International Journal of Salt Lake Research 4: 133-163, 1995. �9 1995 KluwerAcademic Publishers. Printed in the Netherlands.

Effects of heavy metals on the survival of Diacypris compacta (Herbst) (Ostracoda) from the Coorong, South Australia

ANNA BROOKS 1, REHEMA M. W H I T E 1,2 and DAVID C. PATON 1 1 Department of Zoology, University of Adelaide, Adelaide, South Australia 5005, Australia; 2 Department of Zoology and Entomology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa

Abstract. The hypermarine southern Coorong is threatened by proposals to drain relatively fresh surface water and groundwater from adjacent agricultural areas into the Coorong. These influent waters carry moderate loads of heavy metals. Acute toxicity of heavy metals to Diacypris compacta, an abundant ostracod in the Coorong, was measured in the laboratory at 18 ~ in a static system using Coorong water (pH 7.8, salinity 50 ppt). At 4 days (96 h) the mean values of LCs0 for copper, zinc, lead and cadmium respectively were 0.8, 2.1,3. I and 4.3 mg L - 1, and at 8 days the respective mean LCs0s were 0.4, 0.7, 2.2 and 1.1 mg L - i. The effect of two or three metals on mortality was additive in some cases and synergistic in other cases, but generally less than additive. However, in all cases mortality was greater in the presence of two or three metals than in the presence of a single metal. According to ANZECC (1992) guidelines, maximum acceptable concentrations of heavy metals should be no higher than 0.01 • the lowest LCso value. Using the lowest LCs0 values for Diacypris compacta obtained at 8 days, maximum acceptable concentrations in the Coorong would be 4, 5, 9 and 22 #g L - l for copper, zinc, cadmium and lead respectively, the values for zinc and copper falling below those recommended by ANZECC (1992) for marine waters. Reported concentrations of copper and zinc in surface water and groundwater in areas adjacent to the Coorong sometimes exceed these values, hence drainage of these waters into the Coorong represents a significant hazard to the Coorong biota.

Key words: Coorong, heavy metals, acute toxicity, Ostracoda, Diacypris

Introduction

The Coorong is a narrow coastal lagoon system which runs for 120 km from near the mouth of the Murray River towards Kingston, in the South East of South Australia. There are two main lagoons, the North and the South Lagoons, which are joined by a stretch of water 100 m wide known as The Narrows. A gradient of increasing salinity from north to south provides a variety of water conditions ranging from estuarine in the north to hypermarine in the south (Geddes and Butler, 1984; Molsher et al., 1994). This unique lagoon system is conserved within the Coorong National Park and the area has been declared a Wetland of Intemational Significance to Waterfowl under the RAMSAR Agreement. The Coorong supports several hundred thousand birds during the summer and autumn months (Paton, 1982; Jaensch and

134

Barter, 1988). These waterfowl include waterbirds (ducks, terns and grebes) and waders (sandpipers, stilts and plovers), and some species are listed on the Japan Australia Migratory Bird Agreement and the China Australia Migratory Bird Agreement. In addition there are a number of species of fish, several of which are prolific enough to support a small commercial fishing industry (Hall, 1984).

The integrity of the hypermarine southern Coorong is now threatened by proposals to drain surface water and groundwater into the Coorong from adja- cent areas in the Uppe~ South East of South Australia. The agricultural areas of the Upper South East of South Australia suffer from dryland salinization and local surface flooding as a result of extensive vegetation clearance (Bakers Range and Marcollat Watercourses Working Group, 1991; Upper South East Dryland Salinity and Flood Management Plan Steering Committee, 1993). The prefered solution is to drain surplus surface water and some of the saline groundwater into the southern end of the South Lagoon of the Coorong. How- ever, these influxes of predominantly fresh water will reduce salinities in the South Lagoon (CFMI, 1992) and may alter other components of the Coorong ecosystem by introducing heavy metals, nutrients and other contaminants. Heavy metals are of particular concern because of their environmental persis- tence, toxicity at low concentrations and ability to be incorporated into food chains and concentrated by aquatic organisms (Negilski, 1976; Connell and Miller, 1984; ANZECC, 1992). In the Coorong, they could adversely affect the ecosystem through their accumulation in organisms at the lower trophic levels and then on to higher trophic levels including birds and fish.

Agricultural activity in the Upper South East of South Australia has con- tributed to the loads of heavy metals and other nutrients and contaminants in the water bodies in the Upper South East. Soils in the Upper South East of South Australia are deficient in agricultural terms in certain trace elements, especially zinc and copper, and landowners have applied these trace elements directly to the land to increase agricultural production. Superphosphate and single phosphate fertilisers have also been applied. Such fertilisers can also be contaminated with heavy metals with maximum possible concentrations listed by suppliers being quite high (e.g. zinc 200 ppm; copper 140 ppm; cadmium 30 ppm). Thus the total amount of heavy metals applied (delib- erately as trace elements and as impurities with fertiliser) can therefore be substantial, particularly if Department of Agriculture guidelines for frequen- cy and quantity of application of trace elements are exceeded. The history of trace element and fertiliser use in the Upper South East of South Australia is described in more detail by Eco Management Services (1992).

Trace element and fertiliser runoff and leaching, which are exacerbated by application before rain or by single large applications instead of multiple

135

smaller doses, result in water being polluted by heavy metals and nutrients (Connell and Miller, 1984). There are only fragmentary data available to indi- cate current concentrations of heavy metals in surface water and groundwater in the Upper South East of South Australia. However, the figures indicate that concentrations of some heavy metals are quite high, particularly for zinc and copper, as expected from agricultural practices. In surface water, levels of zinc and copper as high as 374 #g L- 1 and 400 #g L- 1 respectively have been recorded, often with high concentrations of both at the same site, although many sites have undetectable levels of either metal (Eco Management Ser- vices, 1992). In groundwater, levels of cadmium were low, and copper and lead were rarely present with maximum levels of 50 and 32 #g L -1 respec- tively. However, the distribution of zinc was patchy with some sites having concentrations as high as 822 #g L -1. These concentrations of heavy met- als approach or exceed the maximum acceptable levels of zinc and copper for fresh and marine water in Australia. ANZECC (1992) recommends that zinc and copper concentrations in freshwater should not exceed 5-50 #g L-1 and 2-5 #g L-1 respectively, depending on water hardness, and in marine water 50 #g L -1 and 5 #g L -1 . Drainage of this contaminated water into the Coorong therefore clearly represents a considerable hazard.

The aim of this study was to determine the potential effects of heavy metals on the Coorong ecosystem by measuring the toxicities of zinc, copper, cadmium and lead to an ostracod, Diacypris compacta. This species was chosen for several reasons. First, crustaceans form an important link in the foodchains of the Coorong. Diacypris compacta occurs throughout the South Lagoon and serves as a major food source for hardyhead fish (Atherinosoma microstoma) which in turn are eaten by a variety of piscivorous birds (terns, pelicans, cormorants, grebes, stilts and avocets; Paton, 1982; 1986; Molsher and Paton, unpubl.). Any change in population densities of this ostracod could affect fish and bird populations. Second, D. compacta is a native species, so toxicity effects for this species are directly relevant to the Coorong. Third, microcrustaceans are known to be sensitive to heavy metal toxicity and may therefore act as useful indicators of heavy metal pollution (Fennikoh et al., 1978; Lake et aL 1979; Connell and Miller, 1984). Fourth, this species was known to be easily maintained in the laboratory.

Natural history of Diacypris and taxonomically similar ostracods

There are more than a hundred species of non-marine ostracods in Australia (De Deckker, 1983). Many are widely distributed across Australia, possibly due to passive dispersal of ostracods and their eggs by migrating waterfowl (Kesling, 1956; De Deckker, 1977). Members of the genus Diacypris are found in ephemeral and permanent salt lakes, and Diacypris compacta has

136

been found in salinities up to 218 ppt, although at salinities above 200 ppt they were very sluggish or dying (De Deckker, 1983).

The mode of locomotion varies between species. Most ostracods are ben- thic but some are planktonic nearly all their lives (Kesling, 1956). Diacypris compacta is an adept swimmer but is also found creeping on halophytes and in the upper few millimetres of lake sediments (De Deckker and Geddes, 1980; De Deckker, 1983; pers. observ.).

Carapace size and ornamentation may vary within ostracod populations. Adults of Australocypris and Diacypris species are much larger during winter than in spring and early summer in the Coorong region (De Deckker, 1983). Minimum size coincides with the time of highest temperature, lowest water depth, and highest salinities in the Coorong (Geddes and Butler, 1984; Geddes, 1987; Molsher et al., 1994). Food supply may also influence carapace size in ostracods (De Deckker, 1983) but little is known of the diet of Diacypris spp. Some ostracods feed on periphyton, others are predatory (e.g. Kesling, 1956; Roca et al., 1993). Diacypris compacta probably gleans periphyton while creeping over halophytes and sediments.

The reproductive biology of Diacypris compacta is poorly documented. In the permanent waters of the southern Coorong, D. compacta is present throughout the year but there are no quantitative data to assess seasonal patterns of abundance. Presumably population densities of Diacypris com- pacta in the southern Coorong are maintained by the continual production and hatching of eggs. Eggs of other Diacypris hatch over a wide range of salinities (18-94 ppt; Geddes, 1976). In nearby ephemeral saline lakes, D. compacta is usually present from June to December when the lakes contain water (De Deckker and Geddes 1980; Williams and Kokkinn, 1988). Popu- lation densities remain more or less constant over this period despite changes in salinity and water temperature (Williams and Kokkinn, 1988).

Materials and methods

Collection and maintenance of animals

Samples ofDiacypris compacta were collected from the water column and the benthos at Villa dei Yumpa near the northern end of the South Lagoon using a 150/zm mesh plankton net (70 cm diameter). Small fish were removed from the samples and the remaining organisms retained. Populations were maintained in the laboratory in open plastic tubs (38 x 27 cm) with Coorong water (8 cm deep) and sediment, and small amounts of aquatic plants and animals. The water was continuously aerated and was topped up with distilled water when necessary, so that a salinity of 50 ppt and a pH of 7.8 were

137

maintained. This salinity is typical of the winter-spring salinities measured at the northern end of the South Lagoon (Molsher et aL, 1994). The tubs were maintained in a constant temperature room at 18 ~ under a 12 h light/12 h dark photoperiod. Light was provided by fluorescent tubes at an irradiance of 10 #tool quanta m-2s - 1 (400-700 nm). Algae (Cladophora sp.) collected from Villa dei Yumpa were kept in water under the same conditions in a separate aquarium in the same room. A stock of Coorong water was kept in darkness for use in the experiments. The initial batch of ostracods was collected from Villa dei Yumpa in September 1992 and the colony was supplemented with additional animals collected in October 1992, February 1993 and July 1993.

The distribution and abundance of Diacypris compacta in the Coorong environment were assessed by sampling the sediment, aquatic plants and water column at Villa dei Yumpa in September 1993 as follows. Sediments were sampled using a cylindrical metal core to remove a plug of sediment (1.5 cm diameter • 1 cm deep) which was then preserved in 10% formalin with rose bengal stain. Epifanna samples were taken by cutting off strands of the aquatic plant Ruppia tuberosa, and transferring them to jars containing 70% ethanol (approximately 50 strands per sample), while samples from the water column were taken with a plankton net (30 • 21 cm mouth, 150 #m mesh) and preserved in 70% ethanol. Plankton sampling consisted of making six sweeps of approximately 1 m back and forth across the same point. Five samples were collected from each of the three sampling sites. For water column and sediment samples, the first sample was taken at a depth of 5 cm (water temperature 16.2 ~ and the rest at 5 m intervals away from the shore, to a final depth of 40 cm (water temperature 14.3 ~ Epifauna samples were all taken at 5 cm depth, since Ruppia tuberosa was not available at the other depths in September 1993. In the laboratory the number of whole ostracods in each sample was counted under a dissecting microscope (4-40 x magnification).

Experimental procedures: preliminary trials

Basic procedure and effect of aeration All experiments were conducted at 18 ~ in the light regime described above, and in all cases six ostracods per dish were used. Survival was compared in two types of static system. In the first, ostracods were placed in 10 mL of Coorong water in 55 mm diameter polystyrene petri dishes and covered. Each dish contained approximately 55 mg of Cladophora. Only vigorously swimming ostracods were used. In the second system, each petri dish had a window cut out and covered with fine mesh, and was placed in 75 mL Coorong water in a glass jar. The water in the jars was continuously aerated

138

so that these animals were exposed to a total of 85 mL of oxygenated water, whereas in the first system only 10 mL of Coorong water was present and this was not aerated during the experiment. As the survival was better in the first system rather than the second, all subsequent experiments were conducted using the former.

Effect of macrophytic algae on ostracod survival Ten petri dishes, each containing about 55 mg of Cladophora, and ten dishes without algae were kept in the above conditions for six days. Survival of ostracods was monitored daily. In all other experiments algae were includ- ed.

Effect of total water volume on ostracod survival Ostracods were placed in petri dishes with algae, and either 5.2 or 10.4 mL of Coorong water (ten dishes of each). Since there was no significant effect of volume on survival, some of the subsequent measurements of LCs0 were made with a total volume of 5.2 mL and some with 10 mL.

Effect of ostracod size on survival Ostracods were not measured, but smaller or larger animals from the popu- lation were transferred to petri dishes using a pasteur pipette. Ten dishes of each ostracod size were used, each containing 10 mL of Coorong water, and mortality was assessed after 4 and 8 days.

Experimental procedure for measuring effects of metals on ostracod survival

Petri dishes contained either 9.8 or 5 mL of Coorong water plus 0.2 mL of distilled water and/or stock metal solution. Ten replicates (dishes) of each concentration were used, and ten controls with no metal were included in each experiment. All stock solutions were 1 g L-1 except for ZnC12 which was 0.76g L -1 (checked using atomic absorption spectroscopy). When necessary these were diluted 10 fold immediately prior to use. The metals used were CdC12, CuSO4, ZnSO4 and ZnC12, and PbNO3. At regular intervals, generally 4 and 8 days, mortality was assessed. Dishes were inspected under a dissecting microscope (4-40 x magnification) and ostracods were scored as alive if any movement of appendages was observed, or dead if no such movement occurred even when dishes were gently shaken. Live ostracods were further classified as swimming vigorously or not. In the initial experiments only one metal was present. In later experiments the effects of two or three metals together were also determined. In all these later experiments a volume of 10 mL was used.

139

Measurements of heavy metal concentrations in experimental dishes

Measurements were made to check whether the metals were precipitated in the Coorong water of the experiments (hence altering their concentration), and whether the presence of algae or ostracods affected the metal concentra- tions. Known amounts of metal were added to petri dishes of Coorong water containing either ostracods and algae, algae, or Coorong water alone. Control dishes with ostracods and algae but no metal were monitored concurrently. These dishes were maintained in the constant temperature room under the above conditions. At day 0 and day 4 the water was collected and assayed for zinc, copper, cadmium and lead using Inductively Coupled Plasma methods. The evaporation of water from the dishes was also determined in this experi- ment by weighing the dishes at 0 and 4 days (and weighing known volumes of Coorong water to determine its density). Five replicates of each treatment were used.

In addition, samples of the Coorong water and of the surface sediment collected from Villa dei Yumpa were analysed to determine current levels of heavy metals at that site. Although sediment was not used in our experimental procedure, we measured whether the presence of Coorong sediment would affect the concentration of metal in solution. Known amounts of heavy metal were added to 10 mL of Coorong water with 6 g wet surface sediment, and after 4 days the water was pipetted off the sediment and analysed for heavy metals. Five replicates of each metal were used.

Calculations of LCsos and statistical analyses

The effects of algae, ostracod size and of the volume of water on survival of control animals were each analysed by analysis of variance. The effect of a heavy metal on mortality of ostracods was assessed by calculating the LCs0, the 'median lethal concentration', which was the concentration at which 50% of the animals died after exposure to that metal. LCs0s were obtained from graphs of the mortality of each treatment relative to the control for that experiment against the log concentration. Percentage mortality relative to the controls (% MRC) was calculated as follows:

%MRC = (treatment number dead - control number dead)x 100

control number alive

LCs0 values were calculated for 4 days (96 h) and for 8 days (192 h) of exposure. Since the graphs of MRC against log concentration were often sigmoidal, the LCs0 was obtained by fitting a line between the two points immediately above and below 50% mortality (see Fig. 1), rather than by fitting a straight line through all the data points.

140

0 z,.,-

0

=z

-0.80

00 ~ / /

-0.60 -0.40 .0.20 0.00 0.20 0.40 0.60 0.80 1.00

I ".- Day4 I Day8 I

Log concentmtion

Fig. 1. Effect of zinc sulphate on mortality of Diacypris compacta, illustrating method used to determine the LCs0 concentrations at 4 and 8 days. Each point represents the mean of 10 replicate dishes, each containing a total volume of 10 mL, 6 ostracods, approximately 55 mg Cladophora sp. and enough ZnSO4 to give Zn concentrations from 0.2 to 9 mg L -l. Values of LCso were obtained from this and similar graphs by reading off the concentration giving 50% mortality relative to controls. In this example the LCs0 was 2.1 mg L -1 at 4 days and 0.5 mg L-t at 8 days.

For experiments in which ostracods were exposed to combinations of 2 or 3 metals, an expected additive mortality was calculated from the mortality in individual metal solutions measured as part of the same experiment. This expected additive mortality was compared with the 95% confidence limits of the actual mortality observed in the presence of combined metals. Met- al effects were considered additive if the calculated additive mortalities fell within these 95% confidence intervals, and either synergistic (more than addi- tive) or antagonistic (less than additive) if they fell outside these confidence intervals.

Differences between the concentrations of metal in Coorong water from dishes with Coorong water alone, water and algae or water with algae and ostracods were analysed by one way analysis of variance to determine whether there was significant uptake of heavy metals by ostracods and algae. Differ- ences between concentrations of heavy metals after addition to water alone and to water with sediment were analysed by t-tests.

Results

Habitat use by Diacypris compacta in the Coorong environment

Diacypris compacta used all three microhabitats in the Coorong with 20 q- 5, 21 -4- 7 and 60 + 15 (mean -t- s.e., n = 5) whole Diacypris compacta being

141

~. T Algae

~ 4 .~. N

R >~ 3

~ 2

1

o

0 1 2 3 4 5 6

. ' Days

Fig. 2. Effect of algae on survival of Diacypris compacta. Each dish contained 5.2 mL of Coorong water with or without approximately 55 mg of algae (Cladophora sp.). Each point represents the mean number of ostracods in ten replicate dishes. Bars show the standard errors.

collected per sample from the water column, off Ruppia tuberosa foliage and from the sediments respectively. No direct comparisons between the numbers present in each of these microhabitats are made since the sampling techniques were not comparable. However, densities of Diacypris compacta from the sediments were equivalent to 34 million ostracods m -3.

Survival of control animals

Survival of control Diacypris was significantly improved (F1,60 = 35.0, p < 0.001) by the addition of algae to each dish (Fig. 2). All subsequent experiments were then conducted with algae present. Over a period of 16 days, survival of control animals in a continuously aerated system using 85 mL containers was significantly lower than in a static system (10 mL dishes, not aerated) (F1,54 = 25.0, p < 0.001). Survival in the static system was still 92% after 16 days (Fig. 3). A static system was therefore used for all toxicity experiments. The survival of control animals in this experiment was higher than the mean over all experiments (Fig. 4).

Survival of control animals was not significantly different between 10.4 mL of Coorong water and 5.2 mL (F1,54 = 3.4, p > 0.05). After 10 days there were 1.7 4- 0.2 (mean + s.e, n = 10) and 0.7 �9 0.2 ostracods per dish surviving in 10.4 and 5.2 mL respectively.

142

6

5

~ 4

E �9 ~ 3

E . = 2

Fig. 3.

static

Continuous aeration "~

0 i i i

0 2 4 6 8 10 12 14 16

Days

Survival of Diacypris compacta in static or continuously aerated conditions. In the static system, ostracods and algae were enclosed in polystyrene petri dishes with 10 mL of Coorong water. In the continuously aerated system, windows were cut in identical petri dishes and covered with fine mesh and the dishes were placed in glass jars containing a total volume of 85 mL aerated Coorong water. Each point represents the mean number of ostracods in 10 replicate dishes. Bars represent standard errors. Survival was significantly greater in the static system than the continuously aerated system (p < 0.001).

Under control conditions there was no significant difference between the

survival of small ostracods and that of large ostracods (Ft,20 = 0.78, p > 0.05). After 8 days there were 4.3 4- 0.5 (mean 4- s.e., n -- 10) and 4.2 4- 0.5

ostracods alive per dish for large and small animals respectively. There was some variation in mortality of control animals between experi-

ments which could not be accounted for by alterations in batch of ostracods or volume of water used, and to avoid masking any effects of season or other factors, experiments were not combined and each was analysed separately. Over all experiments the mean survival of control animals was 85.6% (+ 2.3 s.e., 19 experiments) at day 4 and 68.6% (4- 4.4 s.e., 15 experiments) at day

8 (Fig. 4).

Measurements of metal concentrations in the Coorong

Coorong water taken from Villa dei Yumpa and used in experiments had undetectable levels of zinc, copper, cadmium and lead (Table 1). Only one sample of sediment from the site at Villa dei Yumpa was analysed, and this had undetectable levels of copper, cadmium and lead, but a zinc concentration

of 13.1 mg kg - I dry weight.

143

4 " o

�9 -~ 3

E . = 2

!2 4 5

4 1

i

0 2 4 6 8 10 12 14 16

Days

Fig. 4. Long-term survival of Diacypris compacta in control conditions. Data from control animals (no metal) of all experiments are included. Each experiment had ten replicate dishes with 6 ostracods, approximately 55 mg Cladophora sp and 5.2 or 10 mL Coorong water per dish. Points represent the mean number of ostracods alive per dish. The number of experiments is indicated beside each point. Bars represent standard errors.

Table 1. Concentrations of heavy metals in water and sediment from Villa dei Yumpa in the Coorong. Metals were measured by inductively coupled plasma methods at pH 7.8. Metal concentrations in Coorong water (3 replicates) were undetectable, i.e. values given represent the lower limits of detection by the instruments. Sediment concentrations (one sample only) were also undetectable for all metals except zinc. Sediment dry weight was 70% of wet weight.

Metal Coorong water (mg L - I ) Coorong sediment (mg kg - l dry weight)

Zinc < 0.05 13.1 Copper < 0.01 < 0.77 Cadmium < 0.05 < 3.86 Lead < 0.20 < 15.4

Measurements of metal concentrations in the experimental systems

I n d u c t i v e l y c o u p l e d p l a s m a m e t h o d s were not sens i t ive e n o u g h to g ive rel i -

ab le m e a s u r e m e n t s o f l ead at the concen t r a t i ons w e u sed in l a bo ra to ry expe r i -

men t s , and a t t emp t s to m e a s u r e concen t r a t i ons o f the me ta l by a tomic a bso rp -

144

Table 2. Effects of the presence of algae, ostracods and sediment on heavy metal concentra- tions (rag L-~). All petri dishes contained 10 mL Coorong water and 1 mg L -~ metal and were kept at 18 ~ Some dishes also contained 55 mg algae (Cladophora sp.), 6 ostracods or 6 g of wet Coorong sediment. After 4 days metals in the water were measured by inductively coupled plasma methods at pH 7.8. Groups of 4 dishes, each containing a different metal, were pooled prior to measurement such that expected metal concentrations were 0.25 mg L- ~. Values are the means 4- s.e of 5 replicates. Analysis of variance showed no significant effects of algae or ostracods on metal concentrations (F2,n = 1.69, 0.82 and 2.68 for Cu, Zn and Cd respectively; p > 0.05). However, sediment caused a significant lowering of all metal concentrations in the water (t8 = 3.73, 3.66 and 219 for Cu, Zn and Cd respectively; p < 0.01). In some dishes with sediment the metal concentrations were below the detection limit of the intruments. In order to calculate means, these cases were given a value equal to the concentration limit of the instruments, i.e. the means quoted for the last column are slight overestimates.

Metal Coorong water Coorong water Coorong water, Coorong water alone algae algae and ostracods and sediment

Copper 0.10 -t- 0.02 0.12 4- 0.01 0.14 -4- 0.01 0.02 4- 0.00 Zinc 0.31 4- 0.07 0.20 + 0.03 0.19 4- 0.39 0.04 4- 0.01 Cadmium 0.27 4- 0.00 0.25 4- 0.01 0.27 4- 0.01 0.05 4- 0.00

tion were unsuccessful due to interference of sodium with the flame. Mea-

surements of lead concentrations for the experimental treatments are therefore

not presented in Table 2. Measurements of other heavy metals were made at

the pH of the Coorong water used in the experiments, pH 7.8. These concen- trations therefore represent soluble metal under experimental conditions and

not total metal within the system. (Total metal is generally measured at acid

p H when all metal is in a soluble form).

The effects of the presence of algae or ostracods on heavy metal concen- trations under the experimental conditions are shown in Table 2. After 4 days

there were no significant differences (p > 0.05) in metal concentrations in the

water be tween petri dishes containing Coorong water alone, Coorong water with algae, and Coorong water with algae and ostracods. This was true for

all metals and indicates that the amounts of metal accumulated by algae and ostracods were small relative to the amounts present in solution during the

four day period. I f all the added metal had remained in solution, the expected total concen-

tration of metal in these dishes was 0.25 mg L - l . After 4 days the measured concentrat ions were close to 0.25 mg L - l for zinc and cadmium, indicating

that mos t of the metal added was soluble, but were approximately half this value for copper (Table 2), indicating that much of the copper was not soluble in the exper imenta l conditions (Coorong water at 50 ppt, pH 7.8).

145

" , N " \ , " " ".,.. T - - "-" 0.2

-N "~ , - - ' ' 5

" , ~ "~" " 9

" . ' ~ . . . . : . . . - . - . . . - - . - - .= . . . . . . . . . . . .-=.

0

0 4 8

Days

Fig. 5. Effect of zinc sulphate on survival of Diacypris compacta. Each point represents the mean of ten replicate dishes, each of which contained a total volume of 10 mL, approximately 55 mg Cladophora sp. and enough ZnSO4 to give Zn concentrations from 0 (controls) to 9 mg L -~. Bars represent standard errors.

4 ~5

�9 ~ 3

E

When identical amounts of heavy metal were added to dishes of Coorong water with or without sediment, the concentrations of metals in water with sediment were significantly lower than in water alone (Table 2; copper and zinc: p < 0.01; cadmium p < 0.001), i.e. sediment caused a lowering of the metal concentration in the aqueous phase. Sediments were not used in the LCs0 trials.

Influence of evaporation on metal concentrations in experimental system

Over 4 days the average evaporation of water from each dish containing 10 mL was 0.48 mL + 0.01 (mean + s.e., n = 60). After 4 days of treatment the metal concentration would therefore have increased by 4.8%. If the evaporation rate was relatively constant, the mean metal concentration over the 4 day period would therefore be 2.4% higher than the initial concentration at day 0. This difference is small compared with differences in LCs0 between experiments, and therefore values of LCs0 were not adjusted to take evaporation into account.

LCso values for Diacypris compacta

The number of live ostracods decreased with time and this decrease was more marked at higher concentrations of metal. Examples of the effects of each

146

5 � 9 ~ . ~ ' ' ~ - ~ �9 Control " , , " - . 1 ' , . ~ "- . . .

" " , " '~', T " ' \ . ~ " " - - , 1 - ~ 0.2

0 ,

0 4 8

Days

Fig. 6. Effect of copper sulphate on survival ofDiacypris compacta. Each point represents the mean of ten replicate dishes, each of which contained a total volume of 10 mL, approximately 55 mg CIadophora sp. and enough CuSO4 to give Cu concentrations from 0 (controls) to 1 mg L -~. Bars represent standard errors.

"~ 4 =5

�9 =-> 3

E

"~ 4 =5

�9 ~ 3

E , = 2

�9 \

-..i i , ""t \

0 4 8 12

Days

: Control

~ ' - - 0.1

" " * " " 0.3

�9 0.6

. . . . 3 2,

Fig. 7. Effect of cadmium chloride on survival of Diacypris compacta. Each point represents the mean of ten replicate dishes, each of which contained a total volume of 10 mL, approxi- mately 55 mg CIadophora sp. and enough CdC12 to give Cd concentrations from 0 (controls) to 7 mg L- 1. Bars represent standard errors.

147

6

5

3

E E ~ 2

0

0

Fig. 8.

"~" ~' ~' "~" ~ ' \ "~" ~ " "~ " ~ ----= 0.2C~176

~ - t \

".~N - .=.. 9

~ . . . . , . . . . ' ? . - - . "2": 2 " - - ~ m

l 8

Days

Effect of ]cad nitrate on survival of Diacyprix compacta. Each point represents the mean of ten replicate dishes, each of which contained a total volume of I0 mL, approximately 55 mg Cladophora sp. and enough PbNO3 to give Pb concentrations from 0 (controls) to 9 mg L - l . Bars represent standard errors.

Table 3. Values of LCs0 at day 4 and day 8 for Diacypris compacta exposed to zinc, copper, cadmium or lead.

Metal LCs0 (mg L - l ) LCso (mg L - l ) 8d LCs0 at 4 days (96 h) at 8 days (192 h) as a

Mean 4- s.e (n) Range Mean 4- s.e. (n) Range percentage

of 4d LCs0

Zinc 2.06 (1) 0.66 4- 0.15 (3) 0.49~3.95 32% Copper 0.78 4- 0.24 (2) 0.54-1.03 0.42 4- 0.03 (3) 0.38-0.48 54% Cadmium 4.34 4- 0.88 (4) 2.95-6.86 1.09 4- 0.09 (3) 0.94-1.26 25% Lead 3.10 4- 0.62 (2) 2.48-3.72 2.16 (1) 70%

metal on survival over time are shown in Figs 5 to 8. Several such experiments were performed for each metal, but due to inter-experiment variability in the survival of ostracods, the LCs0 for each experiment was calculated separately. The LC50 was calculated as the concentration of metal giving 50% survival relative to the control survival (see 'Methods' and Fig. 1).

As expected, lower LCs0s were obtained after longer exposure times, with the LCs0 after 8 days ranging from 25 to 70% of that after 4 days (Table 3). Of the four metals, the most toxic was copper, giving the lowest mean LCs0 at

148

6

5

74

.~ 3

2 2

'., - . . . _ _ S ~ "3. " "- "" " , \ .~ ~" - - ~ - CdlO

" "'/ L �9 . \ :, ~ . . . . Zn 0.1

�9 .\ ~ ~ Zn 1 " \ "I

�9 \ " "~" " CdlO:ZnO.l:

1 " ' ,"NL ~ ~ Cdl:Znl

o

0 4 8

Days

Fig. 9. Effects of cadmium chloride and zinc sulphate on survival of Diacypris compacta (Experiment 1). Each point represents the mean of ten replicate dishes, each containing a total volume of 10 mL and approximately 55 mg Cladophora sp. The concentration of Cd ranged from 0 (controls) to 10 mg L -1 and Zn ranged from 0 to 1 mg L -1. Metals were added either alone or together as indicated. Bars represent standard errors.

both day 4 and day 8 (0.8 and 0.4 mg L -1 respectively), even though copper seemed to complex in these experimental conditions (e.g. see Table 2). Zinc was also highly toxic, with slightly higher mean LC50s of 2.1 and 0.7 mg L-1 at day 4 and day 8 respectively. Lead and cadmium were slightly less toxic but all LCs0s were of the same order of magnitude, ranging from 0.4 to 4.0 mg L -1 (Table 3).

Effects o f two or three metals on the survival ofDiacypris compacta

Combinations of zinc, copper and cadmium were used in these experiments. Time courses for ostracod survival in the presence of two metals are shown in Figs 9 to 12, and Fig. 13 shows the effect of all three metals. In each experiment the effects of single metals were also measured and used to cal- culate theoretical additive mortalities. These were compared with the 95% confidence intervals obtained for the actual mortality in the presence of two metals (e.g. Fig. 14) to determine if the effects of two or three metals were additive or not. The data from all these experiments are summarised in Table 4. Some synergistic effects were observed with combinations of zinc and cad- mium, and zinc and copper at both days 4 and 8, but not with combinations of cadmium and copper. Also, with many of the combinations of metal con-

149

N " ~ :,, ~",-~-,-~.'7_....-..--...-~-',,- X "~ ' ~ .~" ~'~"'2 " ~ ' : ' ~ - " ~ - ~ . - . . . . Control

i , . . , "1 ....... zno., "' \ . ' r - .X\ ' \ " x ~ - ~ ' ~ I * ' " Znl

2 , X , " ~ " ~ - - * " Cdl0:Zn.l

�9 ., "~ "" \|,~. - - ~ " " Gdl:Znl

Cdl:ZnO.1 r

0 4 8

Days

Fig. 10. Effects of cadmium chloride and zinc sulphate on survival of Diacypris compacta (Experiment 2). Conditions for this experiment were identical to those given in Fig. 9.

~ 4 ~5

>= 3

E 7 = 2

�9 ---'-- - 2 , . " - , - ' ~ - ~ - . ~ " " " " " 1 ~ - - , '--, - '~ -~,~, ~I'~,-,,"_ "-.,

\ , ' - - ' . " , '1 ' , : \ " , " . . 1

" " - , " ' . "~. T \ ' ~ . , NI

' \ \

N \

, , ' ,~

0 i

0 4

Days

Control

- - " " Zn0.1

- - " - Z n l

- - ' - " Cu0.1

- - ~ " Cut

� 9 Zn1:Cu0,1

Zn0.1:Cul

" "=" " Zn0.t:Cu0.1

Fig. II. Effects of zinc sulphate and copper sulphate on survival of Diacypris compacta. Each point represents the mean of ten replicate dishes, each containing a total volume of 10 mL and approximately, 55 mg Cladophora sp. The concentrations of Zn and Cu ranged from 0 (controls) to 1 mg L -L Metals were added either alone or together as indicated. Bars represent the standard errors.

150

- . ~

5

~ 3

"~E 2 X \ '" 1 " " \ \

\ " , " . \

o

�9 Control

- - * " C d l

" " " " CdlO

- - " " CuO.1

- - " - C u l

- - ' - - CdlO:CMI

" " " " CdlO:CuO.1

�9 Cdl:CUl

4 8

Days

Effects of cadmium chloride and copper sulphate on survival of Diacypris compacta. Fig. 12. Each point represents the mean often replicate dishes, each containing a total volume of 10 mL and approximately 55 mg Cladophora sp. The concentration of Cd ranged from 0 (controls) to 10 mg L - l and Cu ranged from 0 tol mg L - ' . Metals were added alone or together as indicated. Bars represent the standard errors.

"~ 4

�9 ~ 3

E 7=2

- ~ - - - _ ~ , : ~ - . . . . . . . . . ,i, ~ - . : . - ~ - : ~ " ~ . . . . . . . . . . . . . . . . .

- - ' T ' , , 7 - ~ , " ' ' ~ ~ :--" : .- . . . . . . . I

q

0 *

0 4

Days

�9 Control

- - ~ CuO.1

- - " - C d l

- - ~ " ZnO.1

" " � 9 Cu:Cd

Cu:Zn

Cd:Zn

- - " " Cu:Cd:Zn

Fig. 13. Effects of copper sulphate, cadmium chloride and zinc sulphate on survival of Diacypris compacta. Each point represents the mean of ten replicate dishes, each containing a total volume of 10 mL and approximately 55 mg Cladophora sp. Controls contained no metal. The concentrations of Cu and Zn added were 0.1 mg L- l and that of Cd was 1 mg L- 1. Treatments contained either one, two or three of the metals as indicated. Bars represent the standard errors.

151

~ 4

> 3

E = 2

z

i

0 4 8

Days

Fig. 14. Confidence limits for survival of Diacypris compacta exposed to cadmium chloride and copper sulphate. These data are taken from Figure 12 (see legend for Conditions). Squares represent the means of ten replicate dishes. Triangles represent the 95% confidence limits around each mean. From the effects of each metal alone (Fig. 12) we also calculated the predicted number alive if the two metals had additive effects on mortality. If these predicted values fell within the confidence limits then the effect was considered additive. If not, the effects were considered either synergistic or antagonistlc (see Table 4).

centrations, the total mortality was less than additive (though always greater than with a single metal). In some cases, however, there was evidence for synergistic effects. Synergistic interactions were somewhat more likely when medium: low or medium: medium concentrations were combined (6 of 17 combinations) than when low: low concentrations were examined (2 of 14 combinations; Table 4). There was no evidence of greater synergism at day 8 than day 4, indicating that sensitivity to these metal combinations does not increase over time. However, the effects of metal combinations may not be consistent, since a repeat of the experiment with cadmium and zinc did not produce exactly the same effects (Table 4). When ostracods were exposed to three metals (low concentrations of copper, zinc and cadmium together) the mortality was less than the calculated additive mortality (Table 4). However, in cases where the effects of 2 or 3 metals were antagonistic, mortality was still always considerably greater than in the presence of a single metal.

152

Table 4. Effects of two or three metals on ostracod mortality. For each experiment mortality was measured in the presence of single metals and combinations of metals. In all cases the presence of a second metal resulted in higher mortality than when either metal was administered alone. For each combination of metals the actual mortality was compared with an additive mortality calculated by adding the single metal mortalities for the same metal concentration. Values given in the Table are the calculated additive percentage mortality subtracted from the measured percentage mortality. Positive values indicate actual mortality was greater than additive (i.e. synergistic); negative values indicate that the increase in mortality with two or more metals was antagonistic; and * indicates that the calculated additive value was outside the 95% confidence interval of the measured mortality. Low and medium concentrations of zinc and copper were 0.1 and 1 mg L - I and of cadmium were 1 and 10 mg L - l respectively. Each row of data gives data from a separate experiment.

Metals Day 4 Day 8

low:low med:low low:reed med:med low:low med:low low:med

Cd:Zn +5.0* -15.0 -16.6" - -1 .6 +1.7 -8.3*

Cd:Zn -6.6* -10.0" +5.0* - -11.6" 0 -30.0*

Cd:Zn -10.0" -8.3* Zn:Cu -3.3* +6.7* -6.7* - +15.0" +18.3" +5.0*

Zn:Cu -6.7* -8.3* Cd:Cu -13.3" -5.0* +10.0" 0 -41.7"

Cd:Cu -13.3" -10.0"

Zn:Cu:Cd -13.3" -8.4*

(all low)

med:med

- - i

Discussion

LC5o values for Diacypris compacta

Heavy metals are toxic to Diacypris compacta and the LC50 values that we obtained fall within the range of values reported for other aquatic biota. For example, acute sensitivities (often 96 h LC50 values) to copper, zinc, lead and cadmium for a variety of freshwater and marine Crustacea ranged widely from 0.005-9.6, 0.04-58, 0.03-5.5 and 0.005-8.5 mg L -1 for each metal respectively (e.g. Weatherly et al., 1980; Skidmore and Firth, 1983; Connell and Miller, 1984; Hong and Reish, 1987; Hart and Lake, 1987; ANZECC, 1992). Our mean 96 h (4 day) LC50 values forDiacypris compacta in hypermarine Coorong water were 0.8, 2.1, 3.1, and 4.3 mg L- x respectively for the same four metals. This order of relative toxicity for the four metals is also similar to the order reported in other studies (e.g. Weatherly et al. 1980; Skidmore and Firth 1983; Connell and Miller 1984).

153

The lower concentrations of metals for LCs0s at day 8 compared to day 4 indicate an increase in mortality with increased exposure to heavy metals (Table 3). LCs0 concentrations generally decrease with increasing time of exposure, eventually reaching an asymptote (Skidmore and Firth, 1983). In our study this asymptotic LCs0 may not have been reached by day 8 since the few data obtained for longer periods indicated that survival continued to decrease after day 8, more so in the presence of metals than in controls (e.g. Fig. 7). Hence the asymptotic LCs0 values will undoubtedly be lower than those we have reported for 8 days in Table 3.

Experimental procedure

Some acute toxicity studies report higher survival of aquatic biota in static than flow-through systems (Ahsanullah 1976; Negilski, 1976), while others report similar mortality in both (Kemp and Swartz, 1988) or better survival in flow-through systems (e.g. Picketing and Gast, 1972; Gerhardt, 1992; Sun- deram et al., 1992). Although continuous flow tests are generally preferable to static tests, many toxicity studies have successfully used static systems to measure metal toxicities (e.g. Fennikoh et al., 1978; Lake et al., 1979; Hong and Reish, 1987; Meador, 1991). Mearns et aL (1986) showed that a static 10 day test with continuous aeration provided a reproducible labo- ratory sediment toxicity test. The possible problems associated with static systems include low concentrations of dissolved oxygen or the build up of toxic metabolic wastes with time (Skidmore and Firth, 1983). Since survival of control animals was reasonably high in our experiments, environmen- tal conditions were not unfavourable to Diacypris compacta over the 8-day observation period at least. The better survival that we observed under static conditions than continuously aerated conditions (Fig. 3) is consistent with observations that Diacypris in the Coorong is more commonly found in still water than in turbulent water (R. Molsher, pers. comm.).

The effect of macrophytic algae in increasing the survival of control ostra- cods (Fig. 2) may have been because they provided food (possibly peri- phyton), suitable shelter, or because their photosynthetic activity provided a source of oxygen (e.g. Roca et al., 1993). Other water plants have been used successfully to aerate another ostracod, Candona, in the laboratory (Kesling, 1956).

The addition of macrophytic algae to dishes in our study, however, may have affected the observed LCs0 values for Diacypris. Algae can complex heavy metals (Skidmore and Firth, 1983), thus lowering metal concentrations in solution. However, this did not occur to a significant extent in our study since the heavy metal concentrations in dishes with and without algae were not significantly different after 4 days (Table 2). Note, however, that the

154

precision of measurements of metal concentrations in the small volumes of saline water that were tested were such that the amounts of heavy metals accumulated by macrophytic algae, associated periphyton and ostracods could not be determined. Macrophytic algae, periphyton and ostracods are likely to have accumulated heavy metals during the trials (Weatherly et al., 1980; Connell and Miller, 1984; Holmes et al., 1991; Mizutani et al., 1991; Weeks and Rainbow, 1991). Rates of accumulation vary depending on concentrations but even if macrophytic algae accumulated heavy metals at the relatively high rate of i mg kg -1 d - l (Kilgour, 1991; Mizutani et al., 1991; Weeks and Rainbow, 1991; Ringwood, 1991), then 55 mg of Cladophora would have accumulated only 0.2 #g in 4 days and this is not sufficient to significantly alter concentrations of metals in the water. The quantities of metal added to the 10 ml of Coorong water in test chambers varied from 2-100/~g.

Some of the mortality of ostracods in solutions of heavy metals, however, may have been due to toxic effects of heavy metals on algae or periphyton. For example, if heavy metals inhibited the growth of certain periphyton (e.g. various bacteria, microflora) and these were used as food by Diacypris com- pacta, then reductions in food availability may have increased the mortality of D. compacta in test chambers. We found that the presence of macrophytic algae was necessary for acceptable survival of D. compacta in control condi- tions (Fig. 2). Both algae and associated epiphytic bacteria readily accumulate heavy metals (e.g. Holmes et al., 1991) and the growth, reproduction and sur- vival of various species of algae are adversely affected by metals such as copper, zinc and cadmium (Fennikoh et al., 1978; Weatherly et al., 1980; Garvey et al., 1991). However, whether metals acted directly on ostracods or indirectly through algae and periphyton would not matter in an ecosystem such as the Coorong, the mortality of ostracods would still occur.

Differences in the calculated LCs0 values between consecutive experi- ments using the same metal may be due to slight changes in salinity altering the activity of the free metal ion (Blust et al., 1992), or to variation in age, nutritional condition, pathogen or parasite loads between ostracods or season- al effects. Instar stage of animals was not assessed in this study and age has previously been shown to alter susceptibility to toxicity in other aquatic ani- mals (e.g. Connor, 1972; Lake et al., 1979; Weatherly et al., 1980; Ringwood, 1990, 1991; McCahon and Pascoe, 1988). Diacypris compacta varied in size and differences in mean sizes in consecutive experiments may have account- ed for some of the differences in metal toxicity. Lake et al. (1979) found that smaller individuals of the freshwater shrimp Austrochiltonia australis were more sensitive to zinc than larger individuals. Similar size-related differences in sensitivity to heavy metals may have occurred for Diacypris compacta. However, survival of D. compacta under control conditions was similar for

155

small and large individuals. Seasonal variations in sensitivity of freshwater phytoplankton communities to toxicity of copper have been observed, with higher responses in spring to a chronic copper stress (Winner and Owen, 1991), and some seasonal variation (possibly related to seasonal changes in size) may have occurred in our experiments with Diacypris, although a con- sistent seasonal pattern was not evident. Some variation in survival of control animals also occurred between different experiments but these differences did not systematically affect the calculated values of LCs0, since variations in LCs0 values did not show any clear relationship with variations in survival of controls.

Mortality of Diacypris was always greater in the presence of two or three metals than in the presence of a single metal. The results indicated that some combinations of metals can produce synergistic effects but these were not large (Table 4). However, in many cases the effects were antagonistic. Synergistic effects may be the result of the presence of one metal increasing sensitivity to the toxicity of another. Antagonistic effects may arise when the physiological actions of two toxic metals are similar; after a physiological system has been damaged by one metal, further damage to that system by a second metal may not further increase mortality. This effect, however, does not indicate that the metals can cancel each other out. The presence of both of those metals still represents a biological hazard which is greater than the presence of one of those metals alone.

With respect to synergistic, additive, or antagonistic effects, particular metal combinations did not always produce the same results at day 4 and day 8 (Table 4). This may be due to different mechanisms of toxicity with some combinations of metals and concentrations producing an acute mortal- ity response and others a delayed effect. In addition, different results were obtained in two identical experiments with zinc and cadmium. These exper- iments were conducted under the same conditions but seven weeks apart. Differences may reflect subtle seasonal or generational differences in the response of ostracods to multiple toxicants. We conclude that the response to metal combinations varies with changes in metal concentrations, time and ostracods. Often the response is antagonistic, but in some cases synergism is observed. The lack of predictability of response indicates the need for caution in estimating possible biological effects of a metal on ostracods when other metals or toxic compounds are present, even in low concentrations, especially organic forms that are more readily taken up.

Implications for the management of the Coorong

Laboratory bioassays have limitations when extrapolated for the management of a natural resource (Chapman, 1995). Environmental conditions within the

156

laboratory are kept constant so that the effect of the toxicant can be isolated. In a natural ecosystem conditions are constantly changing and the effect of a toxicant may consequently be different. Also, in the laboratory, animals are inadvertently selected for survival in laboratory conditions. Thus the LCs0s and effects of multiple metals obtained in our static laboratory experiments may provide a higher concentration for the LCs0 than in the field, where animals are subjected to natural stresses such as food shortages, predation and changes in salinity, temperature or turbidity. Under stress animals will be more sensitive to the toxic effects of heavy metals.

ANZECC (1992) have recommended 'safe' concentrations for copper, zinc, cadmium and lead in Australia to be 2.0-5.0, 5.0-50.0, 0.2-2.0, and 1.0-5.0 #g L -1 respectively in freshwater, depending on hardness, and 5.0, 50.0, 2.0 and 5.0/zg L-1 respectively in seawater. No recommendations were given for hypermarine environments. A common method used to set 'safe' concentrations for persistent toxicants like heavy metals is to multiply the LCs0s by some factor, usually 1% (e.g. Fennikoh et al., 1978; ANZECC, 1992). 'Safe' concentrations are those that protect all forms of aquatic life through all stages of the life cycle under indefinite exposure. Ideally asymp- totic LCsos should be used in these calculations. Using the lowest values of LCs0 obtained after 8 days exposure, the 'safe' concentrations for Diacypris compacta in the Coorong would be set at 4 #g L -I for copper, 5 #g L -1 for zinc, 9/zg L - l for cadmium and 22 #g L -1 for lead. These calculations suggest that 'safe' concentrations, at least for copper and zinc in the hyper- marine waters of the Coorong are less than those considered 'safe' for marine systems.

Setting the safe limit at 1% of the LCs0 value, however, is entirely arbitrary (Skidmore and Firth, 1983). Concentrations which do not affect survival may still affect development adversely and behavioural effects have been seen at a cadmium concentration one hundred-fold lower than the 144h LCso value for larvae of the midge Glyptotendipes pallens (Heinis et al., 1990). Previous studies cited by McCahon and Pascoe (1991) have shown decreased larval development, and reduced production of Chironomus riparius at concentra- tions of cadmium 5000 times lower than the 48h LCs0 value for fourth instar larvae. Taylor et al. (1991) found that the growth of Chironomus riparius lar- vae was retarded when concentrations of copper exceeded 17 #g L- 1, a tenth of the LCs0 value recorded for these larvae after 10 days, while Beisinger and Christensen (1972) reported a 3 week LCs0 value for zinc of 0.16 mg L- l for Daphnia magna but found a 50% reproductive impairment at 0.070 mg L -1. Measurable effects on the behaviour, growth and reproduction of aquatic biota may also be detected at even lower concentrations of heavy metals over longer time periods.

157

Values of LCs0 for a species also vary with conditions such as salinity, temperature and age, and ideally the sensitivity of test animals to pollutants should be measured under the most unfavourable conditions to which those taxa are likely to be exposed (Skidmore and Firth, 1983; Chapman et aL, 1993). This is particularly relevant to the Coorong since there is great sea- sonal variation in factors such as salinity and temperature (e.g. Geddes and Butler, 1984; Molsher et al., 1994). Also, sensitivity to heavy metals varies considerably (up to 100 fold) between different species living in the same conditions (Skidmore and Firth, 1983). Given this, acute toxicities should be measured for a range of aquatic species, covering phytoplankton, zooplank- ton, benthos, macrophytes and fish in conditions pertinent to the Coorong. The most sensitive species should then be tested under the least favourable conditions using chronic toxicity tests to determine the levels of pollutants which will have no long term effect. Such information is not yet available for the Coorong. Other species may be more sensitive to heavy metals than Diacypris and a range of Coorong biota needs to be thoroughly tested before 'safe' levels can be confidently established for the Coorong ecosystem.

The sensitivity of Diacypris compacta to heavy metals is of concern since concentrations of copper and zinc in the surface water and groundwater due to be diverted to the Coorong from the Upper South East of South Australia exceed these values at times. Maximum concentrations of zinc and copper in surface water were 374 and 400 #g L -1 respectively, and in groundwater the highest recorded values were 822 and 50 #g L -1 respectively (Eco Manage- ment Services, 1992). Concentrations of zinc exceeded 5 #g L -1 in all but 4 of 25 groundwater sites sampled, and also at 7 of 13 surface water sites. Concentrations of cadmium in surface water and groundwater were < 0.2 #g L -a in all but one case while concentrations of lead in surface water were usually < 1 #g L -1 with the highest recorded concentrations being 3 #g L -1 in surface water and 32 #g L -1 in groundwater (Eco Management Services, 1992). Concentrations of heavy metals in this incoming water may need to be even lower than the ANZECC recommendations, if there is any likeli- hood that these heavy metals could accumulate in the Coorong ecosystem. The OECD (1992) have recommended that the concentrations of toxicants in influent waters should not exceed 0.001 • the lowest LCs0 value, if only acute data are available for a few species as is currently the case for the Coorong.

The LCs0 values used above only consider each metal in isolation. Since high concentrations of zinc and copper were usually found together in sur- face waters in the Upper South East of South Australia (Eco Management Services, 1992) calculations of maximum acceptable levels of these metals should take into account the presence of all toxicants. In our laboratory stud-

158

ies, combinations of two or more metals were usually antagonistic, though sometimes synergistic. However, even when the effects of more than one metal were antagonistic, the mortality of Diacypris was always greater than when only one of the metals was present at the same concentration, i.e. the LCs0 for a heavy metal is lower in the presence of other heavy metals. Thus the influx of even low levels of several metals in combination could be a significant hazard, and the presence of pesticide or herbicide residues could exacerbate the response to a low concentration of a heavy metal.

Even very low levels of heavy metals in the drainage water may not adequately protect the Coorong if there is a risk of the chemical composition of Coorong water changing or heavy metals accumulating above safe limits in the sediments and food chains of the Coorong.

Drainage of large quantities of relatively fresh water into the southern Coorong will lower the salinity and possibly alter the pH and hardness of the water as well. These changes may stress the aquatic biota adapted to the hypermarine environment of the southern Coorong and may make them more susceptible to toxicants. In other systems, toxicity of heavy metals increases as salinity decreases (e.g. Jones, 1975; McLusky et al., 1986; De Lisle and Roberts, 1988; Blust et al., 1992; Lin and Dunson, 1993) or increases as salinities rise above or drop below the isosmotic point of aquatic fauna (McLusky and Hagerman, 1987). Freshening of the Coorong, therefore, is likely to increase the toxicity of heavy metals above that reported in this study not only because of direct chemical interactions with the metals but also because of the biological stress on hypermarine biota due to reductions in salinity. Changes in pH, water temperature and water hardness also alter heavy metal tolerances of aquatic animals (e.g. Skidmore and Firth, 1983; Meador, 1991; Gerhardt, 1992) though the direction of these changes varies with the metals and systems being studied.

The composition of organic matter of the lagoons will also influence the toxicity of metals. Toxicity of cadmium depends on dissolved organic car- bon, and in waters with low dissolved organic carbon, sensitive species are very vulnerable (Lawrence and Holoka, 1991). Acute toxicity of cadmium to amphipods was mostly mediated by interstitial and not particle (organic matter) bound cadmium (Kemp and Swartz, 1988). The amount of carbon also affects the amount of ionic copper and thus toxicity; copper complexed as a carbonate is less toxic than a hydroxyl because of altered bioavailability (Meador, 1991). Because of these alleviating effects of organic matter, con- centrations in sediment and not just in water must be considered (Mizutani et al., 1991). In the Coorong this is particularly important because Diacypris was found in the sediment layer as well as the water column. Drainage from agricultural land would be likely to increase the concentration of nutrients

159

and organic compounds in the Coorong and this could complex some heavy metals. However, the metals may be recycled within the system after break- down of organic compounds, and either be released into solution or be taken up by aquatic plants or benthic fauna (Connell and Miller, 1984; Hart and Lake, 1987; Jackson et al., 1991). Furthermore, fish and waterbirds, including waders, may consume the contaminated organic material, and heavy metals would then be released in the acid conditions of the stomach and potentially have toxic effects.

Analysis of Coorong sediment indicated only low or undetectable levels of heavy metals. However, if heavy metals arrive with drainage water they could complex with or precipitate onto the substrate. If so, the Coorong would act as a sink where heavy metals may accumulate to toxic levels over a number of years. Organisms in the sediment could thus be adversely affected, and water turbulence caused by wind or water inputs could release sediment metals back into solution at various times. Interactions between metals, Coorong water, sediments and biota will be complex (e.g. Skidmore and Firth, 1983; Connell and Miller, 1984; Hart and Lake, 1987), and predicting exactly how they will behave in this hypermarine system is not possible without further study.

In previous studies, some populations of aquatic biota have evolved resis- tance to some pollutants. For example, a population of the oligochaete Limnodrilus hoffmeisteri evolved improved resistance to cadmium, nickel and cobalt (Klerks and Bartholomew, 1991). This ability to cope with high- er metal concentrations was due to increased levels of a cadmium binding metaUothionein-like protein and an ability to sequester metals in vesicles and not due to a decrease in cadmium accumulation. This suggests that even if ostracods can acquire a degree of resistance, uptake of heavy metals may not be reduced and these metals will still present a hazard to animals further up the food chain unless each and every species can acquire resistance. Since many of the bird species that use the Coorong use it for only part of the year, this seems highly unlikely.

Given that (1) Diacypris compacta is sensitive to heavy metals; (2) little is known about the behaviour, bioavailability and interactive effects of heavy metals and other substances in the hypermarine and turbulent waters of the Coorong; and (3) the capacity of the Coorong to accumulate and store heavy metals in inert forms is unknown; then the only sensible strategy is to (1) prevent influxes of any water carrying heavy metals; and (2) if water is added then the volumes should be limited so that the current salinity regimes of the Coorong are maintained.

160

Acknowledgements

We thank Robyn Molsher (Depar tment of Zoology, University of Adelaide) for collection and care of the ostracods and for much valuable advice, George

Levay (Water Resources Laboratory, University of South Australia) for mea-

surement of heavy metal concentrations and Elise Wollaston (Depar tment of

Botany, Univers i ty of Adelaide) for identifying the algae. Funds were pro-

vided by the Murray Darling Basin Commiss ion and laboratory space by the Depar tment of Zoology, Universi ty of Adelaide. Sam Lake, Paul Boon,

Michael Kokkinn, Andrew Boulton, and Bill Will iams kindly provided com- ments on the manuscript .

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