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E nviron~! IP c l~oxicology

Arch. Environ. Contain. Toxicol. 20,247-252 (1991)

o n t a m i ~ i o n and

9 J991 Springer-Verlag New York Inc.

Effects of Metals on Early Life Stages of the Brine Shrimp, Artemia: A Developmental Toxicity Assay T h o m a s H. M a c R a e a a n d A m r i t a n s h u S. P a n d e y Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J 1 Abstract. The need for simple, fast and inexpensive tests to

study metal pollution in the marine environment has become more pressing as utilization of coastal waters increases. To address this concern, the influence of four metals, cupric sulfate, lead nitrate, zinc sulfate and nickel sulfate, on emergence and hatching of the brine shrimp, Artemia, has been assessed. Occurrence of these easily recognized developmental milestones provides a convenient method to assay metal effects on development. Copper and lead were about equally toxic, reducing the rate and extent of Artemia development at or below concentrations of 0.1 txM. Zinc was somewhat less toxic than copper and lead, while nickel was the least toxic. Emerging Artemia are much more sensitive to metals than are larvae and adults. Furthermore, in contrast to results obtained with larvae and adults, the toxicity of lead is high when prelarval stages are considered. It is apparent from the findings that use of early stages of Artemia is an alternative to the examination of slower growing animals for the study of metal pollution in coastal marine waters.

It is desirable, as the threat from metal contamination of the environment increases, to have model systems which may be used to a s s a y the impact of metals on all living organisms. To this end, many different animals and cell types have been exploited as bioindicators, either to establish LC50s or to assess the physiological/biochemical consequences of metals (Dinnel et al. 1989; Whaley et al. 1989; Lopez-Arifiguez et al. 1989; Goldstein and Babich 1989). The brine shrimp, Artemia, has been used to study metals and a standardized toxicity test employing second or third instar larvae has been developed (Blust et al. 1988, 1986; Verriopoulos et al. 1987). There are several advantages to using Artemia, including their ready availability, low cost, ease of culture and a large literature describing their morphological, biochemical and molecular characteristics (Warner et al. 1989; MacRae et al. 1988). However, larval and adult Artemia are relatively insensitive to metals and, since they are

I To whom correspondence should be addressed.

not fed during the assay, Lhey are subject to stress in addition to that imposed by the presence of the metals. The high tolerance of Artemia adults and larvae to metals, coupled with the need for an inexpensive system to study pollution of marine environments, has prompted us to consider earlier life stages of Artemia. The organism can be commercially obtained as an encysted, metabolically inactive gastrula, enclosed by a shell impervious to substances other than water or gases. Following incubation in well aerated hatch medium at 28~ during which growth and development occur, the cyst shell cracks. The animal is released from the cyst, as a prenaupluis enclosed within a hatching membrane, by the process of emergence. In the first stage of emergence (termed E l ) there is a progressive extrusion of the prenauplius, an event thought to be at least partially dependent on the generation of an osmotic potential across the hatching membrane (Trotman et al. 1989, 1987). Once the prenauplius has completely emerged, it may remain attached to the cyst shell by the inner cuticular membrane, a stage called E2. The prenauplius may separate from the shell yielding an E3 stage which then hatches with the rupture of the enclosing or hatching membrane. Within a culture of Artemia started by incubating cysts, there is a rise in the number of emerged animals which then declines as the animals hatch. The developmental events of emergence and hatching, which form the basis of the assay results presented herein, were previously described (Rafiee et al. I986; Go et al. 1990). The effects of cadmium (Rafiee et ai. 1986) and mercury (Go et al. 1990) on the postgastrula development of Artemia have been previously examined and a preliminary study of zinc chloride completed (Bagshaw et aI. 1986). In our earlier work, prehatch Artemia were much more sensitive to metals than were free-swimming larvae and adults. The results indicated that Artemia have greater potential for use in the study of metals in the marine environment than previously realized. To substantiate this observation and to determine the s e n s i t i v i t y of A r t e m i a to a wider range of metals, emerging animals were exposed to copper sulfate, lead nitrate, zinc sulfate, and nickel sulfate. The results demonstrate that Artemia are well suited for the bioassay of metals in the marine environment and for examining biochemical/ molecular parameters of metal toxicity.

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Materials and Methods

Results

Preparation and Growth of Artemia

In the absence of metals, about 50-60% of the brine shrimp emerged after 12 h of development at 28~ (Figure 1). Emergence co n t i n u ed during incubation but the number of emerged organisms decreased as hatching occurred (Figures 1, 2) so that by 72 h only a small number of emerged animals remained (Figures 1, 3). For the cysts used in this study about 70-80% underwent development, an amount obtained by adding the number of emerged and hatched animals at 72 h. When Artemia were exposed to metals their patterns of emergence and hatching were altered (Figures I, 2). The overall influence on the rate and extent of development was dependent on the metal and its concentration. For CuSO4 and Pb(NO3)2, the highest number of emerged animals was greater than the control at all concentrations tested, peaking at 16 h or 20 h into the incubation (Figure 1). Such a result does not reflect enhanced emergence but rather that emergence was slowed or halted at the time the cyst cracked open and metals en t er ed the organism. For Z n S Q and NiSO4, the number of emerged animals generally peaked at 12 or 16 h (Figure 1). The effect of zinc and nickel on the final number of emerged animals was much less than for copper and lead (Figures 1, 3), a result that was especially obvious at 1 and 0.1 ixM, indicating that the former two

The Artemia embryos were purchased from San Francisco Bay Brand, Newark, CA, and were of the species A. franciscana. Encysted Artemia were hydrated in distilled water at 4~ for at least 6 h, followed by washing to separate the cysts that float from those that sink. The sinkers were collected on a Buchner funnel and washed with cold distilled water followed by hatch medium (Rafiee et al. 1986). Cupric sulfate (CuSO4), lead nitrate (Ph(NO3)2), zinc sulfate (ZnSO4), and nickel sulfate (NiSO4) were tested for their effects on Artemia development. The final concentrations were 0.01-5.0 p.M for copper sulfate and 0.1 - 10 o~Mfor the other metals. Expressed as p~g/L of metal ions the concentrations were: copper, 0.64- 318; lead, 20.7-2027; zinc, 6.5-654 and nickel 5.9-587. All metals were added to incubations from stock solutions at 0.1 mM in hatch medium and exposure concentrations reported are nominal values. The influence of the metals on Artemia development was determined by incubating 4 replicate samples of 25 hydrated cysts in 5 ml of hatch medium contained within 50 mm diameter plastic petri dishes at 28~ with shaking at 100 rpm. A dissecting microscope was used to determine the number of organisms present at each stage of development at the time intervals shown on the graphs. For emergence and hatching, the mean percentage and standard deviation were determined by dividing the number of organisms at either stage of development by the number of organisms in the test. The range of standard deviations was from 4-12%.

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metals had less of an effect on the emerging animals than the l a t t e r two. At metal c o n c e n t r a t i o n s of 5 IxM or higher, copper and lead were about equally toxic to Artemia, while zinc tended to be less toxic and nickel even less so (Figures 1-3). From these data it was possible to estimate an LCs0 at 72 h, or in other words, a concentration of metal at which 50% of the animals were killed by 72 h. The estimation depends on the observation that brine shrimp which have not hatched by 72 h in the presence of metal are very unlikely to survive and develop. For copper the LC5o was between 0.01 and 0.1 ~M (0.64-6.4 p~g/L), lead was less than 0.1 ~M (20.7 p.g/L), zinc was about 1 p~M (65 ~g/L) and nickel was greater than 10 t~M or 587 p~g/L. By examining the end point of Artemia development it was not always possible to ascertain relative toxicities of metals at all concentrations of interest. This was true for example, when zinc and nickel were compared at 0.1 ~M after 72 h of d e v e l o p m e n t (Figure 3). H o w e v e r , if the hatching and emergence curves were plotted on the same graph, the time when the curves crossed during incubation could be determined (Figure 4). The earlier the curves intersected, the less toxic was the metal, since there had been a reduced effect on the rates of emergence and hatching. For nickel, the curves crossed at about 36 h while for zinc they crossed at 48 h and for lead and copper the curves did not meet during the 72 h incubation. If the concentration of copper was reduced 10-fold to 0.01 ~M, the emergence and hatching curves intersected at about 47 h, still substantially

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Figure 2. The effect of metals on the hatching of Artemia. The metal is indicated in the upper left hand corner of each graph and the concentration (FM) is shown by the number following each curve. The error bars represent standard deviation from the mean

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delayed in relation to the 22 h time point obtained for control incubations in the absence of metal. Emerging Artemia were clearly sensitive to copper and lead at concentrations less than 0.1 FM, or 6.4 and 20.7 ~g/L respectively, of each metal. These types of plots, readily obtainable only when the animal displays two recognizable developmental milestones in relatively rapid succession, define periods of time during which the organism is reacting to and overcoming the metal effects. It will be extremely interesting to determine molecular and biochemical aspects of this adaptive stage.

Discussion

The effects of four metals, CuSO4, Pb(NO3)2, Z n S Q , and NiSO4, on the emergence and hatching of the brine shrimp, Artemia, are described. The results apply directly to the artificial environment established for the experiments and the metal concentrations reported are nominal. The concentration of metals in the solutions can be reduced by adsorption to container walls, loss to the atmosphere by volatilization, and uptake by the organisms (Verriopoulos et at. 1987; Hennig and Greenwood 1981). The net result, if such losses are encountered, is to demonstrate that emergingArtemia are most sensitive to metals than the study indicates. It is unlikely that the use of hatch medium versus sea water has a major effect on metal speciation (Hahne and Kroontje 1973) and toxicity, although this is uncertain since the effect of speciation on the toxicity of these metals to Artemia is un-

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known. Of more significance, in natural sea water, metals complex to organic ligands, thus affecting their availability (Blust et al. 1986) and their toxicity is influenced by suboptimal conditions of pH ( B l u s t e t al. 1988) temperature and salinity (Del Ramo et al. 1987; Voyer and Modica 1990) which exert additional physiological stress on animals. Lead and copper are more inhibitory to developing Art e m i a than is zinc and nickel is the least toxic of the four metals. Although the number of viable organisms remaining after 72 h was not determined, animals which fail to hatch by 72 h are either dead or will die without further apparent development. From this observation, we were able to estimate LCsos as outlined in the Results section. The LCso values are much lower than reported for adult or larval A r t e m i a (Verriopoulos et al. 1987; Sleet and Brendel 1985). In addition, lead, which was the least toxic to adult A r t e m i a , was very toxic to emerging prenauplii. When compared across species boundaries, emerging A r t e m i a are as sensitive to metals as several other animals used in acute toxicity testing of marine or o t h e r e n v i r o n m e n t s (Verriopoulos and Dimas 1988; Nelson et al. 1988; Bodar et al. 1989). Since A r t e m i a survive in sea water, although they are not marine animals p e r se,

they are good candidates as model organisms for the study of metal pollution in marine environments. That this is true is strengthened when characteristics of A r t e m i a , such as their rapid growth, the presence of easily detected developmental milestones, year round availability, ease of storage and their low cost, are considered. The effects of the metals on emergence and hatching parallel those for cadmium (Rafiee et al. 1986) and mercury, suggesting a common mechanism of action for all of the metals. Specifically, the metals may disrupt the generation of an osmotic potential within the cyst which is required for emergence to occur (Trotman et al. 1989, 1987). Since the osmotic potential is thought to be produced by the salt gland N a , K - A T P a s e pump (Trotman et al. 1989) or by the exchange of sodium and calcium (Cheon and Reeves 1988), the primary impact of metals on A r t e m i a may be to perturb ion transport in the membranes of salt glands, a proposition consistent with the known characteristics of metals (Foulkes 1988). Whatever the biochemical basis for the disruption of development, it is clear that the brine shrimp offer significant potential for the study of metals in the marine environment.

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Acknowledgments. The financial support of the Natural Sciences and Engineering Research Council of Canada and the Department of Fisheries and Oceans is gratefully acknowledged.

References Bagshaw JC, Rafiee P, Matthews CO, MacRae TH (1986) Cadmium and zinc reversibly arrest development of Artemia larvae. Bull Environ Contain Toxicol 37:289-296 Blust R, Van der Linden A, Verheyen E, Decleir W (1988) Effect of pH on the biological availability of copper to the brine shrimp Artemiafranciscana. Mar Biol 98:31-38 Blust R, Verheyen E, Doumen C, Decleir W (1986) Effect of corn-

60

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Figure 4. The effect of metals on the rate of Artemia development. The times at which the percent emergence (El and hatching (H) curves cross one another in the absence (control) and presence of metals are shown. The concentration of meted tested is indicated in the upper left hand side of each graph. The error bars represent standard deviations from the mean

plexation by organic ligands on the bioavailability of copper to the brine shrimp Artemia sp. Aquat Toxicol 8:211-221 Bodar CWM, Zee Avd, Voogt PA, Wynne H, Zandee DI (1989) Toxicity of heavy metals to early life stages of Daphnia magna. Ecotoxicol Environ Safety 17:333-338 Cheon J, Reeves JP (1988) Sodium-calcium exchange in membrane vesicles from Artemia. Arch Biochem Biophys 267:736-741 Del Ramo J, Diaz-Mayans J, Torreblanca A, Nunez A (1987) Effects of temperature on the acute toxicity of heavy metals (Cr, Cd, and Hg) to the freshwater crayfish, Procambarus ctarkii (Girard). Bull Environ Contam Toxicol 38:736-741 Dinnel PA, Link JM, Stober QJ, Letourneau MW, Roberts WE (1989) Comparative sensitivity of sea urchin sperm bioassays to metals and pesticides. Arch Environ Contain Toxicol 18:748755

252 Foulkes EC (1988) On the mechanism of transfer of heavy metals across cell membranes. Toxicology 52:263-272 Go EC, Pandey AS, MacRae TH (1990) The effect of inorganic mercury on the emergence and hatching of the brine shrimp, Artemia franciscana. Mar Biol (In press). Goldstein SH, Babich H (1989) Differential effects of arsenite and arsenate to Drosophila melanogaster in a combined adult/developmental toxicity assay. Bull Environ Contain Toxicol 42:276-282 Habne HCH, Kroontje W (1973) Significance of pH and chloride concentration on behavior of heavy metal pollutants: Mercury (II), cadmium (II), zinc (II), and lead (II). J Environ Quality 2:444-450 Hennig HF-KO, Greenwood PJ (1981) The loss of cadmium and zinc from sea water during accumulation experiments: its implication on toxicity threshold concentrations. Mar Pollut Bull 12:47-50 Lopez-Artiguez M, Soria ML, Repetto M (1989) Heavy metals in bivalve molluscs in the Huelva estuary. Bull Environ Contam Toxicol 42:634-642 MacRae TH, Bagshaw JC, Warner AH (1988) Biochemical and cell biology of Artemia. CRC Press Inc, Boca Raton, FL Nelson DA, Miller JE, Calabrese A (1988) Effect of heavy metals on bay scallops, surf clams, and blue mussels in acute and longterm exposures. Arch Environ Contain Toxicol 17:595-600 Persoone G, Sorgeloos P, Roels O, Jaspers E (1980) The brine shrimp Artemia. Vols. 1-3. Universa Press, Wetteren, Belgium Rafiee P, Matthews CO, Bagshaw JC and MacRae TH (1986) Reversible arrest of Artemia development by cadmium. Can J Zool 64:1633-1641

T . H . MacRae and A. S. Pandey Sleet RB, Brendel K (1985) Homogeneous populations of Artemia nauplii and their potential use for in vitro testing in developmental toxicology. Teratog Carcinog Mutagen 5:41-54 Trotman CNA, Gieseg SP, Pirie RS, Tate WP (1989) Developmental abnormalities related to bicarbonate ion status during emergence of Artemia. In: Warner AH, MacRae TH, Bagshaw JC (eds) Cell and molecular biology of Artemia development. Plenum Press, NY, pp 17-28 - (1987) Abnormal development in Artemia: Defective emergence of the prenauplius with bicarbonate deficiency. J Exp Zool 243:225-232 Verriopoulos G, Dimas S (1988) Combined toxicity of copper, cadmium, zinc, lead, nickel, and chrome to the copepod Tisbe hoIothuriae. Bull Environ Contam Toxicol 41:378-384 Verriopoulos G, Moraitou-Apostolopoulou M, MiUiou E (1987) Combined toxicity of four toxicants (Cu, Cr, oil, oil dispersant) to Artemia salina. Bull Environ Contam Toxicol 38:483-490 Voyer RA, Modica G (1990) Influence of salinity and temperature on acute toxicity of cadmium to Mysidopsis bahia Molenock. Arch Environ Contain Toxicol 19:124-131 Warner AH, MacRae TH, Bagshaw JC (1989) Cell and molecular biology of Artemia development. Plenum Press, NY Whaley M, Garcia R, Sy J (1989) Acute bioassays with benthic macroinvertebrates conducted in situ. Bull Environ Contain Toxicol 43:570-575

Manuscript received March 22, 1990 and in revised form June 8, 1990.