Comparison of intermittent and continuous exposure to mercury in the marine mussel, Mytilus edulis: Accumulation and sub-lethal effects

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1 Comparison of intermittent and continuous exposure to mercury in the marine mussel, Mytilus edulis: Accumulation and sub-lethal effects Amachree, D 1,2., Moody, A. J 1 and Handy, R. D 1 University of Plymouth dokuboba.amachree@plymouth.ac.uk Introduction Intermittent exposure is the most likely way aquatic organisms are exposed to environmental chemicals. Unlike the continuous, intermittent event are unpredicted. WQC set from continuous exposure data assume that in an equivalent exposure dose, response of organism in intermittent event is the same as the continuous counterpart. There are concerns that the hazards from intermittent exposure may not be accurately predicted with the existing standard test.

2 Comparison of intermittent and continuous exposure using the standard test Objectives of the study To investigate the pattern of accumulation of mercury in intermittent compared to continuous exposure. Measure toxicity with biological responses such as total glutathione, TBARS, neutral red retention, total haemocyte counts, tissue pathology, tissue and plasma ions, osmotic pressure.

3 Mercury Non essential and highly toxic. In the EU Hg consumption in chlor-alkali plants (190 tonnes/ yr) and dental amalgam (90 tonnes/ yr). Typical concentrations in water bodies < µg/l. Avg conctn in fish µg/g >70% as MeHg. Marine mussel, Mytilus edulis Widely used as a biomonitoring species. Ubiquitous, sedentary, filter feeders. Easy to identify. Can be used for both laboratory transplant and experiments. LC 50 for inorganic Hg in M.edulis mg/l.

4 Experimental design Mussels (n=60/ treatment); semi-static and triplicate design (20 mussels/ tank containing 20L SW). Control (no added Hg, 0 µg/l) or 50 µg/l Hg as HgCl 2 for 14 days. Continuous (daily) ; intermittent exposure (2d exp: 2 clean seawater). 100% water change, ph, salinity, DO, total ammonia, water Hg profile measured daily. 2 mussels/ tank ~ 6/ treatment on days 0, 2, 4, 6, 8, 10, 12, 14 for osmotic pressure,tissue Hg accumulation, tissue and plasma ions. Additionally 6 mussels/ treatment were collected on days 0, 6, 14 for TBARS, total gluthathione, organ pathology, glucose Results

5 Hg concentration in the water sample after 14 day exposure to 0 µg/l control (--) or 50 µg/l Hg as HgCl 2 in continuous ( ) or intermittent ( _ ) exposures. Fig. 1. Data are mean n = 3 samples/ treatment/day µg/l (CE); 47.9 µg/l (IE) Water samples were collected immediately after daily dosing Fig. 2. Data (means ± SEM, n=4-6 mussels/treatment/day). Different letters sig.dif/day. # sig. dif compared to previous day. + sig. dif compared to day 0. ANOVA or Kruskall-Wallis p<0.05.

6 Fig. 3. Data (means ± SEM, n=4 6 mussels/treatment/day). Different letters sig.dif/day. # sig. dif compared to previous day. + sig. dif compared to day 0. ANOVA or Kruskall Wallis p<0.05. Table 1: Haemolymph chemistry and plasma ions (Na +, K + ) concentrations at day 14. Treatments (µg/l) Hg as HgCl 2 Parameters Control Continuous Intermittent Osmotic pressure (mosmol/kg) ± ± ± 20.9 Plasma Na+ (mm) ± ± ± 20.1 Plasma K+ (mm) 11.8 ± ± ± 0.4 Plasma glucose (mm) 0.51 ± ± ± 0.2 Total haemocyte counts (*10 6 cells/ml) 1.90 ± 1.0 a, ± 0.2 #,b, ± 0.7 #,c,+ Neutral Red Retention (OD/mg protein) ± 90.8 #, ± 64.8 #, ± 25.3 #,+ Data (means ± SEM, n=4 6 mussels/treatment/day). Different letters sig.dif/day. # sig. dif compared to previous day. + sig. dif compared to day 0. ANOVA or Kruskall Wallis p<0.05.

7 Table 2: Tissue ions (Na +, K +, Ca 2+, Mg 2+ ) concentrations (µmol/g dry weight tissue) at day 14. Tissues Treatments Na + K + Ca 2+ Mg 2+ (µg/l) Hg as HgCl 2 Gill Control ±137.4 #, ±27.1 ª,# 56.2± ±18.0 #,+ Continuous ±68.4 #, ±12.7 b,#,+ 69.7±6.7 #, ±8.3 #,+ Intermittent ±67.5 #, ±9.8 b,#,+ 64.6±7.4 # 228.2±9.2 #,+ Digestive Control 784.0± ±9.4 a 41.4± ±9.4 #,+ gland Continuous 975.9±89.9 #, ±12.1 b,#,+ 46.2± ±11.3 #,+ Intermittent 914.0±74.5 #, ±12.9 b,#,+ 49.4± ±10.5 #,+ Adductor Control 580.7± ±25.6 a 36.8±4.8 a 88.0 ±9.6 a muscle Continuous 943.2±104.8 #, ±11.5 b,#,+ 62.7±12.4 b,#, ±12.3 b,#,+ Intermittent 895.5± #, ±10.8 b,#,+ 77.0±24.1 b,#, ±21.6 b,#,+ Data (means ± SEM, n=4-6 mussels/treatment/day). Different letters sig.dif/day. # sig. dif compared to previous day. + sig. dif compared to day 0. ANOVA or Kruskall-Wallis p<0.05. Thiobarbituric acid reactive substances (TBARS) concentration in the homogenate from gill control (white bar), continuous (Grid bar) or intermittent (upward diagonal bar) exposures. Fig. 5. Data (means ± SEM, n=4 6 mussels/treatment/day). Different letters sig.dif/day. # sig. dif compared to previous day. + sig. dif compared to day 0. ANOVA or Kruskall Wallis p<0.05.

8 Comparison of the histopathological examinations of the gills Hyp Hyp Oe Continuous Intermittent Key: Hyp = Hyperplasia; Oe = Oedema; Scale bar = 50 µm Comparison of the histopathological examinations of the digestive gland At Dqm Gra continuous intermittent Key: At = Atrophy; Dqm = Desquamation; Gra = Granulocytomas; scale bar = 50 µm

9 Result summary No difference in Hg accumulation pattern apart from the gill in the intermittent exposure. Gill showed step-wise increases corresponding with the exposure mode. Total Hg body burden was less in the intermittent exposure compared to the continuous mode. No difference in haemolymph chemistry in terms of osmoregulation and immunology. No difference in oxidative stress measured by TBARS. Difference in organ pathology. In gill continuous in more damaged. In DG intermittent is the most damaged. Does equivalent peak concentration give equal response in the exposure modes? Implication to risk assessment In equivalent peak concentration, Hg accumulation is less in the intermittent than the continuous exposure. Biological responses between the two exposure regimes are somewhat conflicting. The intermittent are sometimes equalled, less or more than the continuous counterparts. This work provided evidence that the hazard from the intermittent is different from the continuous exposure to Hg within the concentration and duration use here. The risk assessment criteria for continuous may not apply for the intermittent pollution events.

10 References Boxall et al Pest Mgt Sci. 56, Handy, Arch. Env. Contam. and Toxicol. 22, Handy, Comp. Biol. and Pharm. 107, Handy, Can. J. Fish and Aquat. Sci. 52, McCahon and Pascoe Funct. Ecol. 4, Pascoe and Shazili Ecotox and Env. Saf. 12, Acknowledgements Technical support from University of Plymouth staffs