ECOTOXICOLOCY
AND
ENVIRONMENTAL
SAFETY
21,68-79 (1991)
Toxicity of Organic Mercury Compounds Brine Shrimp, Artemia AMRITANSHUSPANDEY
to the Developing
AND THOMAS H.MACRAE
Department of Biology, Dalhousie University, Ha&x,
Nova Scotia, Canada B3H
4Jl
Received January IS, 1990 Mercury can be coupled to a wide variety of organic compounds but there is limited information concerning the influence of such substitutions on the toxicity of mercury within the marine environment. We therefore determined the effectsof six organomercuries on the emergence and hatching of the brine shrimp, Artemia. The relative toxicities of the organic mercuries were unaffected by the ability of the compounds to ionize, whereas the sizes of the compounds appeared to be important. Thus, brine shrimp were equally sensitive to five of the organic mercuries while diphenylmercury, the largest of the organic mercuries tested, was the least toxic. In the presence of 0.1 PM diphenylmercury the final amount of hatching was similar to that in the absence of metal but in this situation there was an easily measured reduction in the rate of development. By determining the rates of emergence and hatching it is apparent that Artemia are adversely affected by organic mercuries at concentrations less than 0.1 pA4, the lowest level examined in this study. The work extends our earlier findings with cadmium and zinc, supporting the proposal that Artemia is an excellent alternative to more complex, slow-growing animals for the study of biochemical/physiological aspects of marine pollution. Q 1991 Academic Press, IIIC.
INTRODUCTION The study of metals within the environment takes many forms including the use of living organisms as bioindicators (Lopez-Antiguez et al., 1989; Goldstein and Babich, 1989; Schoor et al., 1988; Williams and Dusenbery, 1988; Goldberg and Martin, 1983; Bitton, 1983). The amount of metal present and its influence on the physiological processes of organisms are the parameters most commonly examined. The effects of metals on Artemia have been determined under a variety of conditions (Blust et al., 1986, 1988; Persoone and Wells, 1987; Verriopoulos et al., 1987; Jayasekara et al., 1986; Trieff, 1980; Vanhaecke et al., 1980; Saliba and Krzyz, 1976; Brown and Ahsanullah, 197 1) and a standardized toxicity assay using mortality of second or third instar Artemia nauplii as the test criterion is now available (Persoone and Wells, 1987; Vanhaecke et al., 1980). The test provides an effective means of monitoring for the presence of several pollutants but may have limited application in the analysis of toxic effects at the physiological level, especially with sublethal amounts of toxicants, since the organisms are not fed. This limits the length of time the animals can be studied and introduces an additional stress due to hunger, thus complicating the perceived response of the animals to pollutants. Consideration of the life history of Artemia reveals that prelarval organisms are suitable for toxicity studies. Dormant encysted gastrula are readily available in large quantities from commercial suppliers. Once development is reiniated, Artemia undergo a period of relatively synchronous growth in the absence of cell division (Nakanishi et al., 1962; Olson and Clegg, 1978). After 12- 15 hr of incubation, the cyst shell cracks open. The prenauplius, enclosed within a hatching membrane, is released from the 0147-6513/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
68
ORGANIC MERCURY TOXICITY TO
Arfemia
69
shell in a process termed emergence (E), which is characterized by two discrete stages (Rafiee et al., 1986). In the first stage (E,) the prenauplius is progressively extruded from the cyst and in the second stage (E2) the prenauplius is completely outside the cyst but remains attached to the shell by the inner cuticular membrane. The emerged. membrane-encased prenauplius is then usually detached from the shell (sometimes called the E3 stage) and, in a process termed hatching, the membrane ruptures to yield a free-swimming nauplius. Thus, a normally developing population of Artemia undergoes a transient phase characterized by a relatively large number of emerged animals which subsequently declines as the animals go on to hatch. These events are well characterized morphologically (Rafiee et al., 1986) and there is an increasingly large amount of biochemical/molecular information describing Artemia development (Warner et al., 1989; MacRae et al., 1988; Sorgeloos et al., 1987; Wahba and Woodley, 1984; Persoone et al., 1980; Bagshaw and Warner, 1979). Artemia are very sensitive to low concentrations of cadmium and zinc at the early E, stage of development (Rafiee et al., 1986; Bagshaw et al.. 1986) and to several other metals (unpublished data). However, no data existed concerning the sensitivity of the El stage organism to organic mercury, prompting this work and a related study in which the consequence to Artemia of exposure to inorganic mercury is considered (manuscript submitted). It is important to consider organic mercuries since they are released directly into the environment or occur as conversion products of inorganic mercury and they are often more toxic than is inorganic mercury (Wood, 1976; Vallee and Ulmer, 1972). All of the organic mercuries we tested, except one, were equally toxic to Artemia, exhibiting dramatic effects on emergence and hatching at concentrations of 0.1 pA4, the lowest level tested. Artemia were sensitive to concentrations of mercury less than 0.1 PM and the degree of toxicity appeared to be independent of the ability of the tested organomercuries to ionize but at least partially dependent on the size of the compound. The results support our earlier conclusion (Rafiee ef ul., 1986) that Artemia is an excellent animal to use in the development of a bioassay for the study of metals in the marine environment. MATERIALS
AND
METHODS
Embryos Brine shrimp embryos of the species A. franciscana (Clark and Bowen, 1976; Barigozzi, 1974) were obtained from San Francisco Bay Brand (Newark, CA). Preparation
of Animals
The encysted brine shrimp were hydrated in distilled water at 4°C for 12 hr followed by washing to separate the cysts that sink from those that float (Warner et al., 1979: Rafiee et al., 1986). The sinkers were collected on a Biichner funnel and washed with cold distilled water followed by hatch medium. Hatch medium was composed of 422 mM NaCl, 9.4 mA4 KCl, 25.4 mM MgS04, 22.7 mM MgC& , 1.4 mM CaC12, 0.5 mM NaHCO,, and 2.6 mM Na2B407 at pH 8.6 in distilled and deionized (Milli-Q) water (Warner et al., 1979; Rafiee et al., 1986). Mercury speciation and solubility should be similar in hatch medium and seawater while formation of insoluble metal complexes should be minimal (Rafiee et al., 1986; Hahne and Kroontje, 1973).
70 Incubation
PANDEY
AND
MACRAE
of Animals
Six organic mercury compounds including methylmercuric iodide (HgCH&, methylmercuric chloride (HgCH,Cl), methylmercuric bromide (HgCHsBr), dimethylmercury ((CH&Hg), diethylmercury ((C2H&Hg), and diphenylmercury ((C6H&Hg) were tested for their toxic effects on Artemia development at final concentrations of 0.1, 1.0, 5.0, and 10.0 @f. The mercury exposure concentrations are nominal and the amount of mercury in incubations was not determined during the experiments. Reports of mercury loss during incubations (McKenney and Costlow, 1982; Jenne and Avotins, 1975) suggest that the metal is present at concentrations lower than nominal and that brine shrimp are more sensitive to organic mercury than indicated by our results. Eight replicates, consisting of 25 animals in 50-mm-diameter plastic petri plates containing 5 ml of hatch medium, were examined for all concentrations of each metal. To set up the incubations hydrated cysts counted with the aid of a dissecting microscope were placed in the petri plates with a Pasteur pipet and the mercury compounds were added from stock solutions prepared in hatch medium. The plates were then incubated at 28°C with shaking at 100 rpm. At the time intervals shown, the organisms at each stage of development were counted with the aid of a dissecting microscope. The mean percentage and standard deviation of emergence and hatching at each time point were calculated by dividing the number of organisms which were present at each stage of development by the number of organisms in the test. Under normal conditions a point is reached in the growth curve where the percentage of emerged animals drops due to hatching. RESULTS Artemia Development
in the Absence of Mercury
In the absence of added mercury (control conditions) the majority of the cysts cracked open and many of the prenauplii were partially emerged from the shell, a developmental stage known as El (Rafiee et al., 1986) after 12 hr of incubation at 28°C (Fig. 1). By 16 hr, E2 and E3 forms were prevalant and about 15% of the organisms had hatched (Fig. 2), resulting in a reduction in the number of emerged organisms, a trend which continued until by 72 hr almost all emerged organisms had hatched (Figs. 1 and 2). For this particular batch of cysts under the conditions used in these experiments approximately 70% of the organisms underwent development in 72 hr and almost all had hatched in this time. Eflect of Organic Mercuries on Artemia Development The addition of organic mercury compounds at all concentrations tested had a profound effect either on the rate and/or the final extent of Artemia emergence and hatching (Figs. 1 and 2). Since the results for methylmercuric iodide, methylmercuric chloride, and methylmercuric bromide were almost identical the data for the latter two compounds are not plotted but are displayed in Tables 1 and 2. Inspection of the data reveals that in the presence of all the organic mercuries, emerged organisms were visible at 12 hr of incubation but their numbers, exclusive of those for diphenylmercury, were greatly reduced (Fig. 1). This, we believe, reflected the very rapid effect of the organic mercury compounds on emergence and not the penetration of the mercury through the cyst shell. Thus, immediately upon cracking
ORGANIC
MERCURY
TOXICITY
80
80
80
80
Y'
10
1 20
30 40 TlME(Hr)
t 50
a 80
7i080 ' 1
TO .4rfmio
10
20
30 40 TlME(Hr)
50
80
70
B
10
20
30 40 TlME(Hr)
50
80
70
80
80
80
FIG. I. The effects of organic mercury compounds on the emergence of Anmia. The mean percentage and standard deviation of emergence. calculated as the number of emerged organisms at a particular time divided by the total number of organisms tested, for each concentration (@,44) of metal are plotted against the time of incubation in hours. The metal tested is indicated in the upper left-hand corner of each graph and the metal concentration is shown by the number following each curve. The same control curve was used for each metal as it was very similar from one experiment to another, as seen by comparison to control values in Tables I and 2.
of the cyst shell, mercury entered the developing organism and slowed the protrusion of the prenauplius from the cyst. The emerged stages after 12 hr of incubation were then more difficult to detect upon examination with the microscope since the prenauplius had emerged to a much lesser extent than when mercury was absent. By 16 hr, however, the number of emerged organisms for each organic mercury had surpassed the number in the control and, except for diphenylmercury, they reached their maximum values by 16-20 hr of incubation. Thus at a time when animals which had emerged in the absence of mercury were hatching, those animals developing in the presence of mercury were stalled, either reversibly or irreversibly, at the emerged stage. For example, at 1 @I mercury, the highest number of emerged animals was about equal to that seen at mercury concentrations of 10 and 5 pA4, but many more of the organisms were able to overcome the effects of the mercury and go on to hatch. an occurrence still more pronounced at 0.1 &4 (Figs. 1 and 2).
72
PANDEY AND MACRAE
TIME (Hr)
FIG.2. The effectsof organic mercury compounds on the hatching ofdrtetnia. The mean percentage and standard deviation of hatching, calculated as the number of hatched organisms at a particular time divided by the total number of organisms tested, for each concentration (pkI) of metal are plotted against the time of incubation in hours. The metal tested is indicated in the upper left-hand comer of each graph and the metal concentration is shown by the number following each curve. The same control curve was used for each metal as it was very similar from one experiment to another, as seen by comparison to control values in Tables 1 and 2.
Eflect of Diphenylmercury
on Artemia Development
Diphenylmercury, which was the largest of the six organic mercury compounds tested, appeared to be the least toxic. In contrast to the other mercuries, there was no delay in the appearance of emerged organisms with about equal numbers of animals at this developmental stage after 12 hr for all concentrations of diphenylmercury tested (Fig. 1). Moreover, at all concentrations of diphenylmercury, many more of the organisms overcame the toxic effects of the metal and were able to hatch (Fig. 2). By directly comparing the percentage of organisms emerged and hatched at each metal concentration after 72 hr of development (Fig. 3), it is obvious that the final effects of all the metals, except for diphenylmercury, were the same. Diphenylmercury was less toxic at all concentrations tested, except at 1.0 &f, where its effect on the final extent of development at 72 hr was equal to that of the other organic mercuries. Moreover, the toxic effects of diphenylmercury were equal at 1, 5, and 10 pA4, due to its reduced toxicity in comparison to the other organic mercuries at the higher concentrations (Fig. 2).
ORGANIC
MERCURY
TOXICITY
TO
73
Arfemia
TABLE 1 EFFECT
OF METHYLMERCURIC AND
CHLORIDE HATCHING OF
(HgCHXl)
ON EMERGENCE
Artemia
Development time (hr) 12 0 0.1 1.0 5.0 10.0
54+ 8” 428 3k4 6+8 3-t4
0 0.1 1.0 5.0 10.0
2 * 4” 0 0 0 0
16 49 64 66 65 62
Ik + -t + +
20 8 4 4 4 4
15 t4 1+3 0 0 0
35+ 70+ 752 73+ 13+
36 8 I 4 4 4
28+ 8 11 + 12 0 0 0
16 52 61 15 75
48
zk 8 + 8 ~4 k4 t 4
50 + 4 29 -t 4 14 f 4 0 0
60
12
f 4 + 8 z!z4 + 4 k 4
5*4 46 + 8 53 +4 75 + 4 15 k 4
4+4 46 k 8 51+-4 14 Ik 4 75 f 4
51 + 4 31 f 4 21 +4 0 0
62 I!L4 31 + 4 22 zk 2 122 0
64 +- 4 31 + 4 24 + 2 1+2 0
10 46 54 75 15
a The upper half of the table shows the percentage of emerged animals while the lower half shows the percentage of hatched animals.
There was an almost equal amount of emergence and hatching after 72 hr for Artemiu grown in the presence of 0.1 @I diphenylmercury or in its absence (Figs. I 3). This observation was not true for other concentrations of diphenylmercury nor was it true for the other organomercuries when they were tested at 0.1 &4. Moreover, the result suggests that diphenylmercury at 0.1 @I may not be having any effect on Artemia development. There was, however, a drastic slowdown of the developmental process when diphenylmercury was present, best shown by comparing the time when
TABLE 2 EFFECT
OF METHYLMERCURIC BROMIDE AND HATCHING OF
(HgCH,Br)
Development
12
0 0.1 I.0 5.0 10.0
55 + 8” 3+4 7+8 3+4 5-18
0 0.1 1.0 5.0 10.0
0” 0 0 0 0
16 50 61 69 69 66
c?z4 + 4 f 4 f 4 + 4
19 +4 If2 0 0 0
20 34 68 68 13 14
+ 4 iT 4 + 8 AZ4 + 4
38 + 0 7f4 4+8 1 + 1.6 0
ON EMERGENCE
Artemia time
36
(hr)
48
Ilk4 41 Z!Y8 51 k8 72 +- 8 75 l+z 4
11 39 49 72 75
+4 + 8 + 8 f 8 + 4
52 I? 4 26 -t 4 21 k4 2k4 0
58 -+ 4 35 kc 4 23 + 4 224 0
60
12
8? 36+ 48k 712 15+
4 12 8 4 4
5k4 34 f 8 47 + 8 70 + 4 75 f4
63+ 41+ 24+ 35 0
4 8 4 4
67 Y!Z4 42 z!z 8 24 f 4 4+4 0
’ The upper half of the table shows the percentage of emerged animals while the lower half shows the percentage of hatched animals.
74
PANDEY AND MACRAE
0.1 FM
go EMERGED
HATCHEO
80
I
70
+
60 f
t
$50 s a40 L 30
HATCHEC
70 60 +5 z y40 30 20 10 t-L
I)
1
2
4
5
2
3
4
5
MERCUR:
FIG. 3. Comparison of the percentage of organisms emerged and hatched at each metal concentration after 72 hr of development. The graphs allow a direct comparison of the effect of each metal, at each concentration tested, on the final amount of emergence and hatching of Arfemia. The metals are: I, methylmercuric iodide; 2, dimethylmercuxy; 3, diethylmercury; 4, diphenylmercury; 5, no metal. The concentration of metal is indicated in the upper middle portion of each graph. The error bars are the standard deviations of the arithmetic means. Results at 10 pM are the same as those at 5 pM and are not shown.
ORGANIC
MERCURY
TOXICITY
TO
Artemiu
75
the hatching and emergence curves cross one another during the incubation period (Fig. 4). In the absence of metal, crossover occurred at about 26-27 hr into the incubation period, whereas in the presence of 0.1 PM diphenylmercury, crossover occurred at about 39-40 hr a full 13 hr later. For the other organic mercuries, the curves did not cross one another during the 72-hr incubation and they became nearly parallel.
TIME (Hr)
TIME (Hr)
10
20
30 40 50 TIME (Hr)
80
70
80
FIG. 4. Developmental delays induced by organic mercury compounds at a final concentration of 0.1 &l. The percentage of animals emerged and hatched in the absence (control) or the presence of organic mercury compounds at a final concentration of 0. I & is plotted against the time of incubation in hours. The metal tested is shown in the upper left-hand comer of each graph. with control indicating that no metal was added. Since results for methylmercuric iodide, dimethylmercury. and diethylmercury were similar. plots for the latter two compounds are not shown. H. hatched: E. emerged.
76
PANDEY
AND
MACRAE
Determination of the time at which the emergence and hatching curves cross one another is clearly a more sensitive parameter of metal effects than is determination of the final number of hatched animals. In addition, developing Artemia were obviously sensitive to organic mercuries at concentrations less than 0.1 PA,& DISCUSSION All of the organic mercury compounds tested had a significant effect on emergence and hatching of Artemia at 0.1 PM, the lowest concentration tested. In fact, all of the mercuries except diphenylmercury were very similar in their ability to disrupt emergence and hatching, being more toxic to Artemia than is inorganic mercury (manuscript submitted). The results show that emerging Artemia are very sensitive to mercury and demonstrate the usefulness of the animal at this stage of development for metal toxicological studies. To understand the mechanisms of mercury toxicity to cells, we need a better appreciation of the effects of organic derivation on the transport of mercury into cells and on its toxicity once it is inside. Of equal importance is to know the speciation properties, solubilities, and degree of dissociation of organic mercury compounds in the marine environment. For example, Blust et al. (1986, 1988) propose that the availability of copper to living organisms may be due to the presence of two distinct ligands. The ionized forms of copper may react with charged residues on the exterior surface of the membrane influencing their transport whereas those species that are neutral cross membranes by passive diffusion. The same reasoning may apply to mercury. Miura and Imura (1987) cite several studies indicating that methylmercury is particularly toxic because it passes easily through the cell’s cytoplasmic membrane. There is evidence (Muira and Imura, 1987; Christie and Costa, 1984; Wood, 1976) to show that inorganic mercury interacts with phospholipids through fatty acid double bonds to form complexes stable enough to isolate, while methylmercury does not. Such interactions may cause a change in the physical properties of the membrane leading to aberrant cell function. The physical nature of the membrane may also be changed by methylmercury even though its diffusion through the membrane is rapid. Of the six organic mercury compounds we tested, the halides possess a stable carbonmercury bond and an ionic mercury-halogen bond. Thus the halides are capable of existing as ions and this may be their reactive species, whereas the remaining three compounds cannot ionize. Of the nonhalide organic mercuries, a major difference is in their molecular size, which may affect their ability to diffuse through membranes, and they may vary somewhat in their degree of solubility in an aqueous solvent with all these compounds being relatively insoluble. From the comparisons we have done, the ability to ionize seems to have a limited effect on the potential toxicity of the organic mercuries tested. This does not mean, however, that the targets or mechanisms of action of the different types of compounds are the same other than exerting their effects through the mercury acting as a Lewis acid with a strong affinity for a number of ligands including sulfhydryl groups, phosphates, and porphyrins among many others (Miura and Imura, 1987; Wood, 1976; Vallee and Ulmer, 1972; Hughes, 1956). Diphenylmercury may exhibit a reduced effect on Artemia either because it is less soluble in hatch medium than are the other organic mercuries or because its greater size may retard its entry into the cell and its reaction with target sites once inside. Deciding the most important determinants of
ORGANIC
MERCURY
TOXICITY
TO
77
Artcmia
toxicity is difficult as we do not know the chemical species of mercury that is most actively transported by cells or, once inside, the chemical state it assumes. That the emergence of the prenauplius from the cyst is so sensitive to mercury may provide a clue to the primary site of toxic activity, at least for emerging Artemia, and reveal information concerning the emergence process. Trotman et al. (1987, 1989) have shown that the bicarbonate ion plays a role in the energy-dependent generation of the osmotic potential required for emergence of prenauplii from cysts. They propose that in a reduced concentration of bicarbonate, the osmotic potential exterior to the prenauplius is sufficient to crack the cyst shell but is not great enough to overcome the elastic nature of the inner cuticular membrane. The prenauplius either remains partly emerged or it emerges completely but cannot hatch due to the presence of the additional membrane. Mercury and other heavy metals affect the emerging prenauplius in a similar way, indicating that they may disrupt the mechanism required to generate the developmentally critical osmotic potential. It is very possible that the osmotic potential is produced by an Na,K-ATPase pump (Trotman et al., 1980, 1989) such as exists in the larval salt gland (Conte, 1989) or by a sodium-calcium exchange mechanism (Cheon and Reeves, 1988). Since membrane transport mechanisms are sensitive to heavy metals (Allemand et al., 1988; Foulkes, 1988) the disruption of Artemia emergence and hatching we describe herein may be due to metal effects on energy-dependent ion transport. Whatever the mechanism oftoxicity, inhibition ofArtemia emergence and hatching provides the physiological basis of a test system which will be very useful for studying metals in the marine environment. There are several advantages inherent in this approach. The test is very rapid and can easily be finished in 40 hr or less if the crossover point of hatching and emergence curves is considered to be indicative of the toxicity of a test compound at a particular concentration (Fig. 4). It is not necessary to culture organisms to the second instar or to maintain adults, thus eliminating work and expense. Including within the assay a sample with no added toxicant eliminates the problem of batch variability and by preliminary screening, cysts exhibiting good emergence and hatching can be selected. We are able to simultaneously monitor, with relative ease, 100 animals (four plates with 25 animals/plate) at each of five metal concentrations at any one time, demonstrating that the test is simple and easy to perform. The emergence process has proven to be very sensitive to all the heavy metals we have examined (Rafiee et al., 1986; Bagshaw et al., 1986: unpublished data) and for all the organic mercuries there is a significant effect on Artemia at a final metal concentration of 0.1 PM. The comparison presented in Fig. 4 shows that Artemia will respond to even lower mercury concentrations. Finally, with emergence and hatching as assay milestones there is a sufficient period of time to conduct biochemical analyses without the added complications of starvation or food addition. CONCLUSION The results show that developing Artemia are very sensitive to organomercuries and that the size of a mercury compound may affect its toxicity. There is a great potential for the use of Artemia in toxicological studies, providing a much needed simple, eukaryotic system suited to the marine environment. ACKNOWLEDGMENTS The financial support Department of Fisheries
of the Natural Sciences and Engineering and Oceans is gratefully acknowledged.
Research
Council
of Canada
and the
78
PANDEY
AND MACRAE
REFERENCES ALLEMAND, D., DE RENZIS, G., PAYAN, P., GIRARD, J-P., AND VAISSIERE,R. (1988). HgCI,-induced cell injury. Differential effect on membrane-located transport systemsin unfertilized and fertilized sea urchin eggs. Toxicology 50,2 17-230. BAGSHAW, J. C., RAL~EE,P., MATTHEWS, C. O., AND MACRAE, T. H. (1986). Cadmium and zinc reversibly arrest development of Artemia larvae. Bull. Environ. Contam. Toxicol. 37, 289-296. BAGSHAW, J. C., AND WARNER, A. H. (1979). Biochemistry ofArtemia Development. University Microfilms International, Ann Arbor, MI. BARIGOZZI, C. (1974). Artemia, a survey of its significance in genetic problems. Evol. Biol. 7, 221-252. BITTON, G. (1983). Bacterial and biochemical tests for assessingchemical toxicity in the aquatic environment: A review. CRC Crit. Rev. Environ. Control 13, 5 1-67. BLUST, R., VAN DER LINDEN, A., VERHEYEN, E., AND DECLEIR, W. (1988). Effect of pH on the biological availability of copper to the brine shrimp Artemia franciscana. Mar. Biol. 98, 3 l-38. BLUST, R., VERHEYEN, E., DOUMEN, C., AND DECLEIR, W. (1986). Effect of complexation by organic ligands on the bioavailability of copper to the brine shrimp, Artemia sp. Aquat. Toxicol. 8, 2 1 l-22 1. BROWN, B., AND AHSANULLAH, M. (197 1). Effect of heavy metals on mortality and growth. Mar. Polka. Bull. 2, 182-187. CHEON, J., AND REEVES,J. P. (1988). Sodium-calcium exchange in membrane vesicles from Artemia. Arch. Biochem. Biophys. 267, 736-741. CHRISTIE, N. T., AND COSTA, M. (1984). In vitro assessment of the toxicity of metal compounds. IV. Disposition of metals in cells: Interactions with membranes, glutathione, metallothionein and DNA. Biol. Trace Elem. Res. 6, 139-158. CLARK, L. S., AND BOWEN, S. T. (1976). The genetics of Artemia salina. VII. Reproductive isolation. J. Hered. 67,385-388. CONTE, F. P. ( 1989). Molecular biology of larval osmoregulation. In Cell and Molecular Biology ofArtemia Development (A. H. Warner, T. H. MacRae, and J. C. Bagshaw, Eds.), pp. 37 l-376. Plenum, New York. FOULKES, E. C. (1988). On the mechanism of transfer of heavy metals across cell membranes. Toxicology 52,263-272.
GOLDBERG, E. D., AND MARTIN, J. H. (1983). Metals in seawater as recorded by mussels. In Trace Metals in Sea Water, pp. 8 1l-823. Plenum, New York. GOLDSTEIN, S. H., AND BABICH, H. (1989). Differential effectsof arsenite and arsenate to Drosophila melanogaster in a combined adult/developmental toxicity assay.Bull. Environ. Contam. Toxicol. 42,276-282. HAHNE, H. C. H., AND KROONTJE, W. (1973). Significance of pH and chloride concentration on behavior of heavy metal pollutants: Mercury (1 I), cadmium (1 I), zinc (1 l), and lead (11). J. Environ. Qua/. 2, 444-450.
HUGHES,W. L. (1956). A physicochemical rationale for the biological activity of mercury and its compounds. Anal. N. Y. Acad. Sci. 65,454-460. JAYASEKARA, S., BROWN, D. B., AND SHARMA, R. P. (1986). Tolerance to cadmium and cadmium-binding ligands in Great Salt Lake brine shrimp (Artemia salina). Ecotoxicol. Environ. Saf 11, 23-30. JENNE,E. A., AND AVOTINS, P. (1975). The time stability of dissolved mercury in water samples. 1. Literature review. J. Environ. Qual. 4,427-43 1. LOPEZ-ARTIGUEZ, M., SORIA, M. L., AND REPETTO, M. (1989). Heavy metals in bivalve molluscs in the Huelva estuary. Bull. Environ. Contam. Toxicol. 42, 634-642. MACRAE, T. H., BAGSHAW, J. C., AND WARNER, A. H. (1988). Biochemistry and Cell Biology of Artemia. CRC Press, Boca Raton, FL. MCKENNEY, C. L., AND COSTLOW, J. D. (1982). The effects of mercury on developing larvae of Rhithre panopeus harrisii (Gould). I. Interactions of temperature, salinity and mercury on larval development. Estuarine Coastal ShelfSci. 14, 193-213. MIURA, K., AND IMURA, N. (1987). Mechanism of methylmercury cytotoxicity. CRC Crit. Rev. Toxicol. l&161-188.
NAKANISHI, Y. H., IWASAKI, T., OKIGAKI, T., AND KATO, H. (1962). Cytological studies ofArtemia salina. I. Embryonic development without cell multiplication after the blastula stage in encysted dry eggs.Annot. Zool. Japon. 35,223-228. OLSON, C. S., AND CLEGG, J. S. (1978). Cell division during the development of Artemia salina. Wilhelm Roux’s Arch. Dev. Biol. 184, l-13. PERSOONE,G., SORGELOOS,P., ROELS, O., AND JASPERS,E. (1980). The Brine Shrimp Artemia, Vols. I3. Universa Press, Wetteren, Belgium.
ORGANIC
MERCURY
TOXICITY
TO Artemia
79
PERSOONE,G.. AND WELLS, P. G. (1987). Artemia in aquatic toxicology: A review. In Artemia Research and Its Applications (P. Sorgeloos, D. A. Bengtson, W. Decleir. and E. Jaspers, Eds.), pp. 259-275. Universa Press, Wetteren, Belgium. RAFIEE, P., MATTHEWS, C. 0.. BAGSHAW, J. C., AND MACRAE, T. H. (1986). Reversible arrest of Artemia development by cadmium. Canad. J. Zuul.64,1633-1641. SALIBA, L. J., AND KRZYZ, R. M. (1976). Acclimation and tolerance ofArlemia salina to copper salts. hdur. Biol. 38, 231-238. SCHOOR,W. P., WILLIAMS, D. E., AND LECH, J. J. (1988). Combined use ofbiochemical indicators to assess sublethal pollution effects on the gulf killifish (Fundulus grundis). Arch. Environ. Contam. Tnxicol 17. 437-441. SORGELOOS,P., BENGTSON, D. A., DECLEIR, W., AND JASPERS,E. (1987). Arremia Research and Its .4pplications. Universa Press, Wetteren, Belgium. TRIEFF, N. M. (1980). Toxicity of heavy metals, oils and other organics on Artemia. In The Brine Shrimp Artemia (G. Persoone. P. Sorgeloos, 0. Roels, and E. Jaspers. Eds.). pp. 253-262. Universa Press, Wetteren. Belgium. TROTMAN, C. N. A., GIESEG, S. P., PIRIE, R. S., AND TATE, W. P. (1987). Abnormal development in Anemia: Defective emergence of the prenauplius with bicarbonate deficiency. J. E-x-p.Zoo/. 243, 22% 232. TROTMAN, C. N. A.. GIESEG, S. P.. PIRIE, R. S., AND TATE, W. P. (1989). Developmental abnormalities related to bicarbonate ion status during emergence of Artemia. In Cell and Molecular Biology qf;lrtemia Development (A. H. Warner, T. H. MacRae, and J. C. Bagshaw, Eds.). pp. 17-28. Plenum, New York. TROTMAN. C. N. A., MANSHELD, B. C.. AND TATE, W. P. (1980). Inhibition of emergence, hatching. and protein biosynthesis in embryonic Artemia salina. Dev. Biol. 80, l67- 174. VALLEE, B. L.. AND ULMER. D. D. (1972). Biochemical effects of mercury. cadmium and lead. .4nnu. Rev. Biochem. 41,9 I - 128. VANHAECKE, P., PERSOONE,G., CLAUS, C.. AND SORGELOOS,P. (1980). Research on the development of a short term standard toxicity test with Artemia nauplii. In The Brine Shrimp Arfemla, (G. Persoone. P. Sorgeloos. 0. Roels. and E. Jaspers, Eds.), pp. 263-285. Universa Press, Wetteren, Belgium. VERRIOPOULOS, G., MORAITOU-APOSTOLOPOULOU, M., AND MILLIOU, E. (1987). Combined toxicity of four toxicants (Cu, Cr, oil, oil dispersant) to Artemia salina. Bull. Environ. Contam. T~~.xico/.38, 483490. WAHBA, A. J., AND WOODLEY, C. L. (1984). Molecular aspects ofdevelopment in the brine shrimp .4rtemia. In Pr0gre.w in Nucleic Acid Research and Molecular Biology (W. E. Cohn and K. Moldave. Eds.). pp. 221-265. Academic Press, New York. WARNER, A. H., MACRAE, T. H.. AND BAGSHAW, J. C. (I 989). Cell and Molecular Biology of‘.&emia Development. Plenum, New York. WARNER, A. H.. MACRAE, T. H.. AND WAHBA, A. J. (1979). The use of Artemia salinn for developmental studies: Preparation ofembryos, tRNA, ribosomes and initiation factor 2. In Methods in En:n;ymo/ogv(K. Moldave and L. Grossman. Eds.). Vol. 60. pp. 298-3 1 I Academic Press, San Diego, WILLIAMS, P. L.. AND DUSENBERY,D. B. (1988). Using the nematode Caenorhabditis elegans to predict mammalian acute lethality to metallic salts. Toxicol. Indust. Health 4, 469-478. WOOD. J. M. ( 1976). The biochemistry of toxic elements in aqueous systems.In Biochemical and Bioph~tt~iu/ Perspectiws in Marine Biology (D. C. Malins and J. R. Sargent, Eds.). Vol. 3. pp. 407-431. Academic Press. New York.
AND
ENVIRONMENTAL
SAFETY
21,68-79 (1991)
Toxicity of Organic Mercury Compounds Brine Shrimp, Artemia AMRITANSHUSPANDEY
to the Developing
AND THOMAS H.MACRAE
Department of Biology, Dalhousie University, Ha&x,
Nova Scotia, Canada B3H
4Jl
Received January IS, 1990 Mercury can be coupled to a wide variety of organic compounds but there is limited information concerning the influence of such substitutions on the toxicity of mercury within the marine environment. We therefore determined the effectsof six organomercuries on the emergence and hatching of the brine shrimp, Artemia. The relative toxicities of the organic mercuries were unaffected by the ability of the compounds to ionize, whereas the sizes of the compounds appeared to be important. Thus, brine shrimp were equally sensitive to five of the organic mercuries while diphenylmercury, the largest of the organic mercuries tested, was the least toxic. In the presence of 0.1 PM diphenylmercury the final amount of hatching was similar to that in the absence of metal but in this situation there was an easily measured reduction in the rate of development. By determining the rates of emergence and hatching it is apparent that Artemia are adversely affected by organic mercuries at concentrations less than 0.1 pA4, the lowest level examined in this study. The work extends our earlier findings with cadmium and zinc, supporting the proposal that Artemia is an excellent alternative to more complex, slow-growing animals for the study of biochemical/physiological aspects of marine pollution. Q 1991 Academic Press, IIIC.
INTRODUCTION The study of metals within the environment takes many forms including the use of living organisms as bioindicators (Lopez-Antiguez et al., 1989; Goldstein and Babich, 1989; Schoor et al., 1988; Williams and Dusenbery, 1988; Goldberg and Martin, 1983; Bitton, 1983). The amount of metal present and its influence on the physiological processes of organisms are the parameters most commonly examined. The effects of metals on Artemia have been determined under a variety of conditions (Blust et al., 1986, 1988; Persoone and Wells, 1987; Verriopoulos et al., 1987; Jayasekara et al., 1986; Trieff, 1980; Vanhaecke et al., 1980; Saliba and Krzyz, 1976; Brown and Ahsanullah, 197 1) and a standardized toxicity assay using mortality of second or third instar Artemia nauplii as the test criterion is now available (Persoone and Wells, 1987; Vanhaecke et al., 1980). The test provides an effective means of monitoring for the presence of several pollutants but may have limited application in the analysis of toxic effects at the physiological level, especially with sublethal amounts of toxicants, since the organisms are not fed. This limits the length of time the animals can be studied and introduces an additional stress due to hunger, thus complicating the perceived response of the animals to pollutants. Consideration of the life history of Artemia reveals that prelarval organisms are suitable for toxicity studies. Dormant encysted gastrula are readily available in large quantities from commercial suppliers. Once development is reiniated, Artemia undergo a period of relatively synchronous growth in the absence of cell division (Nakanishi et al., 1962; Olson and Clegg, 1978). After 12- 15 hr of incubation, the cyst shell cracks open. The prenauplius, enclosed within a hatching membrane, is released from the 0147-6513/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
68
ORGANIC MERCURY TOXICITY TO
Arfemia
69
shell in a process termed emergence (E), which is characterized by two discrete stages (Rafiee et al., 1986). In the first stage (E,) the prenauplius is progressively extruded from the cyst and in the second stage (E2) the prenauplius is completely outside the cyst but remains attached to the shell by the inner cuticular membrane. The emerged. membrane-encased prenauplius is then usually detached from the shell (sometimes called the E3 stage) and, in a process termed hatching, the membrane ruptures to yield a free-swimming nauplius. Thus, a normally developing population of Artemia undergoes a transient phase characterized by a relatively large number of emerged animals which subsequently declines as the animals go on to hatch. These events are well characterized morphologically (Rafiee et al., 1986) and there is an increasingly large amount of biochemical/molecular information describing Artemia development (Warner et al., 1989; MacRae et al., 1988; Sorgeloos et al., 1987; Wahba and Woodley, 1984; Persoone et al., 1980; Bagshaw and Warner, 1979). Artemia are very sensitive to low concentrations of cadmium and zinc at the early E, stage of development (Rafiee et al., 1986; Bagshaw et al.. 1986) and to several other metals (unpublished data). However, no data existed concerning the sensitivity of the El stage organism to organic mercury, prompting this work and a related study in which the consequence to Artemia of exposure to inorganic mercury is considered (manuscript submitted). It is important to consider organic mercuries since they are released directly into the environment or occur as conversion products of inorganic mercury and they are often more toxic than is inorganic mercury (Wood, 1976; Vallee and Ulmer, 1972). All of the organic mercuries we tested, except one, were equally toxic to Artemia, exhibiting dramatic effects on emergence and hatching at concentrations of 0.1 pA4, the lowest level tested. Artemia were sensitive to concentrations of mercury less than 0.1 PM and the degree of toxicity appeared to be independent of the ability of the tested organomercuries to ionize but at least partially dependent on the size of the compound. The results support our earlier conclusion (Rafiee ef ul., 1986) that Artemia is an excellent animal to use in the development of a bioassay for the study of metals in the marine environment. MATERIALS
AND
METHODS
Embryos Brine shrimp embryos of the species A. franciscana (Clark and Bowen, 1976; Barigozzi, 1974) were obtained from San Francisco Bay Brand (Newark, CA). Preparation
of Animals
The encysted brine shrimp were hydrated in distilled water at 4°C for 12 hr followed by washing to separate the cysts that sink from those that float (Warner et al., 1979: Rafiee et al., 1986). The sinkers were collected on a Biichner funnel and washed with cold distilled water followed by hatch medium. Hatch medium was composed of 422 mM NaCl, 9.4 mA4 KCl, 25.4 mM MgS04, 22.7 mM MgC& , 1.4 mM CaC12, 0.5 mM NaHCO,, and 2.6 mM Na2B407 at pH 8.6 in distilled and deionized (Milli-Q) water (Warner et al., 1979; Rafiee et al., 1986). Mercury speciation and solubility should be similar in hatch medium and seawater while formation of insoluble metal complexes should be minimal (Rafiee et al., 1986; Hahne and Kroontje, 1973).
70 Incubation
PANDEY
AND
MACRAE
of Animals
Six organic mercury compounds including methylmercuric iodide (HgCH&, methylmercuric chloride (HgCH,Cl), methylmercuric bromide (HgCHsBr), dimethylmercury ((CH&Hg), diethylmercury ((C2H&Hg), and diphenylmercury ((C6H&Hg) were tested for their toxic effects on Artemia development at final concentrations of 0.1, 1.0, 5.0, and 10.0 @f. The mercury exposure concentrations are nominal and the amount of mercury in incubations was not determined during the experiments. Reports of mercury loss during incubations (McKenney and Costlow, 1982; Jenne and Avotins, 1975) suggest that the metal is present at concentrations lower than nominal and that brine shrimp are more sensitive to organic mercury than indicated by our results. Eight replicates, consisting of 25 animals in 50-mm-diameter plastic petri plates containing 5 ml of hatch medium, were examined for all concentrations of each metal. To set up the incubations hydrated cysts counted with the aid of a dissecting microscope were placed in the petri plates with a Pasteur pipet and the mercury compounds were added from stock solutions prepared in hatch medium. The plates were then incubated at 28°C with shaking at 100 rpm. At the time intervals shown, the organisms at each stage of development were counted with the aid of a dissecting microscope. The mean percentage and standard deviation of emergence and hatching at each time point were calculated by dividing the number of organisms which were present at each stage of development by the number of organisms in the test. Under normal conditions a point is reached in the growth curve where the percentage of emerged animals drops due to hatching. RESULTS Artemia Development
in the Absence of Mercury
In the absence of added mercury (control conditions) the majority of the cysts cracked open and many of the prenauplii were partially emerged from the shell, a developmental stage known as El (Rafiee et al., 1986) after 12 hr of incubation at 28°C (Fig. 1). By 16 hr, E2 and E3 forms were prevalant and about 15% of the organisms had hatched (Fig. 2), resulting in a reduction in the number of emerged organisms, a trend which continued until by 72 hr almost all emerged organisms had hatched (Figs. 1 and 2). For this particular batch of cysts under the conditions used in these experiments approximately 70% of the organisms underwent development in 72 hr and almost all had hatched in this time. Eflect of Organic Mercuries on Artemia Development The addition of organic mercury compounds at all concentrations tested had a profound effect either on the rate and/or the final extent of Artemia emergence and hatching (Figs. 1 and 2). Since the results for methylmercuric iodide, methylmercuric chloride, and methylmercuric bromide were almost identical the data for the latter two compounds are not plotted but are displayed in Tables 1 and 2. Inspection of the data reveals that in the presence of all the organic mercuries, emerged organisms were visible at 12 hr of incubation but their numbers, exclusive of those for diphenylmercury, were greatly reduced (Fig. 1). This, we believe, reflected the very rapid effect of the organic mercury compounds on emergence and not the penetration of the mercury through the cyst shell. Thus, immediately upon cracking
ORGANIC
MERCURY
TOXICITY
80
80
80
80
Y'
10
1 20
30 40 TlME(Hr)
t 50
a 80
7i080 ' 1
TO .4rfmio
10
20
30 40 TlME(Hr)
50
80
70
B
10
20
30 40 TlME(Hr)
50
80
70
80
80
80
FIG. I. The effects of organic mercury compounds on the emergence of Anmia. The mean percentage and standard deviation of emergence. calculated as the number of emerged organisms at a particular time divided by the total number of organisms tested, for each concentration (@,44) of metal are plotted against the time of incubation in hours. The metal tested is indicated in the upper left-hand corner of each graph and the metal concentration is shown by the number following each curve. The same control curve was used for each metal as it was very similar from one experiment to another, as seen by comparison to control values in Tables I and 2.
of the cyst shell, mercury entered the developing organism and slowed the protrusion of the prenauplius from the cyst. The emerged stages after 12 hr of incubation were then more difficult to detect upon examination with the microscope since the prenauplius had emerged to a much lesser extent than when mercury was absent. By 16 hr, however, the number of emerged organisms for each organic mercury had surpassed the number in the control and, except for diphenylmercury, they reached their maximum values by 16-20 hr of incubation. Thus at a time when animals which had emerged in the absence of mercury were hatching, those animals developing in the presence of mercury were stalled, either reversibly or irreversibly, at the emerged stage. For example, at 1 @I mercury, the highest number of emerged animals was about equal to that seen at mercury concentrations of 10 and 5 pA4, but many more of the organisms were able to overcome the effects of the mercury and go on to hatch. an occurrence still more pronounced at 0.1 &4 (Figs. 1 and 2).
72
PANDEY AND MACRAE
TIME (Hr)
FIG.2. The effectsof organic mercury compounds on the hatching ofdrtetnia. The mean percentage and standard deviation of hatching, calculated as the number of hatched organisms at a particular time divided by the total number of organisms tested, for each concentration (pkI) of metal are plotted against the time of incubation in hours. The metal tested is indicated in the upper left-hand comer of each graph and the metal concentration is shown by the number following each curve. The same control curve was used for each metal as it was very similar from one experiment to another, as seen by comparison to control values in Tables 1 and 2.
Eflect of Diphenylmercury
on Artemia Development
Diphenylmercury, which was the largest of the six organic mercury compounds tested, appeared to be the least toxic. In contrast to the other mercuries, there was no delay in the appearance of emerged organisms with about equal numbers of animals at this developmental stage after 12 hr for all concentrations of diphenylmercury tested (Fig. 1). Moreover, at all concentrations of diphenylmercury, many more of the organisms overcame the toxic effects of the metal and were able to hatch (Fig. 2). By directly comparing the percentage of organisms emerged and hatched at each metal concentration after 72 hr of development (Fig. 3), it is obvious that the final effects of all the metals, except for diphenylmercury, were the same. Diphenylmercury was less toxic at all concentrations tested, except at 1.0 &f, where its effect on the final extent of development at 72 hr was equal to that of the other organic mercuries. Moreover, the toxic effects of diphenylmercury were equal at 1, 5, and 10 pA4, due to its reduced toxicity in comparison to the other organic mercuries at the higher concentrations (Fig. 2).
ORGANIC
MERCURY
TOXICITY
TO
73
Arfemia
TABLE 1 EFFECT
OF METHYLMERCURIC AND
CHLORIDE HATCHING OF
(HgCHXl)
ON EMERGENCE
Artemia
Development time (hr) 12 0 0.1 1.0 5.0 10.0
54+ 8” 428 3k4 6+8 3-t4
0 0.1 1.0 5.0 10.0
2 * 4” 0 0 0 0
16 49 64 66 65 62
Ik + -t + +
20 8 4 4 4 4
15 t4 1+3 0 0 0
35+ 70+ 752 73+ 13+
36 8 I 4 4 4
28+ 8 11 + 12 0 0 0
16 52 61 15 75
48
zk 8 + 8 ~4 k4 t 4
50 + 4 29 -t 4 14 f 4 0 0
60
12
f 4 + 8 z!z4 + 4 k 4
5*4 46 + 8 53 +4 75 + 4 15 k 4
4+4 46 k 8 51+-4 14 Ik 4 75 f 4
51 + 4 31 f 4 21 +4 0 0
62 I!L4 31 + 4 22 zk 2 122 0
64 +- 4 31 + 4 24 + 2 1+2 0
10 46 54 75 15
a The upper half of the table shows the percentage of emerged animals while the lower half shows the percentage of hatched animals.
There was an almost equal amount of emergence and hatching after 72 hr for Artemiu grown in the presence of 0.1 @I diphenylmercury or in its absence (Figs. I 3). This observation was not true for other concentrations of diphenylmercury nor was it true for the other organomercuries when they were tested at 0.1 &4. Moreover, the result suggests that diphenylmercury at 0.1 @I may not be having any effect on Artemia development. There was, however, a drastic slowdown of the developmental process when diphenylmercury was present, best shown by comparing the time when
TABLE 2 EFFECT
OF METHYLMERCURIC BROMIDE AND HATCHING OF
(HgCH,Br)
Development
12
0 0.1 I.0 5.0 10.0
55 + 8” 3+4 7+8 3+4 5-18
0 0.1 1.0 5.0 10.0
0” 0 0 0 0
16 50 61 69 69 66
c?z4 + 4 f 4 f 4 + 4
19 +4 If2 0 0 0
20 34 68 68 13 14
+ 4 iT 4 + 8 AZ4 + 4
38 + 0 7f4 4+8 1 + 1.6 0
ON EMERGENCE
Artemia time
36
(hr)
48
Ilk4 41 Z!Y8 51 k8 72 +- 8 75 l+z 4
11 39 49 72 75
+4 + 8 + 8 f 8 + 4
52 I? 4 26 -t 4 21 k4 2k4 0
58 -+ 4 35 kc 4 23 + 4 224 0
60
12
8? 36+ 48k 712 15+
4 12 8 4 4
5k4 34 f 8 47 + 8 70 + 4 75 f4
63+ 41+ 24+ 35 0
4 8 4 4
67 Y!Z4 42 z!z 8 24 f 4 4+4 0
’ The upper half of the table shows the percentage of emerged animals while the lower half shows the percentage of hatched animals.
74
PANDEY AND MACRAE
0.1 FM
go EMERGED
HATCHEO
80
I
70
+
60 f
t
$50 s a40 L 30
HATCHEC
70 60 +5 z y40 30 20 10 t-L
I)
1
2
4
5
2
3
4
5
MERCUR:
FIG. 3. Comparison of the percentage of organisms emerged and hatched at each metal concentration after 72 hr of development. The graphs allow a direct comparison of the effect of each metal, at each concentration tested, on the final amount of emergence and hatching of Arfemia. The metals are: I, methylmercuric iodide; 2, dimethylmercuxy; 3, diethylmercury; 4, diphenylmercury; 5, no metal. The concentration of metal is indicated in the upper middle portion of each graph. The error bars are the standard deviations of the arithmetic means. Results at 10 pM are the same as those at 5 pM and are not shown.
ORGANIC
MERCURY
TOXICITY
TO
Artemiu
75
the hatching and emergence curves cross one another during the incubation period (Fig. 4). In the absence of metal, crossover occurred at about 26-27 hr into the incubation period, whereas in the presence of 0.1 PM diphenylmercury, crossover occurred at about 39-40 hr a full 13 hr later. For the other organic mercuries, the curves did not cross one another during the 72-hr incubation and they became nearly parallel.
TIME (Hr)
TIME (Hr)
10
20
30 40 50 TIME (Hr)
80
70
80
FIG. 4. Developmental delays induced by organic mercury compounds at a final concentration of 0.1 &l. The percentage of animals emerged and hatched in the absence (control) or the presence of organic mercury compounds at a final concentration of 0. I & is plotted against the time of incubation in hours. The metal tested is shown in the upper left-hand comer of each graph. with control indicating that no metal was added. Since results for methylmercuric iodide, dimethylmercury. and diethylmercury were similar. plots for the latter two compounds are not shown. H. hatched: E. emerged.
76
PANDEY
AND
MACRAE
Determination of the time at which the emergence and hatching curves cross one another is clearly a more sensitive parameter of metal effects than is determination of the final number of hatched animals. In addition, developing Artemia were obviously sensitive to organic mercuries at concentrations less than 0.1 PA,& DISCUSSION All of the organic mercury compounds tested had a significant effect on emergence and hatching of Artemia at 0.1 PM, the lowest concentration tested. In fact, all of the mercuries except diphenylmercury were very similar in their ability to disrupt emergence and hatching, being more toxic to Artemia than is inorganic mercury (manuscript submitted). The results show that emerging Artemia are very sensitive to mercury and demonstrate the usefulness of the animal at this stage of development for metal toxicological studies. To understand the mechanisms of mercury toxicity to cells, we need a better appreciation of the effects of organic derivation on the transport of mercury into cells and on its toxicity once it is inside. Of equal importance is to know the speciation properties, solubilities, and degree of dissociation of organic mercury compounds in the marine environment. For example, Blust et al. (1986, 1988) propose that the availability of copper to living organisms may be due to the presence of two distinct ligands. The ionized forms of copper may react with charged residues on the exterior surface of the membrane influencing their transport whereas those species that are neutral cross membranes by passive diffusion. The same reasoning may apply to mercury. Miura and Imura (1987) cite several studies indicating that methylmercury is particularly toxic because it passes easily through the cell’s cytoplasmic membrane. There is evidence (Muira and Imura, 1987; Christie and Costa, 1984; Wood, 1976) to show that inorganic mercury interacts with phospholipids through fatty acid double bonds to form complexes stable enough to isolate, while methylmercury does not. Such interactions may cause a change in the physical properties of the membrane leading to aberrant cell function. The physical nature of the membrane may also be changed by methylmercury even though its diffusion through the membrane is rapid. Of the six organic mercury compounds we tested, the halides possess a stable carbonmercury bond and an ionic mercury-halogen bond. Thus the halides are capable of existing as ions and this may be their reactive species, whereas the remaining three compounds cannot ionize. Of the nonhalide organic mercuries, a major difference is in their molecular size, which may affect their ability to diffuse through membranes, and they may vary somewhat in their degree of solubility in an aqueous solvent with all these compounds being relatively insoluble. From the comparisons we have done, the ability to ionize seems to have a limited effect on the potential toxicity of the organic mercuries tested. This does not mean, however, that the targets or mechanisms of action of the different types of compounds are the same other than exerting their effects through the mercury acting as a Lewis acid with a strong affinity for a number of ligands including sulfhydryl groups, phosphates, and porphyrins among many others (Miura and Imura, 1987; Wood, 1976; Vallee and Ulmer, 1972; Hughes, 1956). Diphenylmercury may exhibit a reduced effect on Artemia either because it is less soluble in hatch medium than are the other organic mercuries or because its greater size may retard its entry into the cell and its reaction with target sites once inside. Deciding the most important determinants of
ORGANIC
MERCURY
TOXICITY
TO
77
Artcmia
toxicity is difficult as we do not know the chemical species of mercury that is most actively transported by cells or, once inside, the chemical state it assumes. That the emergence of the prenauplius from the cyst is so sensitive to mercury may provide a clue to the primary site of toxic activity, at least for emerging Artemia, and reveal information concerning the emergence process. Trotman et al. (1987, 1989) have shown that the bicarbonate ion plays a role in the energy-dependent generation of the osmotic potential required for emergence of prenauplii from cysts. They propose that in a reduced concentration of bicarbonate, the osmotic potential exterior to the prenauplius is sufficient to crack the cyst shell but is not great enough to overcome the elastic nature of the inner cuticular membrane. The prenauplius either remains partly emerged or it emerges completely but cannot hatch due to the presence of the additional membrane. Mercury and other heavy metals affect the emerging prenauplius in a similar way, indicating that they may disrupt the mechanism required to generate the developmentally critical osmotic potential. It is very possible that the osmotic potential is produced by an Na,K-ATPase pump (Trotman et al., 1980, 1989) such as exists in the larval salt gland (Conte, 1989) or by a sodium-calcium exchange mechanism (Cheon and Reeves, 1988). Since membrane transport mechanisms are sensitive to heavy metals (Allemand et al., 1988; Foulkes, 1988) the disruption of Artemia emergence and hatching we describe herein may be due to metal effects on energy-dependent ion transport. Whatever the mechanism oftoxicity, inhibition ofArtemia emergence and hatching provides the physiological basis of a test system which will be very useful for studying metals in the marine environment. There are several advantages inherent in this approach. The test is very rapid and can easily be finished in 40 hr or less if the crossover point of hatching and emergence curves is considered to be indicative of the toxicity of a test compound at a particular concentration (Fig. 4). It is not necessary to culture organisms to the second instar or to maintain adults, thus eliminating work and expense. Including within the assay a sample with no added toxicant eliminates the problem of batch variability and by preliminary screening, cysts exhibiting good emergence and hatching can be selected. We are able to simultaneously monitor, with relative ease, 100 animals (four plates with 25 animals/plate) at each of five metal concentrations at any one time, demonstrating that the test is simple and easy to perform. The emergence process has proven to be very sensitive to all the heavy metals we have examined (Rafiee et al., 1986; Bagshaw et al., 1986: unpublished data) and for all the organic mercuries there is a significant effect on Artemia at a final metal concentration of 0.1 PM. The comparison presented in Fig. 4 shows that Artemia will respond to even lower mercury concentrations. Finally, with emergence and hatching as assay milestones there is a sufficient period of time to conduct biochemical analyses without the added complications of starvation or food addition. CONCLUSION The results show that developing Artemia are very sensitive to organomercuries and that the size of a mercury compound may affect its toxicity. There is a great potential for the use of Artemia in toxicological studies, providing a much needed simple, eukaryotic system suited to the marine environment. ACKNOWLEDGMENTS The financial support Department of Fisheries
of the Natural Sciences and Engineering and Oceans is gratefully acknowledged.
Research
Council
of Canada
and the
78
PANDEY
AND MACRAE
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