Exp. Eye Rrs. (1990) 50, 443447
LETTER
Nuvan
and Cataracts
TO THE
in Atlantic
The hypothesis that Nuvan is responsible for the increased level of cataract in wild Atlantic salmon on the West coast of Scotland (Fraser, Duncan and Tomlinson, 19 8 9) has attracted a number of criticisms from Dobson and Schuurman (1990). Firstly, Dobson and Schuurman are disturbed by the lack of mention of the manufacturer of Nuvan, CibaGeigy Limited, Basel. A reference was in fact given to Ross and Horsman (1988) who not only gave the manufacturers of Nuvan, but quoted extensive product data and relevant legislation. The trade name Nuvan is used extensively in the press without reference to its manufacturer. We understand that Nuvan is now sold under a Trade name, Aquagard. We did fail to acknowledge the generous‘and willing support shown by Mr William Muir and the crews of Coigach Salmon Fisheries Ltd, and we apologise for this omission. We would take issue with Dobson and Schuurman’s claim that we misquoted references. Apart from Ross and Horsman (1988) quoting reports that overdosing with Nuvan causes ‘blindness and even death ’ of salmon, and further reports of crustacea dying ‘within 30 min of exposure to working concentrations of Nuvan (1 ppm) and within several hours at 0.1 ppm’. They also emphasize that ‘although several studies have been conducted on the toxic effects of Nuvan, little has been published, and most data is therefore not available for independent assessment ‘. In addition to implicating Nuvan in these and other cases involving loss of marine life, they report on losses associated with the use of Neguvon’@Vet (Bayer). Salte, Syvertsen, Kjoennoey and Frode (198 7) similarly described losses associated with trichlorfon which gradually releases dichlorvos. These studies are mentioned in the context of Nuvan use because the active ingredient in Nuvan is dichlorvos, and the batch of trichlorfon described in the study by Salte et al. (198 7) was also considered to be contaminated by dichlorvos. Wooten, Smith and Needham (1982) quoted a 400word paper by Brandal and Egidius (19 7 7) on the oral administration of trichlorfon as Neguvon. The paper reports ‘one of the sublethal effects of the chemical is blindness, which usually occurs from the third day after treatment’. Cataract was not excluded, and we believe cataract is the only form of blindness which could be identified in intact fish without physiological or behavioural experiments (which are not described). Regarding the lack of recorded cataract following dichlorvos feeding, Ross and Horsman (1988) draw attention to the paucity of published data concerning dichlorvos. Since it is known that administration of anticholinesterase can induce cataract both in vivo (Philipson et al., 1979) and in vitro (Michon and 00144835/90/040443+05
$03.00/0
EDITORS
Salmon
(Saho
salar)
Kinoshita, 1968), the lack of data concerning dichlorvos induced cataract is surprising. Our data (Fraser et al., 1989) clearly demonstrate that cataract can be produced in salmon lenses in vitro. Our initial statement that ‘cataract rarely occurs in the wild’ was considered so obvious as not to require a detailed statistical reference. Lens physiologists and anatomists from Brewster (1825) to Iwata et al. (1987) have commented on the optical purity of freshly dissected lenses from a range of fish species. and in the literature on fish diseases prior to 1960 it is in fact impossible to obtain hard statistical data concerning cataract, presumably because it so rarely occurred. However, in a recent critical study of fish in several East coast rivers in Virginia, U.S.A., Hargis and Zwerner (in press) could find no evidence of cataract in unpolluted rivers. Interestingly, this was not the case in the polluted Elizabeth river. Dobson and Schuurman request further details regarding the surveying of cataract in wild salmon. Catches were examined by one of us (P. J.F.) at Coigach Salmon Fisheries, Achiltibuie, Ross-shire, Scotland, over a 5-week period, every summer from 19 77. Salmon were transferred individually using hand nets from the holding compartments of the nets, caught in smooth plastic boxes, and killed carefully. Eyes were examined at sea, immediately before killing in many cases, and always within a few minutes of killing. In 1984, a low number of cataracts was noted. No formal counts were taken, but the incidence was estimated by brief eye inspection at around 5%. Several thousand fish were examined in this and subsequent years. In 198 5, a formal count of a random sample of a whole bag net catch of 77 fish was taken, with 16 (16/77) having cataracts (20.8 %). This figure was considered to be a reasonable estimate based on the observation of several thousand fish. In 1986. 56/139 fish sampled in detail had cataracts, giving 40.3%. Again, eyes of several thousand fish were observed in addition to the detailed count, and the estimate considered reasonable. In 1987 and 1988, detailed counts of 1156/2102 and 1052/1957 fish with cataracts were made (5 5 % and 54’S). Whole catches from several bag nets were used to obtain these data. In 1989, fishing effort was much reduced at Coigach Salmon Fisheries, and 15124 fish with cataracts were counted, although more cataracts were noted in other catches. In 1989, counts were further made from bag nets at a second site at the East side of Scotland on the Moray Firth, with 25/404 (6.25%) fish having cataracts. Fish were transferred from net to boat at Moray Firth nets using conventional fishing techniques, and eyes 0 1990 Academic Press Limited
444
were examined at sea within minutes of killing. Some fish were examined live. The Moray Firth fishery at Macduff was chosen since it sampled fish from East Coast, and it presents useful contrast with the West coast Achiltibuie site. There are four fish farms within a llO-km radius of Macduff, compared with more than 60 within a 1 lo-km radius of Achiltibuie. In tagging studies between 1979 and 1981 involving one of us (P. J.F.), out of c. 800 fish tagged at Achiltibuie and released, none were subsequently recaptured at Macduff (Shearer, 1986). At Macduff, throughout several tagging studies, all tag recaptures, with one exception from a North coast tagging site, came from Moray Firth or East coast (Shearer, 1986). Note also that during the tagging work, no cataracts were noted in Achiltibuie fish, although the condition of each fish was carefully considered before tagging and release. It was not the intention in our paper to review cataractogens in fish, although dietary-related cataracts were certainly of interest. Dobson and Schuurman are misleading in their representation of examples. In a paragraph where ‘many dietary deficiencies causing cataract are not associated with any effect on growth rate or mortality ‘, they quote Richardson, Higgs and Reames (1986) as having described a cataractogenic diet for juvenile chinook salmon. In fact, this paper emphasizes the growth depression. Let us quote from the abstract regarding the cataractogenic diet, (C): ‘Cataracts were detected at day 126 in fish which were fed diet C between days 42 and 84. Fish growth was depressed severely by diet C’. In fact, Akiyama, Mori and Murai (1986) mainly report on a high incidence of scoliosis (spinal curvature) in fish fed a tryptophan-deficient diet, so it is misleading to imply no effects on growth. We have never observed spinal deformities in wild salmon with cataracts. Ultraviolet light certainly has been implicated in cataract formation. In a wild animal, high levels of glutathione in the lens would be expected to mop up free radicals. Dietary interference may reduce or enhance the protective influence of glutathione and also ofvitamins E and niacin (Ross et al., 1982, 1983). Wild salmon migrate in shoals close to the surface, where they utilize UV light for localizing the sun directly, or by means of the pattern of polarized light where cloud cover prevails. Directional swimming would lead to asymmetrical exposure to UV. It is not possible to explain the high incidence of cataract on the West coast compared to a low incidence on the East coast, or the sudden temporal occurrence on the West coast, in terms of UV-induced cataract alone. Exposure to UV light, in addition to cholinesterase inhibitor, may well increase cataract incidence, and might help explain the lack of reports of cholinesterase inhibitor-mediated cataract in laboratory studies with dichlorvos. Dobson and Schuurman further cite Allison (1962b), where lake trout reared as broodstock for
P J FRASER
ETAL.
4-6 yr showed 40-60s incidence of cataract, They identified inflammation in the choroid layer of the eye, leading to oedema which displaced the retina. No such inflammation of choroid layer, or displacement of the retina, has been found in wild salmon with cataracts. Dobson and Schuurman presume that the salmon examined for cataract would be ’ stressed by capture ’ and be ‘susceptible to mechanical injury ‘. They cite the work of Ubels and Edelhauser (1987), showing that abrasions of the cornea of fish caused cataracts. They state that abrasion is likely to occur in salmon caught in nets, and this could be unilateral. The fact that cataracts were not recorded prior to 1984 from the same nets seems to adequately control against possible capture-related artefacts. This is further borne out by the Moray Firth results in 1989, where catching conditions were broadly similar. Furthermore, in a 3 yr unpublished study (1979-1981), involving video monitoring and recording of around 12 hr per day of observation of the behaviour of salmon, in relation to one of the bag nets used for the samples of cataracted fish, no extensive contact with the net liable to lead to abrasion was observed (Muir and Fraser, unpubl. res.). On occasions, shoals of fish have been observed entering the net and have been caught and killed in a few minutes. Such fish show the same high incidence of cataract as those which have accumulated in the net for a longer period. If osmotic stress due to transitory crossings from sea to fresh water were a factor, as implied by the reference to Iwata et al. (1987), then appreciable numbers of cataracts would have been seen before 1984 and in Moray Firth catches. Furthermore, a temporal pattern to the incidence of cataract throughout the main migration time would be expected, corresponding to the pattern of cessation of feeding, development of gonad, and freshwater contact, which takes place throughout the summer in fish from Achiltibuie (Fraser, 1987: Fraser, unpubl. res.). This does not happen. It is not clear where a large population of wild salmon would encounter sublethal concentrations of copper, large variation in pH or thioacetamide. Indeed, such pollution might be considered less likely on the West coast than the East coast. To consider such causes, it would be reasonable to expect at least a demonstration of the existence of a pollutant where it would be encountered by the wild salmon, but Dobson and Schuurman do not do this. There is no evidence of extensive bacterial or viral infection in the wild salmon population. Given the extensive growth of the eye and lens from the smolt to grilse stages, in the 12 months of sea feeding, repaired damage to the lens would show as deep cataract, whereas many of the cataracts observed were anterior or posterior subcapsular. Small, C-shaped opacities deep in the lens, which were observed in quite large numbers of salmon, could have been caused at smolt stage, implying that the causal agent acts both on the smolt and the returning adult stages. There was no evidence of parasite in a seatrout
A REPLY
TO 0. P. DOBSON
AND
445
H. J.SCHUURMAN
smolt with cataract caught at Achiltibuie. There are no reports of large numbers of parasitised wild fish associated with rivers which carry stocks caught at Achiltibuie. Dobson and Schuurman describe a treatment regime involving enclosure of a cage with a tarpaulin and adding dichlorvos as Nuvan to give 1 ppm. In the Data sheet (V) for Aquagard, a second ‘Skirt technique ’ is described which involves twice the volume of Nuvan to be used for the same size of cage. In practice greater concentrations have been used by fish farms. Ross and Horsman (19 8 8) quote one farm operator as follows: ‘Up to 30 cages can be treated together in an hour. No tarpaulin is used. The Nuvan is poured in at one end of the cage unit [group of cages] and is allowed to flow through with the tide, topping up the concentration at each cage. About 300400 ml is used per cage [which are 9 in x 9 m or 12 m x 12 m]. Treatment occurs four or five times per summer ‘. They point out that this would involve approximately 10.5 1 of Nuvan being used in one treatment of a group of 30 cages. If mixing was not uniform, as is likely, then local concentrations in excess of 2 ppm Nuvan would be possible. The Aquagard data sheet (V) states up to three treatments at lo-20 day intervals may be needed, after which ‘the fish should be lice-free for considerable periods if all fish on the site have been simultaneously treated to prevent females from releasing eggs and starting a new infestation cycle’. It further states that ‘dichlorvos is dangerous to many aquatic organisms. The fastest, and therefore most important, detoxification mechanism in coastal waters is dilution which is increased by water movement, including the flushing effect in sea lochs. It is therefore important that under conditions of low water movement, the advice of the relevant water authority is sought before Aquagard is used’. It is misleading for Dobson and Schuurman to state that the treatment solution is ‘allowed out to the open sea ‘, since in many cases, it would have to be carried many miles to exit the sea loch. In Loch Broom, at Achiltibuie, coastal tidal stream speeds are in the order of 125-300 m hr-l. Streams are 50-100 m wide in the coastal zone containing bag nets (matching the length of the leader from shore to trap), and flow is laminar (data from Coigach Salmon Fisheries Ltd). A killing accumulation of Nuvan from a farm net would be expected to be outside a 25-m perimeter in 5-12 min. Dobson and Schuurman quote unpublished work which claims it is not possible to identify dichlorvos above 2 ppb outside this zone. This implies extremely rapid dispersion, or little dispersion with failure to find the tidally displaced Nuvan cloud. In shallow coastal water, dispersion is depth limited, and many observations of tidally borne debris suggest that, at least in coastal currents around salmon nets at Achiltibuie, very little dispersion takes place, other than bulk displacement with the water mass. Dobson and Schuurman question the use of I.34 x I(]-’ M dichlorvos as Nuvan as the starting
They state that this concentration concentration. would kill Atlantic salmon in 15 min. Interestingly, the concentration added to a cage if the Aquagard instructions are followed is a 10 x dilution of the quantity of Aquagard calculated to give the required 2 ppm Aquagard. This will be at an initial concentration of 50 g 1-l or 50000 ppm. Since mixing is mainly by fish movement and oxygen bubbles, and is reckoned to take 5 min, the fish will have considerable exposure to dichlorvos at concentrations above 1 ppm. It is not clear how much dichlorvos normally remains bound in larger droplets of emulsion, nor how droplet size would affect transfer of dichlorvos to organisms. In the size range of cells, dichlorvos concentrations around droplets are liable to exceed 30 ppm. Interaction with tissue will involve several phases, water, lipid, dichlorvos and the carrier in Nuvan. The lack of effect of Nuvan on juvenile stages of sea lice (Wooten et al., 1982) implies that mechanisms other than simple diffusion from aqueous phase may be taking place. It is certainly necessary to establish whether effects would be likely at lower concentrations. It can be seen from Table I that effects on membrane potential of wild salmon lenses are apparent at dichlorvos concentrations down to lo-’ M, confirming the likely sensitivity of the lens system. When a target tissue is accessible, the main effect of concentration of organophosphorus cholinesterase inhibitors is simply on the rate of inactivation of cholinesterase. Since real time effects could be measured by the electrophysiological techniques employed, a concentration was selected to give clear instantaneous effects. Incubation of trout lens for long term in vitro drug toxicity testing is well established (Hikida and Iwata, 1987). and the ratio of sodium to potassium ions is used as an indicator of lens opacillcation. Since the cornea will be a negligible barrier to a molecule as lipid soluble as dichlorvos, the only barrier to passage into the eye from the sea via the cornea will be the diffusional resistance of the stroma, and will be in the order of 1 mm hr-l. The isolated lens is hence not too unrealistic a model for the intact fish. Dobson and Schuurman question the realism of the in vitro lens since, in the intact fish, replenishment of the aqueous humour would be expected. This would initially aid in
TABLE I
The membrune potentials of 16 wild salmon lenses incubated in groups of four overnight at 10.7”C in artificial aqueous humour (AAH) and AAH with lo-‘, 1OW6and 1 Om4M dichlorvos as Nuvan. Concentration of dichlorvos
0
Membrane potential Negative mean mV 90.07 (1.16) (S.E.) mV
lo-?M
1o-6
M
1o-4
75.89
75.35
64.7
(5.70)
(5.44)
(5.23)
M
446
P. J. FRASER
distributing dichlorvos throughout the eyeball, and help it reach the lens via aqueous phase. It would not be involved in possible lipid phase transfer. Once having had its effect, dichlorvos would, of course, reduce the replenishment rate of the aqueous humour, and reduce ocular pressure and fluid flow, which is why cholinesterase inhibitors are used to treat severe cases of glaucoma in humans, despite the side effect of cataract. Dobson and Schuurman do not deduce this obvious effect or anywhere acknowledge that cholinesterase inhibitors could have any effect on the eyeball-d lens, although such effects are well known (see Kaufman, Wiedman and Robinson, 1984). Cataracts exist in farmed salmon (G. H. Rae, pers. commun.). Photographs and verbal descriptions of these cataracts corresponded well to cataracts seen in wild salmon. A proper survey of the geographical distribution and yearly temporal distribution of cataracts in fish farms would be a massive undertaking requiring large resource. Furthermore, such a survey would only be of limited value without knowledge of the temporal pattern of cataract occurrence throughout the Nuvan-using decade. Measures to alleviate dietary deficiencies in the presence of Nuvan treatment might very well have alleviated damage by Nuvan. One reaction to cataract has been to increase levels of vitamins in diets. As Dobson and Schuurman point out, certain vitamins are known to not only prevent cataract formation, but to suppress cataract incidence in the presence of known causal agents. Dobson and Schuurman state
ET AL
that ‘susceptibility to LJV light is ameliorated in diets rich in niacin ’ (Allison, 1960, 1962a). More recently it has been shown that glutathione and vitamin E can prevent glucose (diabetic) cataract in vivo and in vitro (see Ross et al., 1982, 1983). In our opinion, the farmed situation is not an adequate control for the wild fish population, since diets have been tuned to eliminate cataracts and other problems in an environment with continual dichlorvos exposure. Cataracts occur in farmed fish escapeescaught at Achiltibuie, so if Dobson and Schuurman are correct and there are no cataracts in farmed salmon, we have a situation where the farmed fish in cageswould seem to be protected from a causal agent which seemsto be local to the farmed sites. Wild salmon are unique in concentrating in coastal currents for a period of several weeks. No other population of fish could be so threatened with extinction by fishermen operating with fixed trap nets covering SO-100 m from the shore line, that leaders have to be removed for statutory periods on a weekly basis.A cloud of Nuvan releasedfrom a fish farm will simply be carried in the coastal currents which carry the wild salmon. As they move up and down the coast, moving 20-80 km day-l, the wild salmon will certainly be expected to encounter the Nuvan clouds. Hence, considering all the possiblecausesof cataract including those suggestedby Dobson and Schuurman, we would still affirm that Nuvan is the most likely cause of cataract observed in wild salmon at Achiltibuie.
Zoology Department, Aberdeen University, Aberdeen AL39 2TN, U.K.
PETER J. FRASER
School of Biological Sciences, University of East Anglia, University Plain, Norwich NR4 7TJ, U.K.
GEORGE DUNCAN JULIE TOMLINSON
(Received 18 December 1989 and accepted in revised form 27 December 1989) cataract in Atlantic Salmon(Salmo salar). Exp. Eye Res. References Akiyama, T., Mori, K. and Murai, T. (1986). EZffectsof temperatureon the incidenceof scoiiosisand cataract in chum salmonfry causedby tryptophan deficiency. Bull. Jpn Sot. Sci. Fish 52, 2039.
Allison, L. N. (1960). Sunburning fingerling lake trout with ultraviolet light and the effect of a niacin fortified diet. Prog. Fish. Cult. 22. 114-16.
Allison, L. N. (1962a). Cataractamonghatchery rearedlake trout. Prog. Fish. Cult. 24, 155. Allison, L. N. (1962b). Cataract in hatchery lake trout. Trans. Am. Fish. Sot. 92, 34-8.
Brandal, P. 0. and Egidius,E. (1977). Preliminary report on oral treatment against salmon lice, Lepeophtheirus salmonis, with Neguvon. Aquaculture 10, 177-8. Brewster,D. (1825). On the formation of singlemicroscopes from the lensesof fishes.Edinburgh 1. Sci. II, 98-100. Dobson,P. and Schuurman.H. J. (1990). Possiblecausesof
50, 43942.
Fraser,P. J. (1987). Atlantic salmonSalmo salar L. feed in Scottish coastal waters. Aquaculture Fish. Man. 18, 243-7.
Fraser,P. J., Duncan, G. and Tomlinson,J. (1989). Effectsof a cholinesteraseinhibitor on salmonidlens: A possible causefor the increasedincidenceof cataract in salmon (Salmo salar). Exp. Eye Res. 49, 293-8.
Hargis, W. J. and Zwerner, D. E. Someeffectsof sediment borne contaminants on development and cytomorphologyof teleosteye-lensepitheiialcellsand their derivatives. Marine Env. Res. (in press). Hikida, M. and Iwata, S. (1987). In vitro subacute cataractogenic study in rainbow trout lens. I. Pharmacobio. Dyn. 10, 443-8. Iwata, M., Komatsu, S., Collie, N. L., Nishioka, R. S. and Bern, H. A. (1987). Ocular cataract and seawater adaptationin salmonids.Aquaculture 66, 3 15-28. Kaufman, P. L., Wiedman. T. and Robinson,J. R. (1984).
A REPLY
TO D. P. DOBSON
AND
447
H. J. SCHUURMAN
Cholinergics.Pharmacologyof the Eye. HandbookExp. Pharmacol.69, 149-9 1, Michon, J. and Kinoshita,J. H. (1967). Cholinesterase in the lens.Arch. Ophthalmol.77, 804-8. Philipson,B., Kaufman, P. L., Fagerholm,P., Axelsson, U. and Barany, E.H. (1979). Echothiophatecataracts in monkeys: Electron microscopyand microradiography. Arch. Ophthabnol.97, 340-6. Richardson,N. L., Higgs, D. A. and Beames,R. M. (1986). The susceptibility of juvenile chinook salmon (Oncorhynchustschawytscha)to cataract formation in relation to dietary changesin early life. Aquaculture52, 237-43.
Ross, W. M., Creighton, M. O., Stewart-DeHaan, P. J., Sanwal. M., Hirst, M. and Trevithick. J. R. (1982). Modelling cortical cataractogenesis : 3. In vivo effectsof vitamin E on cataractogenesisin diabetic rats, Can.J. Ophthalmol.17, 61-6. Ross,W. M., Creighton, M. 0.. Trevithick, J. R., Stewart DeHaan,P. J. and Sanwal,M. (1983). Modellingcortical cataractogenesis : 6. Induction by glucosein vitro or in diabetic rats: prevention and reversal by glutathione. Exp. EyeRes.37, 559-73.
Ross,A. and Horsman,P. V. (1988). TheUseojNuvan 500 ECin theSalmonFarmingIndustry.Marine Conservation Society: Ross-on-Wye. Salte,R., Syvertsen,C.. Kjoennoey,M. and Frode.F. (1987). Fatal acetylcholinesterase inhibition in salmonidssubject to a routine organophosphate treatment. AquacuZture 61, 173-V. Shearer,W. M. (1986). The exploitation of Atlantic Salmon in Scottishhomewater fisheriesin 1952-1983. In The Status of the Atlantic Salmon in Scotland ITE SymposiumNo. 15 (EdsJenkins.D. and Shearer,W. M.). Pp. 3749. ITE. Ubels,J. L. and Edelhauser,H. F. (1987) Etrectsof cornea1 abrasion on cornea1transparency, aqueous humor composition, and lens of fish. Prog. Fish. Cult. 49, 219-24.
Wooten. R.. Smith, J. W. and Needham, E. A. (1982). Aspects of the biology of the parasitic copepods Lepeophtheirus salmonisand Caliguselongatuson farmed salmonids,and their treatment. Proc. R. Sot. Edin.81B. 185-97.
LETTER
Nuvan
and Cataracts
TO THE
in Atlantic
The hypothesis that Nuvan is responsible for the increased level of cataract in wild Atlantic salmon on the West coast of Scotland (Fraser, Duncan and Tomlinson, 19 8 9) has attracted a number of criticisms from Dobson and Schuurman (1990). Firstly, Dobson and Schuurman are disturbed by the lack of mention of the manufacturer of Nuvan, CibaGeigy Limited, Basel. A reference was in fact given to Ross and Horsman (1988) who not only gave the manufacturers of Nuvan, but quoted extensive product data and relevant legislation. The trade name Nuvan is used extensively in the press without reference to its manufacturer. We understand that Nuvan is now sold under a Trade name, Aquagard. We did fail to acknowledge the generous‘and willing support shown by Mr William Muir and the crews of Coigach Salmon Fisheries Ltd, and we apologise for this omission. We would take issue with Dobson and Schuurman’s claim that we misquoted references. Apart from Ross and Horsman (1988) quoting reports that overdosing with Nuvan causes ‘blindness and even death ’ of salmon, and further reports of crustacea dying ‘within 30 min of exposure to working concentrations of Nuvan (1 ppm) and within several hours at 0.1 ppm’. They also emphasize that ‘although several studies have been conducted on the toxic effects of Nuvan, little has been published, and most data is therefore not available for independent assessment ‘. In addition to implicating Nuvan in these and other cases involving loss of marine life, they report on losses associated with the use of Neguvon’@Vet (Bayer). Salte, Syvertsen, Kjoennoey and Frode (198 7) similarly described losses associated with trichlorfon which gradually releases dichlorvos. These studies are mentioned in the context of Nuvan use because the active ingredient in Nuvan is dichlorvos, and the batch of trichlorfon described in the study by Salte et al. (198 7) was also considered to be contaminated by dichlorvos. Wooten, Smith and Needham (1982) quoted a 400word paper by Brandal and Egidius (19 7 7) on the oral administration of trichlorfon as Neguvon. The paper reports ‘one of the sublethal effects of the chemical is blindness, which usually occurs from the third day after treatment’. Cataract was not excluded, and we believe cataract is the only form of blindness which could be identified in intact fish without physiological or behavioural experiments (which are not described). Regarding the lack of recorded cataract following dichlorvos feeding, Ross and Horsman (1988) draw attention to the paucity of published data concerning dichlorvos. Since it is known that administration of anticholinesterase can induce cataract both in vivo (Philipson et al., 1979) and in vitro (Michon and 00144835/90/040443+05
$03.00/0
EDITORS
Salmon
(Saho
salar)
Kinoshita, 1968), the lack of data concerning dichlorvos induced cataract is surprising. Our data (Fraser et al., 1989) clearly demonstrate that cataract can be produced in salmon lenses in vitro. Our initial statement that ‘cataract rarely occurs in the wild’ was considered so obvious as not to require a detailed statistical reference. Lens physiologists and anatomists from Brewster (1825) to Iwata et al. (1987) have commented on the optical purity of freshly dissected lenses from a range of fish species. and in the literature on fish diseases prior to 1960 it is in fact impossible to obtain hard statistical data concerning cataract, presumably because it so rarely occurred. However, in a recent critical study of fish in several East coast rivers in Virginia, U.S.A., Hargis and Zwerner (in press) could find no evidence of cataract in unpolluted rivers. Interestingly, this was not the case in the polluted Elizabeth river. Dobson and Schuurman request further details regarding the surveying of cataract in wild salmon. Catches were examined by one of us (P. J.F.) at Coigach Salmon Fisheries, Achiltibuie, Ross-shire, Scotland, over a 5-week period, every summer from 19 77. Salmon were transferred individually using hand nets from the holding compartments of the nets, caught in smooth plastic boxes, and killed carefully. Eyes were examined at sea, immediately before killing in many cases, and always within a few minutes of killing. In 1984, a low number of cataracts was noted. No formal counts were taken, but the incidence was estimated by brief eye inspection at around 5%. Several thousand fish were examined in this and subsequent years. In 198 5, a formal count of a random sample of a whole bag net catch of 77 fish was taken, with 16 (16/77) having cataracts (20.8 %). This figure was considered to be a reasonable estimate based on the observation of several thousand fish. In 1986. 56/139 fish sampled in detail had cataracts, giving 40.3%. Again, eyes of several thousand fish were observed in addition to the detailed count, and the estimate considered reasonable. In 1987 and 1988, detailed counts of 1156/2102 and 1052/1957 fish with cataracts were made (5 5 % and 54’S). Whole catches from several bag nets were used to obtain these data. In 1989, fishing effort was much reduced at Coigach Salmon Fisheries, and 15124 fish with cataracts were counted, although more cataracts were noted in other catches. In 1989, counts were further made from bag nets at a second site at the East side of Scotland on the Moray Firth, with 25/404 (6.25%) fish having cataracts. Fish were transferred from net to boat at Moray Firth nets using conventional fishing techniques, and eyes 0 1990 Academic Press Limited
444
were examined at sea within minutes of killing. Some fish were examined live. The Moray Firth fishery at Macduff was chosen since it sampled fish from East Coast, and it presents useful contrast with the West coast Achiltibuie site. There are four fish farms within a llO-km radius of Macduff, compared with more than 60 within a 1 lo-km radius of Achiltibuie. In tagging studies between 1979 and 1981 involving one of us (P. J.F.), out of c. 800 fish tagged at Achiltibuie and released, none were subsequently recaptured at Macduff (Shearer, 1986). At Macduff, throughout several tagging studies, all tag recaptures, with one exception from a North coast tagging site, came from Moray Firth or East coast (Shearer, 1986). Note also that during the tagging work, no cataracts were noted in Achiltibuie fish, although the condition of each fish was carefully considered before tagging and release. It was not the intention in our paper to review cataractogens in fish, although dietary-related cataracts were certainly of interest. Dobson and Schuurman are misleading in their representation of examples. In a paragraph where ‘many dietary deficiencies causing cataract are not associated with any effect on growth rate or mortality ‘, they quote Richardson, Higgs and Reames (1986) as having described a cataractogenic diet for juvenile chinook salmon. In fact, this paper emphasizes the growth depression. Let us quote from the abstract regarding the cataractogenic diet, (C): ‘Cataracts were detected at day 126 in fish which were fed diet C between days 42 and 84. Fish growth was depressed severely by diet C’. In fact, Akiyama, Mori and Murai (1986) mainly report on a high incidence of scoliosis (spinal curvature) in fish fed a tryptophan-deficient diet, so it is misleading to imply no effects on growth. We have never observed spinal deformities in wild salmon with cataracts. Ultraviolet light certainly has been implicated in cataract formation. In a wild animal, high levels of glutathione in the lens would be expected to mop up free radicals. Dietary interference may reduce or enhance the protective influence of glutathione and also ofvitamins E and niacin (Ross et al., 1982, 1983). Wild salmon migrate in shoals close to the surface, where they utilize UV light for localizing the sun directly, or by means of the pattern of polarized light where cloud cover prevails. Directional swimming would lead to asymmetrical exposure to UV. It is not possible to explain the high incidence of cataract on the West coast compared to a low incidence on the East coast, or the sudden temporal occurrence on the West coast, in terms of UV-induced cataract alone. Exposure to UV light, in addition to cholinesterase inhibitor, may well increase cataract incidence, and might help explain the lack of reports of cholinesterase inhibitor-mediated cataract in laboratory studies with dichlorvos. Dobson and Schuurman further cite Allison (1962b), where lake trout reared as broodstock for
P J FRASER
ETAL.
4-6 yr showed 40-60s incidence of cataract, They identified inflammation in the choroid layer of the eye, leading to oedema which displaced the retina. No such inflammation of choroid layer, or displacement of the retina, has been found in wild salmon with cataracts. Dobson and Schuurman presume that the salmon examined for cataract would be ’ stressed by capture ’ and be ‘susceptible to mechanical injury ‘. They cite the work of Ubels and Edelhauser (1987), showing that abrasions of the cornea of fish caused cataracts. They state that abrasion is likely to occur in salmon caught in nets, and this could be unilateral. The fact that cataracts were not recorded prior to 1984 from the same nets seems to adequately control against possible capture-related artefacts. This is further borne out by the Moray Firth results in 1989, where catching conditions were broadly similar. Furthermore, in a 3 yr unpublished study (1979-1981), involving video monitoring and recording of around 12 hr per day of observation of the behaviour of salmon, in relation to one of the bag nets used for the samples of cataracted fish, no extensive contact with the net liable to lead to abrasion was observed (Muir and Fraser, unpubl. res.). On occasions, shoals of fish have been observed entering the net and have been caught and killed in a few minutes. Such fish show the same high incidence of cataract as those which have accumulated in the net for a longer period. If osmotic stress due to transitory crossings from sea to fresh water were a factor, as implied by the reference to Iwata et al. (1987), then appreciable numbers of cataracts would have been seen before 1984 and in Moray Firth catches. Furthermore, a temporal pattern to the incidence of cataract throughout the main migration time would be expected, corresponding to the pattern of cessation of feeding, development of gonad, and freshwater contact, which takes place throughout the summer in fish from Achiltibuie (Fraser, 1987: Fraser, unpubl. res.). This does not happen. It is not clear where a large population of wild salmon would encounter sublethal concentrations of copper, large variation in pH or thioacetamide. Indeed, such pollution might be considered less likely on the West coast than the East coast. To consider such causes, it would be reasonable to expect at least a demonstration of the existence of a pollutant where it would be encountered by the wild salmon, but Dobson and Schuurman do not do this. There is no evidence of extensive bacterial or viral infection in the wild salmon population. Given the extensive growth of the eye and lens from the smolt to grilse stages, in the 12 months of sea feeding, repaired damage to the lens would show as deep cataract, whereas many of the cataracts observed were anterior or posterior subcapsular. Small, C-shaped opacities deep in the lens, which were observed in quite large numbers of salmon, could have been caused at smolt stage, implying that the causal agent acts both on the smolt and the returning adult stages. There was no evidence of parasite in a seatrout
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smolt with cataract caught at Achiltibuie. There are no reports of large numbers of parasitised wild fish associated with rivers which carry stocks caught at Achiltibuie. Dobson and Schuurman describe a treatment regime involving enclosure of a cage with a tarpaulin and adding dichlorvos as Nuvan to give 1 ppm. In the Data sheet (V) for Aquagard, a second ‘Skirt technique ’ is described which involves twice the volume of Nuvan to be used for the same size of cage. In practice greater concentrations have been used by fish farms. Ross and Horsman (19 8 8) quote one farm operator as follows: ‘Up to 30 cages can be treated together in an hour. No tarpaulin is used. The Nuvan is poured in at one end of the cage unit [group of cages] and is allowed to flow through with the tide, topping up the concentration at each cage. About 300400 ml is used per cage [which are 9 in x 9 m or 12 m x 12 m]. Treatment occurs four or five times per summer ‘. They point out that this would involve approximately 10.5 1 of Nuvan being used in one treatment of a group of 30 cages. If mixing was not uniform, as is likely, then local concentrations in excess of 2 ppm Nuvan would be possible. The Aquagard data sheet (V) states up to three treatments at lo-20 day intervals may be needed, after which ‘the fish should be lice-free for considerable periods if all fish on the site have been simultaneously treated to prevent females from releasing eggs and starting a new infestation cycle’. It further states that ‘dichlorvos is dangerous to many aquatic organisms. The fastest, and therefore most important, detoxification mechanism in coastal waters is dilution which is increased by water movement, including the flushing effect in sea lochs. It is therefore important that under conditions of low water movement, the advice of the relevant water authority is sought before Aquagard is used’. It is misleading for Dobson and Schuurman to state that the treatment solution is ‘allowed out to the open sea ‘, since in many cases, it would have to be carried many miles to exit the sea loch. In Loch Broom, at Achiltibuie, coastal tidal stream speeds are in the order of 125-300 m hr-l. Streams are 50-100 m wide in the coastal zone containing bag nets (matching the length of the leader from shore to trap), and flow is laminar (data from Coigach Salmon Fisheries Ltd). A killing accumulation of Nuvan from a farm net would be expected to be outside a 25-m perimeter in 5-12 min. Dobson and Schuurman quote unpublished work which claims it is not possible to identify dichlorvos above 2 ppb outside this zone. This implies extremely rapid dispersion, or little dispersion with failure to find the tidally displaced Nuvan cloud. In shallow coastal water, dispersion is depth limited, and many observations of tidally borne debris suggest that, at least in coastal currents around salmon nets at Achiltibuie, very little dispersion takes place, other than bulk displacement with the water mass. Dobson and Schuurman question the use of I.34 x I(]-’ M dichlorvos as Nuvan as the starting
They state that this concentration concentration. would kill Atlantic salmon in 15 min. Interestingly, the concentration added to a cage if the Aquagard instructions are followed is a 10 x dilution of the quantity of Aquagard calculated to give the required 2 ppm Aquagard. This will be at an initial concentration of 50 g 1-l or 50000 ppm. Since mixing is mainly by fish movement and oxygen bubbles, and is reckoned to take 5 min, the fish will have considerable exposure to dichlorvos at concentrations above 1 ppm. It is not clear how much dichlorvos normally remains bound in larger droplets of emulsion, nor how droplet size would affect transfer of dichlorvos to organisms. In the size range of cells, dichlorvos concentrations around droplets are liable to exceed 30 ppm. Interaction with tissue will involve several phases, water, lipid, dichlorvos and the carrier in Nuvan. The lack of effect of Nuvan on juvenile stages of sea lice (Wooten et al., 1982) implies that mechanisms other than simple diffusion from aqueous phase may be taking place. It is certainly necessary to establish whether effects would be likely at lower concentrations. It can be seen from Table I that effects on membrane potential of wild salmon lenses are apparent at dichlorvos concentrations down to lo-’ M, confirming the likely sensitivity of the lens system. When a target tissue is accessible, the main effect of concentration of organophosphorus cholinesterase inhibitors is simply on the rate of inactivation of cholinesterase. Since real time effects could be measured by the electrophysiological techniques employed, a concentration was selected to give clear instantaneous effects. Incubation of trout lens for long term in vitro drug toxicity testing is well established (Hikida and Iwata, 1987). and the ratio of sodium to potassium ions is used as an indicator of lens opacillcation. Since the cornea will be a negligible barrier to a molecule as lipid soluble as dichlorvos, the only barrier to passage into the eye from the sea via the cornea will be the diffusional resistance of the stroma, and will be in the order of 1 mm hr-l. The isolated lens is hence not too unrealistic a model for the intact fish. Dobson and Schuurman question the realism of the in vitro lens since, in the intact fish, replenishment of the aqueous humour would be expected. This would initially aid in
TABLE I
The membrune potentials of 16 wild salmon lenses incubated in groups of four overnight at 10.7”C in artificial aqueous humour (AAH) and AAH with lo-‘, 1OW6and 1 Om4M dichlorvos as Nuvan. Concentration of dichlorvos
0
Membrane potential Negative mean mV 90.07 (1.16) (S.E.) mV
lo-?M
1o-6
M
1o-4
75.89
75.35
64.7
(5.70)
(5.44)
(5.23)
M
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P. J. FRASER
distributing dichlorvos throughout the eyeball, and help it reach the lens via aqueous phase. It would not be involved in possible lipid phase transfer. Once having had its effect, dichlorvos would, of course, reduce the replenishment rate of the aqueous humour, and reduce ocular pressure and fluid flow, which is why cholinesterase inhibitors are used to treat severe cases of glaucoma in humans, despite the side effect of cataract. Dobson and Schuurman do not deduce this obvious effect or anywhere acknowledge that cholinesterase inhibitors could have any effect on the eyeball-d lens, although such effects are well known (see Kaufman, Wiedman and Robinson, 1984). Cataracts exist in farmed salmon (G. H. Rae, pers. commun.). Photographs and verbal descriptions of these cataracts corresponded well to cataracts seen in wild salmon. A proper survey of the geographical distribution and yearly temporal distribution of cataracts in fish farms would be a massive undertaking requiring large resource. Furthermore, such a survey would only be of limited value without knowledge of the temporal pattern of cataract occurrence throughout the Nuvan-using decade. Measures to alleviate dietary deficiencies in the presence of Nuvan treatment might very well have alleviated damage by Nuvan. One reaction to cataract has been to increase levels of vitamins in diets. As Dobson and Schuurman point out, certain vitamins are known to not only prevent cataract formation, but to suppress cataract incidence in the presence of known causal agents. Dobson and Schuurman state
ET AL
that ‘susceptibility to LJV light is ameliorated in diets rich in niacin ’ (Allison, 1960, 1962a). More recently it has been shown that glutathione and vitamin E can prevent glucose (diabetic) cataract in vivo and in vitro (see Ross et al., 1982, 1983). In our opinion, the farmed situation is not an adequate control for the wild fish population, since diets have been tuned to eliminate cataracts and other problems in an environment with continual dichlorvos exposure. Cataracts occur in farmed fish escapeescaught at Achiltibuie, so if Dobson and Schuurman are correct and there are no cataracts in farmed salmon, we have a situation where the farmed fish in cageswould seem to be protected from a causal agent which seemsto be local to the farmed sites. Wild salmon are unique in concentrating in coastal currents for a period of several weeks. No other population of fish could be so threatened with extinction by fishermen operating with fixed trap nets covering SO-100 m from the shore line, that leaders have to be removed for statutory periods on a weekly basis.A cloud of Nuvan releasedfrom a fish farm will simply be carried in the coastal currents which carry the wild salmon. As they move up and down the coast, moving 20-80 km day-l, the wild salmon will certainly be expected to encounter the Nuvan clouds. Hence, considering all the possiblecausesof cataract including those suggestedby Dobson and Schuurman, we would still affirm that Nuvan is the most likely cause of cataract observed in wild salmon at Achiltibuie.
Zoology Department, Aberdeen University, Aberdeen AL39 2TN, U.K.
PETER J. FRASER
School of Biological Sciences, University of East Anglia, University Plain, Norwich NR4 7TJ, U.K.
GEORGE DUNCAN JULIE TOMLINSON
(Received 18 December 1989 and accepted in revised form 27 December 1989) cataract in Atlantic Salmon(Salmo salar). Exp. Eye Res. References Akiyama, T., Mori, K. and Murai, T. (1986). EZffectsof temperatureon the incidenceof scoiiosisand cataract in chum salmonfry causedby tryptophan deficiency. Bull. Jpn Sot. Sci. Fish 52, 2039.
Allison, L. N. (1960). Sunburning fingerling lake trout with ultraviolet light and the effect of a niacin fortified diet. Prog. Fish. Cult. 22. 114-16.
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Brandal, P. 0. and Egidius,E. (1977). Preliminary report on oral treatment against salmon lice, Lepeophtheirus salmonis, with Neguvon. Aquaculture 10, 177-8. Brewster,D. (1825). On the formation of singlemicroscopes from the lensesof fishes.Edinburgh 1. Sci. II, 98-100. Dobson,P. and Schuurman.H. J. (1990). Possiblecausesof
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Cholinergics.Pharmacologyof the Eye. HandbookExp. Pharmacol.69, 149-9 1, Michon, J. and Kinoshita,J. H. (1967). Cholinesterase in the lens.Arch. Ophthalmol.77, 804-8. Philipson,B., Kaufman, P. L., Fagerholm,P., Axelsson, U. and Barany, E.H. (1979). Echothiophatecataracts in monkeys: Electron microscopyand microradiography. Arch. Ophthabnol.97, 340-6. Richardson,N. L., Higgs, D. A. and Beames,R. M. (1986). The susceptibility of juvenile chinook salmon (Oncorhynchustschawytscha)to cataract formation in relation to dietary changesin early life. Aquaculture52, 237-43.
Ross, W. M., Creighton, M. O., Stewart-DeHaan, P. J., Sanwal. M., Hirst, M. and Trevithick. J. R. (1982). Modelling cortical cataractogenesis : 3. In vivo effectsof vitamin E on cataractogenesisin diabetic rats, Can.J. Ophthalmol.17, 61-6. Ross,W. M., Creighton, M. 0.. Trevithick, J. R., Stewart DeHaan,P. J. and Sanwal,M. (1983). Modellingcortical cataractogenesis : 6. Induction by glucosein vitro or in diabetic rats: prevention and reversal by glutathione. Exp. EyeRes.37, 559-73.
Ross,A. and Horsman,P. V. (1988). TheUseojNuvan 500 ECin theSalmonFarmingIndustry.Marine Conservation Society: Ross-on-Wye. Salte,R., Syvertsen,C.. Kjoennoey,M. and Frode.F. (1987). Fatal acetylcholinesterase inhibition in salmonidssubject to a routine organophosphate treatment. AquacuZture 61, 173-V. Shearer,W. M. (1986). The exploitation of Atlantic Salmon in Scottishhomewater fisheriesin 1952-1983. In The Status of the Atlantic Salmon in Scotland ITE SymposiumNo. 15 (EdsJenkins.D. and Shearer,W. M.). Pp. 3749. ITE. Ubels,J. L. and Edelhauser,H. F. (1987) Etrectsof cornea1 abrasion on cornea1transparency, aqueous humor composition, and lens of fish. Prog. Fish. Cult. 49, 219-24.
Wooten. R.. Smith, J. W. and Needham, E. A. (1982). Aspects of the biology of the parasitic copepods Lepeophtheirus salmonisand Caliguselongatuson farmed salmonids,and their treatment. Proc. R. Sot. Edin.81B. 185-97.
