The effect of selenium on the handling of mercury in the shore crab Carcinus maenas

Marine Pollution Bulletin, Vok 31, Nos 1-3, pp. 78-83, 1995

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Pergamon

0025-326X(95)00028-3

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0025-326X/95 $9.50+ 0.00

The Effect of Selenium on the Handling of Mercury in the Shore Crab Carcinus m a e n a s LISE F O G LARSEN and P O U L BJERREGAARD*

Ecotoxicology Group, Institute of Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark * A u t h o r to w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d .

By means of the radiotracer 2°3Hg the effects of selenite on accumulation, elimination and tissue distribution of low levels of organic and inorganic mercury in the shore crab Carcinus maenas were investigated. Selenite in food or seawater seemed to prompt C. m a e n a s to eat more food contaminated with organic mercury, but did not consistently alter the assimilation efficiency of organic mercury. However, assimilation of organic mercury was higher from homogenized mussel in gelatinous cubes with added organic mercury, than from mussels exposed to organic mercury in seawater. Elimination of injected inorganic mercury from the haemolymph was initially speeded up by selenite in seawater, whereas elimination of injected organic mercury from the haemolymph seemed to be slowed down initially. Selenite in seawater was found to promote transiocation of injected organic mercury to the muscles. Selenite given in the food, but not selenite in the seawater, had the same effect on ingested organic mercury.

Selenium is known to interact with the accumulation and toxicity of mercury in organisms (for reviews see Magos & Webb, 1980; Pelletier, 1985). Recent studies (Bjerregaard & Christensen, 1993) have shown that exposure to waterborne selenite affects the retention and distribution between organs of dietary organic mercury in the shore crab Carcinus maenas, whereas handling of inorganic mercury is less affected. These studies indicated that crabs may handle mercury accumulated in their food items in situ differently from mercury added in the laboratory to a homogenized food source. The present studies were initiated to investigate mercury assimilation efficiencies from different types of food items, mercury kinetics and internal distribution in C. maenas, and the effects of dietary selenium as opposed to waterborne selenium on mercury handling. M a t e r i a l s and M e t h o d s

Experimental animals Mussels Mytilus edulis were collected from Kerteminde Fjord, Funen, Denmark. Shore crabs (C. maenas) 78

were caught in seine nets in Heln~es Bugt, Funen, Denmark. Neither location is contaminated with mercury or selenium. Crabs used in Experiment 1 (March 1993) were caught in October and kept in flowing seawater aquaria at the Marine Biological Station, Bogebjerg, NE Funen. In Experiments 2 and 3 (August and December-January 1993) freshly caught crabs were used.

Preparation of food Soft parts of M. edulis were homogenized in a food processor and mercury and/or selenium solution was added to the homogenate. Then, 13.6 g of commercial gelatine was melted (37°C), and added to 250 ml of mussel homogenate. The mixture was cooled in a grid, yielding solid cubes of approximately 1.7 g. Organic mercury was added as CH3HgC1 in 0.005 M Na2CO 3 solution, inorganic mercury as HgC12 in 1 M HCI solution and selenium as SeO2 in distilled water, SeO~-. For Experiment 2, 100 M. edulis were exposed to 6 × 1012 cpm organic mercury in 25 1 of seawater for 8 h, frozen prior to storage, dissected and partly thawed before they were fed to the crabs. For Experiment 3, 60 mussels were exposed to 8 × 105 cpm organic mercury in 10 1 of seawater for 2.5 h, but not frozen before they were dissected and fed to the crabs.

Chemicals and chemical analyses Inorganic mercury, 2°3HGC12, was obtained from Amersham, England. Methyl mercury, CH~°3HgCI, was prepared from this according to Toribara (1985), with the exceptions that toluene was used instead of benzene, and that the stirring was done either by an Ika-VibraxVXR (Janke & Kunkel, IKA-WEK, Germany) shaking machine or by shaking manually in separating funnels. SeO 2 was obtained from Fluka, Buchs, Switzerland (no. 84900) and added as selenite, SeO~-. Whole body 2°3Hg contents were determined with a Bicron well-type NaI(T1) crystal with a diameter and depth of 7.6 cm. Samples of faeces and tissues were counted for radioactivity on a 1480 Wizard TM 3" automatic gamma counter. Where comparison of data from the two counters was necessary adjustment for counting efficiencies was made. All results were corrected for radioactive decay, but not for self absorption.

Volume 3 l/Numbers 1-3

Exposure procedures The crabs were kept in groups of five in 10 1 polystyrene aquaria (Experiment 1) and individually in 2.5 1 polystyrene aquaria, with the bottom of the aquaria covered with a polystyrene net under which faecal pellets could be collected (Experiments 2 and 3). The water was aerated and no sediment was placed in the aquaria. The crabs were allowed to eat for ca 0.5 h in separate aquaria with 0.5 1 of seawater (Experiments 2 and 3). Salinity and temperature during acclimation and experiments were 20 _+1%o and 14-16°C. Experiment 1 To investigate the effect of selenite on the handling of injected organic and inorganic mercury, two groups of 10 male shore crabs (body wet wt 3 4 + 1 g) were acclimated in the laboratory for 6 days before 1 mg Se 1-1 as SeO 2- (13 ~tM) was added to two aquaria. After 12 days pre-exposure to selenite, 100 ~1 with ca 112 000 cpm 2°3HGC12 ( - 1 2 ng Hg) in crab Ringer (Bjerregaard, 1988) or ca 147 000 cpm CH32°3HgCI ( - 1 9 ng Hg) in 0.005 M Na2CO 3 was injected into the haemolymph of each crab through the arthrodial membrane of the posterior walking leg. Whole body counts were performed and haemolymph (ca 100 ~tl) was sampled through the arthrodial membranes of each crab 0.5, 1, 2, 4, 22 and 33 h after injection of CH32°3HgC1, and 2, 4, 22, 39, 56 and 75 h after injection of 2°3HgC12. Midgut gland, gills, heart and gonads were removed together with samples of carapace, hypodermis and muscle. Their 2°3Hg contents were measured. Experiment 2 To investigate the effect of selenite on the handling of organic mercury assimilated from food, four groups of 6 male shore crabs (body wet wt 3 8 + 2 g) were acclimated for 10 days in the laboratory. The crabs were then fed three times a week for 4 weeks. Group 1 was fed mussels exposed to CH32°3HgC1; group 2 was fed mussel cubes contaminated with CH32°3HgCI and 127 gM selenite (10 mg Se 1-1 as SeO2-); group 3 was fed mussel cubes contaminated with CH32°3HgC1 as was group 4, which was simultaneously exposed to 13 ~M selenite (1 mg Se 1-1 as SeO 2-) in the seawater. MeHg and M e H g + S e cubes contained 18 7 5 6 + 4 3 5 cpm g-~ (23 ng Hg g-l) (n---18) and mussels contained 10 9 5 4 + 1641 cpm g-1 (13 ng Hg g-l) (n--6). Whole body counts were performed before and after each feeding session. To make up a budget of how much the crab actually ate, the water in which the crabs had been eating was filtered and the radioactivity in the remains and in the filtered water was measured. Faecal pellets were collected by means of a pipette and counted three times a week. After 4 weeks, the crabs were dissected into midgut gland, gills, heart and gonads, together with samples of carapace, hypodermis, muscle and haemolymph. Their 2°3Hg contents were measured. Experiment 3 To investigate elimination of organic and inorganic mercury, four groups of six male shore crabs (body wet

wt 2 9 + 2 g) were acclimated for 8 days in the laboratory. The crabs were then fed for five successive days with contaminated food. Group 1 was fed mussels exposed to CH32°3HgCI; group 2 was fed mussel cubes contaminated with CH32°3HgC1 and 127 ~tM selenite (10 mg Se 1-1 as SeO2-); group 3 was fed mussel cubes contaminated with CH32°3HgC1, and group 4 was fed mussel cubes contaminated with 2°3HgCl2. MeHg and M e H g + S e cubes contained 3541 + 82 cpm g-J (4 ng Hg g-l) (n--18), Hg cubes contained 1813 + 73 cpm g-1 (2 ng Hg g-i) ( n - 6 ) and mussels contained 1047+ 140 cpm g-l (1 ng Hg g-l) (n--6). Whole body counts were performed before and after each feeding session. Faecal pellets were collected and counted each day. After 5 days the crabs were fed uncontaminated mussel cubes twice a week for 7 weeks. Whole body counts were performed prior to each feeding session. Faecal pellets from group 4 were collected and counted twice a week throughout the experiment. Faecal pellets from the other groups were collected and counted for the first 4 weeks only Data treatment One- and two-way ANOVA tests were used to evaluate effects of exposure/elimination time and/or selenium treatment. Individual experimental groups were compared by Tukey's multiple comparison test. Regression equations were calculated on the data means. The statistical procedures were carried out with the Macintosh programs SYSTAT 5.1 and Cricket graph 1.3.

Results Experiment 1 All of the crabs survived the exposure and elimination period. Organic mercury was eliminated from the haemolymph very quickly compared to inorganic mercury (Fig. l(a)). Half of the organic mercury detected in the first sample (half an hour after injection) was eliminated within 2-3 h, whereas half of the inorganic mercury was eliminated after 11 and 87 h without and with pre-exposure to selenium, respectively. Selenium in the water initially slowed down the elimination of organic mercury from the haemolymph (p < 0.05) and speeded up the elimination of inorganic mercury (p <0.005), but elimination rates of both mercury forms converged, regardless of exposure to selenite. From 22 h after injection, elimination of inorganic mercury from the haemolymph decreased linearly with time both in the control group ((Hgino~g)--87.53-0.43h; r2--0.99) and in the group pre-exposed to selenite ((Hginorg)-54.14-0.38h; r2=0.99). Elimination of organic mercury from the haemolymph decreased linearly with time from 4 h after injection ((Hgo~g)-----22.86 -- 0.46 h; r 2= 0.99). With preexposure to selenite the concentration reached approximately the same line after ca 22 h ((Hgorg)~17.00 0.26 h (calculated on two points only)). Elimination of inorganic mercury from the whole body after injection seemed to be divided into two compartments (Fig. l(b)). An initial fast part (in this

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Marine Pollution Bulletin (a)

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Carcinus maenas. Distributions of mercury among organs in crabs after injection of inorganic (Hg) and organic mercury (MeHg) into unexposed crabs and crabs pre-exposed to selenite (+Se). Mean :t: SEM for five crabs shown. Gonads (11), heart (ll), midgut gland (m), gills (~l), hypodermis (O), carapace (11), muscles (~) and haemolymph ([]).

Experiment 2 Three of the 24 crabs died during the exposure period. Crabs pre-exposed to selenite in water and food ingested higher amounts of organic mercury than crabs not exposed to selenite (Table 1). Accumulation of 4o organic mercury from the food followed quadratic equations (Fig. 3(a)). Crabs fed mussels exposed to organic mercury in seawater accumulated significantly less than crabs fed homogenized mussels in gelatinous cubes, even though the former source of food had the o 0 20 40 60 80 higher Hg concentrations. Selenite in the food augElimination time (hours) mented the accumulation of organic mercury significantly (p < 0.0005), as did pre-exposure to selenite in Fig. I Carcinus maenas. Retention of inorganic mercury (squares) and organic mercury (circles)in the haemolymph (a) and whole seawater (p < 0.005). Selenite in the food augmented body retention (b) of crabs afterinjectioninto unexposed crabs the accumulation significantly more than pre-exposure (filled symbols) and crabs pre-exposed to selenite (open to selenite in seawater (p < 0.0005). symbols). Mean + S E M for fivecrabs shown. Crabs fed mussels exposed to organic mercury in seawater assimilated significantly less organic mercury than crabs fed homogenized mussels in gelatinous experiment only represented by two observations cubes with added organic mercury (Fig. 3(19)) ((Hgmorg)--104.24-2.12h)), followed by a slower ((Hgorg)-68.8-0.gd; r2~0.77; p <0.05). Selenite in linear decrease ((Hg~org)--96.44-0.21h; r 2 ~ 1 . 0 0 ) . food or seawater did not consistently affect the Pre-exposure to selenite suppressed the first fast part assimilation efficiency ((Hgorg)-- 80.4 - 0.5 d; r 2-- 0.82). Within each group there was no consistent change in but crabs pre-exposed to selenite excreted inorganic the activity in the faecal pellets collected during the mercury with the same rate as the slow excretion from crabs not exposed to selenite ((Hgmorg)--101.06- 0.21 h; exposure period (Table 1), but there was a significant r2~0.97). Results on whole body retention of organic difference between the groups. The concentrations of mercury after injection were missed due to a technical organic mercury in faeces were elevated in groups exposed to selenite .both in food and in seawater problem with the whole body counter. The distributions of inorganic and organic mercury (p < 0.0005), but no significant differences were seen in among the organs were significantly different the percentages of ingested organic mercury collected (p <0.0005) (Fig. 2). Inorganic mercury accumulated in the faeces. Organic mercury accumulated from food was found predominantly in gills and a large fraction was also predominantly in midgut gland and muscles (Fig. 4). found in the midgut gland. Pre-exposure to selenite did not change the distribution significantly. The major part The distribution was not different in crabs that were fed of the injected organic mercury was found in the midgut mussels exposed to organic mercury in seawater comgland and muscle. Only a little was found in the pared to those fed mussel cubes containing organic gills. Selenite significantly augmented the fraction mercury. Selenite accumulated along with organic accumulated in the muscle (p < 0.0005) and it lowered mercury from the food seemed to lower the relative the amount of organic mercury in the hypodermis body burden of organic mercury found in the midgut (p < 0.0005). gland, however, due to very large variability this

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Volume 31/Numbers 1-3 TABLE 1 Contents of organic mercury in faecal pellets collected from crabs offered a diet of 28 5 2 5 + 8 7 0 cpm in cubes and 39 383 :t: 2262 cpm in mussels, and the amount of organic mercury actually ingested. Mean + SEM for meals and five to six crabs in each group is given. * and *** indicate that the difference from the group fed organic mercury 0VleHg) in cubes is significant at the 0.05 and 0.001 level, respectively. Carcinus maenas.

Mercury in faeces (cpm)

Exposure Crabs Crabs Crabs Crabs

fed MeHg cubes fed MeHg exposed mussels fed MeHg + Se cubes exposed to Se in water and fed MeHg cubes

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Fig. 4 Carcinus maenas. Distributions of organic mercury among organs in five crabs fed mussels exposed to organic mercury in seawater (Mussel), five crabs fed mussel cubes containing organic mercury and selenite (MeHg + Se), five crabs fed mussel cubes containing organic mercury (MeHg) and six crabs exposed to selenite in seawater and fed mussel cubes containing organic mercury (Se in seawater). Symbols are explained in Fig. 2.

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Carcinus maenas. Accumulation (a) and percentage retention of total amount (b) of organic mercury ingested by crabs fed mussel cubes containing 35 657+ 1087 cpm or mussels containing 49 229:1:2827 cpm MeHg. Mean + SEM for five to six crabs. O: Crabs fed cubes containing organic mercury and 127 ~tu selenite ((Hgorg)- 13 8 5 3 + 6 5 6 0 d - 7 3 d 2 ; r2-0.98). A: Crabs exposed to 13 ~M selenite in seawater and fed cubes containing organic mercury ((I-lgorg)- 5782 + 5 7 4 1 d - 78 d 2 ; r 2 - 0.98). O: Crabs fed cubes containing organic mercury ((Hgors)-7447 + 4 6 6 6 d - 6 3 d 2 ; r2-0.98. A: Crabs fed mussels exposed to organic mercury in seawater ((Hgorg)-5317+1857d-34d2; r2-0.95; p < 0.0005).

difference was not significant. Selenite in the food lowered the accumulated organic mercury concentration in the hypodermis (p < 0.005) and augmented it in the carapace (p = 0.005). Exposure to selenite in the ambient seawater did not change the content of organic mercury in the midgut gland, but did lower the proportion of the organic mercury in the gills significantly (p = 0.001).

Three of the 24 crabs died during the experiment. Elimination of inorganic mercury given in the food was significantly faster than elimination of organic mercury (p <0.0005). Elimination of inorganic mercury followed an exponential curve with a biological half life of approximately 56 days ((nginorg) ~- 95.8 × 10-°°05d; r2----0.98) (Fig. 5(a)). Organic mercury was eliminated very slowly from the crabs (Fig. 5(a)). Redistribution of the accumulated organic mercury among the tissues may have affected self absorption effects on the counting procedure and thereby produced the values exceeding 100% retention seen during the elimination period (Fig. 5(a)). The highest activity was found in crabs fed mussel cubes containing organic mercury and selenite, followed by the crabs fed mussel cubes with only added organic mercury; the crabs fed mussels exposed to organic mercury in the water had the lowest activity of those exposed to organic mercury. Elimination of organic mercury from crabs fed fresh mussels and crabs fed mussel cubes could not be described quantitatively. The elimination of organic mercury from crabs fed mussel cubes containing both organic mercury and selenite could be described by the exponential equation (Hgorg)----lll.5×10-°'°°°ad; r 2-0.60, between 17 and 48 days elimination, indicating a biological half life of approximately 753 days. 81

Marine Pollution Bulletin 120 ]

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neither assimilation efficiency nor elimination of organic mercury are consistently affected by exposure to selenite• Bjerregaard & Christensen (1993) similarly found that selenite in seawater augmented the retention of organic mercury present in the food, but the release of mercury during the feeding process was not assessed• There was a significant difference between the concentration of organic mercury in crabs fed homogenized mussels in gelatinous cubes containing organic mercury, and crabs fed mussels exposed to organic mercury in seawater. This difference may be due to the greater difficulty, on the part of the crab, in eating the mussel completely, but the difference in retention suggests that the internal handling of organic mercury in the crab might be different• For example, the mercury could be bound in different chemical forms when naturally incorporated, as opposed to being added in solution to a homogenate (Phillips & Buhler, 1978; Bjerregaard & Christensen, 1993).

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Elimination time (days) Fig. 5 Carcinus maenas. Retention of mercury (a) and excretion of mercury in faeces CO) after 5 days of exposure to mercury in the food. Mean + SEM for four to six crabs. ©: Crabs fed mussel cubes containing organic mercury and selenite. O: Crabs fed mussel cubes containing organic mercury. A: Crabs fed mussels exposed to organic mercury in seawater. I1: Crabs fed mussel cubes containing inorganic mercury.

Excretion of inorganic mercury in the faeces (Fig. 5(b)) rose to reach a maximum of 3 days after exposure had been stopped. Thereafter it initially declined quickly ((Hginorg)= 337.6 × 10-°"°386'/; r e-- 0.99) and then, after 17 days of elimination, more slowly ((Hginore)-~ 103.3 × 10-°-°°98a; r e--- 0.83). The concentration of organic mercury in the faeces dropped from the first day of exposure and reached zero around day 10 after exposure ceased. Selenium in food seemed to enhance the concentration of organic mercury in the faeces, and crabs fed fresh mussels excreted least organic mercury in the faeces.

Discussion Accumulation and assimilation efficiency

Seventy to eighty percent of the organic mercury ingested by C. maenas is assimilated. Selenite, in food or water, augments the accumulation of organic mercury in the crabs fed mussel cubes containing organic mercury. Selenite apparently augments the amoUnt of mercury ingested within the food, since 82

The tissue distribution of organic mercury among the tissues is not affected by the type of food, but the presence of selenite does affect the tissue distribution of organic mercury. Most of the organic mercury accumulated from food is found in midgut gland and muscles, and simultaneous exposure to selenite in food seems to lower the amount of organic mercury in the midgut gland, but this is not seen when the exposure to selenite is as the dissolved form in seawater. After injection of organic mercury, the majority was found in midgut gland and muscles. Selenite in seawater augments the amount of organic mercury in the muscles, but does not change the amount of organic mercury in the midgut gland. Bjerregaard & Christensen (1993) found that selenite in seawater augmented the amount of organic mercury accumulated from food in the muscles and lowered it in the midgut gland. Injected inorganic mercury is predominantly found in gills and midgut gland, and selenite shows no effect on the tissue distribution. Bjerregaard & Christensen (1993) found that inorganic mercury absorbed from food is found predominantly in the midgut gland and muscles, and that selenite in seawater at high concentrations of inorganic mercury in the food did alter the distribution, augmenting the amount in gills and lowering it in midgut gland• Elimination

Elimination of inorganic mercury was very fast compared to the elimination of organic mercury, the biological half lives after exposure in food being approximately 56 and 753 days, respectively. The biological half life of inorganic mercury after injection, approximately 9 days, was much faster than that after exposure in food. The biological half life of organic mercury has been estimated by Miettinen et al. (1972) to be 400 + 50 days. Selenite in seawater or food might change the initial elimination rate of ingested organic mercury from C. maenas, but within a time period it stabilizes at the same rate as in the absence of selenite exposure.

Volume 31/Numbers 1-3 The elimination of inorganic mercury from the h a e m o l y m p h is not a reflection of its elimination from the whole animal. The elimination of injected inorganic mercury from the whole animal is slower than elimination from the haemolymph. The effects of selenite on elimination of inorganic mercury from the h a e m o l y m p h and whole body are different. Selenite suppresses the initial fast part of the elimination of inorganic mercury from the whole animal, whereas it speeds up the elimination from the haemolymph. Organic mercury injected into the h a e m o l y m p h is taken up by the tissues very quickly c o m p a r e d to injected inorganic mercury. Selenite in the water initially slowed down the elimination of organic mercury from the h a e m o l y m p h and speeded up the elimination of inorganic mercury. Bjerregaard & Christensen (1993) found that both mercury forms were taken up into the tissues faster in selenite pre-exposed crabs, but Bjerregaard (1988) found that cadmium was taken up into the tissues more slowly in selenite-exposed crabs. The fact that all four groups approach the same elimination rate indicates that, after a period of fast uptake by the tissues, possible binding sites might be saturated. The amount of organic mercury in faeces reflects the amount of organic mercury ingested. Approximately 10 days after exposure to organic mercury in food had

ceased, no more organic mercury was excreted in the faeces. Inorganic mercury was excreted in the faeces for a longer period. The steep drop in Fig. 5(b) might indicate that the gut is being emptied of remaining contaminated food, whereas the slower excretion probably illustrates the actual excretion. The study was supported by grants from the Danish Natural Science Council and the Danish Environment Research programme 19921996. Bjerregaard, P. (1988). Interactions between selenium and cadmium in the haemolymph of the shore crab Carcinus maenas (L.). Aquat. Toxicol. 13, 1-12. Bjerregaard, P. & Christensen, L. (1993). Accumulation of organic and inorganic mercury from food in the tissues of Carcinus maenas: effectof waterborne selenium. Mar Ecol. Prog. Ser. 99, 271-281. Magos, L. & Webb, M. (1980). The interaction of selenium with cadmium and mercury. CRC Crit. Rev. Toxicol. 8, 1-42. Miettinen, J. K., Heyraud, M. & Keckes, S. (1972). Mercury as a hydrospheric pollutant, II. Biological half-time of methyl mercury in four Mediterranean species: a fish, a crab and two molluscs. In Marine Pollution and Sea Life, pp. 1-12. Published by arrangement with FAO, FishingNews (Books) Ltd, London. Pelletier, E. (1985). Mercury-selenium interactions in aquatic organisms: a review.Mar. Environ. Biol. Res. 18, 111-132. Phillips, R. P. & Buhler, D. R. (1978). The relative contributions of methylmercury from food or water in a rainbow trout (Salmo gairdneri) in a controlled laboratory environment. Trans. Am. Fish. Soc. 107,853-961. Toribara, T. Y. (1985). Preparation of CH~2°3HgCIof high specific activity. Int. Z AppZ Radiat. lsotop. 26, 9//3-904.

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