ELSEVIER Aquatic Toxicology 38 (1997) 125-143 Experimental and field studies of effects of dichlorvos exposure on acetylcholinesterase activity in the gills of the mussel, Myths edulis L. J.G. McHenery ‘, G.E. Linley-Adams, D.C. Moore, G.K. Rodger, I.M. Davies* SOAEFD Marine Laboratory, P.O. Box 101, Victoria Road, Aberdeen ABll 9DB, UK Accepted 30 September 1996 Abstract Dichlorvos (DDVP) is an organophosphate medicine, used to treat ectoparasitic sea lice infestations of farmed salmon. Effects of this chemical on a non-target organism were inves- tigated. Experimental exposure of mussels (Mytilus edulis L.) to dichlorvos (DDVP) resulted in changes in gill acetylcholinesterase (AChE) activity with a hermetic increase at lower concentrations, and 50% inhibition at 3.6 pg 1-l after 24 h exposure. Repeat exposures re- sulted in cumulative inhibition. Twenty-four hour exposure to DDVP impaired the ability to close shells (ECsO of 1700 kg 1-l) and caused mortalities at high concentrations (L& of 8200 pg 1-l). AChE levels in mussels collected from sea lochs were indicative of DDVP inhibition related to the quantities of DDVP used. The use of AChE as a sublethal indicator of DDVP usage in the field is discussed. Crown Copyright 0 1997 Published by Elsevier Science B.V. Keywords: Dichlorvos; Myths; Toxicity; Acetylcholinesterase 1. Introduction Infestation of farmed salmon by ectoparasitic copepods can result in severe skin damage in affected fish, which may lead to death through osmoregulatory failure or infection by opportunistic pathogens (Bruno et al., 1990). The main mechanism for controlling such infestations (Costello, 1993; Roth et al., 1993) is by immersing fish *Corresponding author. ‘Present address: Inveresk Research International, Trenent, Musselburgh, Edinburgh EH33 2NE, 1JK. 0166-445X/97/$17.00 Crown Copyright 0 1997 Published by Elsevier Science B.V. PIISO166-445X(96)00834-X
Transcript
ELSEVIER Aquatic Toxicology 38 (1997) 125-143
Experimental and field studies of effects of dichlorvos exposure on acetylcholinesterase activity in the gills of the
SOAEFD Marine Laboratory, P.O. Box 101, Victoria Road, Aberdeen ABll 9DB, UK
Accepted 30 September 1996
Abstract
Dichlorvos (DDVP) is an organophosphate medicine, used to treat ectoparasitic sea lice infestations of farmed salmon. Effects of this chemical on a non-target organism were inves- tigated. Experimental exposure of mussels (Mytilus edulis L.) to dichlorvos (DDVP) resulted in changes in gill acetylcholinesterase (AChE) activity with a hermetic increase at lower
concentrations, and 50% inhibition at 3.6 pg 1-l after 24 h exposure. Repeat exposures re- sulted in cumulative inhibition. Twenty-four hour exposure to DDVP impaired the ability to close shells (ECsO of 1700 kg 1-l) and caused mortalities at high concentrations (L& of
8200 pg 1-l). AChE levels in mussels collected from sea lochs were indicative of DDVP inhibition related to the quantities of DDVP used. The use of AChE as a sublethal indicator of DDVP usage in the field is discussed.
Crown Copyright 0 1997 Published by Elsevier Science B.V.
Infestation of farmed salmon by ectoparasitic copepods can result in severe skin
damage in affected fish, which may lead to death through osmoregulatory failure or infection by opportunistic pathogens (Bruno et al., 1990). The main mechanism for controlling such infestations (Costello, 1993; Roth et al., 1993) is by immersing fish
0166-445X/97/$17.00 Crown Copyright 0 1997 Published by Elsevier Science B.V. PIISO166-445X(96)00834-X
126 J. G. McHencv~~ et dldquatic Toxicology 38 (1997) 125 143
in a bath of Aquagard SLTB (CibaaGeigy Agrochemicals, Cambridgeshire), which is licensed as a veterinary medicine for the treatment of salmon in the UK. The
active ingredient of this formulation is the organophosphate dichlorvos (DDVP), which kills crustaceans by inhibition of their acetylcholinesterase (AChE) activity.
Infested fish are bathed in 1 ppm dichlorvos for 1 h in cages surrounded by tarpau-
lins (Rae, 1979). After treatment, the tarpaulins are removed and dichlorvos is released into the surrounding water, where it is dispersed and diluted (Turrell, 1990; Wells et al., 1990; Dobson and Tack, 1991).
Although concern has been expressed over the potential of the released DDVP to
adversely affect non-target animals in sea lochs (Ross and Horsman, 1988; Ross, 1989; Fraser et al., 1989) community analysis in lochs containing fish farms has
failed to demonstrate changes which can be attributed to dichlorvos use (Murison et
al., 1990) and the acute toxicity of DDVP to non-target organisms (McHenery et al., 1990; McHenery et al., 1991; McHenery et al., 1996). However, there are few field studies of the sublethal effects of DDVP on non-target organisms, arising from
either infrequent exposure to treatment water containing high concentrations of DDVP, or from exposure to lower concentrations for longer periods as the treat-
ment water disperses. The mussel (M~~tilus edulis) is a common component of the wild fauna in sea
lochs where fish farms are present. Shellfish cultivation, in particular mussel farms, can occur in the same lochs as salmon farms, and mussels may be exposed to
DDVP via the treated water released from the fish farm. Mussels therefore are both members of sea loch ecosystems and of commercial importance.
Initial studies of the toxicity of DDVP to mussels (McHenery et al., 1990) showed that exposure to 1000 ug 1-l DDVP for 24 h resulted in no mortalities. There was, however, a sublethal effect, in that the same concentration resulted in a 90% in-
hibition of the AChE activity in the gill. Estimation of AChE activity in marine organisms (fish, Galgani et al., 1992; mussels, Bocquene et al., 1993; Narbonne et al., 1993) has been made under sublethal conditions in field studies, and the results have been related to pollution gradients. Elevations in acetylcholine have been shown to affect the water pumping rate in the mussel (Jones and Richards, 1993), and another organophosphate pesticide affected the ciliary activity in fresh-
water mussel gills (Basha and Swami, 1987). The present study was therefore undertaken to investigate experimentally the
effects of DDVP exposure on the mussel (Mytilus e&&r), including the sublethal effects on shell closure, AChE activity and filtration rate. Field studies of the AChE activity in mussels from four sea lochs on the west coast of Scotland were also carried out, and are presented in relation to both DDVP usage and the laboratory studies. Confidentiality requirements prevent the naming of the lochs, or the pro- vision of maps.
J.G. McHenery et aLlAquatic Toxicology 38 (1997) 125-143 127
2. Materials and methods
2.1. Laboratory studies
2.1.1. Acute toxicity Groups of five adult mussels (2-5 cm) were placed in 1 1 aliquots of aerated
68 pm filtered seawater (FSW) at 10°C in 3 1 Pyrex beakers, and allowed to re- sume filtration. DDVP was added to eight beakers to create a semi-logarithmic concentration gradient of l-10 000 yg l-l, together with an untreated control. After 24 h exposure, the mussels were removed from the beakers and mortalities were recorded. Surviving animals were placed on paper towelling and the time taken to close their shells was recorded. Animals were dissected and portions of gill removed for AChE determination (McHenery et al., 1991).
Gill portions were homogenised with a Teflon pestle in 0.5 ml of ice cold 0.1 M sodium phosphate buffer, pH 8, in centrifuge tubes. The tubes were centri- fuged at 13 OOOg for 10 min at 4°C and supernates dispensed into microtitre plates for assays.
All reagents employed in the AChE microassay were obtained from BDH Ltd. (Poole, UK) and prepared in 0.1 M sodium phosphate buffer, pH 8. Activities were determined for 5 ~1 aliquots of sample supernate diluted to 25 ~1 with buffer in 96-well microtitre plates. Aliquots, 125 ~1, of dithiobisnitrobenzoate (0.6 mM) and acetylthiocholine iodine (1.2 mM) (see Ellman et al. (1961)) were added to each sample and absorbance at 415 nm was recorded immediately in a microplate reader against a reagent blank. Absorbances were read after 5 and 10 min incu- bation at 20°C and the enzyme activity was calculated. One enzyme unit was de- fined as that quantity which produced an increase in AdI of 0.001 units mine1 under the assay conditions.
AChE levels were standardised in terms of specific activity, units per milligram of protein, in each sample supernate. Protein concentrations in all homogenates were measured using an automated dye binding protein assay (Bio-Rad, Rich- mond, CA, USA) in 96-well microtitre plates.
2.1.2. Repeat exposure-recovery studies Groups of mussels were exposed as above to 0, 10, 100 and 1000 pg 1-l DDVP
for 24 h and transferred to running seawater for 7 days. The mussels were then re-exposed to the same concentrations of DDVP for a further 24 h and returned to flowing seawater for a further 7 days. Five mussels were removed from each treatment group at the end of each exposure and recovery period and assayed for AChE activity.
Mussels were exposed to DDVP at 0, 1, 3, 10 and 30 pg 1-l for 8 days with daily replacement of test solutions and assayed for AChE activity.
Eight groups of mussels were exposed to 100 yg 1-l DDVP for 1 h each day over 8 days, and held in 1 1 aliquots of FSW between exposures. After each expo- sure, a group of animals (plus controls) was killed for AChE determination.
128 J.G. McHenery et al.lAquatic Toxicology 38 (1997) 125-143
2.1.3. Salinity efSects
Duplicate groups of mussels were exposed to 0 and 100 ug 1-t DDVP as
above for 24 h. One group at each concentration was exposed to FSW-distilled water (7:3), i.e. at a salinity of 24%. After exposure, AChE levels were deter-
mined.
2.1.4. Size efects
Groups of large (5.0-6.3 cm) and small (1.5-3.0 cm) mussels were exposed to DDVP at 0, 100 and 1000 pg 1-l for 24 h as above, and assayed for AChE activ-
ity.
2.1.5. EfSect of’ DDVP exposure on jiltration rate
Groups of five mussels were exposed to DDVP at 0, 100, 300, 1000 and 3000 p.g 1-l for 24 h. All animals were rinsed in FSW and transferred to beakers containing 1 1 aliquots of aerated FSW. A sixth beaker was prepared with-
out mussels. The marine bacterium Afteromonas 2024 was labelled with (methyl-
3 H)thymidine ( Birkbeck and McHenery, 1982) and added to each beaker
at a concentration of lo7 ml-‘. At 0, 1, 2, 4 and 22 h after the addition of
bacteria, water samples were taken. A 1 ml sample was removed and added to 10 ml of scintillation fluid (Quickzintm, Packard) to determine total radio- activity. A 1.5 ml sample was centrifuged at 13 OOOg for 5 min, and 1 ml
of supernate was added to scintillation fluid to determine soluble label. The difference between the two readings was taken as cell associated label. Disin- tegrations per minute were determined by counting liquid scintillation over a
20 min period. All results are given in terms of the initial nominal concentra-
tions.
2.2. Field studies
Field studies were carried out at four sea lochs (A-D) on the west coast of Scotland. The historical usage of DDVP differed between the sites. The annual amount of DDVP used in the late 1980s was 414 kg, 178 kg, 46 kg and 10 kg in Lochs A-D, respectively.
Samples of mussels were collected where possible, from the cages, near-shore, farm pier and at the far-shore within the sea lochs, and transported in damp sea- weed to the Marine Laboratory, Aberdeen, within 6 h of collection. Animals were placed in running seawater for 1 h, then transferred in groups of five into 1 1 aliquots of FSW. Mussel samples from each site were exposed to DDVP at 0, 10 and 100 pg 1-l for 24 h at 10°C as above and assayed for AChE to investigate the response of the mussels from the different lochs to experimental DDVP exposure. Samples (cage and pier) were obtained from Loch D before fish were treated with DDVP and 24 h after treatment.
J.G. McHenery et aLlAquatic Toxicology 38 (1997) 125-143 129
Time (seconds)
112.5 -
100,o -
815 -
75.0 - /
62,5 - r’ /’ /
50#0 - T ,/
37s /’ -
25.0 -
12.5 -
_s-r-‘id_...._~-...‘+e”
0.0 @-- /fl
I1 III 0 100 1000
DDVP (WV
Fig. 1. Time taken for mussels to close, mean ? SEM, after 24 h exposure to DDVP.
AGhE (unMng\
800 1
n’-- ~-
I
0 1
DDVP @g/l)
Fig. 2. ChE activity per milligram of protein, mean + SEM in gills of mussel exposed to DDVP.
130 J.G. McHenery et aLlAquatic Toxicology 38 (1997) 125-143
3. Results
3.1. Luboratory studies
3.1.1. Acute toxicity
Exposure to DDVP for 24 h resulted in impairment of the ability of mussels to
close their shells (Fig. 1). At concentrations of 3000 pg 1-l and above, mussels lost the ability to retract mantle fringes and close the valves of their shells, with
an ECSo of 1690 ,ug I-’ for this parameter. After 24 h exposure to 10000 pg l-‘, four of the five mussels so exposed had died, LCSo of 8200 pg 1-l.
Gill AChE activity in mussels exposed to 1 pg 1-l DDVP for 24 h was signifi-
cantly higher than in the unexposed control group (t = 7.34, df= 8, P < 0.0005) (Fig. 2). As DDVP concentration increased, there was a concentration-dependent
reduction in activity, with 50% inhibition at 3.6 yg 1-l.
3.1.2. Repeat exposures
After 24 h exposure to DDVP, there was a concentration-dependent reduction
in AChE activity (Fig. 3). Over the 7 day recovery period in flowing seawater, there was a recovery in AChE levels in each group previously exposed to DDVP,
AChE (unitslmg)
800 1
700 -
500 -
1
0.00 5.00 10.00 15.00
Days
Fig. 3. AChE activity per milligram of protein, mean k SEM in gills of mussels exposed to DDVP for
24 h, after 7 days in running seawater, after 24 h further exposure and after a further 7 days in run-
ning seawater (0, 0 pg I-‘; A, 10 pg 1-l; H, 100 pg I-‘; T. 1000 1.18 I-‘).
J.G. McHenery et al.lAquatic Toxicology 38 (1997) 125-143 131
AChE (units /mg)
700 1
DDVP @g/l)
Fig. 4. AChE activity per milligram of protein, mean k SEM in gills of mussels after 8 days daily ex-
posure to DDVP.
although the AChE levels did not recover to the control level over this period.
After the second 24 h exposure to DDVP, the AChE levels were again reduced in the DDVP exposed groups, the degree of inhibition again being concentration de- pendent. After a further 7 days in flowing seawater the AChE levels in the ex-
posed groups had again partially recovered. The degree of recovery after the sec- ond DDVP exposure was not as complete as that after the first exposure.
Repeated daily exposures for 8 days to DDVP at concentrations of l-30 ug 1-l resulted in a highly significant (F= 19.2, df= 4 and 20, P < 0.01) concentration- dependent reduction in AChE activity of 48% to 16% of the untreated control value (Fig. 4).
Daily exposure for 1 h to 100 pg 1-r DDVP resulted in a marked reduction in
mussel gill AChE activity (Fig. 5). After the first exposure, activity had been re- duced to 35% of the unexposed control, with increasing inhibition after each sub- sequent exposure. After five periods of exposure, the activity was reduced to a re- sidual level of 9%, at which it remained over the next three exposures.
3.1.3. Salinity efects After 24 h exposure to 100 pg 1-l DDVP in seawater in which the salinity had
been reduced to 24 %O , there was no significant difference in the degree to which AChE activity was inhibited, (110 ? 15 units mgg’ (mean f SEM), at 24 %O ;
132 J.G. McHenery et allAquatic Toxicology 38 (1997) 125-143
AChE (unitslmg)
700 -
600 -
500 -
f
A’ 1.. . c _.- -
‘-. 400 - -b
Fig. 5. AChE activity per milligram of protein, mean? SEM in gills of mussels exposed to 100 Kg I-’
DDVP for 1 h per day over 8 days (0. 0 pg I-’ ; A, 100 pg I-‘).
104 +_ 8 units mgg’ at 34 X0). The levels in the unexposed controls were not signif-
icantly different either: 548 + 44 units mggl and 480 I!Y 63 units mg-’ in the full
strength and reduced salinity exposures, respectively.
3.1.4. Size effects
Large and small mussels exposed to DDVP for 24 h showed a dose-de- pendent reduction in AChE activity with increasing DDVP concentration. After exposure to 10 ug 1-l DDVP, AChE activities were 147? 10 units mgg’ and
156 ? 12 units mgg’, and 50 f 7 units mgg’ and 33 ? 12 units mgg’ after exposure to 100 ug l-‘, in small and large mussels, respectively. There was no significant difference between the two size groups in the degree of inhibition.
3.1.5. EfSect on filtration rate
The scintillation counts for unfiltered and filtered water samples are given in Table 1. After exposure to DDVP at concentrations up to 3000 ug l-l, which led to marked inhibition of AChE activity in gills, there were no dif- ferences in the rate at which mussels filtered bacteria from water, nor in the rate at which free “H was released into the water after digestion of the bac- teria.
J.G. McHenery et al.lAquatic Toxicology 38 (1997) 125-143 133
Table 1 Results of the effect of exposure of mussels to DDVP on their subsequent rate of filtration of a radio-
The radioactivity (disintegrations per minute, DPM) was counted in the total unfiltered seawater and also
in the supernatant fraction to determine the amount of 3H in the soluble fraction. The cellular compo-
nent (the difference between the amount of label in the unfiltered sample and in the soluble phase)
relating to the radioactivity in the mussels.
3.2. Field studies
The levels of AChE detected in the gills of mussels collected from sea lochs showed indications of decreasing AChE activity with increasing usage of DDVP (Fig. 6). The mussels from the cage site in Loch A, which had the highest usage of
DDVP of the four lochs studied, showed the lowest AChE activity in their gills. The highest AChE activity was found at the far-shore site in Loch D, with the lowest DDVP usage in the study. The mussels from Lochs B and C, with intermediate DDVP usage, were found to have AChE activity levels generally between those of Lochs A and D.
In mussels collected from the cage structures, there was a significant negative correlation (r = -0.978, t = 6.63, df = 2, P < 0.05) between AChE activity and annu- al DDVP usage. The correlations between mean AChE activity and DDVP usage were not significant at the other sites, but there was a suggestion of decreasing
134
AChE ~u~[s~rng)
700 -
600 -
500 -
400 -
300 -
200 -
100 -
J. G. McHenery et dldquatic Toxicology 38 (1997) 125-143
pCO.05 for cage samples
I I I I I I,
10 50 100 150 200 400 DDVP(kg/year)
Fig. 6. AChE activity per milligram of protein, mean IL SEM in gills of mussels from positions in sea lochs vs. annual DDVP usage (0, cage; A. pier; n , near-shore; v, far-shore).
activity with increasing DDVP usage (u = -0.661 and Y = -0.849 for the farm pier
and far-shore sites, respectively). Exposure of mussels from each site in Lochs A-D to lo-100 pg 1-l DDVP
showed inhibition of AChE activity. The degree of inhibition increased slightly in
samples from all sites when exposed to the higher concentration of 100 pg 1-l (Figs. 7-11). Significant inhibition in AChE activity in mussel gills from the four sites in Loch A was found at both 10 pg 1-l (P < 0.025) and 100 pg 1-l (P < 0.05). The order of sites was the same at both concentrations tested; the lowest activity was found in samples from the cage, followed by far-shore, near-shore and pier. The
samples from Loch B also showed significantly lower AChE activity after 24 h in 10 pg 1-l (P < 0.05) and 100 pg 1-l (P < 0.05). The data for samples from Loch C were similar; the mussels from the cage, pier and far-shore showed significant (P < 0.025) AChE inhibition after exposure to both 10 and 100 ,ug 1-l.
The mean levels of AChE activity in the mussel gills from the pier, near- and far- shore sites were similar after experimental exposure to DDVP, irrespective of loch sampled. The pier, near- and far-shore stations showed mussel gill AChE activity in the approximate ranges 300-350 units mg-‘, 350450 units mg-’ and 300- 450 units mg-’ protein for Lochs A, B and C, respectively. The AChE activity in mussel gills from these sites after exposure to 10 pg 1-l DDVP was 75- 125 units mg-‘, 75-175 units mg-’ and 75.-150 units mg-’ protein, and after expo- sure to 100 pg 1-l DDVP was 50-125 units mg-‘, 75-150 units mg-’ and 25- 75 units mg- l protein for Lochs A, B and C, respectively.
J.G. McHenery et al.lAquatic Toxicology 38 (1997) 125-143 135
AChE (unitslmg)
0 1 I I to1
0 10 20 30 40 50 60 70 8090100
Fig. 7. AChE activity per milligram of protein, mean + SEM in gills of mussels collected from Loch A
before DDVP release and after 24 h exposure to DDVP (0, cage; A, pier; W, near-shore; v, far-
shore).
The greatest inhibition of AChE activity in the field samples was found in the mussels from the cage sites in all four lochs (Figs. 7-l 1); however, for these mussels
the degree of further inhibition on experimental exposure was less than in mussels collected at the other sites. The initial AChE activity was higher in Loch C cage
samples than those from the cage sites on Lochs A and B, reflecting the lower usage of DDVP in Loch C. When these Loch C samples were subsequently exposed to
10 pg 1-l and 100 ug 1-r DDVP the inhibition of AChE activity was therefore proportionally greater than in the cage mussel samples from the other two lochs.
As above, the mussels from Loch D (Fig. 11) showed an inhibition when exposed to 10 ug 1-l DDVP, and a further degree of inhibition on exposure to the higher concentration of 100 pg 1-l DDVP. The levels of AChE activity in the mussels from
the cages were lower than those from the other sites irrespective of whether they were sampled before or after treatment. The AChE activity in the mussels from the cage sites was higher than those from cage sites in Lochs A and B, reflecting the lower usage of DDVP in Loch D. The inhibition resulting from the experimental exposure of the Loch D samples was therefore proportionally greater than in the cage samples from Lochs A and B. There was, however, no significant difference (P > 0.05) in the AChE activity in mussels collected at any of the sites in Loch D, before and after the cage had been treated with DDVP when exposed experimen-
tally to 10 ug 1-l and 100 ug 1-l DDVP.
136 J.G. McHenery et al.lAyuatic Toxicology 38 (1997) 125-143
AChE (unitslmg)
500 ‘r
0 ! 0
I I I I IIJII
20 30 40 50 60 70 80 90100 DDVP WV
Fig. 8. AChE activity per milligram of protein, mean + SEM in gills of mussels collected from Loch B
after DDVP release and after 24 h exposure to DDVP (0, cage; A, pier; W, near-shore; T, far-
shore).
4. Discussion
The acute toxicity studies showed that DDVP had a 24 h LCso to mussels of
8200 pg ll’, and an EC so for the ability to retract mantle fringes and close the
valves of 1690 ug I-‘. The treatment concentration of DDVP is 1 mg 1-l for 1 h (Rae, 1979), and after release it is diluted and dispersed in the receiving waters (Turrell, 1990; Wells et al., 1990). It is therefore unlikely that these non-target
Table 2
LCsO values for a variety of species exposed to DDVP
Values are mean 2 SEM (n = 10) AChE activity (units mg-’ protein)
“McHenery et al. (1990).
J.G. McHenery et al.lAquatic Toxicology 38 (1997) 125-143 137
AChE (uniim[)
pco.025
I I I / I1ll,,
0 10 20 30 40 50 60 70 80 90100
Fig. 9. AChE activity per milligram of protein, mean k SEM in gills of mussels collected from Loch
C, after 24 h exposure to DDVP (0, cage; A, pier; T, far-shore).
organisms would be exposed to concentrations near the EC50 or LCsa, or suffer acute toxicity. This is supported by field observations of the effects of DDVP usage on mussels deployed near cages where fish were treated (McHenery, 1990) and by the common observation of mussels growing on salmon cage supports. The LCsa values for some other species are shown in Table 2, illustrating the wide range of toxicities of DDVP to marine organisms, and the relative insensitivity of mussels. Egidius and Master (1987) investigated the sensitivity of the mussel (Mytilus edulis) to trichlorfon (which degrades to DDVP) and DDVP itself, and found them to be less sensitive than crabs or lobsters. No mortalities were reported. Mussels (M. edulis) were exposed to concentrations of dichlorvos from 0.001 to 1 .O ppm (Cusack and Johnson, 1990; Thain et al., 1990). DDVP has been suggested as a therapeutic agent for mussels parasitised by the copepod Mytilicola intestinalis at a dosage of 30 mg 1-l for 2 h (Blateau et al., 1992). The low sensitivity of mussels to organo- phosphate pesticides may be due to an ability to adjust their metabolism (Mohan et al., 1987).
Mussel gill AChE activity showed a concentration-dependent inhibition between 1 pg 1-l and 10 000 pg 1-l) and also under repeat exposure conditions as was found for lobster larvae at concentrations of 0.01-100 pg 1-l (McHenery et al., 1991). The sensitivity of mussel gill AChE activity to DDVP inhibition in vivo was similar to that previously reported for lobster AChE, with 50% inhibition values for 24 h exposures of 3.6 ug 1-l and 2.7 pg l-l, respectively (McHenery et al., 1991). A hermetic increase in gill AChE activity at low DDVP concentrations was also observed in lobster larvae (McHenery et al., 1990) and red crayfish (Repetto et
138 J.G. McHenery et al.lAquatic Toxicology 3X 11997) 125-143
AChE (units/mg)
100 -
50 -
0 I I I I I,,lll 0 10 20 30 40 50 60 70 8090100
Fig. 10. AChE activity per milligram of protein, mean + SEM in gills of mussels collected from Loch
D, before DDVP release and after 24 h exposure to DDVP (0. cage; A, pier).
al., 1988). Exposure to DDVP was reported to increase the growth of mussels (Thain et al., 1990). In general, mussel gill AChE activity showed responses to
experimental DDVP exposure similar to those reported for lobster larvae (McHen- ery et al., 1991). However, the concentrations at which these responses were elicited in mussels were higher than for lobster larvae, reflecting the lower sensitivity of
mussels to DDVP. Repeat exposure of mussels to concentrations of DDVP up to the treatment dose
(1000 pg 1-l) showed that mussel AChE activity can recover to almost pre-exposure levels after one exposure. However, the degree of recovery was not as great after the second 24 h exposure to these concentrations. There is therefore a degree of cumu- lative effect of repeated exposure to DDVP on mussel gill AChE activity, as was
also found with lobster larvae (McHenery et al., 1996). Five exposures of lobster
larvae to 50 pg I-’ for 1 h resulted in AChE inhibition and no mortalities; however, repeated exposure to 50 yg 1-l for 6 h resulted both in AChE inhibition and mor- talities (McHenery et al., 1996). However, no mortalities of mussels were recorded in the present study after various repeat exposures of mussels up to concentrations of 1000 pg 1-1, although AChE activity was inhibited. Exposure of mussels to 3 pg 1-l DDVP or more caused inhibition of their AChE activity in the gills, as above; however, there was no evidence of this affecting either the ability to survive in low-salinity water (up to 100 pg 1-l DDVP), or their filtration rate (up to 3000 j.Lg 1-l).
Treatment of fish with DDVP to remove sea lice is carried out for 1 h and at
J.G. McHenery et al.lAquatic Toxicology 38 (1997) 125-143 139
irregular intervals during a season. If mussels are exposed to concentrations as high as the treatment level (1000 pg 1-l) before dilution and dispersion in the environ- ment, some inhibition of AChE may occur. If no further exposure occurs, this
inhibition would be likely to return to normal within a week. Impairment of the ability of mussels to close at high DDVP exposures could
potentially reduce their survival. The importance of shell closure in the protective
role against organophosphate pesticide toxicity in a freshwater mussel was high- lighted by the studies of Mohan et al. (1987). However, the brief exposures to concentration equal to or less than the treatment level are unlikely to significantly
affect closure times, or vulnerability to predation. The degree of inhibition of AChE activity in the gills from the mussels samples
taken from the four lochs increased towards the treatment cages. The AChE activity
was also shown to decrease with increasing DDVP usage in the lochs, strongly
suggesting that the indigenous mussel populations in Lochs A-D had been affected by the DDVP usage at the fish farms. The lowest mean levels of AChE activity in
field mussel samples was 120 units mgg’ protein, at the cage site in Loch A, where the greatest usage of DDVP had occurred. The experimental data showed that
exposure to 1000 yg I-’ (the treatment dose) for 24 h, or at least three daily ex- posures to 100 pg 1-l DDVP would be necessary to achieve AChE values lower
than 100 units mgg’ protein. The hydrographic characteristics of sea lochs prevent this occurring as a result of a single use of DDVP, but it might arise from the cumulative effects of a series of treatments.
The existence of the gradients of AChE activity away from the cages in all four lochs, irrespective of when DDVP was last used in the lochs (as much as 6 months
in Loch D), supports the suggestion of incomplete recovery between treatments. The experimental studies also indicated that multiple exposures to high concentra-
tions of DDVP for 24 h may result in persistent reduction of AChE activity. It therefore appears that the cumulative effect of lowered AChE activity observed experimentally in the mussel gills is also present in the field samples, and that
recovery may take weeks or months. However, during this recovery period and from the results of the laboratory experiments, there is no evidence to suggest
that the reduction in AChE activity observed would adversely affect the ability of the individuals to survive a reduction in salinity to 24% or to result in reduction of the filtration rate. Indeed fouling of nets and cage structures by mussels remains a concern for fish farmers, indicating that the use of DDVP has not resulted in a widespread reduction in mussel reproduction and settlement.
On exposure of the field mussel samples to 10 and 100 ug 1-l DDVP, the AChE activity showed a concentration-dependent inhibition. In the samples from Loch A, with the highest DDVP usage, the gradient of effect between sites is reflected in both the exposure concentrations; the cage samples showed the greatest inhibition, with lesser effects in the other samples. Most of the other samples showed a similar level of inhibition of AChE activity, irrespective of the sampling site, or loch. The response of the field animals to high dosages of DDVP for 24 h is therefore similar, irrespective of the previous exposure of the animals to DDVP. This corroborates the experimental repeat exposure study which showed that the inhibition level of
140 J. G. McHenery et al.lAyuutic Toxicology 38 (1997) 125-143
AChE (uniis/mg]
1 / I I II
0 10 20 30 40 50 60 70 8090100
Fig. 1 I AChE activity per milligram of protein, mean 2 SEM in gills of mussels collected from Loch
D, after DDVP release and after 24 h exposure to DDVP (0, Cage 1; A, Cage 2; n , pier: T , Pier).
AChE activity remained at a similar level after exposure to 100 pg 1-l DDVP daily
for 8 days. Previous exposure to DDVP did not therefore confer any ability on the mussels to adapt their AChE activity mechanisms in the gills to exposure at high
concentrations, either in the longer-term field situation, or the short term of the
experiments. The relationship between mussel gill AChE activity and the quantities of DDVP
used at different farms and the proximity to the treatment cages supports the use of this measurement of enzyme activity as an indicator of organophosphate exposure (Weirs, 1959; Coppage and Matthews, 1974; McHenery et al., 1991; Galgani et al., 1992). Further exposure of the field samples to 1% and 10% of the treatment dose would not yield any additional information, as the response of the individuals
appears to be similar irrespective of the previous exposure of the mussels. To enhance the usefulness of the mussel as an indicator of low-level DDVP exposure, additional studies would be necessary to investigate the possible existence of a threshold concentration for the inhibition of AChE activity, as was found with the lobster larvae (McHenery et al., 1996) and to define any hermetic response
and its variation with exposure history. The apparent differences in sensitivities of mussel gill AChE to DDVP inhibition
can be accounted for in two ways. The reduced sensitivities in mussels collected from positions close to the treatment cages, most notably in Loch B, may reflect adaptation to exposure. The low initial AChE levels in mussels collected from salmon cages in Lochs B and D were such that there was a lower margin for
J.G. McHenery et aLlAquatic Toxicology 38 (1997) 125-143 141
inhibition, the low levels having been caused by repeated treatment exposures which exceeded the adaptive capacity of the mussels.
Recent field studies suggest that environmental contaminants other than organo- phosphate pesticides can affect AChE in fish (Payne et al., 1996). The variations in AChE in the mussel Mytilus galloprovinciulis collected along a pollution gradient in the Mediterranean (Narbonne et al., 1993), and in Mytilus edulis from around the coastline of France (Bocquene et al., 1993) suggest that this may also be the case with mussels. No changes in fish AChE have been recorded in relation to age, sex and reproduction (Galgani et al., 1992), and in the only mollusc studied for season- al effects on AChE, the oyster Crussostrea virginica, few trends in enzyme activity were observed (Chambers et al., 1979). The lochs in the present study are situated away from significant inputs of sewage, or industrial, agricultural or domestic wastes. The main discharges are from fish farms. It is therefore unlikely that the inhibition of AChE recorded in this paper is due to factors other than the use of DDVP at the fish farms.
5. Conclusions
Mussels exhibit a similar concentration-dependent inhibition, hermetic re- sponse and cumulative inhibition of the acetylcholinesterase enzyme to DDVP to those found in lobster larvae. Both the 24 h LC5a and ECse values (8200 l.rg 1-l and 1700 ltg l-l, respectively) are higher for mussels than for lobster larvae, reflecting the lower sensitivity of mussels. In sea lochs with salmon farms, the field samples showed a reduction of AChE activity in the mussel gills with the following gradient at sampling sites: cages < pier < near < far sampling sites. The level of inhibition of AChE activity in each loch reflected the amount of DDVP used in the four lochs studied. Mussels showed a cumulative inhibition of AChE activity, both in the repeat exposure studies and in field samples. The inhibition of AChE activity in mussel gills is considered to be a useful sublethal indicator of the use of DDVP in aquaculture.
Acknowledgements
The authors acknowledge the co-operation of Marine Harvest Ltd, and thank Ciba-Geigy Agrochemicals for the provision of Aquagard.
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