Comparison of intermittent and
continuous exposure to mercury in
the marine mussel, Mytilus edulis:
Accumulation and sub-lethal effects
Amachree, D1,2., Moody, A. J1 and Handy, R. D1
University of Plymouth
[email protected]
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.
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.
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 <0.0010.003µg/l.
• Avg conctn in fish 0.07 – 0.17 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.
• LC50 for inorganic Hg in M.edulis 0.5 –
2.0 mg/l.
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 HgCl2 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
Hg concentration in the water sample after 14 day
exposure to 0 µg/l control (--) or 50 µg/l Hg as HgCl2 in
continuous (…) or intermittent (_) exposures.
Fig. 1. Data are mean n = 3 samples/ treatment/day. 48.7 µg/l (CE); 47.9 µg/l (IE)
Water samples were collected immediately after daily dosing .
1740.9
1358.8
8.9
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.
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 HgCl2
Parameters
Control
Continuous
Intermittent
Osmotic pressure (mosmol/kg)
1073.3 ± 12.1
1099.8 ± 22.6
1038.3 ± 20.9
Plasma Na+ (mM)
495.5 ± 15.5
495.1 ± 13.8
490.7 ± 20.1
Plasma K+ (mM)
11.8 ± 0.3
12.0 ± 0.8
10.5 ± 0.4
Plasma glucose (mM)
0.51 ± 0.1
0.50 ± 0.1
0.52 ± 0.2
Total haemocyte counts (*106 cells/ml)
1.90 ± 1.0 a,+
2.12 ± 0.2 #,b,+
1.53 ± 0.7 #,c,+
Neutral Red Retention (OD/mg protein)
136.54 ± 90.8 #,+
153.03 ± 64.8 #,+
143.45 ± 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.
Table 2: Tissue ions (Na+, K+, Ca2+, Mg2+)
concentrations (µmol/g dry weight tissue) at day 14.
K+
Ca2+
Mg2+
1598.9±137.4 #,+
267.7±27.1 ª,#
56.2±4.0
229.2±18.0 #,+
Continuous
1682.1±68.4 #,+
346.3±12.7 b,#,+
69.7±6.7 #,+
232.1±8.3 #,+
Intermittent
1599.8±67.5 #,+
323.0±9.8 b,#,+
64.6±7.4 #
228.2±9.2 #,+
Digestive
Control
784.0±79.8 +
241.3±9.4 a
41.4±4.1
119.2±9.4 #,+
gland
Continuous
975.9±89.9 #,+
311.2±12.1 b,#,+
46.2±2.5
141.1±11.3 #,+
Intermittent
914.0±74.5 #,+
291.3±12.9 b,#,+
49.4±8.4 +
131.3±10.5 #,+
Adductor
Control
580.7±74.3
190.5±25.6 a
36.8±4.8 a
88.0 ±9.6 a
muscle
Continuous
943.2±104.8 #,+
268.1±11.5 b,#,+
62.7±12.4 b,#,+
131.1±12.3 b,#,+
Intermittent
895.5±181.69 #,+
261.9±10.8 b,#,+
77.0±24.1 b,#,+
128.1±21.6 b,#,+
Tissues
Treatments
(µg/l) Hg as
HgCl2
Gill
Control
Na+
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.
Comparison of the histopathological examinations of
the gills
Hyp
Hyp
Oe
Intermittent
Continuous
Key: Hyp = Hyperplasia; Oe = Oedema; Scale bar = 50 µm
Comparison of the histopathological examinations of
the digestive gland
At
continuous
Dqm
Gra
intermittent
Key: At = Atrophy; Dqm = Desquamation; Gra = Granulocytomas;
scale bar = 50 µm
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.
References
• Boxall et al. 2002. Pest Mgt Sci. 56, 637-648.
• Handy, 1992. Arch. Env. Contam. and Toxicol. 22, 77-81.
• Handy, 1994. Comp. Biol. and Pharm. 107, 171-184.
• Handy, 1995. Can. J. Fish and Aquat. Sci. 52, 13-22.
• McCahon and Pascoe. 1990. Funct. Ecol. 4, 375-383.
• Pascoe and Shazili. 1986. Ecotox and Env. Saf. 12, 189-198..
Acknowledgements
• Technical support from University of Plymouth staffs