Wat. Sci. Trch. Vol. 38, No.7, pp. 263-270. 1998. IAWQ e 1998 Published by Elsevier Science LId. Printed in Greal Britain. All nghu reserved 0273-1223198519'00 + (H)()
EFFECT OF CADMIUM ON THE EMBRYOS AND JUVENILES OF A TROPICAL FRESHWATER SNAIL, PHYSA ACUTA (DRAPARNAUD, 1805) C. C. C. Cheung and P. K. S. Lam Centre for Environmental Science &: Technology and Department ofBiology &: Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon. Hong Kong
ABSTRACf The effects of cadmium on early life stages of a freshwater snail. Physa acuta (Drapamaud), were investigated. The 24- and 48-hour LCSO for embryos were 1.27 mgll (95% confidence interval: 1.13 • 1.42) and 0.85 mgll (0.71 - 1.01), respectively. The 24- and 48-hour LCso for juveniles were 1.32 mgll (1.13 • 1.S4) and 1.05 mgll (0.81 • 1.36), respectively. An increase in cadmium concentrations at sublethal levels resulted in a significant reduction in embryo growth rate and embryo hatchability. Developmental deformities of the embryos were also observed. The NOEC and LOEC for embryonic growth were 0.32 and 0.50 mgll. respectively, and the LOEC for hatchability was 0.13 mgll. These results suggest that embryonic growth and hatchability arc useful endpoints in chronic toxicity tests. In addition. the potential use of early life stages of pulmonate snails in ecotoxicologicaltests is discussed. e 1998 Published by Elsevier Science Ltd. All rights reserved
KEYWORDS Heavy metal; cadmium; freshwater snail; Physa; early life stages. INTRODUCflON Acute lethality tests have been widely used in providing rapid estimates of the concentrations of test substances that cause direct. irreversible harm to test organisms because of their high reproductibility, simplicity and convenience. Notwithstanding. results of acute tests cannot provide substantive information about the sublethal or cumulative effects of test substances and thus these tests are not predictive of potential chronic toxicity (Macek et al., 1978). Moreover. the toxicant concentrations used in acute tests seldom occur under realistic field conditions, The above shortcomings associated with acute tests make it necessary to supplement these tests with other sublethal tests. In this connection, complete life-cycle or multiple generation chronic toxicity tests have been developed. However. these tests demand high costs, labour. equipment and space. and the time required to get results is very long. usually months to years . Consequently. the usefulness of these tests is limited. To get a more ecologically realistic result from toxicity assessments, tests other than acute or fulllife-cycle tests should be considered. One type of test uses the energy allocation (e.g. scope for growth) of an organism as the endpoint. another uses growth rates of the
C. C. C. CHEUNG and P. K. S. LAM
organisms as a criterion to measure the response of organisms towards environmental stress (Anderson et al., 1980; Gauss et al., 1985; Thomas and McClintock, 1990). Cadmium has been placed on the blacklists of most international pollution conventions by virtue of its toxicity, potential for bio-accumulation and persistence (Taylor, 1983). The world production of cadmium was about 1.5 x I ()4 tons per year during 1971-80 (Moore and Ramamoorthy, 1984), and this figure is likely to have increased in recent years. Cadmium-contalning compounds are widely used in electroplating, as stabilizers and as one of the components of rechargeable batteries. In this study, freshwater snails were selected as the target organism since snails are abundant members of the freshwater commun ity and are estimated to constitute 20% of the diet of some species of freshwater fish (Pennak, 1978). Cadmium toxicity has been previously reported on adult snails of a number of species, e.g, Physa integra (Spehar et al., 1978) and Lymnaea stagnalis (Puymbroeck et al., 1982), using mortality as a criterion. Other ecotoxicologists have studied the effects of cadmium on the "scope for growth" of the adult snail, Brotia hainanensis (Lam, I996a). In addition, the acute effect of cadmium on the embryos and juveniles of snails have also been investigated (e.g. Rehowoldt et al..• 1973; Wier and Walter, 1976; Ravera, 1977). Despite this, very little work has been done on the chronic effects of cadmium on the growth and development of snail embryos . Further research is urgently needed to provide important information on the biological effect of metals on early, and probably more sensitive, life stages of these organisms. The snails used in this study, Physa acuta (Draparnaud, 1805), are commonly found in tropical streams or ponds and are important components in aquatic food webs. Here, the acute toxic effects of cadmium on the embryos and juveniles of P. acuta were studied in static toxicity tests. Specifically, the chronic toxic effects of cadmium on the growth and hatchability of the snail embryos were examined. The 50% Lethal Concentration (LC so), No Observed Effect Concentrations (NOECs) and Lowest Observed Effect Concentrations (LOECs) were estimated. Results of this study will enable a comparison of the relative sensitivity and usefulness of different endpoints towards cadmium. This step is crucial in formulating good, relevant testing protocols for assessing toxic stressors. MATERIALS Test chemicals and o[~anisms The stock cadmium solution was prepared by adding the appropriate amount of cadmium (II) chloride-Ihydrate salt to deionized water. The test concentrations were prepared by dilution of the stock solution of cadmium (500 mgll) with artificial pond water (APW; Lam and Calow, 1989) (conductivity 600 ~-I, pH = 7.4, [Ca2+] = 80 mgll, [Mg2+] = 12.115 mgll, and hardness = 250 mgll CaC03). Snails were collected from flooded furrows in Sai Kung, New Territories, Hong Kong. The snails were maintained on a diet of boiled lettuce in an aquarium filled with aerated APW at 25°C. The snails were checked daily and any egg masses laid, as well as the juvenile snails were carefully collected and put into a beaker containing aerated APWat25°C.
METHODS Acute toxicitY tests usjn~ Phvsa acuta embO'os and juyeniles In this experiment, 96-hour old embryos (with a well defined shell and heartbeat) and 4-day old juvenile snails were used in assessing the acute toxicity of cadmium. Egg masses were collected and examined under a stereomicroscopc for normal appearance and the number of embryos in each egg mass was counted (Millemann et al., 1984). Five nominal test concentrations 0, 1,2, 3,4 mgll cadmium were used. For the embryo test, one egg mass was placed in each 100 m1 glass beaker containing 80 ml of test solution of the appropriate cadmium concentration. Three replicates were used for each test concentration and the control
Cadmium effect on embryos
groups were exposed to APW only. For the juvenile test. 10juvenile snails were placed in each plastic petri dish (4.5 em diameter) containing 12 mI of test solution. The petri dishes were then covered by a fine mesh held in position by a rubber band. Food was not provided during the test period (Wier and Walter. 1976). Three replicates were used for each of the four test concentrations and the control. In both cases. test vessels were aerated continuously throughout the test. The viability of embryos and juvenile snails was checked every 24 hours. For embryos. death was defined as the absence of heartbeat. and for juvenile snails. absence of heartbeat and the lack of foot movement upon stimulation by probing was defined as death (Millemann et al..• 1984). Chronic toxicity test usin!: Physa acuta embr.yos and juyeniles Newly-laid egg masses were used in the embryo test and 4-day old juveniles were used in the juvenile test. They were washed twice with APW and then carefully examined under a stereomicroscope for any abnormalities prior to any experimentation. Dewitt (1954) found that abnormal development frequently occurred in very small embryos so embryos with diameter below 0.1 mm were not used. Moreover. eggs with more than one embryo were also discarded. In both tests. nominal cadmium concentrations were 0.1, 0.2. 0.3. 0.4. 0.5 mg/l and the control contained only APW. The number of replicates. test vessels and volume of test solution in each vessel were the same as in the acute test. The experimental set up was maintained in an environmental chamber at 25°C and 95% humidity with a photoperiod of 12-hour light and 12-hour dark (Masakado and Inaba, 1992). The test solutions were continuously aerated and changed every 72 hours. and the pH and temperature were checked at the time of solution renewal. The sizes of the embryos and juveniles were measured daily and weekly respectively. using a compound microscope with an ocular micrometer. The number of embryos hatched at day 8 and 28 were also recorded. Eggs that did not hatch after 28 days were considered unhatchable , Photographs of some of the embryos exposed to various cadmium concentrations were also taken to illustrate typical developmental abnormalities . Embryos were designated as abnormal if they met at least one of the following three criteria: (I) aberrant gross morphology involving abnormal growth. or gross morphological deformities compared with control embryos at the same developmental stage. (2) abnormal pigmentation of embryo tissues with white or transparent regions in contrast to the normal brown colour, and (3) obvious decomposition and/or arrested development for more than two weeks (Hunter, 1988). Samples of the test solution were taken from each replicate at the time of solution renewal for subsequent determination of actual cadmium levels using atomic absorption spectrophotometry. AA-6501S (furnace). RESULTS
• 24 In eJCpOsure
48 In e"POsure
• 24In eltXJ!U8
Cadmium concentration (mgll)
Figure I Percentagemortality of Physa acuta embryos Figure 2 Percentagemortality of Physa acuta juveniles exposed to various cadmium concentrations exposed to various cadmium concentrations for 24 and 48 hours. Vertical lines are ±.S.E.. for 24 and 48 hours. Vertical lines are ± S.E..
c. c. C. CHEUNG and P. K. S. LAM
Acute toxicity of cadmium on Physa acuta embryos and juveniles The 24- and 48-hour 50% lethal concentrations (LC so) for the embryos and juveniles were determined by probit analysis (Finney, 1989). The 24- and 48-hour LC sOvalues for the embryos were 1.27 mg/I (upper and lower 95% confident limits: 1.42 and 1.13) and 0.85 mg/I cadmium (1.01 and 0.71), respectively (Fig. I). For the juvenile snails, the 24-hour LC so value was 1.32 mg/I (1.54 and 1.13), while the 48-hour LC so was 1.05 mg/l (1.36 and 0.81) (Fig. 2). Chronic tests on Physa acuta embryos and juveniles
Developmental deformities of Physa acuta embryos: At a relatively low cadmium concentration (0.05 mg/l), snail embryos still were of a similar size to the normal embryos of the same stage (Plate Ia) but an abnormally clear region was observed inside the body of an 8 day-old embryo at 0.05 mg/I cadmium (Plate Ib). The growth of embryos at a higher cadmium concentration (0.13mg/l) was impaired and a twisted and compressed shell was observed from an embryo at 0.13 mg/l cadmium (Plate Ic). At an even higher cadmium concentration (0.50 mg/l), the embryonic growth was severely retarded, and an abnormally small embryo size was observed (Plate Id).
Plate Ia. Normal embryo (70x).
Plate Ib. Embryo with an abnormal clear region (arrow) (70x).
Plate Ie. Embryo with deformed and twisted shell (70x).
Abnormally small embryo (70x).
Cadmium effect on embryos
Reduction in growth rate of Physa acuta embryos and juveniles: The growth rate of the embryos and juveniles decreased with increasing cadmium concentrations (Fig. 3). Growth rate was defined using the formula of Simpson and Roe (1939). (log YI - log Yo)
...size at time 0 .. time interval
= size at time t .. geometric growth rate
One-way analysis of variance (ANOVA) revealed that an increase in cadmium concentration caused a significant decrease in the growth rate of snail embryos (FS.12 = 5.518. P < 0.01; Fig. 3). but not juveniles (FS.lI 0.6492. P>O.05). Pair-wise comparisons using Dunnett's tests (Zar, 1984) revealed that the NOEC and LOEC of cadmium on the growth of snail embryos were 0.32 and 0.50 mgIJ.respectively. Reduction in hatchability of Physa acuta embryos: Percentages of embryos hatched decreased with increasing cadmium concentrations (Fig. 4). One-way ANOVA indicated that an increase in cadmium concentration caused a significant decrease in the percentage of embryos hatched (Day 8: FS.11 = 400.44, P < 0.0001; Day 28: FS• lI = 36.70. P < 0.0001). Pair-wise comparisons using Dunnett's tests revealed that exposure to O. I3 mgIJ of cadmium or higher caused significant reduction in the hatching of snail embryos (Fig. 4) which will hatch on day 8 in the absence of cadmium. In addition. hatching was completely inhibited in embryos exposed to cadmium concentrations of 0.2 I mgIJor higher.
0.00+--r---r--"--T--.---4 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Cberwd cactmm CCllICelItJ aIa1 (nv'J) Figure 3 Effects of various cadmium concentrations Figure 4 on the growth rate of Physaacuta embryos. Vertical lines are ± S.D..
0.1 0.2 0.3 0.4 0.5 0.8 CblervedC8ltnlll1\ concentration (rrv'J)
Effects of various cadmium concentrations on the hatchability of Physa acuta embryos. Vertical lines are ± S.D..
It is worth mentioning that most of the previous studies were only concerned with adult snails, very little work has been done on embryos and juvenile snails. In fact, embryo-juvenile toxicity tests possess many advantages such as the relatively short time required. Dave (1993) found that juveniles usually require less time to reach equilibrium and respond quickly especially at low toxicant concentrations . More importantly. early developmental stages are usually more sensitive than adult stages towards toxicants, thus offering a potentially higher degree of protection to the receiving environment.
C. C. C. CHEUNG and P. K. S. LAM
Ecotoxicologists (e.g, Rehwoldt et al., 1973; Wier and Walter, 1976: Ravera, 1977) who studied the responses of snail embryos and juveniles towards cadmium toxicity, generally used LC sOo ST 50 and hatching success as the endpoints, and none of them has used reduction in growth rate as a criterion. Actually, growth of an individual represents an integration of physiological processes which depends on the balance between metabolic demand and supply (Sutcliffe et al., 1981: Gauss et al.• 1985). Mortality-based acute tests are highly reproducible but low in ecological significance, and are unable to predict potential chronic effects of a toxicant at sublethal concentrations (Rand and Petrocelli, 1985). By contrast, complete life cycle tests are low in reproducibility but high in ecological relevance. Growth inhibition is Icnown to be a sub lethal response to chronic toxicant exposure (Kosalwat and Knight, 1987), and is a good compromise between using mortality-based acute tests and chronic tests involving an assessment of fecundity and reproductive ability in ecotoxicological tests. In this study, NOEC and LOEC for the embryonic growth were 0.32 and 0.50 mg/l cadmium, respectively. If hatchability of embryos (hatching success) was used as an endpoint, the LOEC (0.13 mg/l) was much lower. These results indicate that hatchability of snail embryos is more sensitive than embryonic growth . Moreover, the LOEC values obtained can be used to derive the Maximum Allowable Toxicant Concentration (MATC) for cadmium which may be useful in setting water quality standards (Petrocelli, 1985). Undcr cadmium stress, only 7.58% of the embryos hatched at 0.15 mg/l cadmium, and none of the embryos hatched at 0.21 mgll cadmium or above. Moreover, abnormality of P. acuta embryos was observed at cadmium levels as low as 0.05 mg/l, suggesting that cadmium is teratogenic even at low concentrations. Growth and developmental rates are important fitness components (Taylor et al., 1991) and, all other things being equal, a longer developmental period would mean fewer offspring produced per unit time. As a result, even at low cadmium concentration, a significant impact on the population will be expected to occur and this may severely affect the aquatic food chain, and hence, the functioning of the aquatic ecosystem. Results of the acute tests on snail embryos and juvenile snails showed that embryos were more sensitive to cadmium than juveniles. This finding is at variance with other studies which reported that juvenile snails were more sensitive than adult snails as well as embryo snails (Wier and Walter, 1976; Ravera, 1977). A similar difference in sensitivity was also reported for fish alevins and fish embryos (Eaton et al., 1978; Rombough and Garside, 1982). It has been suggested that only a very small percentage of the cadmium ions actually reach the embryos (Beattie and Pascoe, 1978; Michibata, 1981). Indeed. it was found that about 90% of the cadmium ions were associated with the chorion, rather than with the embryo or the yolk. As a consequence, the embryos were less vulnerable to cadmium. By contrast, the larvae/juveniles were not protected by the chorion, and thus were more susceptible to cadmium toxicity. Notwithstanding, the results obtained in this study suggest that Physa acuta embryos are more sensitive than the juveniles. It is conceivable that the relative sensitivity of different life stages may be species-specific, and further investigations will be required to clarify this point. The use of small freshwater pulmonate snails, such as Physa acuta; as target organisms has many other advantages over commonly used freshwater fishes such as Rainbow trout (Salmo gairdneTl). Snails are comparatively more sedentary than fishes so they are more vulnerable to toxic discharges or effluents. While the commonly used water fleas (Daphnia) are restricted to marginal hard water or polluted habitats and show a distinct preference for ponds (Brendonck and Persoone, 1993), snails can be sampled in large quantities with relative ease in a wide range of habitats including lotic and lentic waters (Lam, 1996b). Furthermore, these pulmonates can survive a relatively long period of time without any aeration, and this together with their small size can further simplify the testing protocol (Ravera, 1977). Moreover, since the developmental stages of embryos are easy to identify, other sublethal endpoints such as frequency of abnormal embryos and delay of hatching can also be easily measured, and the impacts of the toxicant assessed (Ravera, 1977). There are additional reasons why freshwater snails are useful as test organisms in ecotoxicological assessments. These pulmonate snails are important detritivores in standing and running waters. Cummins (1974) devised four main categories of invertebrate consumers in streams, and freshwater snails like Physa acuta are classified as "grazer-scrapers" that have mouth parts appropriate for scraping off and consuming the organic layer (composed of attached algae, fungi and dead organic matter) attached to rocks and stones . Indeed, Dudgeon and Yipp (1983) found that Physa acuta in Hong Kong consumes
Cadmiumeffect on embryos
primarily detritus with green algae (unicellular and filamentous) and diatoms as a regular part of the diet. These grazers act as an important energy link between the producers and other members of higher tropic levels. and thus are ecologically meaningful test organisms in ecotoxicological assessments. CONCLUSIONS Embryonic growth and hatchability were significantly affected by cadmium. The NOEC and LOEC for embryo growth were 0.32 and 0.5 mgll cadmium, respectively. The LOEC of cadmium effect on hatching success was 0.13 mgll cadmium. In addition. developmental deformities were observed even at cadmium concentrations as low as 0.05 mgll. These results indicate that hatching success is a more sensitive endpoint in assessing cadmium toxicity than embryo growth rate. Overall. our results suggest that the embryos of Physa acuta are useful in ecotoxicologicaJ assessments. ACKNOWLEDGEMENT This work was funded by research grants from the City University of Hong Kong and the Hong Kong Research Grants Council (No.: CityU0952J96M). REFERENCES Anderson. R. L.• Walbridge. C. F. and Randt J. T. (1980). Survival and growth of Tanytarsus dissimllis (Chironomidae)exposed to copper, cadmium, zinc, and lead. Archivesof Environmental Contamination and Toxicology, 18, 492-496. Beattie. J. H. and Pascoe, D. (1978). Cadmium uptake by Rainbow Trout, Salmo gairdneri, eggs and alevins. Journal of Fish Biology, 13, 631-637. Brendonck, Land Persoone, G. (1993). BiologicaVecological characteristics of large freshwater branchiopods form endorheic regions and consequences for their use in cyst-based toxicity tests , In: Progress in Standardization of Aquatic Toxicity Tests. P. Calow and Soares, Amadeu M. V. M. (eds), CRC Press, Boca Raton. Florida, p. 5. Cummins, K. W. (1974). Structure and function ofstream ecosystems. BioScience, 24, 631-641. Dave. G. (1993). Replicability, Repeatability. and Reproductibility of Embryo-Larval Toxicity Tests with Fish. In: Progress in Standardization of Aquatic Toxicity Tests. P. Calow and Soares. Amadeu M. V. M. (eds), CRC Press, Boca Raton, Florida. pp. 129-157. Dewitt, R. W. (1954). Reproduction.embryonic development, and growth in the pond snail. Physella gyriM Say. Transactions American Microscopical Society73, 124-137. Dudgeon. D. and Yipp, M. W. (1983). The diets of Hong Kong freshwatergastropods. In: The MalacofauM of Hong Kong and Southern China. B. Morton and D. Dudgeon (eds), 1'012, Hong Kong UniversityPress. Hong Kong.pp. 491-509. Eaton. J. G.•McKim. J. M. and Holcombe, G. W. (1978).Metal toxicityto embryos and larvae of seven freshwater fish species. I. Cadmium. Bulletinof Environmental Contamination and Toxicology, 19, 95-103. Finney. D. J. (\989). Probit Analysis.3rd edn, CambridgeUniversity Press. Cambridge. Gauss, J. D.• Woods, P. E.• Winner, R. W. and Skillings. J. H. (1985). Acute toxicity of copper to three life stages of Chironomus tentans as affected by water hardness-alkalinity. Environmental Pollution (SeriesAJ. 37, 149-1S7 Hunter. R. D. (\ 988). Effect of acid water on shells, embryos, and juvenile survival of Planorbella trivolvis (Gastropoda:Pulmonate): a laboratorystudy. Journal ofFreshwater Ecology, 4, 315-327. Kosalwat, P. and Knight. A. W. (I 987}. Chronic toxicity of copper to a partial life cycle of the midge. Chironomus decour. Archivesof Environmental Comaminatian and Toxicology, 16,283-290. Lam. P. K. S. and Calow, P. (1989). Intraspecific life-history variation in Lymnaea peregra (Gastropoda: Pulmonata). n. Environmentalor genetic variance? Journalof AnimalEcology, 58. S89-602. Lam. P. K. S. (l996a ). Sublethal effects of cadmium on the energetics of. tropical freshwater snail, Brotia hainanensis (Brot, 1872). Environmental Toxicology and WaterQuality, 11, 34S-349. Lam, P. K. S. (l996b) . Effect of cadmium on the consumptionand absorptionrates of a tropical freshwatersnail. Radixplicatulus. Chemosphere, 32(1I}. 2127-2132. Macek, K.. Birge. W.• Mayer. F. L.. Buikema. A. L and Makia. A. W. (1978). Toxicological effects. In: Estimating the Hazardof Chemical Substances to Aquatic Life, J. Cairns. K. L. Dickson. A. W. Maki (eds), pp. 27-32. ASTM STP 657. Philadelphia: AmericanSociety for Testing and Materials. Masadado, K. and Inaba, M. (I 992}. Individual differences in growth and access to food in the freshwater snail. Physa QCuta. Journalof Ethology, 10,75-79. Michibata, H. (1981). Uptakeof cadmium by egg of the Teleost Oryzia:latipes.Journalof FishBiology, 19, 691-696. Millemann. R. E.• Santa, J. T.• Jenny. L. F. and Daren. L. D. (1984). Comparative Toxicities of Coal- and Shale-DerivedCrude Oils and a Petroleum-Derived Fuel Oil to the Freshwater Snails Helisoma trivolvis and Physa gyrina. Environmental Pollution (Slries A), 33, 23-38.
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