IMPOSEX INCIDENCE IN Thais tuberose (Roding, 1798) Cronia margariticola (Broderip, 1832) IN THE COASTAL BARANGAYS OF NASIPIT, AGUSAN DEL NORTE, PHILIPPINES

1Fabio C. Ruaza Jr., 2Lilia Z. Boyles and 2Eve V. Fernandez

ABSTRACT

The imposex incidence in Thais tuberose (Roding, 1798) and Cronia margariticola (Broderip, 1832) were studied to assess the incidence of imposex in the coastal Barangays of Nasipit, Agusan del Norte. Almost all sites were showed incidence of imposex development. The most affected and highest incidence of imposex using T. tuberose and C. margariticola is Brgy. Camagong with the value of 73.33% and 66.66% in Incidence of Imposex (IOI); 32.35% and 34.21% in Conversion of Potential Female of Imposex Index (CPFII); and in Relative Penis Length Index ( RPLI) of 72.10 and 61.71% respectively. Followed by Sta. Ana, Apagan, Punta, Amontay have the most cases of imposex. The study identified higher occurrences of imposex near and adjacent the shipyard. Results showed that T. tuberose is more sensitive to imposex compared to C. margariticola, thus, suggesting a better bioindicator.

Keywords: cronia margariticola, thais tuberose, imposex, biondicator

1.0 Introduction

Imposex or pseudohermaphrodism is et al., 1993; Horiguchi et al., 1994). a widespread phenomenon among Tributyltin is the most commonly used prosobranch molluscs characterized by antifouling agent used in paints for the the development and superimposition of underwater hull of large boats (Berge and non-functional male accessory sex Walday, 1999). It was used to kill the organs (i.e vas differences and/or a algae, mussels and barnacles found in penis) on female or juvenile gastropods the hulls of the vessels which is a (Astilla et al., 2005). The incidence of historic problem that hinders the imposex in gastropods is a worldwide operation of marine vessels. concern and has been studied in many Several studies have been conducted, gastropods (e.g. Nias et al., 1993; Minsink et Gibbs and Bryan (1987) confirmed a long al.,1996; Bryan et al.,1993; Schulte-Oehlmann term field and laboratory experiments et al., 1997; Short et al., 1989; Matthiessen et with Nucilla lapillus, exposed to Tributyltin al., 1998; Stroben et al.,1992). This which showed the bioaccumulation within phenomenon is caused by exposure to female correlated with an increase in the Tributyltin compound and results in development of imposex. Also, Matthiessen accumulation of testosterone in the et al., (1998) explained that gastropods gastropods (Gibbs & Bryan, 1996; Nias bioaccumulate TBT and its endocrine

1Surigao del Sur State University 2,3 Caraga State University SDSSU Multidisciplinary Research Journal Vol. 1 No. 2, 2013 73 Imposex Incidence in Thais tuberose (Roding, 1798) Cronia margariticola (Broderip, 1832) in the Coastal Barangays disruptive effects result in elevated TBT pollution in the Philippines. These testosterone levels giving rise to imposex. two species of gastropods are Nias et al., (1993) reported that TBT has present and common in the costal water been the major cause of imposex in N. in Nasipit, Agusan del Norte which has lapillus. Such occurrence would therefore the heavy shipping traffic of marine affect the reproductive capacity of the vessels. Also, it was presumed as the affected population, especially when source of TBT pollution. The outcome of imposex is known to be irreversible this study is to compare the sensitivity of (Blackmore, 2000). Also, Smith (1996) these two species of gastropods in added that severe imposex may result to response to TBT pollution. The use of population decline or even mass imposex standard indices will be extinction. In addition, Mensink et al., established to evaluate the incidence of (1996) concluded that severe stages of imposex in different coastal Barangays of imposex at higher TBT concentrations Nasipit, Agusan del Norte considering can lead to female sterilization and that it is an inexpensive method to death. During the exposure in early life establish occurrence, patterns and stages of high concentration of TBT, a changes in TBT pollution in marine complete sex change might occur environment. (Stroben et al., 1992). Despite the pollution of TBT in the 2.0 Research Design and Methods marine environment, it is still considered a problem worldwide, particularly in 2.1 Study Site seaports although, some countries have led to regulatory measures in using TBT. The nine (9) coastal Barangays of France and United Kingdom prohibit the Nasipit, Agusan del Norte were selected use of TBT paints on vessels under 25 as sampling sites considering the meters in length. In the Philippines, presumed source of organotin pollution there is no monitoring of TBT pollution. (the seaport). The nine sampling stations The use of TBT is very rampant. In the are the Barangay Talisay, Camagong, study of Astilla et al., (2005) in Sta. Ana, Apagan, Punta, Amontay, Mactan Cebu, the first study conducted Aclan, Ata-tahon and Cubi-cubi. The in the Philippines, showed 100% Geographical Positioning System (GPS) development of female penis developed in was used to record the distance and the the population in the Caltex depot. location of each barangays in the study The use of Cronia margariticola by area. Pandey and Evans (1996) in Indonesia and Astilla et al., (2005) in Mactan 2.2 Collection of Samples and Processing Island, Philippines in monitoring TBT in marine waters by documenting imposex Collection of T. tuberose and C. exhibited sensitive to imposex. The use of margariticola were done using a random Thais tuberose by Liu et al., (1997) found sampling method in each station. that imposex in this species is more Sampling was carried out while the tide severe than other species despite similar is at low shore due to the fact that the TBT burdens. The T. tuberose species is the specimen is binding on rocks and in sea first study to be conducted as biomonitoring of grasses further down the shoreline. These

74 SDSSU Multidisciplinary Research Journal Vol. 1 No. 2, 2013 Imposex Incidence in Thais tuberose (Roding, 1798) Cronia margariticola (Broderip, 1832) in the Coastal Barangays gastropods species are found only in the index that quantifies the degree of intertidal zone. All samples were placed imposex in the population and is in a container filled with seawater and obtained from the equation: (Mean length sealed for transit. The collection of of female penis)/(Mean length of male samples was done through handpicking penis) x 100 (Minchin et al., 1996; Gooding from identified stations. To have a et al., 1999 ). reliable data 30 specimens in every site The Conversion of Potential Female was collected (Castro, 2002). The Imposex Index (CPFII) is a new index specimen were preliminarily narcotized developed to calculate the potential with magnesium chloride solution 3.5% females of a given area of population (Huet et al., 1995) and preserved in a converted to imposex. Equation is refrigerator. After 24 hours, the samples obtained in (number of imposex were placed in 10% formalin solution of animals)/(Number of Animals + Number seawater and kept frozen prior to of Imposex Animals) x 100 (Tewari et al., analysis. 2002). The Incidence of Imposex (IOI) was 2.3 Recognition of Sexes calculated using the following formula by Shim, et al., (1999): (Number of Imposex In analysis, the length of each Animals)/(Total Numbers of Females) x gastropod was measured from the apex 100. to the distal end of the siphonal canal Linear Regression Analysis was used using a vernier caliper measured to the to correlate the RPLI vs shell length and nearest 0.1 mm. The shell of the the distance from the source and RPLI. specimen was being crushed with a The degree of imposex is analyzed hammer. The soft part was placed in a through bivariate factors – across petridish for recognition of sexes using sampling locations in the T. tuberose and the dissecting microscopes. Penis length C. margariticola. were measured by the vernier caliper to the nearest 0.1 mm. Males were 3.0 Results and Discussions distinguished by the presence of seminal vesicles and red orange gonad and A total of three hundred seventy (369) females were identified by the presence of Cronia margariticola individuals and of a white egg capsule gland and three hundred sixty nine (370) of Thais albumen gland, and cream–colored tuberose were collected from the nine (9) gonad with yellow ovaries (Smith, 1980). coastal Barangays of Nasipit Agusan del Imposex is established by the presence of Norte. Imposex were detected in two a penis on females located dorsal to the gastropods. As shown in Figure 1, the right tentacle. IOI, CPFII and RPLI in T. tuberose of 35.5%, 16.5%, and 46.8% are relatively 2.4 Data Analysis higher than C. margariticola of 23%, 12.3% and 34.5% respectively in all To determine the occurrence of sampling sites. Moreover, a relatively imposex, the following indices for indirect high RPLI observed, shows a significant TBT biomonitoring were calculated: The development of the penis among female Relative Penis Length Index (RPLI) is an gastropods used in the study; that

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Figure 1. Comparison of the incidence of imposex indices of T. tuberose and C. margariticola is, penis developed among female species factor that can influence the expression is more than half of the actual male of imposex is age. The species that are penis among the populations. The penis longer lived or slower growing may be length of the females are smaller more likely to have high levels of TBT compared to male penis. The longest and thus exhibit imposex. The Incidence pseudopenis was 0.58mm in C. of Imposex (IOI); Conversions of Potential margariticola and 0.62mm in T. tuberose. Female Index (CPFI) and Relative Penis This result was supported by Blackmore Length Index (RPLI) of T. tuberose and C. (2000) who stated that penis size in margariticola are presented in Table 1. females is always smaller in males. The highest Incidence of Imposex (IOI) of The T. tuberose species exhibited high the T. tuberose and C. margariticola is in value of imposex indices. This result was Brgy. Camagong with the value of consistent in the previous studies 73.33% and 66.66%. This is followed by conducted by Liu et al., (1997) in Brgy. Talisay of 66.66% and 41.66% Taiwan, that imposex was much more respectively. Least incidence was severe in Thais species than the other obtained from barangay Cubi-cubi, neogastropoda, despite similar organotin Ata-atahon and Aklan. A marginal burdens. The C. margariticola exhibited degree of imposex was obtained in less imposex incidence due to its Barangay Aklan, 0% in C. margariticola sensitivity in organotin pollution. and 6.89% IOI in T. tuberose. Moreover, According to Tan (1999) the differences Barangays Cubi-cubi and Ata-atahon in habitat, diet, and physiology have showed no incidence of imposex in both been suggested as causes of interspecific gastropods. It should be noted that differences in imposex. In addition, Li barangay Sta. Ana showed a high degree and Collin (2009) stated that another of imposex in terms of IOI (64.00%) for T.

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Table 1. Imposex indices of Thais tuberose and Cronia margariticola across the nine coastal barangays

tuberose but relatively lower in IOI TBT has been known as endocrine disrupter (38.46%) for C. margariticola. (WHO, 1999). There was a possibility The extent of imposex is determined that the wolfian duct designed to become using the index of Conversion of a vagina for females with the influence of Potential Female to Imposex Indices estrogen was disrupted thus imposition (CPFII). The value ranges in Thais of male organ occurred in both C. tuberose and Cronia margariticola are margariticola and T. tuberose. This is 32.35% and 34.21% in Brgy. Camagong supported by Matthiessen and Gibbs to 29.27% and 22.22% in Brgy. Talisay (1998) who reported that bivalves and and Brgy. Sta. Ana has the value of gastropods are especially sensitive to 27.58% and 19.60% respectively. The organotin or the TBT larval stage are Relative Penis Length Index (RPLI), is more vulnerable than adults. The shown on Table 1. Brgy. Camagong had internal anatomy of C. margariticola and the highest percentage value with a total T. tuberose showing the male, female and RPLI of 61.71% and 72.10% followed by imposex test animals are shown on Brgy. Talisay with a total value of Figure 2. 52.12% and 67.06% and Brgy. Sta. Ana This study revealed that the highest with the total value of 52.12% and incidence of imposex barangays are near 60.63% respectively. and adjacent from the seaport. ANOVA Barangay Camagong, b e i n g on the linearity (table 2) in the proximal to the shipyard, has a dense relationship between the indices of shipping traffic, thus the occurrence of imposex and distance from the seaport to biological effects (predominantly sampling sites were established. The imposex) in offshore waters. The dense Incidence of Imposex (IOI) (P < 0.001; r = - traffic of ships logically explains the high 0.89 & - 0.78); Conversion of Potential Female concentration of TBT as antifouling agent Index (P < 0.001; r = - 0.90 & - 0.80); and hydrolyzed in the water. It could be that Relative Penis Length Index (P < 0.001; r = - majority of the collected samples from 0.97 & - 0.80) has significant results in T. Brgy. Camagong are juveniles when the tuberose and C. margariticola respectively. sexual differentiation is on the process,

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1.

2.

a b c Legend: (a) imposex; (b) male; (c) female; (k) kidney; (o) ovary; (p) penis; (te) testes; (tn) tentacles.

Figure 2. Internal structure of (1) C. margariticola and (2) T. tuberose

Table 2. Regression analysis and ANOVA result of imposex indices between shell length and distances

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The highest mean shell length is its proximity to a shipyard where obtained in Brgy. Ata-atahon (mean 2.5 considerable painting and repair activities cm.) in T. tuberose and Brgy. Cubi–cubi take place. In addition, De Castro et al., (2005) (mean 6.3 cm) of C. margariticola. The in Northeast Brazil that the increase in the lowest mean shell length is Brgy. incidence of imposex was probably caused Camagong with a value of 5.38 and 2.10 by the establishment of Pecém Harbor and cm respectively. Using regression its increasing shipping activities. Also, analysis between the Relative Penis Bryan et al., (1987) concluded that there Length Index (RPLI) and imposex shell is a strong relationship between the length (r = -0.62 & - 0.63) are negatively degree of imposex and proximity to correlated in T. tuberose and C. harbors. margariticola respectively. It implies that The use of TBT in marine vessels is as the RPLI increases the shell length very rampant worldwide. In the Philippines, decreases or vice versa. The result of this there is no regulation in the use of study particularly on the mean shell Tributyltin in the shipping activities. The length needs further study. This result is prohibition of TBT in large harbor areas inconsistent to the result conducted by is necessary. For example in the country Li and Collin (2009) that the Acanthais Japan, the regulation prohibiting the use brevidentata, showed that females with of TBT-based antifouling is implemented imposex are larger than normal females. and significant decrease of RPSI was However, several factors trigger the observed (Champ, 2000). The regulation growth of gastropods such as exposure of implementation of prohibition of TBT in to wave action and or in predation the Philippines is necessary considering pressure (Blackmore, 2000). the effect of imposex in the marine This result was supported by environment and health hazard to numerous studies in relation to the human. distance of the sampling locations in presumed source, the seaport. According to 4.0 Conclusion the study of Li and Collin (2009) in Taiwan, that frequency and severity of Imposex were detected in two species imposex declined with distance away of gastropods, the T. tuberose and C. from canal anchorage. In addition, Astilla margariticola. Using the standard et al., (2005) in Pandey and Evans (1996) indices; the IOI, CPFII and RPLI in T. stated that within the port area, they tuberose yielded a value of 35.5%, 16.5%, observed 75% imposex incidence, and 46.8% are relatively higher than C. followed by the site adjacent to the port margariticola of 23%, 12.3% and 34.5% area which had 61% imposex incidence, respectively in all sampling sites. The T. then by fishing villages where 30% tuberose species exhibited high value of imposex incidence was detected, and imposex indices compared to C. margariticola lastly, in remote areas where there were suggesting good bioindicator in TBT some small boat activity which registered pollution. Highest occurrence of imposex 15% imposex. In Mactan Island, are near and adjacent from the seaport Philippines, Astilla et al., (2005) reported such as the Brgy Camagong, Talisay, that Caltex Depot has the most severe Sta. Ana, Apagan, Punta and Amontay. imposex and this can be attributed to ANOVA on the linearity revealed

SDSSU Multidisciplinary Research Journal Vol. 1 No. 2, 2013 79 Imposex Incidence in Thais tuberose (Roding, 1798) Cronia margariticola (Broderip, 1832) in the Coastal Barangays significant difference (P < 0.001) in RPLI, zones. Smithsonian Contributions to CPFII and IOI and distance from the the Marine Sciences, 38, 190- 196. seaport to sampling sites. Champ, M.A (2000). A review of or 5.0 Recommendations organot in regulatory strategies, pending actions, related costs and In view of the result of this study, the benefits. The Science of the Total following are recommended; conduct Environment, 258, 21-71. histological examinations to the affected female tissues; use linear measurements Cleary, J. J. (1991). Organotin in the for comparing shell morphology; use Vas marine surface microlayer and Deference Sequence; look into the sub-surface waters of South West lifecycle of the imposex gastropods and England: relation to toxicity thresholds longer sampling period and all nearby and the UK environmental quality Barangays where seaport are located standard. Marine Environmental should be studied not only in Agusan del Research, 32, 97-101. Norte but in the entire Philippines. De Castro, B., Augusto. C., de Meirelles, References: O. Pinheiro, J.L.,Cascon, H. M. & Barreira, C. R. (2005). The increasing Astilla, M. Q., Suan, K. L. & Liao, L.M. incidence of imposex in Stramonita (2005). Imposex in Cronia margariticola haemastoma (Mollusca: Gastropoda: (Mollusca, Prosobranchia) as a Muricidae) Ceará State, Northeast potential marine pollution biomonitor Brazil. Thalassas, 21 (2), 71-75. around Mactan Island, Central Philippines, San Carlos University Fernandez, M. A., Limaverde, M.A. & Phil. Scientist, 42, 79-93. Castro, A.M. (2002). Occurrence of imposex in the Thais haemastoma: Berge, J.A & Walday,M. (1999). possible evidence of environmental Alternative to the use of TBT as an contamination derived from organotin antifouling agent on the hull of ships compounds in Fortaleza, Brazil. with special reference to methods not Reports in Public Health, 18, 463- involving leaching of toxic compounds 476. to the water. Report No. 098149. Norwegian Institute for Water Gibbs, P.E., Bryan, G. W., Pascoe, P.L. Research, pp. 1-34. & Burt, G.R. (1987). The use of the Dogwhelk Nucella lapillus, as an Blackmore G. (2000). Imposex in Thais indicator of Tributyltin (TBT) clavigera (Neogastropoda) as an indicator contamination. Journal of the Marine of TBT (tributyltin) bioavailability in Biological Association of the United Coastal Waters of Hong Kong. J Moll Kingdom, 67, 507-523. Stud, 66, 1-8. Gibbs, P. E., & Bryan, G. W. (1996). TBT- Carter L. & Colli, R. (2009). Imposex in induced imposex in Neogastropod one of the world’s busiest shipping snails: masculinization to mass

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