title: Evaluation of metallothionein as an ecotoxicological biomarker in Nucella lapillus and Littorina littorea
Creators: name: Leung, Kenneth Mei-Yee
Publisher: name: University of Glasgow
Year: 2000

description: 1) Metallothioneins (MTs) are frequently proposed as biomarkers for metal exposure and toxicity in molluscs. However, various biotic and abiotic factors influencing the rate of MT synthesis, are not well understood. The objectives of this study are to investigate the effects of biotic factors (size, sex, growth rate, nutritional state, prey type) and abiotic factors (temperature, Cd or oxidative exposure) on MT induction in Nucella lapillus and Littorina littorea, and to evaluate the usefulness of applying MT as a monitoring tool. In this study, total MTs in tissue samples were quantified using the silver saturation method. 2) Induction of MT was monitored in N. lapillus during and after exposure to Cd. N. lapillus were exposed to 500 μg Cd 1-1 (2.2% of 96h LC50) for 60 d and then placed into clean seawater for 110 d. The concentration of MT in the whole animal increased during the exposure period, peaked at Day 70, and then declined gradually. The half-life of MT was ca. 40 d. Cd concentration increased throughout the period of exposure and while in clean seawater, levelling off only after Day 120, indicating that Cd concentration could not be regulated by N. lapillus. Highest MT induction and Cd accumulation were found in the Leiblein gland of N. lapillus, suggesting that measurement of MT induction in this tissue may prove useful as a sublethal biological response to Cd contamination. 3) The combined effects of Cd and water temperature on the oxygen consumption rate (MO2) and biochemistry of fasted N. lapillus were investigated. Inhibition of MO2 by Cd increased with increasing temperature and decreasing animal size. Cd exposure caused significant reductions in glycogen concentrations in N. lapillus at both temperatures (5 &amp; 10°C). Cd-exposed N. lapillus showed significantly higher MT concentrations in the Leiblein gland at 10°C but not at 5°C, indicating that MT synthesis is temperature dependent. Reduction in MO2 may be directly linked to Cd-induced mucus production, structural damage to gills and reduction in oxygen carrying capacity of haemocyanin. However, metabolic depression, including low MO2, glycogen stores and activity in Cd-exposed N. lapillus, may be a strategy to minimise the uptake and toxicity of Cd, and energy expenditure to spare energy reserves for detoxification and maintenance. 4) The influences of nutritional state and prey type on the survival, growth, Cd accumulation, MT induction and glycogen stores in N. lapillus were studied. Prolonged starvation and Cd exposure synergistically reduced the survivorship of N. lapillus, but feeding could help N. lapillus to combat Cd toxicity and minimise mortality. Extended fasting also caused tissue wastage, leading to higher concentrations of Cd and MT in tissues, whereas fed animals increased in weight and had lower Cd and MT concentrations because of the tissue dilution effect. Prey type significantly affected growth rate of N. lapillus and indirectly influenced Cd accumulation, MT induction and glycogen stores. Eating mussels promoted better growth and higher glycogen reserves them eating barnacles. Individual growth rate decreased with increasing Cd accumulation. Cd-exposed survivors grew faster and consumed more than control animals, implying that these survivors may have better fitness and greater tolerance to Cd toxicity. 5) Investigation of the effect of hydrogen peroxide (H2O2), and the combined effect of H2O2 and Cd on MT induction and condition index (CI) in N. lapillus was conducted. Exposure to either Cd or H2O2 alone induced synthesis of MT or MT-like proteins in N. lapillus. Exposure to high H2O2 (1000 ppm) alone or combined with Cd, and exposure to Cd (0.50 ppm) or H2O2 (2.0 ppm), resulted in significant weight loss, indicated by a reduction of CL However, CIs of N. lapillus exposed to 0.5 ppm Cd + 2.0 ppm H2O2 or 0.25 ppm Cd + 2.0 ppm H2O2, were similar to that of the control suggesting that Cd antagonistically reduces toxicity caused by H2O2 since Cd-induced MT may have a protective function against hydroxyl radicals.