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Copper toxicity in the marine copepod Tigropus japonicus: Low variability and high reproducibility of repeated acute and life-cycle tests
Marine Pollution Bulletin 57 (2008) 632–636
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Copper toxicity in the marine copepod Tigropus japonicus: Low variability and high reproducibility of repeated acute and life-cycle tests Kevin W.H. Kwok a,b,*, Kenneth M.Y. Leung a,b, Vivien W.W. Bao a,b, Jae-Seong Lee c a
The Swire Institute of Marine Science, Faculty of Science, The University of Hong Kong, Cape d’Aguilar, Shek O, Hong Kong, China Division of Ecology and Biodiversity, School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China c Department of Chemistry, and the National Research Laboratory of Marine Molecular and Environmental Bioscience, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea b
a r t i c l e
i n f o
Keywords: Model organism Coefficient of variation Ecotoxicology
a b s t r a c t The intertidal copeopod Tigriopus japonicus, which is abundant and widely distributed along the coasts of Western Pacific, has been suggested to be a good marine ecotoxicity testing organism. In this study, a series of experiments were conducted to investigate the reproducibility and variability of copper (Cu) sensitivity of T. japonicus so as to evaluate its potential to serve as an appropriate test species. To understand the seasonal variation of Cu sensitivity, individuals of T. japonicus were collected from the field in summer and winter, and subjected to standard 96 h acute (static renewal) toxicity tests. 96 h-LC50 values of T. japonicus collected from the two seasons were marginally different (p = 0.05), with an overall coefficient of variation (CV) of 33%. Most importantly, our results indicated that chronic Cu sensitivity of T. japonicus was highly reproducible. The CVs of intrinsic rates of increase in the population of the control and Cu treatment (10 lg Cu l 1) groups were only 10–11% between 10 runs of a standardised complete life-cycle test. Moreover, different Cu(II) salts generally resulted in a similar 96 h-LC50 value while Cu(I) chloride was consistently slightly less toxic than Cu(II) salts. Given such a high reproducibility of toxic responses, it is advocated to use T. japonicus as a routine testing organism. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Aquatic ecotoxicology is biased towards studying freshwater organisms (Leung et al., 2001; Wheeler et al., 2002). One important step to promote marine ecotoxicological studies is, therefore, to find and establish appropriate marine model organisms. Copepods of the genus Tigriopus, which have a wide geographical distribution, short life-cycle and distinctive developmental stages, have been recommended to be one of the promising candidates (Raisuddin et al., 2007) and were shortlisted in a recent OECD document (OECD, 2006) as a potential marine model organism for ecotoxicology. Copper (Cu) contamination is common and widespread in marine coastal environments (Hall and Anderson, 1999). For example, Cu in marine sediment in relatively unpolluted Hong Kong Eastern waters was about 20 mg kg 1, however, such Cu concentrations could reach a maximum of 4000 mg kg 1 in marine sediments collected from highly urbanized areas such as Victoria Harbour (EPD, 2002). In the United Kingdom, the highest annual mean total Cu concentration in marine waters could be as high as 20 lg l 1 (Mat-
* Corresponding author. Address: Division of Ecology and Biodiversity, School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China. Tel.: +852 28092179; fax: +852 25176082. E-mail address: [email protected] (K.W.H. Kwok). 0025-326X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2008.03.026
thiessen et al., 1999). Undoubtedly, contamination of Cu in saltwater environments is generally higher in harbours and estuaries (Hall and Anderson, 1999). The Cu-based antifouling coating on ship hulls has been one of the major sources of Cu contamination in coastal marine environments (Valkirs et al., 2003). Since the partial ban of organotins in many countries in early 1990s, Cu-based antifouling paints have been regaining their popularity and gradually replacing organotin-based antifouling paints (Voulvoulis et al., 1999; Srinivasan and Swain, 2007). The emission rate of Cu from antifouling paints on boats varies, ranging from 0.2 to 65 lg/cm2/ day, depending on the structure of the paint coating and environmental conditions (Valkirs et al., 2003; Schiff et al., 2004). Anthropogenic input of Cu via smelting and refining, mining, domestic wastewater and sewage sludge dumping also contribute to elevated Cu concentrations in the marine environment (Nriagu and Pacyna, 1988). Cu is an essential micronutrient vital to processes such as cellular respiration, free radical defence and cellular iron metabolism, but at elevated levels Cu is toxic to organisms, especially to aquatic invertebrates. Therefore, the widespread contamination of Cu in marine environments poses potential ecological risks to many saltwater organisms and calls for more ecotoxicological studies to better understand its toxic effects towards various marine species.
K.W.H. Kwok et al. / Marine Pollution Bulletin 57 (2008) 632–636
In many ecotoxicity tests, test organisms were collected from the field, acclimated then dosed with chemicals. While the location and time of collection of these animals were often provided, very few studies investigated the natural variability of chemical sensitivity of these organisms (e.g. Winner et al., 1990). It is well documented that of the same species, individuals within a population could have different physiological capacities to cope with a stressor and such capacities often vary according to seasons (Spicer and Gaston, 1999). Therefore, the sensitivity of test organisms possibly changes with time of collection. Hong Kong makes an ideal place to investigate such an effect, as it has two markedly different seasons. The warm, wet season generally begins in April and lasts until September and the cold, dry season generally occurs from October to March (Morton and Morton, 1983). Test organisms collected at different seasons may adopt different physiological strategies to deal with different natural stresses, which may in turn affect their sensitivities towards trace metals. For ecotoxicity tests of Cu, it is common that different salts (e.g. sulphates, nitrates, chlorides) are used. In general, only the concentration of the Cu cation will be considered to have toxic effect on the test organisms (De Laender et al., 2005). Nonetheless, the use of different metal salts will give different forms (e.g. sulphates and nitrates) and different concentrations of anions (e.g. for each mole of Cu, Cu(II) chloride gives two moles of chlorides while Cu(II) sulphate gives one mole of sulphate) in the test solution. As it has been shown that sulphate and chloride ions can influence the resultant Cu toxicity to aquatic organisms, both anions have been included in the biotic ligand models for predicting Cu toxicity (De Laender et al., 2005). The anions of Cu salts may affect the test results by directly having an effect on the test organisms or indirectly by interacting with the Cu cation and thus influencing its bioavailability. The understanding of chronic toxicity of a substance, especially on population level effects, is crucial to assess the long-term effect of the substance in the ecosystem. Reproducibility of chronic toxicity test results, therefore, is a key aspect of a good model organism for ecotoxicological studies. Tigriopus japonicus is highly abundant and widely distributed along the coasts of Western Pacific (i.e. coasts of China, Taiwan, Korea and Japan) and easy to culture, with a complete life-cycle of about 21 days (Raisuddin et al., 2007). This study primarily aimed to investigate the variability of Cu toxicities to the copepod T. japonicus associated with seasons and different Cu salts. Reproducibility of chronic Cu sensitivity was also investigated using a standardised complete life-cycle assay. The results of these tests can be useful to evaluate the potential of T. japonicus to be used as a model organism for marine ecotoxicology.
2. Materials and methods 2.1. Seasonal variability To investigate seasonal differences in sensitivity of T. japonicus to Cu, cultures of the copepod were collected at Cape d’Aguilar Marine Reserve, Hong Kong in warm-wet season (May to August) and in cold-dry season (October to November), respectively. Cultures were acclimated in laboratory at temperatures of 22–25 °C, salinity of 30–35‰ and a 16 h:8 h light:dark cycle. The animals were fed with finely ground green algae Enteromorpha spp. and commercial phytoplankton concentrate (Kent Marine Phytoplex, US). A total of six 96 h acute tests were conducted: three on warm-wet season populations and three on cold-dry season populations. Acute toxicity tests for individual cultures were conducted after 10 d of acclimation in the laboratory. Ten adult males and 10 adult females were used in each replicate and they were placed in a
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50 ml acid-washed breaker with 25 ml of the testing solution. Three replicates were applied for each Cu concentration. At least six nominal concentrations of Cu(II) sulphate were utilised at each test (0 [control] plus at least five concentrations of Cu ranged from 100 to 2000 lg Cu l 1). Test solutions were prepared by diluting a stock solution (reagent grade Cu(II) sulphate, BDH Ltd.; formula weight, f.w.: 249.7) with appropriate volume of fully aerated (aerated for at least 30 min), freshly made filtered artificial seawater (Sea salt: Tropic Marine, Germany; filter: Glass microfibre filters, GF/C 47 mm Circle, Whatman). Test solutions were renewed every 48 h. Test animals were transferred to the 50 ml beakers with filtered artificial seawater and allowed acclimation for 48 h before starting the test. The animals were not fed from the acclimation period till the test ended. Mortality was checked once every 24 h and defined as copepod’s urosome perpendicular to its prosome (McAllen et al., 1999). 2.2. Effect of different copper salts Effect of different Cu salts on Cu toxicity was tested with T. japonicus obtained from an established laboratory culture collected from Cape d’Aguilar maintained for more than one year. 96 h acute toxicity tests were conducted using five different Cu salts (Cu(II) sulphate, f.w.: 249.7; Cu(II) chloride, f.w.: 170.48; Cu(II) nitrate, f.w.: 241.6; Cu(II) acetate, f.w.: 199.65 and Cu(I) chloride, f.w.: 99.0). All the Cu salts were of reagent grade, supplied by Merck (Cu(I) chloride) and BDH Ltd. (the other four salts). Six nominal Cu concentrations of each salt were used in the test (500, 800, 1000, 1200, 1500 and 0 [control] lg Cu l 1). Acute toxicity tests were conducted as described in Kwok and Leung (2005). In brief, 20 randomly selected adult copepods were placed in 25 ml test solution for each replicate and each concentration treatment was triplicated. All acute tests were conducted at temperature 25 ± 1.0 °C, artificial seawater of salinity 33 ± 0.5‰, pH 8.1–8.4 and a 12 h:12 h light:dark photoperiod. All 96 h-LC50 values and their associated 95% confidence intervals (CI) were estimated using Probit analysis (Finney, 1989). The 96 h-LC50 values of the two different seasons were compared using a non-parametric Mann–Whitney test (SPSS version 12.0, Chicago, IL, USA). Coefficient of variation (%) of the LC50 values for each season was computed to understand the amount of variation within a season and between seasons. 2.3. Repeatability of complete life-cycle tests The complete life-cycle toxicity tests of copper chloride (CuCl2; BDH Ltd.) to T. japonicus were conducted using a modified ASTM protocol (ASTM E-2317-04; ASTM, 2004). In 96-well microplates, <24 h-old nauplii were placed individually in wells loaded with 200 ll of test solution (0 [control] and 10 lg Cu l 1) and 2 ll of algal solution containing 107 Skeletonema costatum cells ml 1. Both treatments had eight replicates and each replicate contained 12 nauplii. To ensure consistency of exposure, copepods were placed in new microwells containing fresh test solutions and algal solution every 96 h. Copepods were monitored daily for life-stage mortality, development and sex determination. When the copepods reached copepodid stage 5, male and female copepods were paired up and mated within each replicate for each treatment. Life-cycle testing was terminated after the release of the second brood of nauplii from each mating pair. Test periods for individual microplate bioassays ranged from 20 to 30 d to account for treatment specific developmental delays. A total of ten runs of life-cycle test were conducted. All life-cycle tests were conducted at temperature 25 ± 1.0 °C, artificial seawater of salinity 30 ± 0.5‰, pH 7.9–8.0 and a 12 h:12 h light:dark photoperiod.
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Duration of larval development (from <24 h nauplii to develop into copepodid stage 1), time to first reproduction (from <24 h nauplii to the development of the first egg brood) and the intrinsic rate of increase (rm) were used as endpoints. The rm was computed as suggested by Walthall and Stark (1997) and is an estimate of the intrinsic population growth rate. When rm = 0, it represents a stable population; when it is negative, it represents a declining population, vice versa. Student’s t-test was used to compare the described endpoints between the control and Cu treatment groups.
the control. Cu(II) salts in generally showed comparable 96 h-LC50 values while Cu(I) chloride was the least toxic to T. japonicus, as shown by the non-overlapping 95% CI with the 96 h-LC50 of other Cu(II) salts. The CV in the LC50 values of different Cu(II) salts was 15% and the CV of LC50 values of all Cu salts used was 19%.
3. Results 96 h-LC50 values of T. japonicus collected from different seasons ranged from 323 to 585 lg Cu l 1 (Fig. 1). For all bioassays, control mortality was less than 5%. Between the two seasonal populations, the overall coefficient of variation (CV) in the LC50 values was 33%. The amount of variation for the warm-wet season population was higher (CV = 27%) than that of the cold-dry season (CV = 14%). The LC50 values of warm-wet season cultures were marginally higher than that of the cold-dry season ones (U = 9, d.f. = 4, p = 0.05). 96 h-LC50 values of T. japonicus exposed to the five Cu salts ranged from 743 to 1175 lg Cu l 1 (Fig. 2). There was no mortality for
Fig. 1. Comparison of copper sensitivity of Tigriopus japonicus collected in summer and winter based on the 96 h-LC50 values (mean and 95% C.I.).
Fig. 2. 96 h-LC50 (mean ± 95% C.I.) of Tigriopus japonicus exposed to different copper salts.
Fig. 3. Results of chronic toxicity tests: (a) larval developmental time, (b) time to first reproduction, and (c) intrinsic rate of increase (rm) of Tigriopus japonicus from nauplii to copepodid stage 1 at two different Cu concentrations; middle bar represents the mean, the box represent the first and third quartile and the error bar represents the 95% C.I.
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K.W.H. Kwok et al. / Marine Pollution Bulletin 57 (2008) 632–636 Table 1 LC50 values of marine copepod Acartia tonsa and freshwater cladoceran Daphnia similis to copper (I) and copper (II) salt Species name
Chemical
LC-50 value (lg Cu l
Marine Copepod Acartia tonsa
Cu(I) chloride Cu(II) nitrate Cu(I) chloride Cu(II) chloride Cuprous oxide Cu(II) sulphate
72 h-LC50 = 58.1 72 h-LC50 = 9.0 96 h-LC50 = 1180 96 h-LC50 = 813 48 h-LC50 = 9.6 48 h-LC50 = 3.5
Tigriopus japonicus Freshwater Cladoceran Daphnia similis
For the chronic life-cycle toxicity tests, overall mortality (mean ± 1SD; n = 10) of T. japonicus in the control and treatment (10 lg Cu l 1) groups were 10.4 ± 4% and 13.1 ± 4%, respectively. Results of life-cycle development and population growth endpoints in general were highly reproducible through ten runs of the chronic toxicity tests. Development of T. japonicus slowed down significantly at 10 lg Cu l 1 (t = 6.05, d.f. = 78, p < 0.001) (Fig. 3a). The amount of variation within the control and the Cu treatment was similar, with a CV of 4–5%. Time to first reproduction was significantly delayed at 10 lg Cu l 1 by about a half day (t = 4.82, d.f. = 78, p < 0.001) (Fig. 3b). The amount of variation within the control and Cu treatment was similar, with a CV around 3%. Intrinsic rate of increase (rm) was significantly lowered at 10 lg Cu l 1 (t = 4.54, d.f. = 78, p < 0.001) (Fig. 3c). The amount of variation within the control and the Cu treatment was similar, with a CV of 10–11%. 4. Discussion Acute Cu sensitivities of T. japonicus populations collected from the two seasons were marginally statistically different. The T. japonicus collected in the warm-wet season showed much higher variability in the 96 h-LC50 values than those collected from the colddry season. As Tigriopus species inhabit supratidal rockpools which are subjected to natural extremes of temperature and salinity, they are necessarily tolerant to such extremes (O’Brien et al., 1988). The variability in Cu sensitivity may be attributable to the variability of physiological state of the test animals. In the warm-wet season, Tigriopus population were subjected to larger fluctuations in temperature, salinity, pH and dissolved oxygen (Chan, 2000). Therefore, the physiological state of the summer Tigriopus population could be more variable. Seasonal differences in cadmium sensitivity have also been observed in amphipods such as Leptocheirus plumulosus and Corophium volutator (McGee et al., 1998; Kater et al., 2000). It was suggested that the seasonal variation might be attributed to food supply, population dynamics and difference between field temperature and laboratory test temperature (Sonsowski et al., 1979; Kater et al., 2000). Free copper(II) ion is generally considered to be a more toxic form of Cu (Hall and Anderson, 1999). In the present study, Cu(I) chloride was found to be less toxic than Cu(II) chloride to T. japonicus by comparing their respective 96 h-LC50 values which are in good agreement with the results obtained from previous studies in marine copepod Acartia tonsa and in freshwater cladoceran Daphnia similis (Table 1). Among other Cu(II) salts, Azenha et al. (1995) demonstrated that a significantly higher amount of Cu bound to cells when using Cu nitrate than using Cu chloride and Cu sulphate, suggesting that the choice of Cu salt may affect the final Cu toxicity. The difference in the toxicity of the Cu(II) salts may be attributable to the different degree of complex-forming by the anions with Cu ions. Complex forming of Cu ions could then reduce the bioavailability and thus the toxicity of Cu (Wright, 1995), as toxicity of Cu is suggested to be mainly caused by free Cu ions in the solution (Sunda and Guillard, 1976). Nevertheless, our results indicated that different Cu(II)
1
)
Source Lussier and Cardin (1985) Sonsowski et al. (1979) Present study Present study De Oliveira-Filho et al. (2004) De Oliveira-Filho et al. (2004)
salts did not exhibit a great difference in their toxicities to T. japonicus. Encouragingly, good reproducibility and low variability (CV = 3– 5%) of the chronic toxicity endpoints based on the time of development and reproduction were observed in T. japonicus. For population level effect (rm), the variability was only slightly higher (CV = 10–11%) for the ten independent tests while the rm was significantly reduced at 10 lg Cu l 1. Unsurprisingly, chronic toxicity of Cu to T. japonicus is much higher when compared with the acute toxicity of Cu(II) chloride (96 h-LC50 = 813 lg Cu l 1), giving a rough acute-chronic ratio of 80. The amount of variability of T. japonicus acute LC50s and chronic toxicity endpoints compares favourably with other established model organisms for ecotoxicity testing. Previous reports indicated that the median within-laboratory LC50 endpoints of nine model species have a CV of 14–30% (US EPA, 2000). In terms of chronic tests, inhibition concentration 50% (IC50) based on larval growth of the sheepshead minnow Cyprinodon variegatus has a CV of 19.2–40.9%, the inland silverside, Menidia beryllina has a CV 6.9– 11.2%, the mysid Mysidopsis bahia 5.8% (US EPA, 2002). The sea urchin Arbacia punctulata fertilization test IC50 has a CV 41.8– 55.1% (US EPA, 2002). Nevertheless additional life-cycle tests with T. japonicus are needed for further investigation of the betweenlaboratory variation and the repeatability of toxicity test using other type of chemicals (e.g. organics). Another insight that could be drawn from this study the variability of test endpoints was generally smaller in the tests using laboratory maintained culture than those using field collected culture. This suggested that acclimation period before the toxicity test is important and longer acclimation period may help in lowering the variability of toxicity test results of T. japonicus. In conclusion, T. japonicus demonstrated high reproducibility in acute and chronic Cu sensitivity. The natural populations of T. japonicus collected between seasons have an overall CV of 33% in the 96 h-LC50 values. However, this source of variability should not be ignored when ecotoxicity tests are conducted, as they are of comparable magnitude to other sources of variability, such as inter-laboratory variability of common toxicity tests typically with a CV of 10–20% under good laboratory practice (e.g. US EPA, 2001). Different Cu(II) salts have a similar toxicity to T. japonicus; however, Cu(I) salt is slight less toxic. Chronic toxicity of Cu on population level response (rm) of T. japonicus is highly reproducible only with a small variability (10–11%). Given such a high reproducibility of toxic responses, it is advocated to use T. japonicus as a routine testing organism for ecotoxicological studies. Acknowledgements The study is substantially supported by the Area of Excellence Scheme (AoE) under the University Grants Committee of the Hong Kong Special Administration Region, China (Project No. AoE/P-04/ 2004). KWHK and VWWB are supported by the AoE and matching funding provided by The University of Hong Kong. The authors thank Amy Zhang and the anonymous reviewers for their helpful comments on this manuscript.
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References American Society for Testing Materials (ASTM), 2004. Standard guide for conducting renewal microplate-based life-cycle toxicity tests with a marine meiobenthic copepod. ASTM Standard No. E-2317-04. ASTM, Philadelphia, PA. Azenha, M., Vasconcelos, M.T., Cabral, J.P.S., 1995. Organic ligands reduce copper toxicity in Pseudomonas syringae. Environmental Toxicology and Chemistry 14, 369–373. Chan, B.K.K., 2000. Diurnal physio-chemical variations in Hong Kong rock pools. Asian Marine Biology 17, 43–54. De Laender, F., De Schamphelaere, K.A.C., Verdonck, F.A.M., Heijerick, D.G., Van Sprang, P.A., Vanrolleghem, P.A., Janssen, C.R., 2005. Simulation of spatial and temporal variability of chronic copper toxicity to Daphnia magna and Pseudokirchneriella subcapitata in Swedish and British surface waters. Human and Ecological Risk Assessment 11, 1177–1191. De Oliveira-Filho, E.C., Lopes, R.M., Paumgartten, F.J.R., 2004. Comparative study on the susceptibility of freshwater species to copper-based pesticides. Chemosphere 56, 369–374. EPD, 2002. Marine Water Quality in Hong Kong in 2002. Environmental Protection Department of Hong Kong SAR Government. Finney, D.J., 1989. Probit Analysis, third ed. Cambridge University Press, Cambridge. Hall Jr., L.W., Anderson, R.D., 1999. A deterministic ecological risk assessment for copper in European saltwater environments. Marine Pollution Bulletin 38, 207– 218. Kater, B.J., Hannewijk, A., Postma, J.F., Dubbeldam, M., 2000. Seasonal changes in acute toxicity of cadmium to amphipod Corophium volutator. Environmental Toxicology and Chemistry 19, 2032–3035. Kwok, K.W.H., Leung, K.M.Y., 2005. Toxicity of antifouling biocides to the intertidal harpacticoid copepod Tigriopus japonicus (Crustacea, Copepoda): effects of temperature and salinity. Marine Pollution Bulletin 51, 830–837. Leung, K.M.Y., Morritt, D., Wheeler, J.R., Whitehouse, P., Sorokin, N., Toy, R., Holt, M., Crane, M., 2001. Can saltwater toxicity be predicted from freshwater data? Marine Pollution Bulletin 42, 1007–1013. Lussier, S.M., Cardin, J.A., 1985. Results of Acute Toxicity Tests Conducted with Copper at ERL, Narragansett. US EPA, Narragansett, RI. Matthiessen, P., Reed, J., Johnson, M., 1999. Sources and potential effects of copper and zinc concentrations in the estuarine waters of Essex and Suffolk, United Kingdom. Marine Pollution Bulletin 38, 908–920. McAllen, R., Taylor, A.C., Davenport, J., 1999. The effects of temperature and oxygen partial pressure on the rate of oxygen consumption of the high-shore rock pool copepod Tigriopus brevicornis. Comparative Biochemistry and Physiology Part A 123, 195–202. McGee, B.L., Wright, D.A., Fisher, D.J., 1998. Biotic factors modifying acute toxicity of aqueous cadmium to eastuarine amphipod Leptocheirus plumulosus. Archives of Environmental Contamination and Toxicology 34, 34–40. Morton, B., Morton, J., 1983. The Sea Shore Ecology of Hong Kong. Hong Kong University Press, Hong Kong. Nriagu, J.O., Pacyna, J.M., 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace-metals. Nature 333, 134–139.
O’Brien, P., Feldman, H., Grill, E.V., Lewis, A.G., 1988. Copper tolerance of the life history stages of the splashpool copepod Tigriopus californicus (Copepoda, Harpacticoida). Marine Ecology Progress Series 44, 59–64. OECD, 2006. Detailed review paper on aquatic arthropods in life cycle toxicity tests with an emphasis on developmental, reproductive and endocrine disruptive effects. OECD Series on Testing and Assessment, Number 55, ENV/JM/ MONO(2006)22, Environment Directorate, Paris: Organisation for Economic Cooperation and Development, Paris, 125 p. Raisuddin, S., Kwok, K.W.H., Leung, K.M.Y., Schlenk, D., Lee, J.-S., 2007. The copepod Tigriopus: a promising marine model organism for ecotoxicology and environmental genomics. Aquatic Toxicology 83, 161–173. Schiff, K., Diehl, D., Valkirs, A., 2004. Copper emissions from antifouling paint on recreational vessels. Marine Pollution Bulletin 48, 371–377. Sonsowski, S.L., Germond, D.J., Gentile, J.H., 1979. The effect of nutrition on the response of field populations of the calanoid copepod Acartia tonsa to copper. Water Research 13, 449–452. Spicer, J.I., Gaston, K.J., 1999. Physiological Diversity and its Ecological Implications. Blackwell Science, Oxford, UK. Srinivasan, M., Swain, G.W., 2007. Managing the use of copper-based antifouling paints. Environmental Management 39, 423–441. Sunda, W.G., Guillard, R.R.L., 1976. The relationship between cupric ion activity and the toxicity of copper to phytoplankton. Journal of Marine Research 34, 511–529. US EPA, 2000. Understanding and accounting for method variability in whole effluent toxicity applications under the national pollutant discharge elimination system. Office of Water, Washington, USA. EPA 833-R-00-003. US EPA, 2001. Final report: Interlaboratory variability study of EPA short-term chronic and acute whole effluent toxicity test methods. Office of Water, Washington, USA. EPA 821-B-01-004. US EPA, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms. Third edition. Office of Water, Washington, USA. EPA 821-R-02-014. Valkirs, A.O., Seligman, P.F., Haslbeck, E., Caso, J.S., 2003. Measurement of copper realse rates from antifouling paint under laboratory and in situ conditions: implications for loading estimation to marine water bodies. Marine Pollution Bulletin 46, 763–779. Voulvoulis, N., Scrimshaw, M.D., Lester, J.N., 1999. Alternative antifouling biocide: a review. Applied Organometal Chemistry 13, 135–143. Walthall, W.K., Stark, J.D., 1997. A comparison of acute mortality and population growth rate as endpoints of toxicological effect. Ecotoxicology and Environmental Safety 37, 45–52. Wheeler, J.R., Leung, K.M.Y., Morritt, D., Whitehouse, P., Sorokin, N., Toy, R., Holt, M., Crane, M., 2002. Freshwater to saltwater toxicity extrapolation using species sensitivity distributions. Environmental Toxicology and Chemistry 21, 2459– 2467. Winner, R.W., Owen, H.A., Moore, M.V., 1990. Seasonal variability in the sensitivity of freshwater lentic communities to a chronic copper stress. Aquatic Toxicology 17, 75–92. Wright, D.A., 1995. Trace metal and major ion interactions in aquatic animals. Marine Pollution Bulletin 31, 8–18.
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Copper toxicity in the marine copepod Tigropus japonicus: Low variability and high reproducibility of repeated acute and life-cycle tests Kevin W.H. Kwok a,b,*, Kenneth M.Y. Leung a,b, Vivien W.W. Bao a,b, Jae-Seong Lee c a
The Swire Institute of Marine Science, Faculty of Science, The University of Hong Kong, Cape d’Aguilar, Shek O, Hong Kong, China Division of Ecology and Biodiversity, School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China c Department of Chemistry, and the National Research Laboratory of Marine Molecular and Environmental Bioscience, College of Natural Sciences, Hanyang University, Seoul 133-791, South Korea b
a r t i c l e
i n f o
Keywords: Model organism Coefficient of variation Ecotoxicology
a b s t r a c t The intertidal copeopod Tigriopus japonicus, which is abundant and widely distributed along the coasts of Western Pacific, has been suggested to be a good marine ecotoxicity testing organism. In this study, a series of experiments were conducted to investigate the reproducibility and variability of copper (Cu) sensitivity of T. japonicus so as to evaluate its potential to serve as an appropriate test species. To understand the seasonal variation of Cu sensitivity, individuals of T. japonicus were collected from the field in summer and winter, and subjected to standard 96 h acute (static renewal) toxicity tests. 96 h-LC50 values of T. japonicus collected from the two seasons were marginally different (p = 0.05), with an overall coefficient of variation (CV) of 33%. Most importantly, our results indicated that chronic Cu sensitivity of T. japonicus was highly reproducible. The CVs of intrinsic rates of increase in the population of the control and Cu treatment (10 lg Cu l 1) groups were only 10–11% between 10 runs of a standardised complete life-cycle test. Moreover, different Cu(II) salts generally resulted in a similar 96 h-LC50 value while Cu(I) chloride was consistently slightly less toxic than Cu(II) salts. Given such a high reproducibility of toxic responses, it is advocated to use T. japonicus as a routine testing organism. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Aquatic ecotoxicology is biased towards studying freshwater organisms (Leung et al., 2001; Wheeler et al., 2002). One important step to promote marine ecotoxicological studies is, therefore, to find and establish appropriate marine model organisms. Copepods of the genus Tigriopus, which have a wide geographical distribution, short life-cycle and distinctive developmental stages, have been recommended to be one of the promising candidates (Raisuddin et al., 2007) and were shortlisted in a recent OECD document (OECD, 2006) as a potential marine model organism for ecotoxicology. Copper (Cu) contamination is common and widespread in marine coastal environments (Hall and Anderson, 1999). For example, Cu in marine sediment in relatively unpolluted Hong Kong Eastern waters was about 20 mg kg 1, however, such Cu concentrations could reach a maximum of 4000 mg kg 1 in marine sediments collected from highly urbanized areas such as Victoria Harbour (EPD, 2002). In the United Kingdom, the highest annual mean total Cu concentration in marine waters could be as high as 20 lg l 1 (Mat-
* Corresponding author. Address: Division of Ecology and Biodiversity, School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China. Tel.: +852 28092179; fax: +852 25176082. E-mail address: [email protected] (K.W.H. Kwok). 0025-326X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2008.03.026
thiessen et al., 1999). Undoubtedly, contamination of Cu in saltwater environments is generally higher in harbours and estuaries (Hall and Anderson, 1999). The Cu-based antifouling coating on ship hulls has been one of the major sources of Cu contamination in coastal marine environments (Valkirs et al., 2003). Since the partial ban of organotins in many countries in early 1990s, Cu-based antifouling paints have been regaining their popularity and gradually replacing organotin-based antifouling paints (Voulvoulis et al., 1999; Srinivasan and Swain, 2007). The emission rate of Cu from antifouling paints on boats varies, ranging from 0.2 to 65 lg/cm2/ day, depending on the structure of the paint coating and environmental conditions (Valkirs et al., 2003; Schiff et al., 2004). Anthropogenic input of Cu via smelting and refining, mining, domestic wastewater and sewage sludge dumping also contribute to elevated Cu concentrations in the marine environment (Nriagu and Pacyna, 1988). Cu is an essential micronutrient vital to processes such as cellular respiration, free radical defence and cellular iron metabolism, but at elevated levels Cu is toxic to organisms, especially to aquatic invertebrates. Therefore, the widespread contamination of Cu in marine environments poses potential ecological risks to many saltwater organisms and calls for more ecotoxicological studies to better understand its toxic effects towards various marine species.
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In many ecotoxicity tests, test organisms were collected from the field, acclimated then dosed with chemicals. While the location and time of collection of these animals were often provided, very few studies investigated the natural variability of chemical sensitivity of these organisms (e.g. Winner et al., 1990). It is well documented that of the same species, individuals within a population could have different physiological capacities to cope with a stressor and such capacities often vary according to seasons (Spicer and Gaston, 1999). Therefore, the sensitivity of test organisms possibly changes with time of collection. Hong Kong makes an ideal place to investigate such an effect, as it has two markedly different seasons. The warm, wet season generally begins in April and lasts until September and the cold, dry season generally occurs from October to March (Morton and Morton, 1983). Test organisms collected at different seasons may adopt different physiological strategies to deal with different natural stresses, which may in turn affect their sensitivities towards trace metals. For ecotoxicity tests of Cu, it is common that different salts (e.g. sulphates, nitrates, chlorides) are used. In general, only the concentration of the Cu cation will be considered to have toxic effect on the test organisms (De Laender et al., 2005). Nonetheless, the use of different metal salts will give different forms (e.g. sulphates and nitrates) and different concentrations of anions (e.g. for each mole of Cu, Cu(II) chloride gives two moles of chlorides while Cu(II) sulphate gives one mole of sulphate) in the test solution. As it has been shown that sulphate and chloride ions can influence the resultant Cu toxicity to aquatic organisms, both anions have been included in the biotic ligand models for predicting Cu toxicity (De Laender et al., 2005). The anions of Cu salts may affect the test results by directly having an effect on the test organisms or indirectly by interacting with the Cu cation and thus influencing its bioavailability. The understanding of chronic toxicity of a substance, especially on population level effects, is crucial to assess the long-term effect of the substance in the ecosystem. Reproducibility of chronic toxicity test results, therefore, is a key aspect of a good model organism for ecotoxicological studies. Tigriopus japonicus is highly abundant and widely distributed along the coasts of Western Pacific (i.e. coasts of China, Taiwan, Korea and Japan) and easy to culture, with a complete life-cycle of about 21 days (Raisuddin et al., 2007). This study primarily aimed to investigate the variability of Cu toxicities to the copepod T. japonicus associated with seasons and different Cu salts. Reproducibility of chronic Cu sensitivity was also investigated using a standardised complete life-cycle assay. The results of these tests can be useful to evaluate the potential of T. japonicus to be used as a model organism for marine ecotoxicology.
2. Materials and methods 2.1. Seasonal variability To investigate seasonal differences in sensitivity of T. japonicus to Cu, cultures of the copepod were collected at Cape d’Aguilar Marine Reserve, Hong Kong in warm-wet season (May to August) and in cold-dry season (October to November), respectively. Cultures were acclimated in laboratory at temperatures of 22–25 °C, salinity of 30–35‰ and a 16 h:8 h light:dark cycle. The animals were fed with finely ground green algae Enteromorpha spp. and commercial phytoplankton concentrate (Kent Marine Phytoplex, US). A total of six 96 h acute tests were conducted: three on warm-wet season populations and three on cold-dry season populations. Acute toxicity tests for individual cultures were conducted after 10 d of acclimation in the laboratory. Ten adult males and 10 adult females were used in each replicate and they were placed in a
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50 ml acid-washed breaker with 25 ml of the testing solution. Three replicates were applied for each Cu concentration. At least six nominal concentrations of Cu(II) sulphate were utilised at each test (0 [control] plus at least five concentrations of Cu ranged from 100 to 2000 lg Cu l 1). Test solutions were prepared by diluting a stock solution (reagent grade Cu(II) sulphate, BDH Ltd.; formula weight, f.w.: 249.7) with appropriate volume of fully aerated (aerated for at least 30 min), freshly made filtered artificial seawater (Sea salt: Tropic Marine, Germany; filter: Glass microfibre filters, GF/C 47 mm Circle, Whatman). Test solutions were renewed every 48 h. Test animals were transferred to the 50 ml beakers with filtered artificial seawater and allowed acclimation for 48 h before starting the test. The animals were not fed from the acclimation period till the test ended. Mortality was checked once every 24 h and defined as copepod’s urosome perpendicular to its prosome (McAllen et al., 1999). 2.2. Effect of different copper salts Effect of different Cu salts on Cu toxicity was tested with T. japonicus obtained from an established laboratory culture collected from Cape d’Aguilar maintained for more than one year. 96 h acute toxicity tests were conducted using five different Cu salts (Cu(II) sulphate, f.w.: 249.7; Cu(II) chloride, f.w.: 170.48; Cu(II) nitrate, f.w.: 241.6; Cu(II) acetate, f.w.: 199.65 and Cu(I) chloride, f.w.: 99.0). All the Cu salts were of reagent grade, supplied by Merck (Cu(I) chloride) and BDH Ltd. (the other four salts). Six nominal Cu concentrations of each salt were used in the test (500, 800, 1000, 1200, 1500 and 0 [control] lg Cu l 1). Acute toxicity tests were conducted as described in Kwok and Leung (2005). In brief, 20 randomly selected adult copepods were placed in 25 ml test solution for each replicate and each concentration treatment was triplicated. All acute tests were conducted at temperature 25 ± 1.0 °C, artificial seawater of salinity 33 ± 0.5‰, pH 8.1–8.4 and a 12 h:12 h light:dark photoperiod. All 96 h-LC50 values and their associated 95% confidence intervals (CI) were estimated using Probit analysis (Finney, 1989). The 96 h-LC50 values of the two different seasons were compared using a non-parametric Mann–Whitney test (SPSS version 12.0, Chicago, IL, USA). Coefficient of variation (%) of the LC50 values for each season was computed to understand the amount of variation within a season and between seasons. 2.3. Repeatability of complete life-cycle tests The complete life-cycle toxicity tests of copper chloride (CuCl2; BDH Ltd.) to T. japonicus were conducted using a modified ASTM protocol (ASTM E-2317-04; ASTM, 2004). In 96-well microplates, <24 h-old nauplii were placed individually in wells loaded with 200 ll of test solution (0 [control] and 10 lg Cu l 1) and 2 ll of algal solution containing 107 Skeletonema costatum cells ml 1. Both treatments had eight replicates and each replicate contained 12 nauplii. To ensure consistency of exposure, copepods were placed in new microwells containing fresh test solutions and algal solution every 96 h. Copepods were monitored daily for life-stage mortality, development and sex determination. When the copepods reached copepodid stage 5, male and female copepods were paired up and mated within each replicate for each treatment. Life-cycle testing was terminated after the release of the second brood of nauplii from each mating pair. Test periods for individual microplate bioassays ranged from 20 to 30 d to account for treatment specific developmental delays. A total of ten runs of life-cycle test were conducted. All life-cycle tests were conducted at temperature 25 ± 1.0 °C, artificial seawater of salinity 30 ± 0.5‰, pH 7.9–8.0 and a 12 h:12 h light:dark photoperiod.
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Duration of larval development (from <24 h nauplii to develop into copepodid stage 1), time to first reproduction (from <24 h nauplii to the development of the first egg brood) and the intrinsic rate of increase (rm) were used as endpoints. The rm was computed as suggested by Walthall and Stark (1997) and is an estimate of the intrinsic population growth rate. When rm = 0, it represents a stable population; when it is negative, it represents a declining population, vice versa. Student’s t-test was used to compare the described endpoints between the control and Cu treatment groups.
the control. Cu(II) salts in generally showed comparable 96 h-LC50 values while Cu(I) chloride was the least toxic to T. japonicus, as shown by the non-overlapping 95% CI with the 96 h-LC50 of other Cu(II) salts. The CV in the LC50 values of different Cu(II) salts was 15% and the CV of LC50 values of all Cu salts used was 19%.
3. Results 96 h-LC50 values of T. japonicus collected from different seasons ranged from 323 to 585 lg Cu l 1 (Fig. 1). For all bioassays, control mortality was less than 5%. Between the two seasonal populations, the overall coefficient of variation (CV) in the LC50 values was 33%. The amount of variation for the warm-wet season population was higher (CV = 27%) than that of the cold-dry season (CV = 14%). The LC50 values of warm-wet season cultures were marginally higher than that of the cold-dry season ones (U = 9, d.f. = 4, p = 0.05). 96 h-LC50 values of T. japonicus exposed to the five Cu salts ranged from 743 to 1175 lg Cu l 1 (Fig. 2). There was no mortality for
Fig. 1. Comparison of copper sensitivity of Tigriopus japonicus collected in summer and winter based on the 96 h-LC50 values (mean and 95% C.I.).
Fig. 2. 96 h-LC50 (mean ± 95% C.I.) of Tigriopus japonicus exposed to different copper salts.
Fig. 3. Results of chronic toxicity tests: (a) larval developmental time, (b) time to first reproduction, and (c) intrinsic rate of increase (rm) of Tigriopus japonicus from nauplii to copepodid stage 1 at two different Cu concentrations; middle bar represents the mean, the box represent the first and third quartile and the error bar represents the 95% C.I.
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K.W.H. Kwok et al. / Marine Pollution Bulletin 57 (2008) 632–636 Table 1 LC50 values of marine copepod Acartia tonsa and freshwater cladoceran Daphnia similis to copper (I) and copper (II) salt Species name
Chemical
LC-50 value (lg Cu l
Marine Copepod Acartia tonsa
Cu(I) chloride Cu(II) nitrate Cu(I) chloride Cu(II) chloride Cuprous oxide Cu(II) sulphate
72 h-LC50 = 58.1 72 h-LC50 = 9.0 96 h-LC50 = 1180 96 h-LC50 = 813 48 h-LC50 = 9.6 48 h-LC50 = 3.5
Tigriopus japonicus Freshwater Cladoceran Daphnia similis
For the chronic life-cycle toxicity tests, overall mortality (mean ± 1SD; n = 10) of T. japonicus in the control and treatment (10 lg Cu l 1) groups were 10.4 ± 4% and 13.1 ± 4%, respectively. Results of life-cycle development and population growth endpoints in general were highly reproducible through ten runs of the chronic toxicity tests. Development of T. japonicus slowed down significantly at 10 lg Cu l 1 (t = 6.05, d.f. = 78, p < 0.001) (Fig. 3a). The amount of variation within the control and the Cu treatment was similar, with a CV of 4–5%. Time to first reproduction was significantly delayed at 10 lg Cu l 1 by about a half day (t = 4.82, d.f. = 78, p < 0.001) (Fig. 3b). The amount of variation within the control and Cu treatment was similar, with a CV around 3%. Intrinsic rate of increase (rm) was significantly lowered at 10 lg Cu l 1 (t = 4.54, d.f. = 78, p < 0.001) (Fig. 3c). The amount of variation within the control and the Cu treatment was similar, with a CV of 10–11%. 4. Discussion Acute Cu sensitivities of T. japonicus populations collected from the two seasons were marginally statistically different. The T. japonicus collected in the warm-wet season showed much higher variability in the 96 h-LC50 values than those collected from the colddry season. As Tigriopus species inhabit supratidal rockpools which are subjected to natural extremes of temperature and salinity, they are necessarily tolerant to such extremes (O’Brien et al., 1988). The variability in Cu sensitivity may be attributable to the variability of physiological state of the test animals. In the warm-wet season, Tigriopus population were subjected to larger fluctuations in temperature, salinity, pH and dissolved oxygen (Chan, 2000). Therefore, the physiological state of the summer Tigriopus population could be more variable. Seasonal differences in cadmium sensitivity have also been observed in amphipods such as Leptocheirus plumulosus and Corophium volutator (McGee et al., 1998; Kater et al., 2000). It was suggested that the seasonal variation might be attributed to food supply, population dynamics and difference between field temperature and laboratory test temperature (Sonsowski et al., 1979; Kater et al., 2000). Free copper(II) ion is generally considered to be a more toxic form of Cu (Hall and Anderson, 1999). In the present study, Cu(I) chloride was found to be less toxic than Cu(II) chloride to T. japonicus by comparing their respective 96 h-LC50 values which are in good agreement with the results obtained from previous studies in marine copepod Acartia tonsa and in freshwater cladoceran Daphnia similis (Table 1). Among other Cu(II) salts, Azenha et al. (1995) demonstrated that a significantly higher amount of Cu bound to cells when using Cu nitrate than using Cu chloride and Cu sulphate, suggesting that the choice of Cu salt may affect the final Cu toxicity. The difference in the toxicity of the Cu(II) salts may be attributable to the different degree of complex-forming by the anions with Cu ions. Complex forming of Cu ions could then reduce the bioavailability and thus the toxicity of Cu (Wright, 1995), as toxicity of Cu is suggested to be mainly caused by free Cu ions in the solution (Sunda and Guillard, 1976). Nevertheless, our results indicated that different Cu(II)
1
)
Source Lussier and Cardin (1985) Sonsowski et al. (1979) Present study Present study De Oliveira-Filho et al. (2004) De Oliveira-Filho et al. (2004)
salts did not exhibit a great difference in their toxicities to T. japonicus. Encouragingly, good reproducibility and low variability (CV = 3– 5%) of the chronic toxicity endpoints based on the time of development and reproduction were observed in T. japonicus. For population level effect (rm), the variability was only slightly higher (CV = 10–11%) for the ten independent tests while the rm was significantly reduced at 10 lg Cu l 1. Unsurprisingly, chronic toxicity of Cu to T. japonicus is much higher when compared with the acute toxicity of Cu(II) chloride (96 h-LC50 = 813 lg Cu l 1), giving a rough acute-chronic ratio of 80. The amount of variability of T. japonicus acute LC50s and chronic toxicity endpoints compares favourably with other established model organisms for ecotoxicity testing. Previous reports indicated that the median within-laboratory LC50 endpoints of nine model species have a CV of 14–30% (US EPA, 2000). In terms of chronic tests, inhibition concentration 50% (IC50) based on larval growth of the sheepshead minnow Cyprinodon variegatus has a CV of 19.2–40.9%, the inland silverside, Menidia beryllina has a CV 6.9– 11.2%, the mysid Mysidopsis bahia 5.8% (US EPA, 2002). The sea urchin Arbacia punctulata fertilization test IC50 has a CV 41.8– 55.1% (US EPA, 2002). Nevertheless additional life-cycle tests with T. japonicus are needed for further investigation of the betweenlaboratory variation and the repeatability of toxicity test using other type of chemicals (e.g. organics). Another insight that could be drawn from this study the variability of test endpoints was generally smaller in the tests using laboratory maintained culture than those using field collected culture. This suggested that acclimation period before the toxicity test is important and longer acclimation period may help in lowering the variability of toxicity test results of T. japonicus. In conclusion, T. japonicus demonstrated high reproducibility in acute and chronic Cu sensitivity. The natural populations of T. japonicus collected between seasons have an overall CV of 33% in the 96 h-LC50 values. However, this source of variability should not be ignored when ecotoxicity tests are conducted, as they are of comparable magnitude to other sources of variability, such as inter-laboratory variability of common toxicity tests typically with a CV of 10–20% under good laboratory practice (e.g. US EPA, 2001). Different Cu(II) salts have a similar toxicity to T. japonicus; however, Cu(I) salt is slight less toxic. Chronic toxicity of Cu on population level response (rm) of T. japonicus is highly reproducible only with a small variability (10–11%). Given such a high reproducibility of toxic responses, it is advocated to use T. japonicus as a routine testing organism for ecotoxicological studies. Acknowledgements The study is substantially supported by the Area of Excellence Scheme (AoE) under the University Grants Committee of the Hong Kong Special Administration Region, China (Project No. AoE/P-04/ 2004). KWHK and VWWB are supported by the AoE and matching funding provided by The University of Hong Kong. The authors thank Amy Zhang and the anonymous reviewers for their helpful comments on this manuscript.
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References American Society for Testing Materials (ASTM), 2004. Standard guide for conducting renewal microplate-based life-cycle toxicity tests with a marine meiobenthic copepod. ASTM Standard No. E-2317-04. ASTM, Philadelphia, PA. Azenha, M., Vasconcelos, M.T., Cabral, J.P.S., 1995. Organic ligands reduce copper toxicity in Pseudomonas syringae. Environmental Toxicology and Chemistry 14, 369–373. Chan, B.K.K., 2000. Diurnal physio-chemical variations in Hong Kong rock pools. Asian Marine Biology 17, 43–54. De Laender, F., De Schamphelaere, K.A.C., Verdonck, F.A.M., Heijerick, D.G., Van Sprang, P.A., Vanrolleghem, P.A., Janssen, C.R., 2005. Simulation of spatial and temporal variability of chronic copper toxicity to Daphnia magna and Pseudokirchneriella subcapitata in Swedish and British surface waters. Human and Ecological Risk Assessment 11, 1177–1191. De Oliveira-Filho, E.C., Lopes, R.M., Paumgartten, F.J.R., 2004. Comparative study on the susceptibility of freshwater species to copper-based pesticides. Chemosphere 56, 369–374. EPD, 2002. Marine Water Quality in Hong Kong in 2002. Environmental Protection Department of Hong Kong SAR Government. Finney, D.J., 1989. Probit Analysis, third ed. Cambridge University Press, Cambridge. Hall Jr., L.W., Anderson, R.D., 1999. A deterministic ecological risk assessment for copper in European saltwater environments. Marine Pollution Bulletin 38, 207– 218. Kater, B.J., Hannewijk, A., Postma, J.F., Dubbeldam, M., 2000. Seasonal changes in acute toxicity of cadmium to amphipod Corophium volutator. Environmental Toxicology and Chemistry 19, 2032–3035. Kwok, K.W.H., Leung, K.M.Y., 2005. Toxicity of antifouling biocides to the intertidal harpacticoid copepod Tigriopus japonicus (Crustacea, Copepoda): effects of temperature and salinity. Marine Pollution Bulletin 51, 830–837. Leung, K.M.Y., Morritt, D., Wheeler, J.R., Whitehouse, P., Sorokin, N., Toy, R., Holt, M., Crane, M., 2001. Can saltwater toxicity be predicted from freshwater data? Marine Pollution Bulletin 42, 1007–1013. Lussier, S.M., Cardin, J.A., 1985. Results of Acute Toxicity Tests Conducted with Copper at ERL, Narragansett. US EPA, Narragansett, RI. Matthiessen, P., Reed, J., Johnson, M., 1999. Sources and potential effects of copper and zinc concentrations in the estuarine waters of Essex and Suffolk, United Kingdom. Marine Pollution Bulletin 38, 908–920. McAllen, R., Taylor, A.C., Davenport, J., 1999. The effects of temperature and oxygen partial pressure on the rate of oxygen consumption of the high-shore rock pool copepod Tigriopus brevicornis. Comparative Biochemistry and Physiology Part A 123, 195–202. McGee, B.L., Wright, D.A., Fisher, D.J., 1998. Biotic factors modifying acute toxicity of aqueous cadmium to eastuarine amphipod Leptocheirus plumulosus. Archives of Environmental Contamination and Toxicology 34, 34–40. Morton, B., Morton, J., 1983. The Sea Shore Ecology of Hong Kong. Hong Kong University Press, Hong Kong. Nriagu, J.O., Pacyna, J.M., 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace-metals. Nature 333, 134–139.
O’Brien, P., Feldman, H., Grill, E.V., Lewis, A.G., 1988. Copper tolerance of the life history stages of the splashpool copepod Tigriopus californicus (Copepoda, Harpacticoida). Marine Ecology Progress Series 44, 59–64. OECD, 2006. Detailed review paper on aquatic arthropods in life cycle toxicity tests with an emphasis on developmental, reproductive and endocrine disruptive effects. OECD Series on Testing and Assessment, Number 55, ENV/JM/ MONO(2006)22, Environment Directorate, Paris: Organisation for Economic Cooperation and Development, Paris, 125 p. Raisuddin, S., Kwok, K.W.H., Leung, K.M.Y., Schlenk, D., Lee, J.-S., 2007. The copepod Tigriopus: a promising marine model organism for ecotoxicology and environmental genomics. Aquatic Toxicology 83, 161–173. Schiff, K., Diehl, D., Valkirs, A., 2004. Copper emissions from antifouling paint on recreational vessels. Marine Pollution Bulletin 48, 371–377. Sonsowski, S.L., Germond, D.J., Gentile, J.H., 1979. The effect of nutrition on the response of field populations of the calanoid copepod Acartia tonsa to copper. Water Research 13, 449–452. Spicer, J.I., Gaston, K.J., 1999. Physiological Diversity and its Ecological Implications. Blackwell Science, Oxford, UK. Srinivasan, M., Swain, G.W., 2007. Managing the use of copper-based antifouling paints. Environmental Management 39, 423–441. Sunda, W.G., Guillard, R.R.L., 1976. The relationship between cupric ion activity and the toxicity of copper to phytoplankton. Journal of Marine Research 34, 511–529. US EPA, 2000. Understanding and accounting for method variability in whole effluent toxicity applications under the national pollutant discharge elimination system. Office of Water, Washington, USA. EPA 833-R-00-003. US EPA, 2001. Final report: Interlaboratory variability study of EPA short-term chronic and acute whole effluent toxicity test methods. Office of Water, Washington, USA. EPA 821-B-01-004. US EPA, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms. Third edition. Office of Water, Washington, USA. EPA 821-R-02-014. Valkirs, A.O., Seligman, P.F., Haslbeck, E., Caso, J.S., 2003. Measurement of copper realse rates from antifouling paint under laboratory and in situ conditions: implications for loading estimation to marine water bodies. Marine Pollution Bulletin 46, 763–779. Voulvoulis, N., Scrimshaw, M.D., Lester, J.N., 1999. Alternative antifouling biocide: a review. Applied Organometal Chemistry 13, 135–143. Walthall, W.K., Stark, J.D., 1997. A comparison of acute mortality and population growth rate as endpoints of toxicological effect. Ecotoxicology and Environmental Safety 37, 45–52. Wheeler, J.R., Leung, K.M.Y., Morritt, D., Whitehouse, P., Sorokin, N., Toy, R., Holt, M., Crane, M., 2002. Freshwater to saltwater toxicity extrapolation using species sensitivity distributions. Environmental Toxicology and Chemistry 21, 2459– 2467. Winner, R.W., Owen, H.A., Moore, M.V., 1990. Seasonal variability in the sensitivity of freshwater lentic communities to a chronic copper stress. Aquatic Toxicology 17, 75–92. Wright, D.A., 1995. Trace metal and major ion interactions in aquatic animals. Marine Pollution Bulletin 31, 8–18.