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pH-Dependent uranium toxicity to freshwater hydra
The Science of the Total Environment, 125 (1992) 159-173 Elsevier Science Publishers B.V., Amsterdam
159
pH-Dependent uranium toxicity to freshwater hydra R.V. H y n e a, G . D . R i p p o n a a n d G. Ellender b
aOffice of the Supervising Scientist, Alligator Rivers Region Research Institute, Jabiru, N.T. 0886, Australia bSchool of Dental Science, University of Melbourne, Parkville, Victoria 3052, Australia ABSTRACT A research program was initiated to characterise the major toxicants in water from Retention Pond 2 (RP2) at Ranger uranium mine. RP2 water was bioassayed using local hydra species, with water from Magela Creek used as a control and diluent (pH 6.1-6.7, conductivity 12-20 #S/cm). The bioassay had survival and population growth after 4-6 days as endpoints. Uranium was identified as the major toxicant in RP2 water, and further studies were carried out to assess its toxicity. At pH 8.5, in the presence of bicarbonate, RP2 water containing uranium at concentrations up to 3900 ppb, and uranium at 1000 ppb added to Magela Creek water, did not affect survival or population growth. However, RP2 water made to 32% with diluent resulting in a pH of 8.0, and uranium concentrations _ 200 ppb in Magela Creek water held at a normal pH of 6.5, did affect survival and population growth. In the absence of uranium in Magela Creek water, the hydra population growth or survival after 6 days was not affected by pH 5.0-8.0 and 5.0-8.5, respectively, pH was adjusted by adding acetic acid or sodium bicarbonate which also increased the conductivity. Nevertheless, an increase in the conductivity > 400 #S/cm affected the population number only after 6 days. This pH-specific effect of uranium toxicity is discussed in relation to the uranyl carbonate complexes of uranium formed in solution.
Key words: Hydra; uranium; toxicity; pH INTRODUCTION T h e R a n g e r u r a n i u m m i n e is o n a lease site s u r r o u n d e d b y K a k a d u N a t i o n a l P a r k in tropical n o r t h e r n Australia. As p a r t o f the m i n e ' s w a t e r strategy it has a series o f r e t e n t i o n p o n d s which act as sediment traps a n d r u n o f f catc h m e n t s in the wet season. T h e s e p o n d s have differing chemical w a t e r qualities, differing r e t e n t i o n capacities, a n d t h r o u g h r e g u l a t o r y criteria, differing f r e q u e n c y in their possibility o f release. F o r example, release o f R e t e n tion P o n d 2 ( R P 2 ) water, w h i c h has a very high u r a n i u m a n d m a n g a n e s e c o n t e n t (Allison a n d H o l d w a y , 1988; H y n e , 1990), into the M a g e l a C r e e k is o n l y p e r m i t t e d in a n e x c e p t i o n a l l y high rainfall wet season. This c o r r e s p o n d s to a f r e q u e n c y o f a b o u t o n c e in e v e r y 10 years p r o v i d i n g o t h e r r e g u l a t o r y c o n d i t i o n s are also met.
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Past mine waste water releases were made in accordance with regulatory criteria. These regulatory criteria, however, did not require biological testing of the waste water prior to release. This was despite substantial accumulated knowledge that only biological evidence could indicate the potential impact of the released water on any ecosystem (e.g. Cairns and Van Der Schalie, 1980). Because of this lack of biological data, we explored the nature of toxicity of these retention pond waters using locally collected species in bioassays developed in this laboratory. Freshwater hydra are sensitive to a number of environmental pollutants including metals (Browne and Davis, 1977; Stebbing and Pomroy, 1978) and some organic compounds (Lue and De La Cruz, 1978; Slooff et al., 1983). Bioassays using hydra asexual reproductive population growth as an endpoint are a sensitive and precise way to measure the effect of pollutants (Stebbing and Pomroy, 1978; Lue and De La Cruz, 1978). A bioassay using two hydra species collected from Magela Creek was developed based on the procedure described by Stebbing and Pomroy (1978). Preliminary experiments showed that uranium added to creek water at concentrations equal to, or greater than, 200 ppb can be detected by the inhibition of population growth of Hydra viridissima after 4 days (Allison and Holdway, 1988). However, when the hydra bioassay was used to assess the toxicity of water from RP2 containing high concentrations of uranium (3900 ppb), there was no effect on hydra population growth after 4 days. As waste water discharged from Ranger flows into river systems of Kakadu National Park, which has been entered onto the World Heritage List and the Convention on Wetlands of International Importance, any releases need to be carefully controlled to avoid environmental damage. The purpose of this study was to investigate the apparent anomaly between the high sensitivity of hydra to uranium salts added to Magela Creek water, and the low sensitivity of hydra to uranium present in a complex water from a mine retention pond. MATERIALS
AND
METHODS
Dilution and test waters
The dilution water used in most experiments was Magela Creek water (Hart et al., 1987), and during the wet season was collected near Georgetown Billabong (Humphrey et al., 1990) upstream of the Ranger uranium mine waste water discharge pipe outlet. The pH of the Magela Creek water ranged from 6.1 to 6.7 and the conductivity varied from 12 to 20 #S/cm during the experimental period. During the dry season, one experiment was undertaken and the dilution water was collected from Buffalo Billabong, a permanent water-body seasonally connected with Magela Creek. The Buffalo Billabong
pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
161
water collected had a pH of 6.5 and conductivity of 25/zS/cm. All dilution waters were collected in clean plastic bottles on the morning of the day the bioassay was to commence and filtered through a coarse filter (Whatman No. 1 filter paper) to remove wild zooplankton. The dilution water used in a test was also used as the control water. RP2 water was collected from the mine site as close as it was practicable to the start of the bioassay in a clean plastic bottle. The water was filtered through a coarse filter and various dilutions prepared. All waters were stored at 4°C until needed.
Hydra bioassay The test species was either H. viridissima or H. vulgaris (Cnidaria, Hydrozoa). The test animals were obtained from laboratory stocks reared in the same water that would be used as dilution and control water in the toxicity test. All animals selected were mature and asexually reproducing by budding. Only hydroids with a tentacled bud were selected as test organisms; a hydroid is a term used in this paper to describe an individual animal with or without buds. Five asexually reproducing test animals were covered with 30 ml of test solution in plastic Petri dishes (90 m m diameter). Each treatment was repeated in triplicate. The test solution was changed each day for fresh test solution of the same concentration and temperature. Daily observations were made of the degree of tentacle contraction (not contracted, partially contracted, or fully contracted) and changes in the number of intact hydroids in each treatment. Conductivity and pH of each test solution was also recorded daily. Test cultures were fed daily and maintained at 30°C with a 12-h photoperiod.
Hydra feeding and cleaning Hydra were fed by individually feeding each hydroid with live brine shrimp nauplii (Artemia salina) made up in the appropriate dilution water, and placed into each Petri dish using a glass Pasteur pipette. The nauplii were made up in the appropriate test solution. Feeding was ad libitum for 30 min after which the Petri dishes were cleaned and test solutions changed. Since the majority of individual H. viridissima adhere to the surfaces of the Petri dish, Petri dishes were cleaned in the following manner. First, the contents of each Petri dish are swirled to dislodge any uneaten brine shrimp and regurgitated food pellets. Each Petri dish is then drained of its contents and a fresh 10-ml aliquot of the appropriate test solution is added and the swirling/draining procedure repeated. Any hydra subsequently dislodged when the Petri dish was drained were carefully picked up in water using a clean
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glass Pasteur pipette and returned to the test Petri dish. A fresh 30-ml aliquot of the appropriate test solution was then added to the test Petri dish and any remaining brine shrimp or unhatched eggs individually removed by pipette. Since most individuals of H. vulgaris float on the water surface by means of a gas bubble produced by the pedal disk, these hydra are simply pipetted into a clean Petri dish containing a fresh 30 ml of the appropriate test solution leaving behind the uneaten brine shrimp and waste food pellets. Any hydra that are attached are gently removed with a clean glass Pasteur pipette and transferred to the new Petri dish.
Statistical analysis Statistical significance is determined for the number of intact hydroids between each test solution and the control over each observation period. The data were tested to ensure that the numbers of intact hydroids are normally distributed and their variances are homogenous. This was done by examining scatter-plots of predicted values for the dependent variable against the residual of a regression analysis (Tabachnick and Fidell, 1983). Another test for the dependent variable's normal distribution was carried out using computed normal scores from Blom's formula and plotting these scores against their actual value (SAS Institute Inc., 1985). Should the data not be normally distributed they were appropriately transformed to allow valid statistical analysis. Thereafter, a two-way Analysis of Variance (ANOVA) was performed with replicates and concentration as the main factors. If replicates were not significantly different, they were pooled and further analysis carried out using a two-way ANOVA, with waste concentration and observation time kept as the main factors. The effect of the test solutions was also examined by a one-way ANOVA with treatment water concentration as the main factor. If the analyses showed that the treatment effects were significant (P _< 0.05), then individual differences between means were assessed using the Least Square Means Test (for the two-way ANOVA) or Fisher's leastsignificant-difference test (for the one-way ANOVA) using the mean square error from the analysis of variance. The probability level for indicating detection of differences was 0.05.
Added uranyl sulphate and treatments A stock solution of uranyl sulphate was made by adding 40 mg of O2SO4" 3H20 to 2 litres of the dilution water. A dilution series of this stock solution was made with dilution water to give the desired concentration of added uranium. These test solutions were stored at 4°C until required.
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pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
A stock solution of uranyl sulphate and manganese sulphate was made by adding 40 mg of UO2SO4" 3H20 and 7 mg of MnSO4- H20 to 2 litres of the dilution water. A dilution series of this stock solution was made with dilution water to give the desired concentration of added uranium and manganese. In experiments where the pH required adjustment, sodium bicarbonate, sodium carbonate, tris (hydroxymethyl) methylamine (Tris) or acetic acid was added to the test solution to achieve the desired pH. The pH of the test solutions was adjusted as desired and stored at 4°C until required. In experiments where the conductivity required adjustment, sodium chloride was added to the water to achieve the desired conductivity.
Water chemistry analysis The concentrations of metals added to the test waters were confirmed by analysis using inductively coupled plasma-mass spectrometry. In some experiments though, uranium was determined by Scintrex Time Delay Fluorimetry and manganese was determined by Graphite Furnace Atomic Absorption Spectrometry.
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Time (days) Fig. 1. Hydra viridissima population growth when e×posedto various concentrations of added uranium to Magela Creek water over 5 days. Conductivity and pH were not significantly affected by the addition of the uranium. Each point is the mean of three replicates. Points with a common alphabetical superscript (within each daily measurement only) are not significantly different (P < 0.05, A N O V A ) from the control
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ET AL.
RESULTS
Toxicity of uranium to two hydra species H. viridissima were exposed to various concentrations of uranium added to water collected from Magela Creek through the 1989-1990 wet season. Significant inhibition in the population growth was obtained with uranium at a concentration of 200 ppb or greater, relative to the controls (Fig. 1). Conductivity and pH were not significantly affected by the addition of uranium. Similar results were obtained when H. viridissima were exposed to uranium added to water collected from Buffalo Billabong in the 1990 Dry season. There was a significant reduction in the population growth when uranium was 150 ppb or greater. H. vulgaris also showed a distinct wet to dry seasonal change in sensitivity to added uranium with the lowest observed effect concentrations (LOEC) varying from 550 to 400 ppb, respectively. Toxicity of water from ranger RP2 In December 1989, prior to the commencement of the wet season, bioassays on RP2 water from Ranger uranium mine showed that 100% RP2 water did not affect H. viridissima's population growth over a 4-day period
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pH DEPENDENT
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165
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Fig. 3. The effect of uranium (1000 ppb) and manganese (100 ppb) on the population number of Hydra viridissima over 4 days at different pHs. Each point is the mean of three replicates.
despite the presence of high concentrations of uranium (3900 ppb). However, if the RP2 water was diluted with diluent water and then tested for toxicity, a strong inhibition of population growth was observed (Fig. 2). The observed toxicity was associated with a reduction in the pH of the diluted RP2 water.
Effect of manganese on the toxicity of uranium The addition of uranium (1000 ppb) and manganese (100 ppb) to Magela Creek water, and pH adjusted with sodium bicarbonate, gave a similar effect to that observed with RP2 water (Fig. 3 compared with Fig. 2). That is, there was no significant effect of added uranium and manganese at an elevated pH of 8.6 on H. viridissima's population growth over 4 days. In contrast, at a pH of 6.6 there was a marked inhibition of population growth. These effects were very similar to that of the 100% RP2 water sample. After 1 day of addition of the uranium and manganese to Magela Creek water at a pH of 8.6 a sample of water was collected. An aliquot of this sample was filtered through a 0.45-tzm filter. Both unfiltered and filtered water were chemically analysed for uranium and manganese. Similar results for both samples were obtained. The reduced toxicity of water at pH 8.6 appeared not to be due to precipitation of uranium from solution with manganese dioxide since both
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R.V. H Y N E ET AL.
4
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Time (days) Fig. 5. The effect of pH on Hydra viridissima's population growth rate over 6 days. Magela Creek water pH was adjusted as described in the legend to Fig. 4. Each point is the mean of three replicates.
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pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
unfiltered and filtered water had similar uranium and manganese concentrations.
pH-Dependance of uranium toxicity The pH-dependent effect of uranium toxicity to H. viridissima was examined by assessing the extent of population growth inhibition by a known toxic dose of uranium (900 ppb). The uranium was added to Magela Creek water and the pH adjusted with acetic acid, sodium bicarbonate, tris (hydroxymethyl) methylamine (Tris) or sodium carbonate to achieve the desired pH. If Tris was used to buffer the uranium treatment to pH 8.5, a strong inhibition of population growth was observed compared with a Trisbuffered control water at pH 8.5. But when the pH was adjusted to 8.5 with bicarbonate, no significant effect of uranium was observed on population growth over 4 days compared with the control (Fig. 4). In contrast to this effect of uranium and bicarbonate, for treatments adjusted to pH 8.0 or less, or adjusted to pH 9.0 with carbonate, there was a strong inhibition of population growth (Fig. 4). The uranium still had a strong inhibitory effect on population growth if the pH was adjusted to pH 5.0 with acetic acid.
51 i Magela Creek Control pH 6.1 Conductivlty 12 uS/cm Added NaCl
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Fig. 6. The effect of increased conductivity on Hydra viridissima's population growth rate of over 6 days. The conductivity of the Magela Creek water was increased by the addition of sodium chloride. Each point is the mean of three replicates. Points with a common alphabetical superscript (within each daily measurement) are not significantly different (P < 0.05, ANOVA) from control.
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Effect of water pH on hydra population growth The effect of pH alone on population growth of H. viridissima over 6 days was determined by adjusting the pH of the Magela Creek water with acetic acid, sodium bicarbonate or sodium carbonate. Over the pH range 7.0-8.0, the population growth was not significantly different from the Magela Creek control water (pH 6.7). At pH 8.5 there was a small but significant reduction in population growth while at pH 9.0 hydra survival was severely impaired (Fig. 5). A further study showed that over the pH range 5.0-7.0 population growth was also not significantly different from the Magela Creek control.
Effect of increased conductivity on hydra pH Adjustment of test waters caused a change in conductivity with the addition of concentrated acidic or alkaline solutions. Therefore, the effect of increased conductivity alone on H. viridissima population growth over 6 days was examined by adding sodium chloride to the Magela Creek water (Fig. 6). On day 4 conductivities of 600 ttS/cm or greater inhibited population growth rate while by day 6 conductivities of 400 ~S/cm or greater inhibited population growth. DISCUSSION Uranium concentration in the surface waters of streams and rivers within Kakadu National Park is typically less than 1 ppb (Hart et al., 1987). Since uranium is non-essential for biological processes and is generally toxic at elevated concentrations (Berlin and Rudell, 1989; Leggett, 1979), concerns for the protection of aquatic biota exposed to contaminated waters must be considered. Ionic strength and composition are known to affect hydra size and asexual reproduction as observed by population growth (Lesh-Laurie, 1982). Koblick and Epp (1975) have previously shown that increasing osmolarity produces a progressive decrease in the population growth of hydra. This decrease was also accompanied by a decrease in the tissue potassium content (Epp and Koblick, 1977). Results from the present study show that low concentrations of uranium in diluent water ( >_ 200 ppb) have a direct inhibitory effect on the population growth of hydra. This inhibitory effect could not be attributed to an increase in the conductivity of the test water for there was no significant difference between conductivities of the test solutions. But, the toxicity of the uranium (900 ppb) to hydra, in diluent water and in the absence on manganese, was shown to be pH-dependent in the presence of bicarbonate. At pH 8.5,
pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
169
uranium in bicarbonate buffer was found to have no significant effect on H. viridissima population growth over 4 days. Bicarbonate-buffered undiluted RP2 water (pH = 8.6) also showed no significant decrease in H. viridissima population growth over 4 days. In contrast, Tris-buffered water with added uranium remained toxic at pH 8.5 compared with an appropriate Trisbuffered control water. This absence of toxicity of uranium at a pH of 8.5 in bicarbonate buffered RP2 water and Magela Creek water could be caused by the formation of uranyl carbonate complexes. At pH 8.5, the dominant uranyl carbonate complex in a typical soft water such as Magela Creek water would be the tricarbonate UO2(CO3)3 4- complex described by Langmuir (1978), in his definitive speciation model of uranyl-carbonate complexes. This negatively charged and extremely soluble ionic complex would experience charge conflicts at cell membrane surfaces which would result in decreased uranium adsorption. In soft water and at a lower pH range of 6-7, the predominant forms of uranium in Langmuir's speciation model are the divalent cationic species UO22÷ or the neutral uranyl carbonate complex UO:CO3, which would be more membrane permeable. Similar interaction between pH and carbonate ion concentration affecting uranium uptake by a marine algae has been reported (Horikoshi et al., 1979). The mine waste water from RP2 would have a complex water chemistry, and many geochemical changes could possibly contribute also to changes in its toxicity. RP2 water would also be expected to contribute a positive nutrient effect on the hydra as well as the negative toxic effect of uranium. However, uranium is present in RP2 water at concentrations an order of magnitude higher than any other toxic element. With dilution during a release of RP2 water into a receiving stream, the toxicity of uranium would then be expected to be the dominant effect. The concentration of soluble manganese in natural waters is also known to vary due to changes in pH of the water (Vuceta and Morgan, 1978). This could possibly affect changes in toxicity of any complex water (Stauber and Florence, 1985). The possible synergistic (or antagonistic) effect of manganese (Mn 2÷) on the toxicity of added uranium was investigated. However, manganese was found not to significantly modify the effect of uranium on H. viridissima population growth at either pH 6.6 or 8.6. In this study uranium was found to be very toxic to hydra within the pH range of 5-7. Nevertheless, within this pH range, pH-adjusted Magela Creek water (no added uranium) showed no adverse effects on the population growth of H. viridissima compared with Magela Creek control water with a pH of 6.7. At a high pH of 9.0, Magela Creek water, with or without uranium (900 ppb), was toxic to H. viridissima. However, this pH is beyond the normal physiological range for hydra and so was not investigated further.
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Like many other metals uranium is accumulated by various aquatic organisms, particularly by fungi, algae, bacteria and invertebrates (Standberg et al., 1981; Nakajima et al., 1981; Ellis and Ahsanullah, 1984; Galun et al., 1987). Uranium has a high affinity for proteins and lipids at the pH found intracellularly, thus preventing reversal desorption. Concentration factors for algae in the range 102-103 based on wet weight have been reported (Pribil and Marvan, 1976; Anonymous, 1983). Uranium can accumulate in fungi, algae and bacteria to relatively high concentrations without apparent toxic consequences. A wide spectrum of results has been obtained in studies concerned with the site of uptake of uranium in microogranisms. The extent of variability was demonstrated by Strandberg et al. (1981) who showed that uranium accumulated extracellularly on the surface of a species of yeast while there was a rapid and massive intracellular accumulation of uranium by a species of bacteria. Galun et al. (1987) showed that fungus can accumulate high concentrations of uranium both intracellularly and extracellularly on the surface, depending on the experimental conditions. Intracellular accumulation of uranium has also been documented in the lysosomes, macrophages, gill epithelia, hindgut epithelia and hepatopancreas cells of molluscs and crustacea exposed to uranium contaminated sea water or food (Chassard-Bouchard, 1983, 1984). Hence, it is now well documented that substantial amounts of uranium may be accumulated inside some types of cells. This accumulated uranium may or may not have a toxicological effect on the cell. Any development of toxicity will depend upon whether the uranium binds to a protein and interferes in its function or whether it is detoxified by the formation of a uranium phosphate microgranule, as observed in the crab (ChassardBouchard, 1983). Uranium has also been shown to inhibit ATPase (Nechay et al., 1980; Kramer et al., 1986), an enzyme located on the plasma membrane of the epidermal cell layer of hydra (Lentz and Barrnett, 1961; Macklin, 1967) and which is involved in osmoregulation and other active transport processes. Uranium has been reported to form a strong association with collagen (Anselme et al., 1990), which is present in the nematocyts of hydra (Blanquet and Lenhoff, 1966). Any binding of uranium with collagen in the nematocyts of hydra may interfere with the hydra's feeding response to prey. There has been very little information published on the toxicity of uranium to aquatic organisms. Tarzwell and Henderson (1960) using fathead minnows reported 96-h LCs0s for uranyl ion in soft water (pH 7.4, hardness 20 mg/1 as CaCO3), which was introduced as uranyl nitrate or uranyl acetate, as 3.1 and 3.7 ppm, respectively. Poston et al. (1984) obtained significant reduction in survival of Daphnia magna after 5 days at a uranium concentration of 520 ppb. Ahsanullah and Williams (1986) reported a reduction in the growth of a marine amphipod, Allorchestes compressa, by uranium at 2 ppm
pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
171
after 4 weeks. In contrast, after 4 days we found the L O E C for H. viridissima to be 150 ppb in the dry season, or 200 ppb in the wet season. Further, H. vulgaris had LOECs for the dry and wet season of 400 and 550 ppb, respectively. This seasonal difference in uranium toxicity may be due to seasonal changes in water quality (Hart and McGregor, 1980), or perhaps due to subtle changes in composition, which are o f an antagonistic/synergistic nature. The toxicological mechanism by which uranium affects the survival and population growth o f hydra, and the ultrastructural localisation of uranium absorbed by the hydra, is currently being investigated. ACKNOWLEDGEMENTS We thank Stephanie technical assistance and Uranium Mines Pty Ltd also thank Helen Allison sity of California, Irvine, species.
H u n t and Jane Summerton for their excellent Peter Cusbert for the chemical analyses. Ranger supplied the chemical analyses of RP2 water. We and Professor Richard D. Campbell of the Univerfor their help in the identification o f the two hydra
REFERENCES Anonymous, 1983. Guidelines for Surface Water Quality. Vol. 1, Inorganic Chemical Substances, Inland Water Directorate, Water Quality Branch, Ottawa, Canada. Anselme, K., K. Julliard and S. Blaineau, 1990. Degradation of metal-labelled collagen implants: ultrastructural and X-ray microanalysis, Tissue Cell, 22: 81-91. Ahsanullah, M. and A.R. Williams, 1986. Effect of uranium on growth and reproduction of the marine amphipod Allorchestes compressa, Mar. Biol., 93: 459-464. Allison, H.E. and D.A. Holdway, Supervising Scientist for the Alligator Rivers Region, Alligator Rivers Region Research Institute, Annual Research Summary for 1987-1988, Australian Government Publishing Service, Canberra, p. 68. Berlin, M., and B. RudeI1, 1979. In: L. Friberg, G.F. Nordberg and V.B. Vouk, (Eds), Handbook on the Toxicology of Metals. Elsevier, Amsterdam, p. 647. Blanquet, R. and H.M. Lenhoff, 1966. A disulfide-linkedcollagenous protein of nematocyst capsules, Science, 154: 152-153. Browne, C.L. and L.E. Davis, 1977. Cellular mechanisms of stimulation of bud production in Hydra by low levels of inorganic lead compounds, Cell Tissue Res., 177: 555-570. Cairns, J. and W.H. Van Der Schalie, 1980. Biological monitoring Part I -- early warning systems, Water Res., 14:1179-1196. Chassard-Bouchard, C., 1983. Cellular and subcellular localization of uranium in the crab Carcinus maenas: a microanalytical study, Mar. Pollut. Bull., 14: 133-136. Chassard-Bouchard, C., 1984. Lysosomes and pollution, Biol. Cell, 51: 15A. Ellis, W.R. and M. Ahsanullah, 1984. The use of nuclear techniques to investigate the levels of uranium in marine waters and its uptake and distribution by marine biota, Nucl. Tracks Radiat. Measmt, 8: 437-441.
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Epp, L.G. and D.C. Koblick, 1977. Relationship of intracellular potassium to asexual reproduction in Hydra, J. exp. Biol., 69: 45-51. Galun, M., S.M. Siegel, M.I. Cannon, B.Z. Siegel and E. Galun, 1987. Ultrastructural localization of uranium biosorption in Penicillium digitatum by stem x-ray microanalysis, Environ. Pollut., 43: 209-218. Hart, B.T. and R.J. McGregor, 1980. Limnological survey of eight billabongs in the Magela Creek catchment, Northern Territory, Aust. J. Mar. Freshwater Res., 31: 611-626. Hart, B.T., E.M. Ottaway and B.N. Noller, 1987. Magela Creek systems, Northern Australia. I. 1982-3 Wet season water quality, Aust. J. Mar. Freshwater Res., 38: 261-288. Horikoshi, T., A. Nakajima and T. Sakaguchi, 1979. Uptake of uranium from sea water by Synechococcus elongatus, J. Ferment. Technol., 57: 191-194. Humphrey, C.L., K.A. Bishop and V.M. Brown, 1990. Use of biological monitoring in the assessment of effects of mining wastes on aquatic ecosystems of the Alligator Rivers Region, tropical northern Australia, Environ. Monit. Assess., 14: 139-181. Hyne, R.V., 1990. In: R.A. McGill and K.A. Malatt, (Eds), Workshop on Biological Toxicity Testing as a Regulatory Mechanism. Northern Territory Department of Mines and Energy, Darwin, p. 81. Koblick, D.C. and L.G. Epp, 1975. Control of growth of Hydra cultures by tissue potassium, Comp. Biochem. Physiol., 50A: 387-389. Kramer, H.J., H.C. Gonick and E. Lu, 1986. In vitro inhibition of Na-K-ATPase by trace metals: relation to renal and cardiovascular damage, Nephron, 44: 329-336. Langmuir, D. 1978. Uranium solution-mineral equilibria at low temperatures with applications to sedimentry ore deposits, Geochim. Cosmochim Acta, 42: 547-569. Leggett, R.W. 1989. The behaviour of chemical toxicity of uranium in the kidney: a reassessment, Health Phys., 57: 365-383. Lentz, T.L. and R.J. Barrnett, 1961. Enzyme histochemistry of hydra, J. Exp. Zool., 147: 125-147. Lesh-Laurie, G.E., 1982. In: F.W. Harrison and R.R. Cowden, (Eds), Developmental Biology of Freshwater Invertebrates. Allan R. Liss, New York, p. 69. Lue, K.Y and A.A. De La Cruz, 1978. Mirex incorporation in the environment: toxicity in Hydra, Bull. Environ. Contam. Toxicol., 19: 412-416. Macklin, M. 1967. Osmotic regulations in hydra: sodium and calcium localization and source of electrical potential. J. Cell. Physiol., 70: 191-196. Nakajima, A., T. Horikoshi and T. Sakaguchi, 1981. Distribution and chemical state of heavy metal ions absorbed by Chlorella cells, Agric. Biol. Chem., 45: 903-908. Nechay, B.R., J.D. Thompson and J.P. Saunders, 1980. Inhibition by uranyl nitrate of adenosine triphosphatases derived from animal and human tissues, Toxicol. Appl. Pharmacol., 53: 410-419. Poston, T.M., R.W. Hanf and M.A. Simmons, 1984. Toxicity of uranium to Daphnia magna, Water, Air, Soil Pollut., 22: 289-298. Pribil, S. and P. Marvan, 1976. Accumulation of uranium by the chlorococcal alga Scenedesmus quadricauda, Arch. Hydrobiol. Suppl. Algol. Stud., (15) 49: 214-225. SAS Institute Inc., 1985. SAS User's Guide: Statistics, version 5 edition. Cary, NC, USA, p. 449. Slooff, W., J.H. Canton and J.L.M. Hermens, 1983. Comparison of the susceptibility of 22 freshwater species to 15 chemical compounds. 1. (Sub) acute toxicity tests. Aquat. Toxicol., 4: 113-128. Stauber, J.L. and T.M. Florence, 1985. Interactions of copper and manganese: a mechanism
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by which manganese alleviates copper toxicity to the marine diatom, Nitzschia closterium (Ehrenberg), W. Smith. Aquat. Toxicol., 7: 241-254. Stebbing, A.R.D. and A.J. Pomroy, 1978. A sublethal technique for assessing the effects of contaminants using Hydra littoralis, Water Res., 12: 631-635. Strandberg, G.W., S.E. Shumate and J.R. Parrott, 1981. Micorbial cells as biosorbents for heavy metals: accumulation of uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa, Appl. Environ. Microbiol., 41: 237-245. Tabachnick, B.G. and L.S. Fidell, 1983. Using multivariate statistics. Harper and Row, New York, p. 93. Tarzwell, C.M. and C. Henderson, 1960. Toxicity of less common metals to fishes, Ind. Wastes, 5: 12. Vuceta, J. and J.J. Morgan, 1978. Chemical modelling of trace metals in fresh waters: role of complexation and adsorption, Environ. Sci. Technol., 12: 1302-1309.
159
pH-Dependent uranium toxicity to freshwater hydra R.V. H y n e a, G . D . R i p p o n a a n d G. Ellender b
aOffice of the Supervising Scientist, Alligator Rivers Region Research Institute, Jabiru, N.T. 0886, Australia bSchool of Dental Science, University of Melbourne, Parkville, Victoria 3052, Australia ABSTRACT A research program was initiated to characterise the major toxicants in water from Retention Pond 2 (RP2) at Ranger uranium mine. RP2 water was bioassayed using local hydra species, with water from Magela Creek used as a control and diluent (pH 6.1-6.7, conductivity 12-20 #S/cm). The bioassay had survival and population growth after 4-6 days as endpoints. Uranium was identified as the major toxicant in RP2 water, and further studies were carried out to assess its toxicity. At pH 8.5, in the presence of bicarbonate, RP2 water containing uranium at concentrations up to 3900 ppb, and uranium at 1000 ppb added to Magela Creek water, did not affect survival or population growth. However, RP2 water made to 32% with diluent resulting in a pH of 8.0, and uranium concentrations _ 200 ppb in Magela Creek water held at a normal pH of 6.5, did affect survival and population growth. In the absence of uranium in Magela Creek water, the hydra population growth or survival after 6 days was not affected by pH 5.0-8.0 and 5.0-8.5, respectively, pH was adjusted by adding acetic acid or sodium bicarbonate which also increased the conductivity. Nevertheless, an increase in the conductivity > 400 #S/cm affected the population number only after 6 days. This pH-specific effect of uranium toxicity is discussed in relation to the uranyl carbonate complexes of uranium formed in solution.
Key words: Hydra; uranium; toxicity; pH INTRODUCTION T h e R a n g e r u r a n i u m m i n e is o n a lease site s u r r o u n d e d b y K a k a d u N a t i o n a l P a r k in tropical n o r t h e r n Australia. As p a r t o f the m i n e ' s w a t e r strategy it has a series o f r e t e n t i o n p o n d s which act as sediment traps a n d r u n o f f catc h m e n t s in the wet season. T h e s e p o n d s have differing chemical w a t e r qualities, differing r e t e n t i o n capacities, a n d t h r o u g h r e g u l a t o r y criteria, differing f r e q u e n c y in their possibility o f release. F o r example, release o f R e t e n tion P o n d 2 ( R P 2 ) water, w h i c h has a very high u r a n i u m a n d m a n g a n e s e c o n t e n t (Allison a n d H o l d w a y , 1988; H y n e , 1990), into the M a g e l a C r e e k is o n l y p e r m i t t e d in a n e x c e p t i o n a l l y high rainfall wet season. This c o r r e s p o n d s to a f r e q u e n c y o f a b o u t o n c e in e v e r y 10 years p r o v i d i n g o t h e r r e g u l a t o r y c o n d i t i o n s are also met.
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Past mine waste water releases were made in accordance with regulatory criteria. These regulatory criteria, however, did not require biological testing of the waste water prior to release. This was despite substantial accumulated knowledge that only biological evidence could indicate the potential impact of the released water on any ecosystem (e.g. Cairns and Van Der Schalie, 1980). Because of this lack of biological data, we explored the nature of toxicity of these retention pond waters using locally collected species in bioassays developed in this laboratory. Freshwater hydra are sensitive to a number of environmental pollutants including metals (Browne and Davis, 1977; Stebbing and Pomroy, 1978) and some organic compounds (Lue and De La Cruz, 1978; Slooff et al., 1983). Bioassays using hydra asexual reproductive population growth as an endpoint are a sensitive and precise way to measure the effect of pollutants (Stebbing and Pomroy, 1978; Lue and De La Cruz, 1978). A bioassay using two hydra species collected from Magela Creek was developed based on the procedure described by Stebbing and Pomroy (1978). Preliminary experiments showed that uranium added to creek water at concentrations equal to, or greater than, 200 ppb can be detected by the inhibition of population growth of Hydra viridissima after 4 days (Allison and Holdway, 1988). However, when the hydra bioassay was used to assess the toxicity of water from RP2 containing high concentrations of uranium (3900 ppb), there was no effect on hydra population growth after 4 days. As waste water discharged from Ranger flows into river systems of Kakadu National Park, which has been entered onto the World Heritage List and the Convention on Wetlands of International Importance, any releases need to be carefully controlled to avoid environmental damage. The purpose of this study was to investigate the apparent anomaly between the high sensitivity of hydra to uranium salts added to Magela Creek water, and the low sensitivity of hydra to uranium present in a complex water from a mine retention pond. MATERIALS
AND
METHODS
Dilution and test waters
The dilution water used in most experiments was Magela Creek water (Hart et al., 1987), and during the wet season was collected near Georgetown Billabong (Humphrey et al., 1990) upstream of the Ranger uranium mine waste water discharge pipe outlet. The pH of the Magela Creek water ranged from 6.1 to 6.7 and the conductivity varied from 12 to 20 #S/cm during the experimental period. During the dry season, one experiment was undertaken and the dilution water was collected from Buffalo Billabong, a permanent water-body seasonally connected with Magela Creek. The Buffalo Billabong
pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
161
water collected had a pH of 6.5 and conductivity of 25/zS/cm. All dilution waters were collected in clean plastic bottles on the morning of the day the bioassay was to commence and filtered through a coarse filter (Whatman No. 1 filter paper) to remove wild zooplankton. The dilution water used in a test was also used as the control water. RP2 water was collected from the mine site as close as it was practicable to the start of the bioassay in a clean plastic bottle. The water was filtered through a coarse filter and various dilutions prepared. All waters were stored at 4°C until needed.
Hydra bioassay The test species was either H. viridissima or H. vulgaris (Cnidaria, Hydrozoa). The test animals were obtained from laboratory stocks reared in the same water that would be used as dilution and control water in the toxicity test. All animals selected were mature and asexually reproducing by budding. Only hydroids with a tentacled bud were selected as test organisms; a hydroid is a term used in this paper to describe an individual animal with or without buds. Five asexually reproducing test animals were covered with 30 ml of test solution in plastic Petri dishes (90 m m diameter). Each treatment was repeated in triplicate. The test solution was changed each day for fresh test solution of the same concentration and temperature. Daily observations were made of the degree of tentacle contraction (not contracted, partially contracted, or fully contracted) and changes in the number of intact hydroids in each treatment. Conductivity and pH of each test solution was also recorded daily. Test cultures were fed daily and maintained at 30°C with a 12-h photoperiod.
Hydra feeding and cleaning Hydra were fed by individually feeding each hydroid with live brine shrimp nauplii (Artemia salina) made up in the appropriate dilution water, and placed into each Petri dish using a glass Pasteur pipette. The nauplii were made up in the appropriate test solution. Feeding was ad libitum for 30 min after which the Petri dishes were cleaned and test solutions changed. Since the majority of individual H. viridissima adhere to the surfaces of the Petri dish, Petri dishes were cleaned in the following manner. First, the contents of each Petri dish are swirled to dislodge any uneaten brine shrimp and regurgitated food pellets. Each Petri dish is then drained of its contents and a fresh 10-ml aliquot of the appropriate test solution is added and the swirling/draining procedure repeated. Any hydra subsequently dislodged when the Petri dish was drained were carefully picked up in water using a clean
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glass Pasteur pipette and returned to the test Petri dish. A fresh 30-ml aliquot of the appropriate test solution was then added to the test Petri dish and any remaining brine shrimp or unhatched eggs individually removed by pipette. Since most individuals of H. vulgaris float on the water surface by means of a gas bubble produced by the pedal disk, these hydra are simply pipetted into a clean Petri dish containing a fresh 30 ml of the appropriate test solution leaving behind the uneaten brine shrimp and waste food pellets. Any hydra that are attached are gently removed with a clean glass Pasteur pipette and transferred to the new Petri dish.
Statistical analysis Statistical significance is determined for the number of intact hydroids between each test solution and the control over each observation period. The data were tested to ensure that the numbers of intact hydroids are normally distributed and their variances are homogenous. This was done by examining scatter-plots of predicted values for the dependent variable against the residual of a regression analysis (Tabachnick and Fidell, 1983). Another test for the dependent variable's normal distribution was carried out using computed normal scores from Blom's formula and plotting these scores against their actual value (SAS Institute Inc., 1985). Should the data not be normally distributed they were appropriately transformed to allow valid statistical analysis. Thereafter, a two-way Analysis of Variance (ANOVA) was performed with replicates and concentration as the main factors. If replicates were not significantly different, they were pooled and further analysis carried out using a two-way ANOVA, with waste concentration and observation time kept as the main factors. The effect of the test solutions was also examined by a one-way ANOVA with treatment water concentration as the main factor. If the analyses showed that the treatment effects were significant (P _< 0.05), then individual differences between means were assessed using the Least Square Means Test (for the two-way ANOVA) or Fisher's leastsignificant-difference test (for the one-way ANOVA) using the mean square error from the analysis of variance. The probability level for indicating detection of differences was 0.05.
Added uranyl sulphate and treatments A stock solution of uranyl sulphate was made by adding 40 mg of O2SO4" 3H20 to 2 litres of the dilution water. A dilution series of this stock solution was made with dilution water to give the desired concentration of added uranium. These test solutions were stored at 4°C until required.
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pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
A stock solution of uranyl sulphate and manganese sulphate was made by adding 40 mg of UO2SO4" 3H20 and 7 mg of MnSO4- H20 to 2 litres of the dilution water. A dilution series of this stock solution was made with dilution water to give the desired concentration of added uranium and manganese. In experiments where the pH required adjustment, sodium bicarbonate, sodium carbonate, tris (hydroxymethyl) methylamine (Tris) or acetic acid was added to the test solution to achieve the desired pH. The pH of the test solutions was adjusted as desired and stored at 4°C until required. In experiments where the conductivity required adjustment, sodium chloride was added to the water to achieve the desired conductivity.
Water chemistry analysis The concentrations of metals added to the test waters were confirmed by analysis using inductively coupled plasma-mass spectrometry. In some experiments though, uranium was determined by Scintrex Time Delay Fluorimetry and manganese was determined by Graphite Furnace Atomic Absorption Spectrometry.
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164
R.V. HYNE
ET AL.
RESULTS
Toxicity of uranium to two hydra species H. viridissima were exposed to various concentrations of uranium added to water collected from Magela Creek through the 1989-1990 wet season. Significant inhibition in the population growth was obtained with uranium at a concentration of 200 ppb or greater, relative to the controls (Fig. 1). Conductivity and pH were not significantly affected by the addition of uranium. Similar results were obtained when H. viridissima were exposed to uranium added to water collected from Buffalo Billabong in the 1990 Dry season. There was a significant reduction in the population growth when uranium was 150 ppb or greater. H. vulgaris also showed a distinct wet to dry seasonal change in sensitivity to added uranium with the lowest observed effect concentrations (LOEC) varying from 550 to 400 ppb, respectively. Toxicity of water from ranger RP2 In December 1989, prior to the commencement of the wet season, bioassays on RP2 water from Ranger uranium mine showed that 100% RP2 water did not affect H. viridissima's population growth over a 4-day period
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pH DEPENDENT
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HYDRA
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despite the presence of high concentrations of uranium (3900 ppb). However, if the RP2 water was diluted with diluent water and then tested for toxicity, a strong inhibition of population growth was observed (Fig. 2). The observed toxicity was associated with a reduction in the pH of the diluted RP2 water.
Effect of manganese on the toxicity of uranium The addition of uranium (1000 ppb) and manganese (100 ppb) to Magela Creek water, and pH adjusted with sodium bicarbonate, gave a similar effect to that observed with RP2 water (Fig. 3 compared with Fig. 2). That is, there was no significant effect of added uranium and manganese at an elevated pH of 8.6 on H. viridissima's population growth over 4 days. In contrast, at a pH of 6.6 there was a marked inhibition of population growth. These effects were very similar to that of the 100% RP2 water sample. After 1 day of addition of the uranium and manganese to Magela Creek water at a pH of 8.6 a sample of water was collected. An aliquot of this sample was filtered through a 0.45-tzm filter. Both unfiltered and filtered water were chemically analysed for uranium and manganese. Similar results for both samples were obtained. The reduced toxicity of water at pH 8.6 appeared not to be due to precipitation of uranium from solution with manganese dioxide since both
166
R.V. H Y N E ET AL.
4
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167
pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
unfiltered and filtered water had similar uranium and manganese concentrations.
pH-Dependance of uranium toxicity The pH-dependent effect of uranium toxicity to H. viridissima was examined by assessing the extent of population growth inhibition by a known toxic dose of uranium (900 ppb). The uranium was added to Magela Creek water and the pH adjusted with acetic acid, sodium bicarbonate, tris (hydroxymethyl) methylamine (Tris) or sodium carbonate to achieve the desired pH. If Tris was used to buffer the uranium treatment to pH 8.5, a strong inhibition of population growth was observed compared with a Trisbuffered control water at pH 8.5. But when the pH was adjusted to 8.5 with bicarbonate, no significant effect of uranium was observed on population growth over 4 days compared with the control (Fig. 4). In contrast to this effect of uranium and bicarbonate, for treatments adjusted to pH 8.0 or less, or adjusted to pH 9.0 with carbonate, there was a strong inhibition of population growth (Fig. 4). The uranium still had a strong inhibitory effect on population growth if the pH was adjusted to pH 5.0 with acetic acid.
51 i Magela Creek Control pH 6.1 Conductivlty 12 uS/cm Added NaCl
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i
i
i
i
I
2
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(days)
Fig. 6. The effect of increased conductivity on Hydra viridissima's population growth rate of over 6 days. The conductivity of the Magela Creek water was increased by the addition of sodium chloride. Each point is the mean of three replicates. Points with a common alphabetical superscript (within each daily measurement) are not significantly different (P < 0.05, ANOVA) from control.
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R.v. HYNE ET AL.
Effect of water pH on hydra population growth The effect of pH alone on population growth of H. viridissima over 6 days was determined by adjusting the pH of the Magela Creek water with acetic acid, sodium bicarbonate or sodium carbonate. Over the pH range 7.0-8.0, the population growth was not significantly different from the Magela Creek control water (pH 6.7). At pH 8.5 there was a small but significant reduction in population growth while at pH 9.0 hydra survival was severely impaired (Fig. 5). A further study showed that over the pH range 5.0-7.0 population growth was also not significantly different from the Magela Creek control.
Effect of increased conductivity on hydra pH Adjustment of test waters caused a change in conductivity with the addition of concentrated acidic or alkaline solutions. Therefore, the effect of increased conductivity alone on H. viridissima population growth over 6 days was examined by adding sodium chloride to the Magela Creek water (Fig. 6). On day 4 conductivities of 600 ttS/cm or greater inhibited population growth rate while by day 6 conductivities of 400 ~S/cm or greater inhibited population growth. DISCUSSION Uranium concentration in the surface waters of streams and rivers within Kakadu National Park is typically less than 1 ppb (Hart et al., 1987). Since uranium is non-essential for biological processes and is generally toxic at elevated concentrations (Berlin and Rudell, 1989; Leggett, 1979), concerns for the protection of aquatic biota exposed to contaminated waters must be considered. Ionic strength and composition are known to affect hydra size and asexual reproduction as observed by population growth (Lesh-Laurie, 1982). Koblick and Epp (1975) have previously shown that increasing osmolarity produces a progressive decrease in the population growth of hydra. This decrease was also accompanied by a decrease in the tissue potassium content (Epp and Koblick, 1977). Results from the present study show that low concentrations of uranium in diluent water ( >_ 200 ppb) have a direct inhibitory effect on the population growth of hydra. This inhibitory effect could not be attributed to an increase in the conductivity of the test water for there was no significant difference between conductivities of the test solutions. But, the toxicity of the uranium (900 ppb) to hydra, in diluent water and in the absence on manganese, was shown to be pH-dependent in the presence of bicarbonate. At pH 8.5,
pH DEPENDENT URANIUM TOXICITY TO FRESHWATER HYDRA
169
uranium in bicarbonate buffer was found to have no significant effect on H. viridissima population growth over 4 days. Bicarbonate-buffered undiluted RP2 water (pH = 8.6) also showed no significant decrease in H. viridissima population growth over 4 days. In contrast, Tris-buffered water with added uranium remained toxic at pH 8.5 compared with an appropriate Trisbuffered control water. This absence of toxicity of uranium at a pH of 8.5 in bicarbonate buffered RP2 water and Magela Creek water could be caused by the formation of uranyl carbonate complexes. At pH 8.5, the dominant uranyl carbonate complex in a typical soft water such as Magela Creek water would be the tricarbonate UO2(CO3)3 4- complex described by Langmuir (1978), in his definitive speciation model of uranyl-carbonate complexes. This negatively charged and extremely soluble ionic complex would experience charge conflicts at cell membrane surfaces which would result in decreased uranium adsorption. In soft water and at a lower pH range of 6-7, the predominant forms of uranium in Langmuir's speciation model are the divalent cationic species UO22÷ or the neutral uranyl carbonate complex UO:CO3, which would be more membrane permeable. Similar interaction between pH and carbonate ion concentration affecting uranium uptake by a marine algae has been reported (Horikoshi et al., 1979). The mine waste water from RP2 would have a complex water chemistry, and many geochemical changes could possibly contribute also to changes in its toxicity. RP2 water would also be expected to contribute a positive nutrient effect on the hydra as well as the negative toxic effect of uranium. However, uranium is present in RP2 water at concentrations an order of magnitude higher than any other toxic element. With dilution during a release of RP2 water into a receiving stream, the toxicity of uranium would then be expected to be the dominant effect. The concentration of soluble manganese in natural waters is also known to vary due to changes in pH of the water (Vuceta and Morgan, 1978). This could possibly affect changes in toxicity of any complex water (Stauber and Florence, 1985). The possible synergistic (or antagonistic) effect of manganese (Mn 2÷) on the toxicity of added uranium was investigated. However, manganese was found not to significantly modify the effect of uranium on H. viridissima population growth at either pH 6.6 or 8.6. In this study uranium was found to be very toxic to hydra within the pH range of 5-7. Nevertheless, within this pH range, pH-adjusted Magela Creek water (no added uranium) showed no adverse effects on the population growth of H. viridissima compared with Magela Creek control water with a pH of 6.7. At a high pH of 9.0, Magela Creek water, with or without uranium (900 ppb), was toxic to H. viridissima. However, this pH is beyond the normal physiological range for hydra and so was not investigated further.
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Like many other metals uranium is accumulated by various aquatic organisms, particularly by fungi, algae, bacteria and invertebrates (Standberg et al., 1981; Nakajima et al., 1981; Ellis and Ahsanullah, 1984; Galun et al., 1987). Uranium has a high affinity for proteins and lipids at the pH found intracellularly, thus preventing reversal desorption. Concentration factors for algae in the range 102-103 based on wet weight have been reported (Pribil and Marvan, 1976; Anonymous, 1983). Uranium can accumulate in fungi, algae and bacteria to relatively high concentrations without apparent toxic consequences. A wide spectrum of results has been obtained in studies concerned with the site of uptake of uranium in microogranisms. The extent of variability was demonstrated by Strandberg et al. (1981) who showed that uranium accumulated extracellularly on the surface of a species of yeast while there was a rapid and massive intracellular accumulation of uranium by a species of bacteria. Galun et al. (1987) showed that fungus can accumulate high concentrations of uranium both intracellularly and extracellularly on the surface, depending on the experimental conditions. Intracellular accumulation of uranium has also been documented in the lysosomes, macrophages, gill epithelia, hindgut epithelia and hepatopancreas cells of molluscs and crustacea exposed to uranium contaminated sea water or food (Chassard-Bouchard, 1983, 1984). Hence, it is now well documented that substantial amounts of uranium may be accumulated inside some types of cells. This accumulated uranium may or may not have a toxicological effect on the cell. Any development of toxicity will depend upon whether the uranium binds to a protein and interferes in its function or whether it is detoxified by the formation of a uranium phosphate microgranule, as observed in the crab (ChassardBouchard, 1983). Uranium has also been shown to inhibit ATPase (Nechay et al., 1980; Kramer et al., 1986), an enzyme located on the plasma membrane of the epidermal cell layer of hydra (Lentz and Barrnett, 1961; Macklin, 1967) and which is involved in osmoregulation and other active transport processes. Uranium has been reported to form a strong association with collagen (Anselme et al., 1990), which is present in the nematocyts of hydra (Blanquet and Lenhoff, 1966). Any binding of uranium with collagen in the nematocyts of hydra may interfere with the hydra's feeding response to prey. There has been very little information published on the toxicity of uranium to aquatic organisms. Tarzwell and Henderson (1960) using fathead minnows reported 96-h LCs0s for uranyl ion in soft water (pH 7.4, hardness 20 mg/1 as CaCO3), which was introduced as uranyl nitrate or uranyl acetate, as 3.1 and 3.7 ppm, respectively. Poston et al. (1984) obtained significant reduction in survival of Daphnia magna after 5 days at a uranium concentration of 520 ppb. Ahsanullah and Williams (1986) reported a reduction in the growth of a marine amphipod, Allorchestes compressa, by uranium at 2 ppm
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after 4 weeks. In contrast, after 4 days we found the L O E C for H. viridissima to be 150 ppb in the dry season, or 200 ppb in the wet season. Further, H. vulgaris had LOECs for the dry and wet season of 400 and 550 ppb, respectively. This seasonal difference in uranium toxicity may be due to seasonal changes in water quality (Hart and McGregor, 1980), or perhaps due to subtle changes in composition, which are o f an antagonistic/synergistic nature. The toxicological mechanism by which uranium affects the survival and population growth o f hydra, and the ultrastructural localisation of uranium absorbed by the hydra, is currently being investigated. ACKNOWLEDGEMENTS We thank Stephanie technical assistance and Uranium Mines Pty Ltd also thank Helen Allison sity of California, Irvine, species.
H u n t and Jane Summerton for their excellent Peter Cusbert for the chemical analyses. Ranger supplied the chemical analyses of RP2 water. We and Professor Richard D. Campbell of the Univerfor their help in the identification o f the two hydra
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