Imposex measurement in the dogwhelk Nucella lapillus (L.)—temporal aspects of specimen preparation† Annagh Minchin*a and Ian M. Daviesa aFRS Marine Laboratory, PO Box 101, Victoria Road, Torry, Aberdeen, AB11 9DB. Fax: +44 1224 295511; E-mail: minchina@marlab.ac.uk; daviesim@marlab.ac.uk Received 9th April 1999, Accepted 30th April 1999 The measurement of the Relative Penis Size Index (RPSI) in the dogwhelk Nucella lapillus (L.) depends upon reliable observations of the length of the penis (PL).Following removal of soft tissue from the shell, PL increases with time leading to a progressively greater over-estimation of the RPSI. The error is proportionally greater at low RPSI values. To retain comparability with previous data, it is suggested that dogwhelks should be examined as soon as possible after shell crushing.for no more than five days. Each shell height was measured Introduction to the nearest 0.1 mm and shells were then crushed and soft The use of tributyltin (TBT) compounds as antifouling agents tissues removed. The sex and VDS of each snail were recorded. since the 1970s led to impacts on non-target organisms.1,2 The PL of each snail was measured at intervals of 45 min Exposure to TBT results in the imposition of male sexual through a working day (6 h, nine observations of each snail ) characteristics (imposex) on female neogastropod snails.3 In to 0.1 mm using a binocular microscope equipped with an severe cases, this can lead to sterility in some species (e.g., the eyepiece graticule.1 These data were converted into millimetres dogwhelk, Nucella lapillus).4 Imposex measurements have been for analysis. Random individual Nucella from the population used widely in surveys of the biological eVects of TBT.4–7 were measured repeatedly over a period of 30 min, up to six Imposex has been adopted by the Oslo and Paris Commission measurements per penis, to account for analytical error.(OSPAR) as a component of their international Joint Assessment and Monitoring Programme (JAMP).The revised OSPAR protocols recommend that snails should be stored live and that the soft tissues should be removed from the shell Results (without narcotisation) prior to examination by microscope.8 The VDSI value for adult dogwhelks from Skatey Shore was Two indices are used to express the degree of imposex; the 3.96 and that for juveniles was 4.0, with an initial RPSI value Vas Deferens Sequence Index (VDSI) and the Relative Penis in adults of 2.5%.The mean shell height of adults was Size Index (RPSI). The Vas Deferens Sequence consists of a 25.48 mm (s 1.45) and of juveniles was 24.98 mm (s 1.60). progression of seven stages from unaVected to sterile females Measurements of PL over 30 min varied by ±0.1 mm and and the VDSI (mean VDS of females) gives an indication of demonstrated a high level of repeatability in relation to both the reproductive competency of the population.The RPSI is the mean PL in the population studied and the change calculated as the cube of the mean length of the penis in induced by delay in the analysis (see below).females (PLF) divided by that in males (PLM), multiplied by a There was a general tendency for PL of both male (PLM) hundred. Reliable measurements of PL are therefore important and female (PLF) Nucella to increase with time after removal as they have direct impact on the calculated RPSI values. of the animals from their shells. The average PLM increased It is common laboratory practice to prepare batches of from 2.71 (s 0.44) to 3.19 (s 0.44) mm and PLF from 0.79 (s snails (e.g., by sampling location) for examination and some 0.31) to 1.35 (s 0.29) mm after 6 h.The average PLM of juvenile time may therefore elapse between removal from the shell and males increased from 1.55 (s 0.39) to 1.93 (s 0.49) mm and measurement of the PL. This may result in variation in penis that of juvenile females from 0.69 (s 0.20) to 0.99 (s 0.23) mm length among individual Nucella, due to the time elapsed from after the same period.The increases mainly took place during the removal of soft tissue from the shell. As continuation of the first three hours, with relatively small changes between an investigation of sample preparation/conservation,9 this four and 6 h (Fig. 1). The increase in PL was proportionally paper examines the changes in PL with time in Nucella which much greater (Fig. 2) in animals with a small penis (e.g., adult have been removed from their shells and assesses the females showed an average increase by a factor of 1.7) than significance of these changes for RPSI determination. in those with a relatively large penis (e.g., adult males, factor of 1.2).Juveniles tended to show less of an increase in PL Methods than did adults of similar initial PL (Fig. 3). Linear regressions were fitted to the relationships between N. lapillus were collected from below the mid-shore level at the initial penis length (PL0) and the penis length after 6 h Skatey Shore, Stonehaven, east Scotland on 30 January (100 (PL6) for both adult (Fig. 4) and juvenile dogwhelks: juveniles) and 4 June (200 adults) 1998. They were maintained at ambient temperature in flowing sea water at the laboratory Juveniles: PL6=1.052 PL0+0.270 (R2=0.9430, n=99) †©Crown copyright. Adults: PL6=0.948 PL0+0.611 (R2=0.9640, n=200) J. Environ. Monit., 1999, 1, 239–241 239Fig. 1 Changes in the ratio of mean penis length at time t (PL-t) to Fig. 4 Relationship between the ratio of RPSI values calculated after initial mean penis length (PL-0) over a period of 6 h for adult and 6 h (RPSI6) to the initial RPSI value (RPSI0) and the initial RPSI juvenile Nucella lapillus. value for adult Nucella lapillus. of imposex in adults (RPSI 10–50%), RPSI would be increased to 17–57% (i.e., factors of 1.7–1.14), but at low RPSI values (<2%) the proportional changes would be very much greater.Huet10 studied the eVect of narcotisation of Nucella by MgCl2 solution before examination. Several field studies have utilised this procedure.3,10,11 Narcotisation led to an extension of the penis that was proportionally greater at high penis lengths. The RPSI of narcotised Nucella was approximately 80% of that of non-narcotised snails over a wide range of RPSI values, and Huet10 suggested that a conversion factor of 0.8 could be used.While narcotisation leads to under estimation of the RPSI, delay in the measurement of untreated snails leads to over-estimation. Fig. 2 Relationship between the ratio of penis length after 6 h (PL-6) The observations in this report suggest that the most to initial penis length (PL-0) and initial penis length for adult and juvenile Nucella lapillus.reproducible results would probably be obtained from snails which had been left out of the shell for a period in excess of four hours. However, most published data have been obtained Discussion from fresh material, as recommended in the OSPAR guidelines Imposex analysis, generally, only requires one PL measurement and adopted in recent international laboratory performance and not repeated measurements of the same individual over studies under QUASIMEME.12,13 In order to retain compartime.The time between removal from the shell and the ability with previous data, it is advisable to examine dogwhelks measurement of the PL will aVect RPSI calculations as, once as soon as possible after extraction from the shell.It is not removed from their shells, PL in snails increased. Juvenile and recommended that PL be ‘corrected’ to some standard elapsed adult females show more of an increase than males. This time by the application of regression equations, such as those resulted in changes in RPSI from initial values of 2.5% in developed above.adults and 9.0% in juveniles, to 7.5% and 13.7%, respectively, over the 6 h period. The corresponding changes in Relative Conclusion Penis Length Index (RPLI) were from 29.3% and 44.8% to 42.2% and 51.5% over the same interval and probably reflect Penis lengths, and hence the RPSI values, of Nucella increase gradual relaxation and senescence of the snails. with time after removal from the shell.Penis lengths stabilise The regression equation above can be used to estimate the after three to four hours, but to retain comparability with potential eVect of this aspect of sample handling on popu- previous data, it is recommended that all snails be measured lations showing a range of RPSI values. At intermediate levels as soon as possible after removal from the shell.Acknowledgements We would like to thank Matt Gubbins, Patricia Barrett and Gavin Grewar for their help with the collection and processing of samples. References 1 G. W. Bryan, P. E. Gibbs, L. G. Hummerstone and G. R. Burt, J. Mar. Biol. Assoc. UK, 1988, 66, 611. 2 E. Stroben, J. Oehlmann, U. Schulte-Oehlmann and P. Fioroni, Malacol. Rev. (1996), Suppl. 6 (Molluscan Reprod.), 1996, 173. 3 J. Oehlmann, E. Stroben and P. Fioroni, J. Molluscan Stud., 1991, 57, 375. Fig. 3 Correlation between penis length measured after 6 h (PL-6) and 4 P. E. Gibbs, G. W. Bryan, P. L. Pascoe and G. R. Burt, J. Mar. Biol. Assoc. UK, 1987, 67 507. initial penis length, for adult and juvenile Nucella lapillus. 240 J. Environ. Monit., 1999, 1, 239–2415 G. W. Bryan and P. E. Gibbs, in Metal Ecotoxicology: Concepts 10 M. Huet, P. Fioroni, J. Oehlmann and E. Stroben, Hydrobiol., 1995, 309, 29. and Applications, ed. M. C. Newman and A. W. McInthosh, Lewis, Ann Arbor, 1991, pp. 323–361. 11 A. Minchin and D. Minchin, Environ. Technol., 1997, 18, 1225. 12 I. M. Davies, QUASIMEME Bulletin, 1998, Issue 5, pp. 19–20. 6 D. Minchin, J. Oehlmann, C. B. Duggan, E. Stroben and M. Keatinge, Mar. Pollut. Bull., 1995, 30(10), 633. 13 QUASIMEME, Report of exercise 359, BE1 Fisheries Research Services, Marine Laboratory, Aberdeen, 1998, 7 pp. 7 J. Svavarsson and H. Skarphe�dinsdo� ttir, Sarsia, 80(1), 35. 8 I. M. Davies, A. Minchin and M. Harding, Marine Laboratory, Aberdeen Report No 9/97, 1997, 33 pp. Paper 9/02836F 9 A. Minchin and I. M. Davies, J. Environ. Monit., 1999, 1(2), 203. J. Environ. Monit., 1999, 1, 239–241