The use of imposex in Nucella lapillus to assess tributyltin pollution in Carlingford Lough

Volume:6

Issue:2

Year: 2007

Mr Paul Mallon1 BSc (Hons) MCIEH and Dr Naran Manga2

1 Environmental Health Officer, Newcastle-under-Lyme Borough Council, Staffordshire

2 Lecturer in Environmental Health, University of Ulster, Northern Ireland

Correspondence: Mr Paul Mallon, Food and Safety Section, Environmental Health Department, Newcastle-under-Lyme Borough

Council, Civic Offices, Merrial Street, Newcastle-under-Lyme, Staffordshire ST5 2AG. Telephone: 01782 742552. E-mail:

Paul.Mallon@newcastle-staffs.gov.uk

Abstract

The condition known as imposex in the female of the marine mollusc Nucella Lapillus is used widely as a biological indicator of tributyltin (TBT) contamination in the marine environment. TBT, an organic compound containing the metal tin, is an effective biocide used in antifouling paints applied to the underwater hull of large boats. It is banned from use on vessels less than 25 metres in length.

This paper provides a review of the literature and reports on a field study which focuses on Carlingford Lough, a cross-border water body, bounded by Counties Down and Armagh in Northern Ireland and County Louth in the Republic of Ireland. It is used by both smaller pleasure craft and large commercial ships.

Two hundred samples were taken from four sites (n = 50 at each site) and the female population of Nucella Lapillus examined. 100% of the female specimens showed the imposex phenomenon indicating various levels of TBT contamination. The intensity of imposex was higher on the Southern shore of the lough. The difference in average imposex values recorded between the Northern and Southern shores suggests that the Southern shore is more polluted with TBT. This may be partially explained by the way in which the tide flushes in the lough where material from the Northern shore may be lifted and deposited on the Southern shore.

This study shows that despite the partial ban on the use of TBT in antifouling coatings on ships the marine environment is still affected some 20 years on.

Carlingford Lough is used to cultivate oysters and there are mussel beds at various points along both the Northern and Southern shores. Shellfish which eat food contaminated with TBT, will result in TBT accumulation in the organism, and thus it will bio-accumulate in the food chain causing a potential food safety problem.

Key words: Carlingford Lough; Environmental health; Nucella Lapillus; Tributyltin (TBT); Imposex; Vas Deferens Sequence Index (VDSI).

Introduction

Tributyltin (TBT) is an organic compound containing the metal tin. It is used as a broad-spectrum killer of algae, fungi, insects and mites, and since the seventies TBT has been used as an effective biocide in antifouling paints. It is now the most commonly used antifouling agent used in paint for the underwater hull of large boats (Berge and Walday, 1999). Fouling is the unwanted growth of biological material e.g. barnacles, algae or molluscs on the water-immersed surface of the vessel. Alziue (1998) stated that fouling leads to problems such as loss of ship speed, excess fuel consumption, spreading of corrosion pits and increased weight of float structures.

Antifouling paint regulation is a genuine public policy concern because it increases the cost of maritime commercial shipping and thus influences the price that the public pays for vessel shipped common goods.

TBT is one of the most toxic substances that have been introduced to natural waters (Nicholson and Evans, 1997; Stewart and de Mora, 1990). During the last decade, organic tin compounds have received much attention because of their strong effects on marine organisms at low concentrations. Unintended environmental effects from TBT in antifouling paints were first found in Arcon Bay in France in the late 1970s in the Pacific oyster (Crassostrea gigas) (Nicholson and Evans, 1997). Since then, effects of organotin compounds have been reported for a variety of other marine species (fish, gastropods, crustaceans,  echinoderms, microalgae) at concentrations thatmay be found in anthropogenically influenced areas (Walday et al., 1997).

The effects are particularly evident on neogastropod snails, where females develop male characteristics (penis and vas deferens), an irreversible phenomenon termed imposex (Gibbs and Bryan 1986; 1987, ten Hallers- Tjabbes et al., 1994; Skarphédinsdóttir et al., 1996; Walday et al., 1997; Berge et al., 1997; Svavarsson, 2000; Svavarsson et al., 2001). Several countries have banned the use of TBT for small boats, with France being the first to ban its use in 1985 followed closely by the UK in 1987, but international regulations still allow restricted use.

Tin organic compounds mainly result in pollution of water since the TBT compounds very easily bind themselves to particles and organic material in water thus accumulating in the sediments. TBT breaks down quickly in water with a half-life of only a few days, but can remain for a long time in sediments, especially in cold climates (Stewart and de Mora, 1990). Contaminated sediments are therefore potential environmental reservoirs for TBT, andmay continue to be a source long after the industrial use of TBT has been curtailed (AMAP, 1997 and 2002). Continued use of TBT on large ships is probably the main source for new TBT to aquatic environments.

Degradation of TBT 

It is well established that TBT degrades by a stepwise debutylation pathway to less toxic breakdown products dibutyl-, monobutyl- and finally inorganic tin. Laboratory studies have shown that wide varieties of agents are capable of cleaving the tin-carbon bond. In aquatic environments, the most relevant processes are likely to be photochemical cleavage and biological cleavage by micro organisms. There are indications that TBT has a low persistence in the water column. However, as TBT shows a tendency to accumulate in sediments, TBT degradation processes in sediments are more likely to control the overall persistence of TBT in the environment (Stewart and de Mora, 1990).

TBT has a short residence time in natural waters, with a half-life in the range of several days to weeks as mentioned above. The rate of disappearance of TBT from the water column is the sum of physical removal processes, and chemical and biological degradation, as well as simple dilution due to water flow.

Biological processes are likely to be the most important mechanism for the decomposition of TBT in the marine environment. Photolysis by sunlight is not a major contributor to environmental TBT degradation (Stewart and de Mora, 1990). Temperature is another factor that affects the degradation rate in waters; TBT degradation proceeds more slowly at lower temperatures. It has been reported that TBT half-life at 5°C is around 60 days whereas at 20°C it is approximately 6 days. Stewart and de Mora (1990) have suggested that colder temperatures inhibit the growth of TBT-degradingmicroorganisms. TBT shows a tendency for both bioconcentration and sediment accumulation. Hence, the short residence time of TBT in the water column alone is unlikely to be an adequate indication of the environmental hazard posed by TBT (Stewart and de Mora, 1990).

In natural watercourses, TBT adheres to particles, then settles and accumulates in bottom sediments. It is effectively bound to organic and inorganic solids. Binding also occurs with organic substances dissolved in water.

As a rule, TBT is not present in a dissolved state in watercourses. Because of its binding tendency and limited dissolution, TBT and its degradation products (dibutyltin, DBT, and monobutyltin, MBT), in practice spread in waterways only when bound to solids.

Toxicity of TBT

TBT is toxic to a broad range of organisms and this is also the basis for its success as an antifouling agent. It is harmful especially to primitive maritime organisms, causing for example, disturbances in reproduction and growth. TBT is more harmful to younger than to older organisms.

The antifouling activity of all types of TBT coatings is based on the leaching of TBT to the water and the toxic/repulsive effect on foulers.

The problem with TBT is that the threshold for harmful effects (hormone disrupter) is extremely low in certain molluscs. Chronic effects in oysters, mussels and crustaceans are observed at exposure levels of less than 1 mg TBT/l while the most sensitive species (dogwhelk snails, e.g. Nucella Lapillus and Buccinum undatum) show sub-lethal effects at concentrations of only a few ng TBT/l or less (AMAP, 1997; Svavarsson, 2000; Jakobsen and Asmund, 2000). A “no-effect” concentration of TBT in water has not yet been quantified.

At these extremely low concentrations, dogwhelk snail’s exhibit imposex that affects reproduction, and this phenomenon will be further discussed later on. Effects are observed in non-target organisms in non-target habitats worldwide (Svavarsson and Skarphédinsdóttir, 1995; Berge and Walday, 1999). Although effects have been observed most frequently along shores adjacent to obvious sources such as marinas or harbours and has been associated with both pleasure boats and commercial shipping, the problem has been defined as long-term and global (Svavarsson and Skarphédinsdóttir, 1995; AMAP, 1997 and 2002). TBT concentrations in natural waters are subject to high temporal and spatial variability, especially when tidal flushing is present (Stewart and de Mora, 1990).

TBT and imposex

The development of male primary sexual characteristics in female gastropods is known as imposex, also called pseudohermaphroditism. The phenomenon is caused by exposure to organotin compounds and results in accumulation of male hormone (testosterone) in the gastropods. TBT is fat-soluble, and therefore accumulates in organisms. Severe stages of imposex at higher TBT concentrations can lead to female sterilization and death (Mensink et al., 1996) and it is believed to account for the extinction and local disappearance of the dogwhelk in severely contaminated areas (Skarphédinsdóttir et al., 1995; Mensink et al., 1996). Imposex has most extensively been studied in the dogwhelk Nucella Lapillus, but effects of TBT have been observed in 70-100 species of gastropods worldwide (Svavarsson and Skarphédinsdóttir, 1995; Berge et al., 1997).

Since the introduction of restrictions on the use of organotin-based antifouling paints around the world, there has been much effort and need to assess the effectiveness of these restrictions (Evans et al., 1995). As there are many difficulties in chemically measuring organotins in the environment (Cleary, 1991; Foale, 1993), changes in imposex levels have been the main assessment tool used (Evans et al., 1995).

Sensitivity to changes in nutrient levels have been described by Gibbs et al. (1999) who reported a massive kill of N. Lapillus in Bude Bay, North Cornwall, and suggested that the mass mortalities may have been caused by eutrophication and summer algal blooms linked to a new sewage outfall in the area.

Occurrence and levels of TBT in the environment 

Despite restrictions on the use of TBT in many countries in the late 1980s, occurrence of imposex is still extensive in most oceans and has increased in some areas (Svavarsson et al., 2001). The effects of organotins are mostly seen in areas with extensive shipping activity (harbours, shipyards, shipping lanes) (Svavarsson et al., 2001). Due to often slow degradation of TBT and its derivatives (dibutyltin (DBT) and monobutyltin (MBT)), these are accumulated in the sediments at the harbour and may be present for a long time.

It has been suggested that the high levels of imposex in dogwhelks aroundmarine European shipping and fishing ports are unlikely to decline until TBT is banned on all vessels (Minchin et al., 1995). Even then, there is the possibility of a continued contamination as TBT is persistent in sediments (Bryan & Gibbs, 1991; Hawkins et al., 1994).

It has been shown that TBT levels in sediments are closely related to the organic content because TBT adsorbs to organic material (Jacobsen and Asmund, 2000), but there seems not to be an obvious single factor responsible for the varying concentrations of TBT in the different harbour sediments. Factors like distance to shipyard and quays, shipping traffic intensities, water renewal, bottom current regime and sedimentation are together responsible for the highly variable concentrations (Berge et al., 1997; Green et al., 2001).

The first detailed survey of imposex was of the dogwhelk N. Lapillus in southwest England in the mid- 1980s (Bryan et. al. 1986). The study found that the imposex condition and TBT contamination was severe in areas of high boating activity. In themost severe cases, there was female sterility and premature death. There was an absence of juveniles in some populations and in the worst affected areas, the species became locally extinct.

Smith, (1996) concluded that following restrictions in New Zealand, areas subject only to pleasure craft displayed a decline in the frequency of imposex. However, in harbours that were subject to both pleasure craft and commercial vessels, imposex frequency did not decrease, although the severity of imposex did.

A study of imposex in Thais Orbita (Neogastropoda) along the New South Wales (NSW) coast, Australia, found imposex was still widespread 10 years after a partial ban on TBT based antifouling paints (Gibson & Wilson, 2003). Transplant experiments carried out showed that imposex was induced in T. Orbita over a 9 week period in Sydney Harbour. A comparison of data collected shortly after the introduction of restrictions on TBT revealed a general decline in imposex frequency occurring at open sites, with the exception of one site located within harbour/bay areas, which did not display a decline in the severity of imposex.

TBT not only affects N. Lapillus, it is also known to affect other organisms (Svavarsson and Skarphédinsdóttir, 1995; Berge et al., 1997). As imposex is a phenomenon in the dogwhelk, intersex is a similar phenomenon known to affect the periwinkle (Littorina Littorea). Bauer et al. (1993) was the first to carry out work on intersex in Littorina Littorea and since this work, other studies have been conducted by Oehlmann et al. (1994) and Bauer et al. (1995).

Intersex in the periwinkle has been used in several surveys in Ireland, e.g. Cork Harbour, Killybegs Harbour, Richies Bay and Ballagan Shore by Minchin, et al., 1996, 1997 and also in Germany (Oehlmann et al., 1996; Bauer et al., 1997) and is now being considered, alongside the imposex response in Nucella Lapillus, for the biological TBT effect monitoring in coastal waters. Studies showed that the partial ban was less effective where the use of TBT based anti-foulants has continued on vessels over 25 metres in length.

Nogueira et al. (2003) conducted a study on the levels of TBT in sediments from selected stations located in the upper Tagus River from the Tagus Estuary Nature Reserve, Portugal. He found that the persistent TBT contamination observed in the sediments from this important European ecosystem was primarily related to shipping activities as well as the naval industrial complexes located downstream.

Aim of study

The overall aim of this study was to assess the relative effects of TBT pollution on N. Lapillus on the Northern and Southern shores of Carlingford Lough, Northern Ireland. This was to be assessed by measurement of imposex levels in N. Lapillus using the Vas Deferens Sequence Index (VDSI). The study aims to show that the site closest to the port will show a higher degree of imposex than that of sites further away from the port.

Methods

Approximately 200 specimens (n = 50 at each site) of N. Lapillus were collected by hand from the identified sites along the North & South shores, between February and March 2006 (Figure 1.0). Sampling was carried out whilst the tide was at low shore due to the Nucella binding to rocks further down the shoreline. They were placed in a container containing sea water and sealed for transit.

It should be noted that whilst sampling was undertaken, the specimens collected from the Southern shore were younger than those collected from the Northern shore. This assumption was made on observation of the shell size of the collected specimens. Generally for Nucella, the smaller the shell size the younger the organism.

Sites were located at varying distances from areas of boating traffic (harbours & marinas etc.). Following collection, specimens were transported to the laboratory for immediate analysis or stored in a refrigerator at approximately 4°C until analysis took place.

Site description

Carlingford Lough is the most southern of the five sea Loughs around the east coastline of Northern Ireland. It is a cross-border water body, bounded by County Down and County Armagh in Northern Ireland and by County Louth in the Republic of Ireland. It is a narrow sea Lough surrounded by mountains. The northern shore lies in Northern Ireland and includes the most significant mudflats in the Lough and an area of saltmarsh. These provide important feeding areas for wintering the lightbellied Brent goose (Branta bernicla hrota) of the Canada/Ireland population. At the mouth of the lough are several small rock and shingle Islands which are of importance to breeding terns, which feed in the shallow waters of the lough.

Carlingford Lough supports a wide variety of aquaculture and fishing interests, including cultivation of oysters, mussels and clams as well as lobster and crab potting. To date, studies focusing on Carlingford Lough have largely centered around the use of the Lough as a major site for mariculture, and on sewage disposal activities in the lough.

The position chosen for the study is an ideal site as it rests on a shipping corridor to both the North and South of the island of Ireland. Extensive works were undertaken to the lough between the months of August and October 2005. This included dredging of the lough and sediment removal at the Warrenpoint Marina area. This INTERREG funded project was aimed at improving the water depths in the lough and expanding docking facilities at theWarrenpoint Port. With this information in mind, it is expected that imposex prevalence will be quite high.

Dissection of N. Lapillus

As both male and imposexed female dogwhelks have a penis the removal of the outer shell is required to ascertain the sex of the animal. The technique adopted involved crushing the shell with a small vice secured to a bench and removing the tissue from within. The tissue was then placed under a dissection microscope in a Petri dish of seawater to prevent desiccation before sexing took place.

Recognition of the sexes

The dogwhelks were sexed solely on the presence of a spermingesting gland,which is only found in females. This brown/red band is situated at the posterior of female dogwhelks, immediately behind the capsule gland (Fig. 2). The degree of imposex is classified by either the Relative Penis Size Index (RPSI) or the Vas Deferens Sequence Index (VDSI). VDSI recognises the development of a vas deferens (sperm duct) in a female in 7 stages Figure 3.0 (Gibbs et al., 1987); stage 0= normal individuals, stage  1= proximal section of vas deferens is formed, stage 2= initiation of penis development and further development of vas deferens, stage 3= formation of a small penis and development of the distal section of the vas deferens, stage 4= involves mainly fusion of the vas deferens, stage 5= overgrowth of vas deferens on the genital papilla and hence female sterilely, and stage 6= aborted egg capsules can be seen in the capsule gland.

Figure 2.0 After Gibbs et al. (1987). Nucella Lapillus. External features of mature male (A) and mature female (B) after removal of the shell. Abbreviations: ag, albumen gland; cg, capsule gland; cm, columella muscle; dg, digestive gland; f, foot; hg, hypobranchial gland; k, kidney; me, mantle edge; o, operculum; ov, ovary; p, penis; pr, prostate; rg, rectal gland; rt, right tentacle; sg, sperm-ingesting gland; t, testis.

Individual snails with a VDSI of 5 or 6 are prevented from releasing egg capsules from their genital papilla and are sterile. Bennett, (2004) concluded that the VDSI is a more consistent method of measuring imposex in N. Lapillus than RSPI.

Figure 3.0 Nucella lapillus. Stages in the development of imposex based on Vas Deferens Sequence (VDSI). Abbreviations: a, anus; b, blister; gp, genital papilla; n, nodule; p,  penis; v, vulva; vd, vas deferens. (After Gibbs and Bryan, 1986).

 Table 1.0 Table showing the observed stages of imposex demonstrated by the female dogwhelks

It does not depend on a penis but it measures the development of a vas deferens, which can indicate reproductive capability.

Results

Table 1.0 shows the varying degree of imposex stages observed at each site. Results obtained show that at each station, of the 50 specimens collected, 100% of the female dogwhelk population demonstrated the imposex phenomenon at varying stages.

The VDSI of individual Nucella ranged from 1 to 5. This shows that there were specimens, which were incapable of reproducing.

The average VDSI values for adults at Warrenpoint sites 1 and 2 were 4.00 and 2.46 respectively while at Omeath the values were 4.71 and 3.42. The average VDSI values for Warrenpoint and Omeath overall were 3.23 and 4.07 respectively. At each shore, site 1 (that closest to the harbour area), showed a higher average VDSI value than that of site 2 (further away from the harbour area).

Discussion

The fact that Nucella Lapillus demonstrated the imposex phenomenon means that TBT is present in Carlingford Lough. This would support other studies that TBT is still around some years after the partial banning of its use (e.g. Smith, 1996 and Walday, et al., 1997). Work carried out by Minchin et al. (1995) found that imposex in N. Lapillus was unlikely to decline until TBT was banned on all vessels. Vessels >25m in length can still use TBT based paints and since Warrenpoint is a busy port, large   vessels use it. This may contribute to TBT levels around the Lough and subsequently imposex in Nucella.

The difference in average value of VDSI between Warrenpoint andOmeath 3.23 and 4.07 respectively would suggest that the Omeath site was more polluted with TBT. Thismay be partially explained by theway inwhich the tide flushes in the lough. A study of the nutrient inputs and trophic status of Carlingford Lough by Taylor et al., (1999), which found that the lough was not eutrophic, also suggested that the flushing pattern of the Lough is one which the tide enters the lough on the Northern shore and leaves the Lough on the Southern Shore. This is illustrated on Figure 4.0. The large arrow shows the current pattern generated by the flowpattern of the tide. This model would suggest thatmaterial fromthe Northern shore is lifted and deposited on the Southern shore. This reason may explain the higher VDSI value at the Omeath site. During dredging of the lough and sediment removal at the Warrenpoint marina, elevated levels of TBT from agitated sediment would have been carried round with the tide to the Southern shore. This may also support the observation made on sampling that the numbers of Nucella were extremely low at Omeath compared to those observed at Warrenpoint. Another important observation made was that no signs of breeding were present at Omeath whilst egg capsules were observed atWarrenpoint.

It may be hypothesised that the population of N. Lapillus at Omeath was wiped out or at least severely declined as a consequence of the works carried out on the Lough. Bryan et al. (1986) suggested that the percentage of females in a locality falls with increasing degree of imposex, which in turn puts additional pressure on the population. As noted in the method section the Omeath site specimens were younger and TBT is more harmful to the young. The high VDSI value at Omeath site 1 would support this statement as the high degree of imposex may account for the decline in numbers of Nucella due to increased pressure placed on breeding females.

Alzieu, (1991) and Dyrynda, (1992) blamed the near collapse of oyster farming in parts of western France and southern England during the 1980s on TBT in the aquatic environment. The first direct evidence of TBT contamination in Irish waters was detected byMinchin & Duggan, (1986). They discovered in the Owenboy Estuary, Cork that the pacific oyster (Crassostrea gigas) were found to have distorted shells and this was linked to TBT contamination.

The TBT contamination is greater in the sites nearer to most obvious sources. However, this differs slightly from the findings of Bryan et al. (1986) who found dogwhelks with high RPSI from coastal areas in the south of England (e.g. Plymouth) which indicated that levels of imposex were consistently high and remained elevated for distances of 5-10 kilometres along adjacent open coastlines.

As imposex is associated with the use of TBT in antifouling paints and, since TBT paints were banned in vessels less than 25min length in 1987, a decrease in the level of imposex would be expected in each subsequent year. Reports of imposex values (from UK and Ireland) for N. Lapillus following TBT restrictions have indicated that whelk populations have generally recovered from imposex, with only major ports remaining as imposex “hot spots” (Evans et al. 1991, Evans et al. 1996 & Miller et al. 1999).

As the samples collected from all sites but one had an average VDSI of less than 4, they could be assumed to be reproductive. Site 1 at Omeath showed an  average VDSI of 4.71 which is closer to 5 and therefore gives cause for concern. The higher VDSI value would suggest that the population were perhaps not fully infertile, but nevertheless the elevated TBT concentrations may have had an impact on the reproductive capacity of the dogwhelks. This shows that there are elevated levels of TBT around this site. This may be explained again by the way, in which the tide flushes the lough as discussed earlier.

As already stated both these sites are close to a  busy commercial port, which is used on a daily basis by a number of large vessels. TBT into the marine environment might therefore be fairly constant. The few pleasure craft launched locally in the spring and summer would have little effect at the sites.

It can be assumed from these findings that TBT in the water column is not the only factor influencing imposex at each site. The food source (e.g. barnacles andmussels) at each site can alter the uptake of TBT into the dogwhelk. Different food sources will accumulate different levels of heavy metals.

The study also suggests that both dumping and dredging, or the effects of tidal transport of re-suspended sediments, are likely the main sources for the TBT contamination observed in the upper estuary. Although the TBT contents found in the sediments studied are low relative to concentrations reported from other parts of the world, they appeared to represent a potential risk for some resident marine populations.

A review of the persistence of TBT in aquatic organisms by Maguire (2000) following the introduction of Canada’s Toxic Substances Management Policy showed that due to the long persistence of TBT in sediments there may be a “legacy problem” in sediments in some locations of Canada for perhaps 20 to 30 years after a total ban. This may help to explain why TBT is still impacting marine organisms in Carlingford Lough some 20 years after the partial ban.

Although effects of TBT on humans are not clear, several incidents of human exposure to the biocide have been reported. Underwear treated with TBT has caused severe skin irritation to its wearers. Shipyard workers exposed to TBT dust and vapours, while repairing a submarine, developed breathing problems, skin irritation, headaches, colds, flu, fatigue, dizziness, and stomach aches (US EPA, 1985). TBT exposure can also irritate the eyes and mucous membranes and prolonged exposure may cause liver and kidney damage.

Carlingford Lough is used to cultivate oysters and there are mussel beds at various points along both the Northern and Southern shores. Fish and shellfish which eat food that may be contaminated with TBT, will result in TBT accumulation in the organism, and thus it will bioaccumulate in the food chain (Kannan & Falandysz, 1997) causing a potential food safety problem.

A tolerable daily intake (TDI) of 15μg of TBT per person per day for a 60kg person has been set out, however there is no recognised TDI for total butyltins or other organotins. Concentrations of butyltin residues have been determined in muscle tissue of fish collected at local markets in Asian and Oceanian countries. This study found that the intake of butyltins by humans via consumption of fish in these countries was considerably less than that of the tolerable daily intake amount. Although this may suggest that there may be no need for concern about eating fish contaminated with TBT or other organotins, it must be stressed that as mentioned earlier in this study, TBT bioaccumultes in organisms and therefore there is no way of knowing if this accumulation is having an effect on public health in the long term.

Conclusions

• All of the female samples of Nucella Lapillus taken from Carlingford Lough showed evidence of the imposex phenomenon indicating TBT contamination.
• This supports the conclusions in other studies that TBT is still around several years after the partial banning of its use in anti-fouling paints for smaller vessels.
• The intensity (i.e. frequency and severity) of imposex was generally higher on the Southern shore of the lough. The difference in average values of VDSI recorded Northern shore (Warrenpoint = 3.23) and the Southern Shore (Omeath = 4.07) suggests that the Omeath site is more polluted with TBT. This may be partially explained by the way in which the tide flushes in the lough where material from the Northern shore may be lifted and deposited on the Southern shore.
• There were no signs of breeding within the Nucella Lapillus population at the Omeath sites whilst egg capsules were observed at the Warrenpoint sites indicating that breeding was taking place.
• TBT is still a widespread problem and is posing a threat to sensitive species, at least in coastal areas where shipping traffic intensities are high and in the vicinity of harbours.
• Shellfish and fish which eat food contaminated with TBT will result in TBT accumulation in the organism, and thus it will bio-accumulate in the food chain causing a potential food safety problem.
• A significant reduction in the overall occurrence and unintended effects of organotin compounds may only be achieved by extending regulations on the use of such compounds in anti-fouling paints to include a ban also on their use on large vessels in the United Kingdom and the Republic of Ireland.

Acknowledgments

The authors would like to thank Dr Sam Irwin, School of Environmental Sciences, University of Ulster, for his expert knowledge, support and use of his research laboratory and Dr Stephanie Bennett, Environment and Heritage Service (Northern Ireland) for her comments on paper content.

References

Alzieu, C., (1998). Tributyltin: case study of a chronic contaminant in the coastal environment, Ocean and Coastal Management Vol. 40, pp. 23–26.

Alzieu, C., (1991). Environmental Problems caused by TBT in France: assessment, regulations, prospects. Marine Environmental Research 32, 7-17.

AMAP, (2002). Arctic Pollution 2002. Arctic Monitoring and Assessment Programme, Oslo, pp. 1-111.

AMAP, (1997). Arctic Pollution Issues; a State of the Arctic Environment Report. Arctic Monitoring and Assessment Programme, Oslo, pp. 1-171.

Bauer, B., Brumm-Scholz, M., Deutsch, U., Fioroni, P., Ide, I., Liebe, S., Oehlmann, J., Stroben E. & Watermann, B., (1993). Basisstudie zur Erfassung pathologischer Effekte von Unterwasseranstrichen bei Schnecken der niedersächsischen Nordseeküste. Final report for ‘Aktion Seeklar’ e.V. (Hamburg) and ‘WWF Germany’ (Frankfurt/M.), Ahrensburg andMünster, pp.105.

Bauer, B., Fioroni, P., Ide I., Liebe, S., Oehlmann, J., Stroben E. & Watermann, B. (1995). TBT effects on the female genital system of Littorina littorea: a possible indicator of tributyltin pollution. Hydrobiologia 309: 15–27.

Bauer, B., Fioroni, P., Schulte-Oehlmann, U., Oehlmann J. & Kalbfus, W. (1997). the use of Littorina littorea for tributyltin (TBT) effect monitoring – results from the German TBT survey 1994/95 and laboratory experiments. Environmental Pollution. 96: 299–309.

Bennett, S.C., (2004). Imposex in the marine mollusc Nucella Lapillus and its relationship with the digenean parasite Parorchis acanthus. PhD Thesis University of Ulster, (Jordanstown Campus).

Berge, J.A and Walday, M. (1999). Alternatives to the use of TBT as an antifouling agent on the hull of ships with special reference to methods not involving leaching of toxic compounds to the water. Report No. O-98149. Norwegian Institute for Water Research, pp. 1-34.

Berge, J.A., Berglind, L., Brevik, E.M., Følsvik, N., Green, N., Knutzen, J., Konieczny, R. and Walday, M. (1997). Levels and environmental effects of TBT in marine organisms and sediment from the Norwegian coast. A summary Report. Norwegian State Pollution Monitoring Program Report No 693/97. Norwegian Institute for Water Research, pp. 1-36.

Bryan, G.W., Gibbs, P. E., Hummerstone, L. G., & Burt, G. R. (1986). The decline of the gastropod Nucella Lapillus around south-west England: evidence for the effect of tributyltin from antifouling paints. Journal of the Marine Biological Association UK, 66, 611–640.

Bryan, G.W., & Gibbs, P.E., (1991). Impact of low concentrations of tributyltin (TBT) on marine organisms: a review. In: Metal ecotoxicology: concepts and applications, (ed. M.C. Newman & A.W. McIntosh), pp. 323-361. Boston: Lewis Publishers Inc.

Cleary, J.J. (1991). Organotin in the marine surface microlayer and sub-surface waters of south west England: relation to toxicity thresholds and the UK environmental quality standard. Marine Environmental Research, 32, 97-101.

Dyrynda, E.A. (1992). Incidence of abnormal shell thickening in the pacific oyster Crassostrea gigas in Poole Harbour (UK), subsequent to the 1987 restrictions. Marine Pollution Bulletin 24, 156-163.

Evans, S. M., Hutton, A., Kendall, M. A., & Samosir, A. M. (1991). Recovery in populations of dogwhelks Nucella Lapillus (L.) suffering from imposex. Marine Pollution Bulletin, 22, 331–333.

Evans, S. M., Leksono, T., & McKinnell, P. D, (1995). Tributyltin pollution: a diminishing problem following legislation limiting the use of TBT-based antifouling paints. Marine Pollution Bulletin, 30, 14-21.

Foale, S. (1993). An evaluation of the potential of gastropod imposex as a bioindicator of tributyltin pollution in Port Phillip Bay, Victoria. Marine Pollution Bulletin, 26, 546–552.

Gibbs, P. E., & Bryan, G.W. (1986). Reproductive failure in populations of the dog-whelk, Nucella Lapillus, caused by imposex induced by tributyltin from antifouling paints. Journal of the Marine Biological Association UK, 66, 767–777.

Gibbs, P.E., Bryan, G.W., Pascoe, P.L. & Burt, G.R., (1987). ‘The use of the dog-whelk, Nucella Lapillus, as an indicator of tributyltin (TBT) contamination’, Journal of the Marine Biological Association of the United Kingdom, vol. 67, pp. 507–523.

Gibbs, P.E., Green, J.C. & Pascoe, P.C., (1999). A massive summer kill of the dog-whelk, Nucella Lapillus, on the north Cornwall coast in 1995: freak or forerunner? Journal of the Marine Biological Association, UK. 79, 103-109.

Gibson, C.P. and Wilson, S.P., (2003). Imposex Still evident in eastern Australia 10 years after tributyltin restrictions. Marine Environmental Research. 55. 101- 112.

Green, N.W., Helland, A., Hylland, K., Knutzen, J. and Walday, M. (2001): Joint Assessment and Monitoring Programme (JAMP). Overvåkning av miljøgifter i marine sedimenter og organismer 1981-1999. Statlig program for forurensningsovervåkning. Overvåkningsrapport nr. 819/01 TA-nr. 1797/2001, pp. 1-191.

Hawkins, S.J., Proud, S.V., Spence, S.K. & Southward, A.J., (1994). From the individual to the community and beyond: water quality, stress indicators and key species  in coastal systems. In Water quality and stress indicators in marine and freshwater ecosystems: linking levels of organisation (individuals, populations, communities) (ed. D.W. Sutcliffe), 35-62. Ambleside, UK: Freshwater Biological Association.

Jacobsen, J. A. and Asmund, G. (2000). TBT in marine sediments and bluemussels (Mytilus edilus) fromcentralwest Greenland. The Science of the Total Environment 245, pp. 131-136.

Kannan, K. and Falandysz, J. (1997). Butlytin residues in sediment fish, fish-eating birds, harbour porpoise and human tissues from the polish coast of the Baltic Sea. Baseline 34, 203-207.

Maguire, R. J., (2000). Review of the Persistence, Bioaccumulation and Toxicity of Tributyltin in Aquatic Environments in Relation to Canada’s Toxic Substances Management Policy. Water Quality Research Journal of Canada, Vol. 35; No. 4; 663-679.

Mensink, B.P., ten Hallers-Tjabbes, C.C, Kralt, J., Freriks, I.L. and Boon, J.P. (1996). Assessment of Imposex in the Common Whelk, Buccinum undatum (L.) from the Eastern Scheldt, The Netherlands. Marine Environmental Research Vol. 41, No. 4, pp. 315-325.

Minchin, D., Bauer, B., Oehlmann, J., Schulte- Oehlmann, U., & Duggan, C. B. (1997). Biological indicators used to map organotin contamination from a fishing port, Killybegs, Ireland. Marine Pollution Bulletin, 34, 235–243.

Minchin, D. & Duggan, C.B. (1986). Organotin contamination in Irish waters. ICES CM1986/F:48: 1-8.

Minchin, D., Oehlmann, J., Duggan, C.B., Stroben, E., & Keatinge, M. (1995). Marine TBT antifouling contamination in Ireland, following legislation in 1987. Marine Pollution Bulletin 30(10): 633-639.

Minchin, D., Stroben, E., Oehlmann, J., Bauer, B., Duggan C.B. & Keatinge, M. (1996). Biological indicators used to map organotin contamination in Cork Harbour, Ireland, Marine Pollution Bulletin 32: 188–195.

Nicholson, G. J. and Evans, S. M. (1997). Anthropogenic Impacts on the Stock of the Common Whelk Buccinum undatum (L.). Marine Environmental Research, Vol. 44, No. 3, pp. 305-314.

Northern Ireland Neighbourhood Information Service (NINIS), (2006). Available on-line at: www.ninis.nisra.gov.uk/mapxtreme/DetailedMaps/Ward/95VV23.jpg [accessed 05/03/06]

Nogueira, J.M.F., Simplicio, B., Florencio M.H. and Bettencourt A.M.M. (2003). Levels of Tributyltin in Sediments from Tagus Estuary Nature Reserve. Estuaries Vol 26; No. 3., p 798-802.

Oehlmann, J., (1994). Imposex bei Muriciden (Gastropoda, Prosobranchia). Eine ökotoxikologische Untersuchung zu TBTEffekten, Cuvillier Verlag, Göttingen, 167pp.

Oehlmann, J., Ide, J., Bauer, B., Watermann, B., Schulte-Oehlmann, U., Liebe, S. & Fioroni, P. (1996). Erfassung morphound histopathologischer Effekte von Organozinnverbindungen auf marine Mollusken und Prüfung ihrer Verwendbarkeit für ein zukünftiges biologisches Effektmonitoring. Final report of R. & D. project 102 40 303/01, Umweltbundesamt, Berlin, 194 pp.

Skarphédinsdóttir, H., Ólafsdóttir, K., Svavarsson, J. and Jóhannesson, T. (1996). Seasonal Fluctiations of Tributyltin (TBT) and Dibutyltin (DBT) in the Dogwhelk, Nucella Lapillus (L.) and the BlueMussel,Mytilus edulis L., in Icelandic Waters. Marine Pollution Bulletin Vol. 32, No. 4, pp. 358-361.

Smith, P.J. (1996). Selective decline in imposex levels in the dogwhelk Lepsiella scpbina following a ban on the use of TBT antifoulants in New Zealand. Marine Pollution Bulletin, 32, 362-365.

Stewart, C. and de Mora, S.J. (1990). A review of the degradation of tri(n-butyl)tin in the marine environment. Environmental Technology Vol. 11, pp. 565-570.

Svavarsson, J. (2000). Imposex in dogwhelk (Nucella Lapillus) due to TBT Contamination: Improvement at High Latitudes. Marine Pollution Bulletin Vol. 40, No. 11, pp. 893-897.

Svavarsson, J., Gram no, Å., Eke Lund, R. and Spear, J. (2001). Occurrence and Effects of Organotins on Adult Common Whelk (Buccinum undatum) (Mollusca, Gastropoda) in Harbours and in a Simulated Dredging Situation. Marine Pollution Bulletin Vol. 42, No. 5, pp. 370-376.

Svavarsson, J. and Skarphédinsdóttir, H. (1995). Imposex in the dogwhelk Nucella Lapillus (L.) in Icelandic waters. Sarsia 80, pp. 35-40.

Taylor, J.E., Charlesworth, M and Service, M. (1999). Nutrient inputs and trophic status of Carlingford Lough. Queens University Belfast/DOE (N.I) Report. Available online at: www.ecowin.org/smile/documents/Carlingford%20Lough%20trophic%20status.pdf [accessed 22/03/06]

ten Haller-Tjabbes, C.C., Everaarts, J.M., Mensink, B.P. and Boon, J.P. (1996). The Decline of the North Sea Whelk (Buccinum undatum L.) Between 1970 and 1990: A Natural or a Human-Induced Event? Marine Ecology, 17 (1-3), pp. 333-343.

ten Hallers-Tjabbes, C.C., Kemp, J.F. and Boon, J.P. (1994). Imposex inWhelks (Buccinum undatum) fromthe Open North Sea: Relation to Shipping Traffic Intensities. Marine Pollution Bulletin Vol. 28, No. 5, pp. 311-313.

United States Environmental Protection Agency (US EPA) (1985). Office of Pesticide Programs. Tributyltin support document.

Walday, M., Berge, J. A. and Følsvik, N. (1997). Imposex og nivåer av organotin hos populasjoner av purpursnegl (Nucella Lapillus) I Norge. Norwegian State Pollution Monitoring Program Report No 694/97. Norwegian Institute for Water Research, pp. 1-28.

Wilson, S.P., Ahsanullah, M. & Thompson, G.B. (1993). ‘Imposex in neogastropods: an indicator of tributyltin contamination in eastern Australia’, Marine Pollution Bulletin, vol. 26, pp. 44–48.