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Marine Pollution Bulletin, Vol. 36, No. 10, pp. 840-843, 1998 Pergamon © 1998 Elsevier Science Ltd

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Imposex in Nucella lapiUus from TBT Contamination in South and South-West Wales: A Continuing Problem around Ports EDWARD MORGAN, JOHN MURPHY and ROISiN LYONS School of Applied Sciences, University of Glamorgan, Llantwit Rd, Trefforest, Pontypridd CF371DL, UK

Tributyltin compounds (TBTs) which have entered the marine environment as a result of their use in anti- fouling paints on boats and aquaculture equipment have been described as being among the most toxic compounds ever produced (Goldberg, 1986). In particular, these have exerted dramatic effects on populations of some molluscs and at least 72 species of gastropod molluscs are known to be affected by TBTs at many locations world-wide (Evans et al., 1995).

Imposex, which is the development of male sex characteristics in female gonochoristic gastropod molluscs in response to TBT pollution (Blaber, 1970) has been studied thoroughly in the European dogwhelk Nucella lapillus, and this species is now widely used as a biomonitor of TBT levels in seawater (Stroben et al., 1995; Gibbs and Bryan, 1996). Geographically separated populations taken from over 10 ° of latitude respond uniformly to TBT (Gibbs et al., 1991). Imposex in N. tapillus is fully developed at ambient TBT concentrations of 1-2 ng 1 1, and at ___3 ng l- females are fully sterilised (Gibbs and Bryan, 1996). Populations of N. lapillus became extinct in some areas near boatyards, marinas and sheltered estuaries when TBTs attained high concentrations in the local environ- ment (Langston et al., 1990).

840

As the environmental effects of TBT became known during the 1980s, governments around the world intro- duced legislation to tackle the problem. In July 1987, the United Kingdom Government banned the use of TBT-based anti-fouling paints for vessels <25 m in length and also its use for aquaculture, a measure aimed at reducing TBT concentrations around marinas and in estuaries. However, TBT from larger vessels which were excluded from the 1987 ban also impacts on populations of marine organisms, as shown by surveys of dogwhelks around Sullom Voe in the Shetland Isles, a deep-water oil and gas tanker port (Davies and Bailey, 1991; Moore et al., 1996).

In south and south-west Wales there are a number of ports of varying commercial importance (Fig. 1). In the west, Milford Haven is an important deep-water port serving tankers bringing oil into and out of refineries. Pembroke Dock is a commercial ferry port for Ireland. Further east, Swansea, Neath, Port Talbot, Barry, Cardiff, Newport and Avonmouth are active ports, though those in South Wales have been of decreasing importance in recent years with the decline of the local heavy industry. The shipping traffic to these ports is a potential source of TBT for the Severn Estuary and Bristol Channel.

The coastal area of south and south-west Wales from the Severn Estuary to the outer Bristol Channel has been the subject of numerous studies over the last 30 years, the result being that the regional chemistry, ecology and hydrology are now well understood (DOE, 1973; Morris, 1984; Owens, 1984; Warwick, 1984; Apte et al., 1990). However, there have been no in-depth studies of TBT levels in the region. One study (Loretto et al., 1994) assessed imposex in N. lapillus populations at Nash point, located between Southerndown and Llantwit Major (Fig. 1) and a second investigation examined dogwhelks from several sites in North Cornwall, Devon and Somerset (Bryan et al., 1986). The aim of the present study was to assess the levels of imposex in N. lapillus populations of south and south- west Wales by means of the relative penis size index, the vas deferens sequence index and the percentage incidence of imposex in females. These data were used to relate the observed levels of imposex to potential TBT sources and to provide a baseline from which other studies can assess change in these populations. Ambient TBT concentrations in the seawater of the region may be determined from the levels of imposex (Stroben et al., 1995) to provide an indication of whether the United Kingdom environmental quality standard (EQS) of 2 ng 1-1 has been attained in this region.

Five N. lapillus populations were sampled from in and around Milford Haven and South Pembrokeshire during October and November 1996. During this time, an extensive search was made of the Milford Haven waterway to establish the presence of N. lapillus populations. A further six samples were taken from the

Volume 36/Number 10/October 1998

Pembroke

Dock

3.62 I 4 ~ ~ 0 6 I 19.6% 3.05 Dale Fort 19.9%

West Angle • 21.8%

Freshwater West

Carmarthen Ba.

'2.87 0.92% Gower

~, " "1.40 / 4.12 0.04% 7.5% Kitchen Corn Stackpole

Quay 4.00 9.61%

Limeslade Bristol Channel

Swansea

3.93 12.33%

Rest Bay

Neath

~ o r t Talbot Cardiff

/ ~ m o r g a n Ba 5

3.06 4.10%

Southerndown

2.77 1.52%

Llantwit Major

Newport

~" Severn Estuary

3.42 4.84% Cold Knap

Fig. 1 VDSI values (plain text), RPSI% (in italics) and site locations in south and south-west Wales of sampled Nucella lapillus populations.

G l a m o r g a n and G o w e r pen insu la coast l ines dur ing July and Augus t 1997. The locat ions of these sites and the sampl ing da tes are given in Tab le 1 and Fig. 1. Much of the coas t l ine be tween South P e m b r o k e s h i r e and the G o w e r pen insu la is of a sandy or m u d d y na tu re and is not sui table for N. lapillus. Swansea Bay presen ts s imilar p rob lems . To the e a s t , sal ini ty is the l imit ing fac tor in the Severn Estuary, with Cold Knap at Barry be ing the fur thes t site that dogwhelks p e n e t r a t e on the nor th shore.

At each site, be tween 60 and 80 N. lapillus were s amp led at low water . These were m a t u r e animals possess ing a th ickened shell r im and a row of tee th just

inside the ape r tu re (Feare , 1970). Each individual was sexed and the degree of imposex was assessed by the me thods of Bryan et al. (1986) and Gibbs and Bryan (1987). The re la t ive penis size index (RPSI ) ca lcula tes the relat ive size of penes in females and males f rom a given sample , as (average cubed female penis length/ average cubed male penis length) x 100% (Bryan et al., 1986). Imposex d e v e l o p m e n t in f emale N. lapillus has been d iv ided into six vas defe rens sequence (VDS) s tages (Gibbs and Bryan, 1987). A female with a V D S stage of 5 or 6 has impa i r ed r ep roduc t ive capaci ty with a b locked pal l ia l oviduct which of ten eventual ly bursts (s tage 6). Thus, popu la t ions with large numbers of

TABLE 1

Locations, dates sampled, numbers of adult toothed male and female Nucella lapUlus assessed, and percentage incidence of imposex in females from populations sampled in south and south-west Wales

Site Location Date sampled No. of males No. of females (% incidence)

Dale Fort 51 ° 41.9' N 5 ° 08.7' W 29/09/96 39 21 (100%) West Angle Bay 51 ° 40.6' N 5 ° 06.7' W 20/10/96 38 22 (100%) Freshwater West 51 ° 38' N 5 ° 03' W 27/10/96 25 35 (100%) Stackpole Quay 51 ° 37.2' N 4 ° 53.6' W 02/11/96 35 25 (100%) Lydstep Bay 51 ° 38.3' N 4 ° 45.2' W 02/11/96 30 30 (97%) Kitchen Corner 51 ° 33.5' N 4 ° 18.4' W 27/07/97 30 30 (70%) Limeslade Bay 51 ° 35' N 3 ° 59' W 16/08/97 45 35 (100%) Rest Bay 51 ° 29.1' N 3 ° 43.2' W 31/07/97 31 45 (100%) Southerndown 51 ° 26.2' N 3 ° 36.2' W 23/08/97 30 30 (100%) Llantwit Major 51 ° 24.3' N 3 ° 29.7' W 11/08/97 45 35 (100%) Cold Knap 51 ° 23.1' N 3 ° 15.2' W 24/07/97 30 33 (100%)

841

females in the later stages of imposex development are often in decline and are characterised by a lack of juveniles and a biased sex ratio. The vas deferens sequence index (VDSI) for each population was calculated as the mean VDS value (Gibbs and Bryan, 1987) for the sample of female N. lapillus at each site.

In the extensive search of Milford Haven waterway for N. lapillus, only three populations were found, where previously many were present (Crothers, 1985). However, viable populations with juveniles and egg masses were found at many sites both east and west of the mouth of Milford Haven. This area was badly affected by the Sea Empress oil disaster when around 70000 tonnes of oil was lost after the vessel ran aground in February 1996. This and other oil spills may have eradicated some of the populations within Milford Haven, and it is not possible to attribute the current poor status of the N. lapillus populations within Milford Haven to TBT alone. However, the popula- tions outside Milford Haven (which were also contami- nated by large quantities of oil) seem to have survived in spite of this. Populations of N. lapillus at all other sites also had large numbers of juveniles present, indicating active breeding within the last few years.

Table 1 shows the number of males and females sampled and the percentage incidence of imposex in females for the 11 populations studied. Only the population at Kitchen Corner on the tip of the Gower peninsula contained a significant number of unaffected females (70% females with imposex), with all the other populations registering 100% incidence of imposex, except for Lydstep Bay (97%). The quantitative expres- sions of imposex calculated for this study (RPSI and VDSI) are shown in Fig. 1. The RPSI values were high for the sites close to or within Milford Haven (Dale Fort, West Angle and Freshwater West) at around 20%, with progressively lower RPSI values at Stackpole Quay (7.5%), Lydstep Bay (0.92%) and Kitchen Corner (0.04%) to the east. Further east a more complicated pattern was apparent, as higher RPSI values were obtained at Limeslade (9.61%) and Rest Bay (12.33%), these possibly resulting from shipping activity at Swansea, Neath and Port Talbot docks. Lower RPSI values were obtained further up the Severn Estuary at Southerndown (4.10%), Llantwit Major (1.52%) and Cold Knap (4.84%). A similar pattern of imposex development was obtained for VDSI, with the lowest values again being obtained at Lydstep Bay (2.87) and Kitchen Corner (1.40) and high values of VDSI in and around Milford Haven (Dale Fort 3.62, West Angle 3.05, Freshwater West 4.06 and Stackpole Quay 4.12) and Swansea Bay (Limeslade 4.00 and Rest Bay 3.93). The Stackpole Quay popula- tion had the highest VDSI value (4.12), indicating that some females at these sites were sterile, as was the case for nearby Freshwater West (VDSI of 4.06). More variable VDSI values were found for the sites furthest up the Severn Estuary (Southerndown 3.06, Llantwit

842

Marine Pollution Bulletin

Major 2.77 and Cold Knap 3.42), possibly reflecting localized TBT input.

The incidence and levels of imposex found throughout the region sampled indicate that the highest levels of TBT contamination are confined to the areas of shipping activity, as expected. Samples from the sites close to Milford Haven waterway exhibited high levels of imposex with VDSI~4 in four sites, RPSI values close to 20% in three sites, and with all the females affected. VDSI values around or above 4 imply that some of the females have impaired repro- ductive potential (exhibiting VDS stages 5 or 6). These values of VDSI indicate ambient TBT concentrations of approximately 3-4 ng1-1 (Stroben et al., 1995). Currents flowing past the entrance to Milford Haven waterway run both east and west on the ebb tide (The Admiralty, 1974). Water exiting Milford Haven carries elevated levels of TBT, presumably causing the high imposex values found at the Dale Fort, West Angle, Freshwater West and Stackpole Quay sites, close to the entrance of Milford Haven waterway. These levels of imposex are slightly lower than those determined in 1995 at sites immediately adjacent to the oil terminal of Sullom Voe in the Shetlands, for which the highest recorded value of RPSI was 31%. However, they are comparable with many the values of the sites near the same terminal, for which the average RPSI value was 23.2% (Moore et al., 1996). Unfortunately, few dogwhelk populations are now present within Milford Haven that could be assessed.

The N. lapillus population at Lydstep Bay exhibited lower values of RPSI (0.92%) than those at the other sites nearby which are closer to the entrance to Milford Haven. Lydstep Bay is at the edge of Carmarthen Bay, where it is protected by the South Pembrokeshire coast and Caldey Island from potentially TBT-laden water discharging from Milford Haven waterway. The population at Kitchen Corner on the other side of Carmarthen Bay exhibited the lowest levels of incidence (70% females with imposex), the lowest RPSI (0.04%) and the lowest VDSI value (1.40) of all the populations sampled. RPSI values of less than 1% are used as a general indicator of sites being remote from TBT sources (Bryan et al., 1986; Bailey and Davies, 1989). Therefore, the population at Kitchen Corner may be considered to be only marginally affected by TBT. The VDSI value of 1.40 indicates an ambient TBT concentration of _< 1 ng 1-1 (Stroben et al., 1995).

Within the Severn Estuary and Bristol Channel there are large quantities of sediment both in suspension (up to 100 g !-1 at certain points in the tidal cycle) and as deposits, (an estimated 270 x 106 tonnes; DoE, 1973). This represents a large potential sink for TBT, as it is readily adsorbed onto sediment particles (Langston and Pope, 1995). However, most studies of TBT degra- dation indicate half-lives of the order of days or months (Seligman et al., 1996), although this may be

Volume 36/Number 10/October 1998

longer in anae rob ic condi t ions . It seems unl ikely that T B T emi t t ed pr io r to the legislat ive cont ro l s r emains a d s o r b e d on these sed iments and this is t he re fo re not cons ide red to be a significant source affect ing cu r ren t popu la t ions of N. lapillus.

Limes lade and Rest Bay ( the two sites closest to the por t s of Swansea, Nea th and Por t Ta lbo t ) con ta ined popu la t ions which are cons iderab ly af fec ted by TBT with VDSI values of 4.00 and 3.93 and RPSI values of 9.61% and 12.33%, respect ively. These V D S I values indicate ambien t T B T concen t ra t ions in this a rea of ~ 3 n g 1 - 1 (S t roben et al., 1995). Res idua l cur rents move wa te r in a c i rcular pa t t e rn a r o u n d Swansea Bay (Uncles , 1984). Any T B T from ships in this a rea would the re fo re be avai lable at these sites. F u r t h e r east a long the relat ively rura l G l a m o r g a n coast l ine, the values of imposex dec reased at Sou the rndown (RPSI 4.1%, V D S I 3.06) and were lower again at Llantwit M a j o r (RPSI 1.52%, VDSI 2.77), a l though all females at bo th sites were affected. A t Cold Knap nea r Barry docks, imposex increased slightly ( R P S I 4.84%, VDSI 3.42). These V D S I values indicate T B T concen t ra t ions of 1 - 2 ng l -~ (S t roben et al., 1995) in the ou te r par t s of the Severn Estuary. Whi ls t these TBT levels are not par t icu lar ly high, it would a p p e a r that they may be due to a local ised source of TBT assoc ia ted with dock activities.

Some repor t s of imposex values among N. lapillus

popu la t ions in Scot land and the Nor th Sea have conc luded that overal l TBT levels are decreas ing (Evans et al., 1994, 1995, 1996). However , por t s in the Shet lands , Norway, the Tyne Es tuary and Cork H a r b o u r which are used by large commerc ia l vessels are well known hot -spots of T B T pol lu t ion (Bailey and Davies, 1989; Davies and Bailey, 1991; Minchin et al,,

1996; M o o r e et al., 1996). This would also seem to be t rue of south and south-west Wales . Whils t none of the popu la t ions examined were steri le, many of those close to por t s exhibi ted levels of imposex which indicate ambien t TBT concen t ra t ions well above the Un i t ed Kingdom EQS of 2 ng 1- ~ for TBT. As it is now more than 10 years since the par t ia l ban on TBT was intro- duced, T B T from the ant i - foul ing paints on large ships would seem to be the most l ikely source of such effects and N. lapillus popu la t ions close to por ts will thus be af fec ted for the fo reseeab le future.

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Blaber, S. J. M. (19711) The occurrence of a penis-like outgrowth behind the right tentacle in spent females of Nucella lapillus (L.). Proceedings'of the Malacogical Society of London 39, 231-233.

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Evans, S. M., Hawkins, S. T., Porter, J. and Samosir, A. M. 11994) Recovery of dogwhelk populations on the Isle of Cumbrae, Scotland following legislation limiting the use of TBT as an anti- foulant. Marine Pollution Bulletin 28, 15-17.

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

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Gibbs, P. E. and Bryan, G. W. (1987) The use of the dogwhelk Nucella lapillus, as an indicator of tributyltin (TBT) contamination. Journal of the Marine Biological Association Of the United Kingdom 67, 507-523.

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Gibbs, P. E., Bryan, G. W. and Pascoe, P. L. (1991) TBT-induced imposex in the dogwhelk Nucella lapillus: geographical uniformity of the response and effects. Marine Environmental Research 32, 79-87.

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Moore, J. J., Little, A. E., Harding, M. J. C., Rodger, G. K. and Davies, 1. M. (1996) Surveys of Dogwhelks Nucella lapillus in the vicinity of Sullom Voe, Shetland, August 1995. Oil Pollution Research Unit OPRU/3/96. Neyland, Milford Haven.

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