Environmental Pollution 134 (2005) 377–383 www.elsevier.com/locate/envpol
No evidence for a critical salinity threshold for growth and reproduction in the freshwater snail Physa acuta Ben J. Keﬀord*, Dayanthi Nugegoda Biotechnology and Environmental Biology, School of Applied Science, RMIT University, PO Box 71, Bundoora 3083, Vic, Australia Received 12 May 2004; accepted 17 September 2004
Responses of snails to increasing salinity were non-linear. Abstract The growth and reproduction of the freshwater snail Physa acuta (Gastropoda: Physidae) were measured at various salinity levels (growth: distilled water, 50, 100, 500, 1000 and 5000 mS/cm; reproduction: deionized water, 100, 500, 1000 and 3000 mS/cm) established using the artiﬁcial sea salt, Ocean Nature. This was done to examine the assumption that there is no direct eﬀect of salinity on freshwater animals until a threshold, beyond which sub-lethal eﬀects, such as reduction in growth and reproduction, will occur. Growth of P. acuta was maximal in terms of live and dry mass at salinity levels 500–1000 mS/cm. The number of eggs produced per snail per day was maximal between 100 and 1000 mS/cm. Results show that rather than a threshold response to salinity, small rises in salinity (from low levels) can produce increased growth and reproduction until a maximum is reached. Beyond this salinity, further increases result in a decrease in growth and reproduction. Studies on the growth of freshwater invertebrates and ﬁsh have generally shown a similar lack of a threshold response. The implications for assessing the eﬀects of salinisation on freshwater organisms need to be further considered. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Low salinity; Concentration–response curve; Sub-lethal; Macroinvertebrates
1. Introduction Increasing salinity (the concentration of dissolved inorganic ions) in rivers and wetlands is a serious environmental problem on all inhabited continents (Williams, 1987), and is likely to aﬀect aquatic organisms (Hart et al., 1990, 1991). Recently, there has been interest in the lethal eﬀects of high salinity on freshwater macroinvertebrates (Goetsch and Palmer, 1997; Williams and Williams, 1998; Blasius and Merritt, 2002; Keﬀord et al., 2002, 2003, 2004a,b). It has been assumed that as salinity increases individuals of a species
* Corresponding author. Tel.: C61 3 9925 7126; fax: C61 3 9925 7110. E-mail address: ben.keﬀ[email protected]
(B.J. Keﬀord). 0269-7491/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2004.09.018
will experience no eﬀect until a threshold is reached (Hart et al., 1991). Rises in salinity above this threshold will produce reductions in biological performance, such as growth and reproduction, which we shall refer to as sub-lethal eﬀects. If salinity increases further, greater levels of the sub-lethal eﬀect will occur, until death results. With a threshold response, changes in salinity below this threshold will have no direct eﬀect on freshwater animals. Despite a widely assumed threshold response, most freshwater ﬁsh do not have maximum growth in freshwater but in water with elevated salinity, see review by Boeuf and Payan (2001). Indeed inverted U-shaped concentration–response curves (the relationship between a substance’s concentration and the eﬀect on a species) are common across a range of taxa and toxicants – referred to as hormesis and essentiality (Chapman,
B.J. Keﬀord, D. Nugegoda / Environmental Pollution 134 (2005) 377–383
1998) and have long been accepted for community level impacts from a variety of stressors (Odum et al., 1979). If an inverted U-shaped concentration–response holds for freshwater organisms exposed to changes in salinity, then small increases in salinity (from a low concentration) will result in an increase in biological performance until a concentration where biological performance is maximized. Further increases in salinity will then result in a decrease in biological performance until death occurs. Therefore with such a response curve, there is no minimum salinity below which changes in salinity will not directly aﬀect freshwater animals and any change in salinity can potentially aﬀect freshwater animals. In this paper, we consider whether low salinity levels aﬀect growth and reproduction of an introduced freshwater snail: Physa acuta Draparnaud (Gastropoda: Physidae). Growth and reproduction were chosen because they are ecologically relevant and sub-lethal. This species was chosen because it can usually be collected in large numbers (compared to most other species), grown in the laboratory and is widely distributed.
2. Materials and methods Experiments were conducted in an aquarium at an ambient air temperature of 20 C. Salinity was expressed as electrical conductivity or EC (mS/cm) adjusted to 25 C as it is the most common measure of salinity and is rapid and cheap to determine (Williams and Sherwood, 1994). We used the synthetic sea salt, Ocean Nature (Aquasonic, Wauchope, NSW), which has ionic proportions similar to sea water, as the salt source. It was used because most inland Australian waters have ionic proportions similar to sea water (Bayly and Williams, 1973). The relationship between EC (mS/cm) and total dissolved solids (TDS, in mg/L) and osmolarity (Osmol/kg H2O) for Ocean Nature salt is TDS Z 0.754 EC and osmolarity Z 18.4 EC (Keﬀord et al., 2003). Snails were transported in water from their collection site and kept in an aquarium in that water and fed coslettuce for 5–10 days to acclimate. During experiments, a standard and arbitrary feeding of 0.1–0.2 g (per snail) of frozen cos-lettuce (that had been thawed and rinsed in distilled water) twice per week was conducted. Test solutions were changed once per week, where un-eaten lettuce was discarded. 2.1. Growth In August 2001, P. acuta was collected from the Barwon River at Pollocksford Bridge (S 38 08#; E 144 11#), in south-west Victoria, Australia where the EC was
880 mS/cm. Small snails (live weight range 2.1–9.9 mg) were chosen as they had good scope for growth. Live masses of P. acuta were measured to 0.1 mg after carefully removing excess external water with tissue paper. Repeat measures of the same snail after re-submerging showed this to be satisfactory. Based on a pilot study, six salinity treatments were prepared (distilled water, 50, 100, 500, 1000 and 5000 mS/cm) by dissolving Ocean Nature salt in distilled water. There were 10 replicate snails per treatment housed separately in approximately 480 mL of water. Snails were held individually so the mass of each could be tracked. Snails were allocated randomly to treatments. After 2 days in their respective treatments, snails were weighed and this was taken as the starting live weight, to exclude any change in the mass due to initial osmotic eﬀects. Fourteen and 28 days later the live mass of individuals was determined. The experiment was then terminated and the dry mass of individuals measured after oven drying at 60 C for 5 days and cooling in a desiccator. The live growth rate of individual snails was calculated as (ﬁnal mass ÿ initial mass)/(number of days between measurements). 2.2. Reproduction In July 2003, P. acuta was collected from the Campaspe River at the Kyneton-Heathcote Road (S 37 23#; E 144 31#) in central Victoria where the EC was 940 mS/cm. Large snails (live mass range 13–81 mg) relative to those at this site were used so that they would be reproductively mature. Treatments were prepared by dissolving Ocean Nature in deionized (DI) water. Because few large P. acuta were available, only ﬁve treatments were used (DI water, 100, 500, 1000 and 3000 mS/cm). The highest salinity treatment used in this experiment was 3000 mS/cm, instead of 5000 mS/cm in the growth experiment, because P. acuta has reduced reproduction at this salinity relative to around 100 mS/ cm (T. Paradise, RMIT University, pers. comm.). Each treatment included four replicate 500-mL containers, each containing four randomly allocated P. acuta. Over 14 days, egg masses were searched for daily and removed. The number of eggs in each mass counted under a dissection microscope and expressed as the number of eggs per snail per day. 2.3. Analysis Any snails that died during an experiment were excluded from subsequent analysis. Diﬀerences between treatments were analysed using standard single factor ANOVAs with multiple comparisons conducted using Tukey HSD tests. Where appropriate, data were log10 transformed to improve the assumptions of normality and homogeneity of variance.
B.J. Keﬀord, D. Nugegoda / Environmental Pollution 134 (2005) 377–383
There was no evidence that initial live mass of P. acuta diﬀered between treatments (F Z 0.53, df Z 5 and 54, P Z 0.752). Growth occurred in all treatments (Fig. 1a,b) and was maximal in the 500 and 1000 mS/cm treatments and the diﬀerences between treatments were highly statistically signiﬁcant (over 14 days: F Z 12.8, df Z 5 and 46, P Z 0.00000008; over 28 days: F Z 8.4, df Z 5 and 39, P Z 0.00002). A similar pattern emerged for the ﬁnal dry mass (Fig. 2, F Z 7.6, df Z 5 and 39, P Z 0.00005). The percentage of weight lost through
Dry mass (mg)
Treatment (µS/cm) Fig. 2. Mean ﬁnal dry weight of individual Physa acuta after 30 days in each treatment.
Growth rate (mg/d)
drying was similar in all treatments except at 5000 mS/ cm where there was less mass lost (mean G standard error: 75 G 0.71% and 70 G 1.7%, respectively; F Z 3.5, df Z 5 and 39, P Z 0.011).
c,d b,c a,b
The similar the DI in the
trend in egg production (per snail per day) was to that of growth. Egg production was lowest in water and 3000 mS/cm treatments and maximal intermediate salinity treatment of 500 mS/cm
Growth rate (mg/d)
Log10 Eggs per snail per day
# *, # #
0.0 Dist. H2O
Treatment (µS/cm) Treatment (µS/cm)
Fig. 1. Mean growth rate of individual Physa acuta in terms of live mass in each treatment over: (a) 14 days and (b) 28 days. Dist. H2O Z distilled water. Error bars are standard errors of the mean and diﬀerent letters symbolize treatments with signiﬁcant diﬀerence at the 0.05 level (Tukey HSD Multiple Comparisons).
Fig. 3. Mean egg production in Physa acuta in each treatment. DI Z deionized water. The * and # indicate signiﬁcant diﬀerences, at the 0.05 level, between a treatment and the deionized water and 3000 mS/cm treatments, respectively. Pairwise comparisons of diﬀerent treatments not conducted because of insuﬃcient power and inﬂated probability of a type-two error.
B.J. Keﬀord, D. Nugegoda / Environmental Pollution 134 (2005) 377–383
(Fig. 3). Diﬀerences between treatments were statistically signiﬁcant (log10 transformed, F Z 3.4, df Z 4 and 15, P Z 0.035).
4. Discussion P. acuta displayed maximum growth and reproduction in intermediate salinity treatments rather than in low salinity treatments as predicted by the threshold response hypothesis (Hart et al., 1991). Duncan (1966) also showed that the freshwater parthenogenetic snail Potamopyrgus antipodarum moved faster and produced more oﬀspring at intermediate salinity. More recently, two clones of this species have been shown to have higher growth rates at intermediate salinity, and one of these clones (that tends to be associated with brackish coastal locations) also had greatest reproductive output at intermediate salinity (Jacobsen and Forbes, 1997). Similarly, most freshwater ﬁsh have maximum growth at salinity levels above normal freshwater (Boeuf and Payan, 2001). Carbon assimilation in Daphnia magna is maximum at intermediate salinity (Yang and He, 1997). Therefore, maximum biological performance at intermediate salinity appears in a number of distantly related freshwater animals. The water content of P. acuta was unaﬀected by treatments %1000 mS/cm but was lower in the 5000 mS/ cm-treatment. Similarly, tissue hydration of freshwater snail Pomacea bridgesi (Ampullaridae) was constant in treatments %8300 mS/cm but was reduced in a 11 000 mS/cm-treatment (Jordan and Deaton, 1999). The bi-valve Lampsilis teres (Unionidae), however, maintained its tissue hydration in the highest treatment of 22 000 mS/cm (Jordan and Deaton, 1999). 4.1. Potential physiological mechanism The physiological and/or biochemical reasons for the diﬀerences in growth and reproduction were not investigated but a number of potential explanations exist. Jacobsen and Forbes (1997) found that the feeding rate in a clone of P. antipodarum that tended to be associated with brackish water was highest at intermediate salinities, while the feeding rate of the more freshwater clone was not reduced by low salinity. Calow and Forbes (1998) argue that the main eﬀect of salinity on reproduction observed by Jacobsen and Forbes (1997) was due to changes in the feeding rate of P. antipodarum and not other physiological processes. However, for growth some other physiological processes appear to be involved (Calow and Forbes, 1998). Salinity is a measure of a mixture of major ions ÿ 2ÿ (NaC, Ca2C, Mg2C, KC, Clÿ, SO2ÿ 4 , HCO3 and CO3 ) that are essential to living organisms. In waters of low salinity there is the possibility that deﬁciencies in these
elements may produce sub-lethal responses in freshwater animals. In molluscs, limited Ca2C at low salinities may increase the energetic cost of shell formation (Palmer, 1992) as may low CO2ÿ concentrations (Wilbur and 3 Saleuddin, 1983). Furthermore, growth and reproduction of several freshwater snail species increased with increasing Ca2C concentration at least up to a threshold concentration (Madsen, 1987; Brodersen and Madsen, 2003). Although Biomphalaria pfeiﬀeri grow and reproduce in water with only calcium bicarbonate added, both growth and reproduction are improved by the addition of Mg2C, NaC or KC (Nduku and Harrison, 1976). Furthermore, of three planorbids, all of which had increased growth and reproduction with increasing Ca2C concentration (Madsen, 1987), one had an inverted U-shaped concentration–response curve for growth in shell length and another had this response curve for egg production when the concentration of sodium chloride was varied in a test medium without altering Ca2C (Madsen, 1990). Therefore, low concentrations of common ions other than Ca2C can aﬀect growth and reproduction in some freshwater gastropods. Ca2C (and other ions) tends to increase as salinity rises, especially in Australian inland waters, which have ionic proportions similar to sea water (Bayly and Williams, 1973). Thus, the eﬀect of deﬁciency of Ca2C (or other ions) at low salinity is diﬃcult to separate from eﬀects of salinity, as least in Australian inland waters. Freshwater animals maintain internal concentrations of major ions at much higher levels than in the surrounding water (Rankin and Davenport, 1981), in doing so they expend energy (Potts, 1954) and in the freshwater snail Lymnaea stagnalis NaC, KC, Ca2C and Clÿ are actively transported (Schlichter, 1981). It has often been assumed that energy expenditure on osmoregulation by aquatic animals would be lowest at salinity levels where the internal osmotic concentration equaled that of the external concentration, called the isosmotic point. If this is the case, at the isosmotic point there should be more energy available for other uses, including growth and reproduction, and this could potentially explain the greater growth and/or reproduction of P. acuta (and other freshwater animals) at intermediate salinity levels. Hunter (1964) contends that freshwater molluscs use relatively little energy on osmoregulation. There are several reasons why it is diﬃcult to evaluate the importance of the energetics of osmoregulation in explaining salinity – growth and reproduction relationships; inconsistent estimates of the proportion of energy used on osmoregulation; inconsistencies in respiration–salinity relationships; confounding eﬀects of changes in animals’ activity; and recent advancements in physiology. Hunter (1964) bases his assessment on calculations by Potts (1954) who estimated that three freshwater invertebrate species (a bi-valve and two decapods) used
B.J. Keﬀord, D. Nugegoda / Environmental Pollution 134 (2005) 377–383
only 0.5–1.2% of total metabolic energy on osmoregulation. Croghan (1961), however, criticised Potts’ calculations and argued that energy expenditure on osmoregulation of freshwater animals could be an important component of their total energy use with ecological consequences. Freshwater species of Garmmarus (Amphipoda) tend to have respiration rates per unit mass of tissue of about 60–65% higher than brackish and marine species, which may be the result of increased osmoregulation demands in freshwater (Sutcliﬀe, 1984). However, calculations of the proportion of energy used in active transport of ions in Garmmarus pulex (a freshwater species) are lower (11– 21%) and no increase in respiration was observed in subsequent experiments (Sutcliﬀe, 1984). Further complicating the situation is the lack of a consistent salinity–respiration response in a range of invertebrates. While studies of invertebrates have observed respiration to be lowest at the isosmotic point (for example, Wheatly and McMahon, 1983), a wide variety of salinity–respiration relationships appear to exist. Respiration has been observed: (1) to decrease with increasing salinity in an intertidal gastropod (Cheung and Lam, 1995), three copepod species (King, 1976) and in three amphipod species (Dorgelo, 1973); (2) to be constant with varying salinity in a plecopteran (Kapoor, 1979) and an ephemeropteran (Beaver, 1990); (3) to be constant except at low salinity levels in some freshwater hirudinoidea (Linton et al., 1982) and in a brackish water bi-valve (Rao et al., 1987); (4) to be maximum at intermediate salinities in a freshwater snail (Duncan, 1966), and (5) to be complex in a freshwater hirudinoidea (Reynoldson and Davies, 1980). The lack of a consistent response in invertebrates makes it diﬃcult to suggest what salinity level will have the least osmoregulatory demands. Duncan (1966) observed that respiration in P. antipodarum was maximum in intermediate salinity treatments. The diﬀerences in respiration would, however, appear to be due to the activity of the snails (as indicated by their speed of movement). Thus, diﬀerences in respiration at diﬀerent salinities may not necessarily be due to osmoregulation. A complex interplay between osmoregulation and the oxygen carrying capacity and pH of the haemolymph (Cameron and Iwama, 1989; Heisler, 1989; Henry and Weatly, 1992) has now been acknowledged. Additionally, organic ions can play a role in regulating the osmolarity of the haemolymph of aquatic animals (Goolish and Burton, 1989; Patrick and Bradley, 2000) including freshwater gastropods (Jordan and Deaton, 1999). At least, in ﬁsh there are hormonal changes following changes in salinity (Boeuf and Payan, 2001), which likely have energetic demands (Morgan et al., 1997). Metabolic costs while acclimating to changes in salinity can be diﬀerent to the metabolic costs once gastropods (Cheung
and Lam, 1995), amphipods (Sutcliﬀe, 1984) and ﬁsh (Iwama et al., 1997; Morgan et al., 1997) have fully adapted to that salinity. These additional complexities make predictions about how respiration will change with varying salinities more diﬃcult. Finally, Styczynska-Jurewicz (1972) reports a freezing point depression of the haemolymph from freshwater acclimatized P. acuta of 0.2D C, which is approximately equivalent to about 5000 mS/cm. This suggests that either our P. acuta had a more dilute haemolymph or growth and reproduction were not maximum at the isosmotic point. 5. Concluding comments The standard view is that aquatic animals are unlikely to be aﬀected by changes in salinity below approximately 1500 mS/cm (Hart et al., 1991; Nielsen et al., 2003). Our results and a full appraisal of the literature suggest that maximum growth and reproduction in freshwater animals occur in intermediate salinities. While a number of plausible physiological mechanisms exist to explain such responses, none appear to be demonstrated conclusively. Salinity levels have been measured as low as 13 mS/cm in natural waters (Kalﬀ, 2002: p.207). Levels lower than those which resulted in the maximum growth rates of P. acuta are common in south-east Australia, despite other water bodies having high salinity (Vicwaterdata, 2004) and P. acuta occurs at EC below levels where optimal growth occurs (BJK, pers. obs.). The measurement of sub-lethal eﬀects in toxicity testing is now common (OEDC, 1996; ASTM, 1998). It is not known whether reductions in reproduction and/or growth at low salinities measured in this and other studies would aﬀect populations or communities in nature (see Calow and Forbes, 2003). Nevertheless, it seems reasonable that if sub-lethal eﬀects can have ecological eﬀects when caused by high levels of a pollutant, including salinity, they can also be caused by low levels. This should be further investigated. Acknowledgments This work was funded by Land and Water Australia (LWA) project no VCE 17 and LWA and Murray Darling Basin Commission project no RMI 12. BJK completed this work while in receipt of a PhD scholarship from RMIT University. We also wish to thank Richard Marchant and Wilson Lennard for comments on the manuscript, Shanaugh McKay for assistance in the laboratory, Natalie Burfurd and Liliana Zalizniak for proof reading, Colin Clay for assistance collecting P. acuta and Tim O’Brien and Tom Ryan for the use of the aquarium.
B.J. Keﬀord, D. Nugegoda / Environmental Pollution 134 (2005) 377–383
References ASTM, 1998. Annual Book of ASTM Standards, Water and Environmental Technology, Biological Eﬀects and Environmental Fate, Biotechnology; Pesticides, vol. 11.05. American Society for Testing and Materials, West Coshohocken. Bayly, I.A.E., Williams, W.D., 1973. Inland Waters and Their Ecology. Longman Australia Pty Limited, Hawthorn. Beaver, C.J.O.P., 1990. Respiratory rate of mayﬂy nymphs in water with diﬀering oxygen and ionic concentrations. In: Campbell, I.C. (Ed.), Mayﬂies and Stoneﬂies: Life Histories and Biology. Proceedings of the ﬁfth International Ephemeroptera Conference and the ninth International Plecoptera Conference. Kluwer Academic Publishers, Dordrecht, pp. 105–107. Blasius, B.J., Merritt, R.W., 2002. Field and laboratory investigations on the eﬀects of road salt (NaCl) on stream macroinvertebrate communities. Environmental Pollution 120, 219–231. Boeuf, G., Payan, P., 2001. How should salinity inﬂuence ﬁsh growth? Comparative Biochemistry and Physiology C 130, 411–423. Brodersen, J., Madsen, H., 2003. The eﬀect of calcium concentration on the crushing resistance, weight and size of Biomphalaria sudanica (Gastropoda: Planorbidae). Hydrobiologia 490, 181–186. Calow, P., Forbes, V.E., 1998. How do physiological responses to stress translate into ecological and evolutionary processes? Comparative Biochemistry and Physiology A 120, 11–16. Calow, P., Forbes, V.E., 2003. Does ecotoxicology inform ecological risk assessment? Environmental Science and Technology 37, 146A–151A. Cameron, J.N., Iwama, G.K., 1989. Compromises between ionic regulation and acid–base regulation in aquatic animals. Canadian Journal of Zoology 67, 3078–3084. Chapman, P.M., 1998. New and emerging issues in ecotoxicology – the shape of testing to come? Australasian Journal of Ecotoxicology 4, 1–7. Cheung, S.G., Lam, S.W., 1995. Eﬀect of salinity, temperature and acclimation on oxygen consumption of Nassarius festivus (Powys, 1835) (Gastropoda: Nassariidae). Comparative Biochemistry and Physiology A 111, 625–631. Croghan, P.C., 1961. Competition and mechanisms of osmotic adaptation. Symposia of the Society for Experimental Biology 15, 156–166. Dorgelo, J., 1973. Comparative ecophysiology of Gammarids (Crustacea: Amphipoda) from marine, brackish and freshwater habitats exposed to the inﬂuence of salinity–temperature combinations: III. Oxygen uptake. Netherlands Journal of Sea Research 7, 253–266. Duncan, A., 1966. The oxygen consumption of Potamopygus jenkinsi (Smith) (Prosobranchiata) in diﬀerent temperatures and salinities. Verhandlungen Internatnationale Vereinigung fu¨r Theoretische und Angewandte Limnologie 16, 1739–1751. Goetsch, P.A., Palmer, C.G., 1997. Salinity tolerance of selected macroinvertebrates of the Sabie River, Kruger National Park, South Africa. Archives Environmental Contamination and Toxicology 32, 32–41. Goolish, E.M., Burton, R.S., 1989. Energetics of osmoregulation in an intertidal copepod: eﬀects of anoxia and lipid reserves on the pattern of free amino acids accumulation. Functional Ecology 3, 81–89. Hart, B., Bailey, P., Edwards, P., Hortle, K., James, K., McMahon, A., Meredith, C., Swadling, K., 1990. Eﬀects of salinity on river, stream and wetland ecosystems in Victoria, Australia. Water Research 24, 1103–1117. Hart, B., Bailey, P., Edwards, P., Hortle, K., James, K., McMahon, A., Meredith, C., Swadling, K., 1991. A review of salt sensitivity of Australian freshwater biota. Hydrobiologia 210, 105–144. Heisler, N., 1989. Interactions between gas exchange, metabolism, and ion transport in animals: an overview. Canadian Journal of Zoology 67, 2923–2935.
Henry, R.P., Weatly, M.G., 1992. Interaction of respiration, ion regulation, and acid–base balance in the everyday life of aquatic crustaceans. American Zoology 32, 407–416. Hunter, W.R., 1964. Physiological aspects of ecology in nonmarine molluscs. In: Wilbur, K.M., Yonge, C.M. (Eds.), Physiology of Mollusca, vol. 1. Academic Press, New York, pp. 83–126. Iwama, G.K., Takemura, A., Takano, K., 1997. Oxygen consumption rates in tilapia in fresh water, sea water, and hypersaline sea water. Journal of Fish Biology 51, 886–894. Jacobsen, R., Forbes, V.E., 1997. Clonal variation in life-history traits and feeding rates in the gastropod, Potamopyrgus antipodarum: performance across a salinity gradient. Functional Ecology 11, 260–267. Jordan, P.J., Deaton, L.E., 1999. Osmotic regulation and salinity tolerance in the freshwater snail Pomacea bridgesi and the freshwater clam Lampsilis teres. Comparative Biochemistry and Physiology A 122, 199–205. Kalﬀ, J., 2002. Limnology of Inland Water Ecosystems. Prentice Hall, Upper Saddle River, New Jersey. Kapoor, N.N., 1979. Osmotic regulation and salinity tolerance of the stoneﬂy nymph, Paragnetina media. Journal of Insect Physiology 25, 17–20. Keﬀord, B.J., Papas, P.J., Crowther, D., Nugegoda, D., 2002. Are salts toxicants? Australasian Journal of Ecotoxicology 8, 63–68. Keﬀord, B.J., Papas, P.J., Metzeling, L., Nugegoda, D., 2004a. Do laboratory salinity tolerances of freshwater animals correspond with their ﬁeld salinity? Environmental Pollution 129, 355–362. Keﬀord, B.J., Dalton, A., Palmer, C.G., Nugegoda, D., 2004b. The salinity tolerance of eggs and hatchlings of selected aquatic macroinvertebrates in south-east Australia and South Africa. Hydrobiologia 517, 179–192. Keﬀord, B.J., Papas, P.J., Nugegoda, D., 2003. Relative salinity tolerance of macroinvertebrates from the Barwon River, Victoria, Australia. Marine and Freshwater Research 54, 755–765. King, I.M., 1976. Respiration in three species of the Genus Gladioferens (Copepoda: Calanoida). Australian Journal of Marine and Freshwater Research 27, 529–532. Linton, L.R., Davies, R.W., Wrona, F.J., 1982. Osmotic and respirometric responses of two species of Hirudinoidea to changes in water chemistry. Comparative Biochemistry and Physiology A 71, 243–247. Madsen, H., 1987. Eﬀect of calcium concentration on growth and egg laying of Helisoma duryi, Biomphalaria alexandrina, B. camerunensis and Bulinus truncatus (Gastropoda: Planorbidae). Journal of Applied Ecology 24, 823–836. Madsen, H., 1990. Eﬀect of sodium chloride concentration on growth and egg laying of Helisoma duryi, Biomphalaria alexandrina and Bulinus trancatus (Gastropoda: Planorbidae). Journal of Molluscan Studies 56, 181–187. Morgan, J.D., Sakamoto, T., Grau, E.G., Iwama, G.K., 1997. Physiological and respiratory responses of the Mozambique Tilapia (Oreochromis mossambicus) to salinity acclimation. Comparative Biochemistry and Physiology A 117, 391–398. Nduku, W.K., Harrison, A.D., 1976. Calcium as a limiting factor in the biology of Biomphalaria pfeiﬀeri (Krauss), (Gastropoda: Planorbidae). Hydrobiologia 49, 143–170. Nielsen, D.L., Brock, M.A., Rees, G.A., Baldwin, D.S., 2003. The eﬀect of increasing salinity on freshwater ecosystems in Australia. Australian Journal of Botany 51, 655–665. Palmer, A.R., 1992. Calciﬁcation in marine molluscs: how costly is it? Proceedings of the National Academy of Science of the United States of America 89, 1379–1382. Patrick, M.L., Bradley, T.J., 2000. The physiology of salinity tolerance in larvae of two species of Culex mosquitoes: the role of compatible solutes. The Journal of Experimental Biology 203, 821–830.
B.J. Keﬀord, D. Nugegoda / Environmental Pollution 134 (2005) 377–383 Potts, W.T.W., 1954. The energetics of osmotic regulation in brackishand fresh-water animals. Journal of Experimental Biology 31, 618– 630. Odum, E.P., Flinn, J.T., Franz, E.H., 1979. Perturbation theory and the subsidy-stress gradient. BioScience 29, 349–352. OEDC, 1996. Guidelines for Testing of Chemicals. Organization for Economic Cooperation and Development, Paris. Rankin, J.C., Davenport, J.A., 1981. Animal Osmoregulation. Blackie, Glasgow. Rao, Y.P., Devi, V.U., Rao, D.G.V.P., 1987. Respiration of a fouling mollusc Mytilopsis sallei (Recluz) in relation to diﬀerent salinities. Mahasagar-Bulletin on the National Institute of Oceanography 20, 139–143. Reynoldson, T.B., Davies, R.W., 1980. A comparative study of weight regulation in Nephelopsis obscura and Erpobdella punctata (Hirudinoidea). Comparative Biochemistry and Physiology A 66, 711–714. Schlichter, L.C., 1981. Ion relations of haemolymph, pallial ﬂuid, and mucus of Lymnaea stagnalis. Canadian Journal of Zoology 59, 605–613. Styczynska-Jurewicz, E., 1972. Fecundity, survival and haemolymph concentration of Physa acuta Drap. (Gastropoda, Pulmonata) and Tubifex tubifex Mull. (Oligochaeta, Tubiﬁcidae) in relation to salinity of external medium. Polskie Archiwum Hydrobiologii 19, 223–234.
Sutcliﬀe, D.W., 1984. Quantitative aspects of oxygen uptake by Gammarus (Crustacean, Amphipoda): a critical review. Freshwater Biology 14, 443–489. Vicwaterdata, 2004. Victorian Water Resources Data Warehouse. Victoria, Australia. Available from http://www.vicwaterdata.net/. Wheatly, M.G., McMahon, B.R., 1983. Respiration and ionicregulation in the euryhaline crayﬁsh Pacifastacus leniusculus on exposure to high salinity: an overview. In: Freshwater Crayﬁsh. 5. Papers from the Fifth International Symposium on Freshwater Crayﬁsh, Davis, California, pp. 43–55. Wilbur, K.M., Saleuddin, A.S.M., 1983. Shell formation. In: Saleuddin, A.S.M., Wilbur, K.M. (Eds.), Physiology. In: The Mollusca, vol. 4. Academic Press, New York, pp. 235–287. Williams, D.D., Williams, N.E., 1998. Aquatic insects in an estuarine environment: densities, distribution and salinity tolerance. Freshwater Biology 39, 411–421. Williams, W.D., 1987. Salinization of rivers and streams: an important environmental hazard. Ambio 16, 180–185. Williams, W.D., Sherwood, J.E., 1994. Deﬁnition and measurement of salinity in salt lakes. International Journal of Salt Lake Research 3, 53–63. Yang, H., He, Z., 1997. The eﬀect of salinity on assimilation metabolism, growth and carbon budget of Daphnia magna. Journal of Fishery Sciences of China 4, 33–38.