate 340/~g g-~, 95% CL, 548, 210 for 10 mg standard dry wt animal) in samples collected in September 1989 from the vicinity of Devoran (Restronguet Creek). This is the highest value for copper in O. gammarellus collected from the field known to our group, and Weeks has found copper-rich granules in the ventral caeca of these amphipods. Thus the equivalent N. Queensferry March sample at 218 ~g gis copper-rich by any standard, and equivalent copper concentrations in amphipods from Sandend (188/tg g-~), Sillo Craig (170~g g I) and St Andrews (145/~g g ~) all exceeded our own Restronguet Creek figure. The copper concentration of the sample from Bergen in Norway (148/~g g-~) is similarly indicative of high copper bioavailability (for reasons which are presently obscure, but which might relate to boating sources). Balls (1985) found an inverse correlation between salinity and dissolved copper concentration in North Sea coastal waters, suggesting a freshwater origin for this element emanating from sedimentary and/or anthropogenic inputs. Balls and Topping (1987, see also Davies, 1987) report that, although dissolved copper concentrations in the outer Firth of Forth are very similar to those in coastal waters outside the Firth ( ~ 0.20/~g dm 3), dissolved copper concentrations in the inner part of the Firth show considerable variation ( < 0.2-0.7/tg dm 3). This variability is especially evident in the Edinburgh to Cockenzie area where the major industrial and sewage-associated inputs are located (Balls and Topping, 1987). Similarly, copper concentrations in the sediments in the same area were higher and more variable than elsewhere in the Firth and beyond (Balls and Topping, 1987). These authors estimated that river flow (both freshwater from sewage works and outfalls, and industrial freshwater) from the south shore of the Firth provide 75 000 kg Cu year- ~ to

ORCHESTIA GAMMARELLUS: BIOMONITOR FOR COPPER AND ZINC

235

the Firth of Forth, as opposed to freshwater sources further west which provide 35 000 kg Cu year -~. Thus there is considerable anthropogenic input of copper into the Firth of Forth. Davies and Pirie (1980) found the highest copper levels in Scottish mussels (Mytilus edulis) from the upper reaches of the Firth of Forth. The high, but variable, bioavailability at North Queensferry cannot be attributed to any industrial input in the immediate vicinity and seems most likely to be caused by chronic build-up derived from the local boatyard, e.g. from antifouling paint removal and application activities, with residues building-up in the small embayment at the sampled site. The Rosyth naval dockyard is close to this site too and it should be recalled that Rainbow et al. (1989) also found high levels of copper in the Clyde at sites (Loch Long, Holy Loch) in the proximity of naval bases. (Whether the scuttled naval ships in Scapa Flow contribute in any way to the variation in copper in the Orkney samples is unknown.) It is pertinent in this context that Bailey and Davies (1987) reported the highest incidences of imposex in dogwhelks (Nucella lapillus) - - brought about by exposure to tributyltin from antifouling paints in fishing harbours, marinas (including N. Queensferry) and boatyards in the Firth of Forth. The high copper bioavailability at other mainland sites may relate in some degree to local copper-rich mineralogy since copper ore has been mined in The Mearns inland from Montrose (Wilson, 1921; Atkinson, 1987), although there is also some industrial input of copper to the Tay estuary (R. Alcock, Tay RPB, personal communication, 1990). It is noteworthy that Davies and Pirie (1980) also reported elevated copper levels in intertidal mussels from this area. Interestingly, the elevated level of copper at Alness (132 #g g-~) was for a site adjacent to the outfall from a whisky distillery (see Rainbow et al., 1989). The Skinflats samples had surprisingly low levels of copper and zinc, coming as they did from a site adjacent to the major I.C.I. industrial complex at Grangemouth. This site, however, was a most atypical beach-hopper habitat with scarce (possibly additionally stressed) O. gammarellus living under minimal marine flotsam and burrowing in soil mostly beneath discarded rubbish on a grassy sward adjacent to extensive mudflats. Although these mudflats contain high levels of heavy metals (Davies, 1987), nothing has been published on levels of contamination in fringing terrestrial vegetation which perforce will be a major food of these O. gammarellus (see Moore and Francis, 1985). It should be recalled, also, that Taylor and Spicer (1986) found the ionic composition of the haemolymph of O. gammarellus taken from high on the shore (among grass above MHWS) to be very different (reduced) from that from animals in the middle of their distribution. Unfortunately they did not measure copper and zinc. -

-

236

P.G. M O O R E ET AL,

In the case of zinc, the estimated concentration of a 10mg dry weight Orchestia gammarellus from Restronguet Creek was 392 ~g g-~ (Rainbow et al., 1989), the next highest value recorded in this species being 246 ~g g-t in amphipods from Holy Loch. All six samples from N. Queensferry in this study have equivalent zinc concentrations which fall between these figures, indicating remarkably high year-round zinc bioavailabilities at this site. Equivalent zinc concentrations at Eyemouth (246 #g g-~ ), N. Kessock (242/~g g ~) and Hopeman (233/~g g-~) are all similarly indicative of high zinc availability on a national scale, as is that from the Bergen sample (218 #g g- ~). The Eyemouth sample was taken within the harbour, which also houses a boat-builder's yard, again with resultant potential for accumulation of metals from antifouling painting activities. The Hopeman site is also a harbour. (Copper levels for these two sites were not excessive however.) Elevated levels at Bergen may be similarly attributable inter alia to antifouling leachings from heavy commercial and pleasure boat activity. Harbours and t~ords are likely to be poorly flushed and hence represent conservative 'sinks' for metal contaminants. Levels of zinc in Scottish mussels reported by Davies and Pirie (1980) were generally comparable nationwide, with the exception of elevated levels in the Firth of Forth. It is plausible "then to conclude that these elevated zinc bioavailabilities are anthropogenic in origin, particularly - - from the present surveys - - at N. Queensferry. The Forth River Purification Board (FRPB) routinely monitors samples of water, sediment, mussels and seaweed (Fucus vesiculosus) along the length of the Firth of Forth. It is interesting that the particular N. Queensferry hotspot identified here using Orchestia gammarellus as a biomonitor had not been detected previously by these other means. Although mussels are known to have deficiencies as biomonitors of copper and zinc (see references in Rainbow et al., 1989) it should be conceded that high concentrations of these metals were also reported in mussels from the Inner Forth by Davies and Pirie (1980). Mitton (1984) though discovered that hotspots in the copper concentrations of Firth of Forth F. vesiculosus were not necessarily indicated by local M. edulis. Since supralittoral talitrids feed upon decaying macro-algae from which they certainly assimilate zinc with a high degree of efficiency (Weeks and Rainbow, 1990), it seems likely that talitrids would magnify any pattern of metal enrichment in local seaweeds. The nearest FRPB routine sampling site for F. vesiculosus is outside the bay sampled here and did not show particularly enhanced levels (Dr A. Griffiths, FRPB, personal communication, 1990), suggesting that contamination at the O. gammarellus site is very localized. Even so, as shown here, it is a feature which is consistent year-round, suggestive of a chronic, contained source. In fact the major drift alga at the

ORCHESTIA GAMMARELLUS: BIOMON|TOR FOR COPPER AND ZINC

237

N. Queensferry site was Ascophyllum nodosum (reflecting an extensive attached bed downshore, indicative of wave-sheltered conditions). Elsewhere, this long-lived alga has been shown (Haug et al., 1974) to accumulate copper and zinc and this is the most likely route through which the amphipods acquired their high levels at N. Queensferry, the low-energy sheltered muddy embayment there acting as a pollutant 'sink'. Mitton (1984) found an analogous localized accumulation of copper in deposited sediments from the sheltered eastern corner of the nearby Dalgety Bay. Once again then (see also Rainbow et al., 1989) these findings highlight the utility of Orchestia gammarellus as a sensitive trace metal biomonitor for these metals. Having now a broad spectrum of established algal, molluscan and crustacean sentinel species available to researchers should expedite trend monitoring of trace metals in coastal ecosystems, given the known deficiencies of water sampling (highlighted most recently by Balls, 1989). Samples were taken from the Orkney Islands (inter alia) because cadmium had been reported to be a local problem there in edible crabs (Cancerpagurus) (Topping, 1973; Davies et al., 1981; Davies, 1985). No detectable cadmium was found, however, in O. gammarellus anywhere in the east coast survey, even at Skinflats near where cadmium used to be discharged subtidally by I.C.I. Grangemouth (Davies, 1987). Cadmium is now a controlled substance and the status of the Forth estuary with respect to this metal is now improving (Dr A. Griffiths, personal communication, 1990). Balls (1985) identified the Humber estuary as the significant east coast source of cadmium to the North Sea, but residual currents would carry such contamination south rather than north (Hill, 1973). ACKNOWLEDGEMENTS Brian Smith provided us with invaluable assistance. Valuable comments from Dr D. McLusky (Stirling University), Dr A. Griffiths and Dr M. Elliott (Forth River Purification Board) aided in the planning or interpretation stages of the project. The extensive hospitality and co-operation of the Orkney Marine Biology Unit (Mr J.A. Simpson and Miss Inga Seatter) during field work in Orkney was greatly appreciated. Financial support from the Institute of Petroleum is gratefully acknowledged. REFERENCES Atkinson, R.L., 1987. Copper and Copper Mining. Shire Publications Ltd, Aylesbury, pp. 132. Bailey, S.K. and I.M. Davies, 1987. Tributyltin contamination in the Firth of Forth. Proc. R. Soc. Edinburgh, 93B: 561-562.

238

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Balls, P.W., 1985. Copper, lead and cadmium in coastal waters of the western North Sea. Mar. Chem., 15: 363-378. Balls, P.W., 1989. Trend monitoring of dissolved trace metals in coastal sea water - - a waste of effort. Mar. Pollut. Bull., 20: 546-548. Balls, P.W. and G. Topping, 1987. The influence of inputs to the Firth of Forth on concentrations of trace metals in coastal waters. Environ. Pollut., 45: 159-172. Davies, I.M., 1985. Marine pollution in Orkney. Proc. R. Soc. Edinburgh, 87B: 105-112. Davies, I.M., 1987. Trace metals and organohalogen compounds in the Forth, Scotland. Proc. R. Soc. Edinburgh, 93B: 315-326. Davies, I.M. and J.M. Pirie, 1980. Evaluation of a "mussel watch" project for heavy metals in Scottish coastal waters. Mar. Biol., 57: 87-93. Davies, I.M., G. Topping, W.C. Graham, C.R. Falconer, A.D. McIntosh and D. Saward, 1981. Field and experimental studies on cadmium in the edible crab Cancer pagurus, Mar. Biol., 64:291-297. Haug, A., S. Melsom and S. Omang, 1974. Estimation of heavy metal pollution in two Norwegian fjord areas by analysis of the brown alga Ascophyllum nodosum. Environ. Pollut., 7: 179-192. Hill, H.W., 1973. Currents and water masses, ln: E.D. Goldberg (Ed.), North Sea Science. MIT Press, Cambridge, Mass., pp. 17-42. Mitton, P.A., 1984. An environmental assessment of copper pollution in a marine bay. Unpublished M.Sc. Project Report, Napier College, Edinburgh, pp. 1-124. Moore, P.G. and C.H. Francis, 1985. Some observations on food and feeding of the supralittoral beach-hopper Orchestia gammarellus (Pallas) (Crustacea: Amphipoda). Ophelia, 24: 183-197. Moore, P.G. and P.S. Rainbow, 1987. Copper and zinc in an ecological series of talitroidean Amphipoda (Crustacea). Oecologia, 73: 120-126. Rainbow, P.S. and P.G. Moore, 1986. Comparative metal analyses in amphipod crustaceans. Hydrobiologia, 141: 273-289. Rainbow, P.S. and P.G. Moore, 1990. Seasonal variation in copper and zinc concentrations in three talitrid amphipods. Hydrobiologia, 196: 65-72. Rainbow, P.S., P.G. Moore and D. Watson, 1989. Talitrid amphipods as biomonitors for copper and zinc. Estuarine Coastal Shelf Sci., 28: 567-582. Taylor, A.C. and J.I. Spicer, 1986. Oxygen-transporting properties of the blood of two semi-terrestrial amphipods, Orchestia gammarellus (Pallas) and O. mediterranea (Costa), J. Exp. Mar. Biol. Ecol., 97: 135-150. Topping, G., 1973. Heavy metals in shellfish from Scottish waters. Aquaculture, 1: 379-384. Weeks, J.M. and P.S. Rainbow, 1990. A dual-labelling technique to measure the relative assimilation efficiencies of invertebrates taking up trace metals from food. Functional Ecol., 4:711-717. White, S i . and P.S. Rainbow, 1985. On the metabolic requirements for copper and zinc in molluscs and crustaceans. Mar. Environ. Res., 16: 215-229. Wilson, G.V., 1921. Special reports on the mineral resources of Great Britain. The lead, zinc, copper and nickel ores of Scotland. Mem. Geol. Surv. Scotland, 17: 1-159.