Jones, 1981) and crabs (Sloan et al., 1974) is rapid (B1/2 = a few weeks), and .... half-life of inorganic mercury in the dungeness crab (Cancer magister). J. Fish.
Marine Biology 86, 55-62 (1985)
Marine ooo,.,o- B i o l o g y ............... 9 Springer-Verlag1985
Accumulation, elimination and chemical speciation of mercury in the bivalves Mytilus edulis and Macoma baithica H. U. Riisg/u'd ~, T. Kiorboe 2, F. Mohlenberg 3, I. Drab~ek 4, and P. Pheiffer Madsen 4 1 Institute of Biology, Odense University; Campusvej 55, DK-5230 Odeuse M, Denmark 2 Danish Institute for Fisheries and Marine Research; Charlottenlund Castle, DK-2920 Charlottenlund, Denmark 3 Marine Pollution Laboratory, National Agency of Environmental Protection; Kavalergfirden 6, DK-2920 Charlottenlund, Denmark 4 Danish Isotope Centre; Skelb~ekgade 2, DK-1717 Copenhagen V, Denmark
Abstract Mussels ( M y i l u s edulis) transferred in net bags from clean to chronically mercury polluted water readily accumulated mercury during an exposure period of three months. Growth of the transplanted mussels had a "diluting" effect on the mercury concentration, but the absolute weight of mercury uptake increased throughout the entire period, though there was a tendency for decreased efficiency of the removal of mercury per liter of water filtered by the mussels. Mussels were also translocated from polluted to clean (laboratory) water to depurate mercury. The biological half-lives of mercury was 293 d for M. edulis from the chronically polluted area in contrast to only 53 d for mussels from a temporary massive mercury polluted area near a chemical deposit. In both cases about 75% of the total mercury in the mussels was inorganic, and it is suggested that both inorganic and organic mercury species were immobilized in mussels from the long-term mercury polluted area, whereas the immobilization capacity was exceeded in the short-term mercury exposed mussels near the chemical deposit. Very slow elimination of mercury was observed in the deposit-feeding bivalve Macoma balthica from the chronically polluted area, and about 6% of the total mercury was methyl-+ phenyl-mercury. This is more than three times lower than found in M. edulis from the same collecting site. A pronounced difference in the mercury speciation (i,e., total mercury, total organic mercury, methyl-mercury and phenyl-mercury) in M. edulis from the two mercury polluted areas is thought to reflect the different character of the mercury pollution in the two areas.
Introduction During the last 10 to 15 years bivalve molluscs have been commonly used to monitor mercury in the marine environment. In spite of this development very little information
exists about the chemical forms of mercury in bivalves from chronically mercury polluted waters, and knowledge about the dynamics of mercury in bivalves has mostly been obtained from laboratory studies. Only very few field studies (cf. Davies and Pirie, 1978; Eganhouse and Young, 1978; Okazaki and Panietz, 1981) have been designed to measure the mercury kinetics in bivalves; though several laboratory studies have shown that, for example, short exposure times in mercury contaminated water can result in relatively short biological half-lives (cf. Miettinen et al., 1970; {)nlti etal., 1970), this may not be representative of the elimination rates in bivalves from chronically polluted waters (cf. Fowler et al., 1978). The aim of the present work has been to acquire information on the chemical forms of mercury in relation to the accumulation and elimination patterns of mercury in bivalves collected in or transplanted to two mercury "hot spot" areas: (1) near an abandoned chemical factory ("Cheminova") on the west coast of the Limfjord, and (2) near a chemical deposit in the dunes on the Danish west coast (see Fig. 1). The "Cheminova" factory produced mercury-containing fungicides from 1956-1962. Mercury was discharged by process waste water and was accidentally released into the Nissum Broad at least on two occasions (Kiorboe et al., 1983). Further, several tons of phenyl-mercuri-acetate and ethyl-mercuri-chloride and waste products from the production of these mercury compounds were placed, together with other chemicals, in a deposit near groyne No. 42 on the west side of the Harboore Tange. Due to leaching of mercury and other chemicals via ground water to the North Sea, most of the chemical deposit was removed during the summer of 1981. This lead, however, to a temporary increased leakage of mercury, which resulted in marked concentration increase of this element in the resident mussel population (RiisgMd, 1984). The two "hot spot" areas were considered especially suitable for basic studies of mercury dynamics in the marine environment due to the different character of the
H.U. Riisg~rd et aL: Hg accumulation, elimination and speciation in bivalves
56
A\
\
\
\t
B __
r
\
\/
\
\
,,,,+,
\ .]'++"/7
q
"
2++-'/"'~
'
-
.
+`..
. {IOKM I
x
-
Struer~
Fig. 1. Map showing the sampling localities for bivalves in the Nissum Broad near the old "Cheminova" factory and on groyne No. 42 near the chemical deposit on the west coast of Harboore Tange. The positions for accumulation experiments with transplanted mussels (A and B) are indicated on Fig. 1C mercury pollution in the two areas. The present paper emphasizes the need for such field studies.
Materials and methods
Collection of Mytilus edulis for mercury analyses Mussels were collected from groyne No. 42 on the west side of the Harboore Tange in February 1982 and in the Nissum Broad near the old "Cheminova" factory in December 1983 (see Fig. 1). In the laboratory the mussels were placed in aquaria with constantly flowing sea water for 24 h so that they could clear their guts. The mussels were then frozen until the analyses for total mercury and organic mercury species could be made.
suspended at two localities (see Fig. 1, Sites A and B), at a depth of 4 to 6 m about 200 m offshore from the former "Cheminova" factory+. On Day 0 and at intervals during the following 66 d, net bags were collected three times by SCUBA divers, and samples of about 50 mussels from each locality were taken for mercury analysis. The mean temperature of the water was about 15 ~
Calculation of filtration rates For calculating the filtration rate (F, 1 h -i) from the dry flesh weight (W, g) of transplanted Mytilus edulis, the following formula was used: F=7.45 W T M (Mohlenberg and Riisggtrd, 1979). Wet weights of soft parts were converted to dry weights by dividing the wet weights by a factor of 5.7.
Mercury accumulation in transplanted mussels Mercury elimination experiments In July 1981, Mytilus edulis were collected in the Logstor Broad in the central part of the Limfjord. The dominant size class (22.6 +-1.2 mm) was selected out and placed in commercially manufactured, cylinder-shaped net bags made of polypropylene fibers with a mesh size of 0.5 to 1.0 cm (Bohle, 1974; RiisgMd and Poulsen, 1981). The net bags, each holding about 100 mussels, were placed in an aquarium for 3 d with constantly flowing sea water to allow the mussels to attach bysally. The net bags were then
In June 1981, Mytilus edulis were collected near the abandoned "Cheminova" factory. In the laboratory 120 individuals (34.7 • 1.1 mm) were placed in an aquarium with aerated, constant-flow sea water. On Day 0 and after 9, 15, 30, 33 and 37 d in clean water, 20 mussels were removed for mercury analysis. In February 1982, mussels were collected from the littoral zone on groyne No. 42 at the Harboore Tange. In
H. U. Riisggtrd et al.: Hg accumulation, elimination and speciation in bivalves the laboratory the mussels were placed in an aquarium containing continuous-flow, aerated sea water. On Day 0 and after 10, 17, 26, 32 and 47 d in clean water, 20 mussels were removed for mercury analysis. Loss of mercury from the deposit-feeding bivalve Macoma balthica was measured on two occasions: (1) In August 1981 about 80 individuals were collected in the Nissum Broad close to the old "Cheminova" factory. In the laboratory the bivalves were allowed to bury themselves in clean sediments in aquaria with aerated, continuous-flow sea water. On Day 0 and after 13, 20, 34, 63 and l18d in clean water, samples (6-16 specimens) were removed, and deep frozen until required for analyses. (2) In July 1982, 200 bivalves were again collected near the old "Cheminova" factory site. The experiment was set up as above. On Day 0 and during the next 244 d, ten clams were removed 17 times for analyses for total mercury and organic mercury species. Weight loss or growth in experimental clams were minimized through moderate feeding with benthic diatoms grown by illuminating the aquaria. In all elimination experiments temperature (14~ ~ and salinity (27-32%0 S) were almost identical to the conditions at the collection sites, when they were sampled. Mercury in leaking water In February 1982 a sample of contaminated leaking ground water was taken from a hole dug in the sandy beach between the chemical deposit near groyne No. 42 and the North Sea. After sedimentation of sand and other particulates, the water was kept frozen until analyses of total mercury and total organic mercury could be made.
57
and displace mercury bound to sulphur. The organic mercury bromide complexes were then extracted with toluene, from which the mercury complexes were extracted with a cystein acetate solution. This solution was again extracted with toluene. The resultant toluene phase was analyzed for its mercury content (i.e. organic mercury of the original sample) by neutron activation analysis. Two standards and one blank sample were run together with each analysis series. The detection limit for a sample of 2 g was 0.1 ng g-~.
Methyl-mercury plus phenyl-mercury The sum of methyl-mercury and phenyl-mercury was determined using the exchange reaction between their chlorides and the ratioactive 131I- (Star~ and Pr~ilovfi, 1976a, b; Star~ etaL, 1978; Drab~ek and Carlsen, 1984). Copper sulphate was added to the homogenized mussels and methyl-Hg and phenyl-Hg were extracted several times, ending up in toluene. To the toluene phase 131I- in an aqueous ascorbic acid solution was added, causing an exchange between the chloride ofmethyl-Hg-C1 and phenyl-Hg-C1 in the toluene phase and 131I- in the water phase. The exchange reaction has been shown to be selective, fast, and quantitative as long as [C1-]/[I-] N 600; after only one minute the measured activity in the toluene phase is proportional to the concentration of the sum of methyl-Hg and phenyl-Hg. The concentration was determined by comparison with standards treated similarly to the samples. The detection limit for a sample of 2 g was approximately 5 ng g-1.
Phenyl-mercury Chemical analyses
Total mercury The thawed bivalves were opened by cutting the posterior adductor muscle and placed on their edge for 5 rain to allow the water to drain off. The soft parts were then removed with a Teflon spatula, homogenized in a Teflon blender, and analyzed for mercury by neutron activation analysis (Jensen and Carlsen, 1978) or by flameless atomic absorption spectrophotometry (Perkin Elmer Model MHS20) according to AOAC Methods (1980) and Perkin Elmer (1981). The detection limits were 0.1 and 1 ngg -x wet weight for neutron activation analysis and atomic absorption spectrophotometry, respectively. The accuracy in both cases was better than 10%.
Phenyl-mercury was determined using the isotope exchange reaction between the mercury of phenyl-mercury chloride ng
Hg,g -1
500-
~ A 3oo.
100
Total organic mercury The principle of the method is described in Drab~ek and Carlsen (1984). The homogenized samples were treated with a bromide solution, acidified with sulphuric acid, and copper sulphate was added in order to mask any free sulphydryl groups
I
10
I
I
30
I
I
50
I
i
70 Days
Fig. 2. Mytilus edulis. Accumulation of mercury in two groups of mussels (A and B) transplanted in net bags to the Nissum Broad near the old "Cheminova" factory
H.U. Riisg~rd et al.: Hg accumulation, elimination and speciation in bivalves
58
and radioactive 2~ (Drabaek and Carlsen, 1984). The reaction was carried out on homogenized mussels after addition of copper-sulphate and hydrochloric acid. After exchange phenylJ~ was isolated by extraction with toluene, and the radioactivity in the toluene phase was measured. The concentration was determined by
comparison with similarly treated standards. The exchange reaction has been shown to be very selective also against methyl-mercury (cf. StarS~ and Prfi~ilov~i, 1976). The detection limit for a sample of 2 g was 0.1 ng g-1.
Results ng Hg .ind. "1 900 t
7OC
500
300
100
I
I~
I
10
I
I
30
[
50
t
I
70
Days
Fig. 3. Mytilus edulis. Accumulation of mercury in two groups of mussels (A and B) transplanted in net bags to the Nissum Broad near the old "Cheminova" factory
Mussels (Mytilus edulis) transferred from clean water to the polluted Nissum Broad near the former "Cheminova" factory readily accumulated mercury. The mercury concentration increased rapidly at the beginning of the exposure period, but leveled off with time (see Fig. 2). However, the transplanted mussels grew during the exposure period and increased their body mass by more than seven times, and the production of new tissues thus had a "diluting" effect on the mercury concentration in the soft parts of the mussels. Hence, the absolute weight of mercury accumulated did not level off during the latter part of the exposure period (see Fig. 3). Mean daily mercury accumulation rates, calculated filtration rates, and amount of accumulated mercury per liter of water filtered are shown in Table 1. The mean amount of mercury accumulated per liter of sea water filtered by the mussels was 0.21+0.05 ng and 0.20+0.03 ng in Groups A and B, respectively. There is, however, a tendency for decreased efficiency of the removal of mercury with increasing body mass, which is most pronounced in the Group A. In these mussels the efficiency fell from 0.27 ng Hg accumulated per liter filtered (Days 0-17) to 0.17 ng Hg per liter (Days 30-66). The deputation of mercury, expressed as a percentage of the total mercury content on Day 0, is shown as a function of time in a semi-logarithmic plot on Fig. 4. The biological half-life, assuming an exponential decrease of mercury content, was 293 d for Mytilus edulis from the old factory site and 53 d for M. edulis from groyne No. 42.
Hg retention % 100-
80
~
o
--..~_ ~ ~ . . ~ 9
60
o
~~293
_ __
old factory: y = l O l x e " 2 . 3 7 x l O ' 3 x t days
gr, No.42:y~101 x e -1"32 x 10"2x t B 1/2--53 days
9
40
s'o Days
Fig. 4. Mytilus edulis. Elimination of mercury in mussels collected near the old "Cheminova" factory and on groyne No. 42. On Day 0 the mussels were transferred to clean water. The regression fines are shown together with the equations for the fines and the biological half-lives (Bw). Mean wet weight of soft parts during the deputation period was 0.98+0.09 g and 0.157+__0.028 g for mussels from the old factory and groyne No. 42, respectively. The mercury concentration (ng Hg g-1 wet weight of soft parts) on Day 0 was 1340 ng Fig g-J for mussels from groyne No. 42 and 2518 ng Hg g-1 for mussels from the old factory collecting site
H, U, Riisghrd et al.: Hg accumulation, elimination and speciation in bivalves
59
Hg retention% 1000 ~ 0 9
Au~ O~
9
9
0
0
9
5040-
9
! Jul 1982
30205~)
i
100
i
150
200
250 "~ Days
Fig. 5. Macoma bahhica. Efimination of mercury in bivalves collected near the old "Cheminova" factory in August 1981 (9 and in July 1982 (e). On Day 0 the clams were transferred to clean water. The regression lines are shown. Mean wet weight of soft parts during the depuration period was 0.34___0.03 g and 0.44 + 0.04 g for clams collected in August 1981 and July 1982, respectively. The mercury concentration was 1530 ng Hg g-1 wet weight of soft parts on Day 0 for clams collected in August 1981. Mercury concentrations in clams collected in July 1982 are shown in Table 2 Table 1. Mytilus edulis. Accumulation of mercury in relation to exposure time and filtration rate in two groups (A and B) of mussels hung up in net begs in the Nissum Broad near the former "Cheminova" factory Group Exposure time
Total Hg Mean wet accumulated weight of per mussel soft parts
(d)
(ng)
(g)
Mean daily Mean weight daily Hg specific Hg uptake per uptake mussel (ng g - ' ) (ng)
Total volume of water filtered per mussel (1)
Hg accumul. per liter of water filtered (ng)
A
0 - 17 17-30 30 - 66
164.2 131.5 479.6
0.48 0.90 1.67
20.2 11.2 8.0
9.7 10.1 13.3
600 668 2,792
0.27 0.20 0.17
B
0 - 17 17-30 30 - 66
129.4 116.9 563.3
0.45 0.83 1.61
16.9 10.8 9.8
7.6 9.0 15.7
571 644 2,717
0.23 0.18 0.21
Table2. Macoma balthica. Total mercury and methyl-+phenyl-mercury ( m e - H g + p h e - H g ) in mussels collected in the Nissum Broad near the old " C h e m i n o v a " factory in July 1982. On Day 0 the bivalves were placed in aquaria with clean sediments in continuous-flowing sea water. N u m b e r of clams in each sample was 10. Dashes indicate no data Days in clean water
Wet weight of soft parts (g)
Total Hg conc. (ng g-1 wet wt)
Me-Hg + phe-Hg* conc. (ng g-1 wet wt)
Me-Hg + phe-Hg as % of total Hg
0 1 3 6 11 15 20 27 39 48 64 85 106 127 148 190 244
0.49 0.50 051 0.47 0.44 0.37 0.47 0.40 0.43 0.43 0.39 0,37 0.43 0.43 0.43 0.44 0,39
1 398 1 376 1 225 1 109 1 413 1 265 1 164 1 066 1 222 1 195 1 175 1 516
91 46 112 < 90 < 90 84 113 87 < 90 63 58 54 < 90
