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Keywords:

  • Tributyltin (TBT);
  • Hermit crabs;
  • Gonad atrophy;
  • Pollution

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Tributyltin (TBT) contamination affects the reproductive system of many species of invertebrates worldwide. The present study was designed to evaluate the effects of exposure to TBT pollution on the reproduction of the hermit crab Clibanarius vittatus. An orthogonal experiment was designed with two treatments: contamination (with or without TBT in the food) and crab sex (males and females). The animals were reared in the laboratory for nine months, and macroscopic and histological analyses of reproductive organs were carried out after the end of the experiment. Tributyltin was recorded in exposed crabs, but no morphological alterations were detected in the gonads of males, regardless of whether they were exposed to TBT. In contrast, females exposed to TBT displayed disorganization and atrophy of their ovaries, thus directly affecting reproduction in this hermit crab species. This effect observed in female hermit crabs may harm populations located in harbor regions, where TBT concentration is high, even after the worldwide TBT ban. Environ. Toxicol. Chem. 2012;31:632–638. © 2011 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Organotin compounds are characterized by a tin atom linked to one or more carbon chains (Sn-C). The number of Sn-C bonds affects the properties of organotin compounds, where the maximum biological activity is attributed to trisubstituted organotins (n = 3) 1, 2. Because of this biological activity, triorganotins are used commercially as biocides in many parts of the world 2. One of these potent triorganotins is tributyltin (TBT), which was developed in the 1970s and was used in antifouling paints to prevent sessile organisms from colonizing boat hulls, increasing water friction and therefore fuel consumption 3.

Organotin compounds, similar to other organic pollutants, can accumulate in many marine organisms from plankton to vertebrates, including mammals 4. These substances cause serious effects even in nontarget species, such as a decrease in oyster production 5, inhibition of growth in microalgae 6, reduction in fecundity and juvenile production in polychaetes 7, and development of imposex in mollusk species 8–10. Imposex is the main effect recorded around the world; it is characterized by a superimposition of male genital organs such as the penis and vas deferens on female gastropods 11. This abnormality has been recorded in approximately 120 mollusk species worldwide, including several that occur in Brazilian waters 8–10. This abnormal development of male sexual characters may sterilize females, as reported by Shi et al. 12 for the prosobranch gastropod Cantharus cecillei (Philippi, 1844) in China.

Because of these negative effects, TBT was regulated in many countries in the 1980s and 1990s. The regulations have been successful in reducing the toxic threat posed by TBT in many locations around the world. Thus, the International Maritime Organization proposed prohibiting all antifouling uses of TBT by 2003 and the presence of TBT on ship hulls by 2008 13.

Despite the strictures on TBT use in many parts of the world 5, 14, 15 and recently in Brazilian waters 16, contamination of the environment and biota continues to be recorded 17, 18, including in hermit crabs 19.

Hermit crabs are anomuran crustaceans that use gastropod shells to protect their soft and fragile abdomens from predation and environmental stress 20. Clibanarius vittatus (Bosc, 1802) is a typical hermit crab of estuaries, the type of habitat in which the greatest concentrations of this pollutant have been recorded 2, 21, 22, including in Brazilian estuaries 17, 19, because of the concentration of harbor activities. Different populations of this species were recorded as having high concentrations of this pollutant in the natural environment 19, and a recent study demonstrated that this hermit crab assimilates TBT mostly from food and shows a rapid depuration rate, making it a good bioindicator of recent TBT contamination 23. In addition to their capacity to accumulate TBT and serve as an indicator of environmental pollution through measurement of the concentration of this pollutant in their tissues, hermit crabs may be directly affected by TBT, especially in the reproductive system. In the present study, an orthogonal experiment was designed to analyze the possible negative effects of exposure to TBT pollution in the reproductive system of hermit crabs using C. vittatus as a model.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Animal samples

The hermit crabs used in the present study were collected in July 2009 (60 males and 60 females). All individuals were measured for the cephalothorax shield length, by means of a caliper (0.05 mm). To obtain males and females uncontaminated by TBT, specimens were collected in the Cananéia Estuary (25°00′35.7″S and 47°55′29.4″W), São Paulo State, Brazil. This area was chosen because its population of C. vittatus does not show detectable levels of TBT 19.

Experimental design

In the experiment, a total of 120 hermit crabs were used in an orthogonal design with two treatments: contamination, with and without TBT in the food; and hermit crab sex. Groups of 30 males and 30 females were used in each treatment, with and without TBT. In the treatment with TBT, the crabs were fed a ration developed for shrimp farming, mixed with TBT, to supply 180 ng Sn/g TBT/day. The crabs were maintained in aquaria (42 × 30 × 30 cm) with aerated seawater (salinity 33 psu and under ambient conditions of light and a temperature approximately 25°C). In each aquarium, six crabs were kept isolated by polyvinyl chloride tubes, allowing each animal to receive an exact amount of food (Fig. 1) during the nine months of the experiment, a period of time that allowed them to molt several times.

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Figure 1. Deployment of hermit crabs in the aquarium during the nine months of the experiments. (A) Plastic polyvinyl chloride structure constructed to isolate each hermit crab. (B) Distribution of the hermit crabs in the aquarium, relative to the position of the aerating pump (black arrow).

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At the end of the experiment, the crabs were anesthetized by thermal shock in cold seawater, removed from the shells, and carefully inspected for gonopore morphology. The crabs were then dissected, and the anatomy of the reproductive tracts was inspected and checked for the presence of parasites. Gonads and hepatopancreas of 15 individuals from each treatment were labeled and stored frozen in aluminum-foil packages until TBT determination, to investigate whether the pollutant offered in the food was assimilated. The reproductive tracts of five randomly selected individuals from each treatment were fixed in 4% paraformaldehyde for further histological procedures.

Histological procedures

The reproductive tracts of five males and five females randomly chosen from each treatment were fixed in 4% paraformaldehyde for 48 h. The samples were buffered with 0.2 M phosphate buffer (pH 7.4) and washed twice for 1 h. They were then dehydrated in an ascending ethanol series (70–95%) for 20 min each, and embedded in 2-hydroxyethyl methacrylate resin (Leica historesin kit) for 72 h at 4°C. After polymerization, the blocks were sectioned in a Leica RM2245 microtome. The 5- to 7-µm sections were mounted on slides and stained with hematoxylin and eosin according to Junqueira and Junqueira 24, avoiding xylene and ethanol baths 25.

The size of spermatozoa produced by five randomly selected males (20 from each individual, totaling 100 spermatozoa for each treatment) from both treatments with and without TBT was measured. The size of the spermatozoa was measured from the apical portion of the operculum to the basis of the perforatorium (µm), using Leica IM50 software coupled to a Leica DM2000 microscope at 100× magnification. The sizes of spermatozoa from males treated with TBT and without TBT were compared by t tests, with the significance level of p < 0.05.

Chemical procedures for TBT determination

The concentration of TBT was determined by the experimental procedure adapted from Limaverde et al. 26 developed for hermit crab tissues by Sant'Anna et al. 23. The first extraction procedure involves hydrochloric acid and methanol, followed by extraction with toluene as the solvent, and complexation with 0.1% ammonium pyrrolidine dithiocarbamate following derivatization using Grignard reagent. The organic fraction was removed, purified in an aluminum oxide column, and eluted with hexane. The final extract was concentrated to 0.1 ml, and tetrabutyltin was added as an internal standard. The recovery was assessed by a method using a surrogate added at the beginning of the analytical process for all samples. Tributyltin was analyzed in a gas chromatograph Varian 3800 equipped with a pulsed flame photometric detector using a tin filter (390 nm) and a VF5 capillary column (30 m × 0.25 mm; Varian). The detection limit was 0.8 ng Sn/g, and the quantification limit was 2.64 ng Sn/g (w/w). The amount of TBT assimilated by individuals (males and females) was tested by t tests, with the significance level of p < 0.05, because this pollutant was not detected in the treatment without TBT.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

The overall mortality rate at the end of the experiment was 17.5%, with 99 animals surviving: 27 males and 24 females in the treatment with TBT, and 25 males and 23 females in the treatment without TBT. The sizes of the individuals that survived are shown in Table 1. In the TBT group, males and females assimilated the contaminant in similar amounts (males = 62.00 ± 21.07, and females = 42.33 ± 6.80 ng Sn/g; t = 1.5383; df = 4; p = 0.1987); in the treatments without TBT, this compound was not detected by chromatographic analysis.

Table 1. Size (cephalothoracic shield length in millimeters) of the hermit crabs in the first day and after nine months of experiment
SexWith TBTWithout TBT
Day 1After 9 monthsDay 1After 9 months
  1. TBT = tributyltin.

Males7.73 ± 0.728.43 ± 0.667.62 ± 0.608.35 ± 0.64
Females6.22 ± 0.716.65 ± 0.736.00 ± 0.656.74 ± 0.70

Examining the gross anatomy showed that both male (testis and vas deferens) and female (ovaries) reproductive systems were macroscopically normal. Parasites were not found in any individuals. Ovarian histology for females without TBT in the food showed normal characteristics of oogenesis, with previtellogenic and vitellogenic oocytes (Fig. 2A–D). Of the five females treated with TBT that were analyzed, three showed different levels of ovarian disorganization, tending to atrophy (Figs. 3A–D). The vitellogenic oocytes showed an irregular form and rupture of the membrane, including free yolk granules (Fig. 3E–G). Many spaces surrounded by follicle cells were evident, and these were identified as atresic follicles (Fig. 3H). In all TBT-treated females, large numbers of hemocytes were identified near irregular oocytes, free yolk granules, and atresic follicles (Fig. 3E–H). The other two females analyzed did not show any other structural alteration (Fig. 3D). However, the degenerative effect of TBT is apparently related to the individual characteristics of each female, because two females showed normal oogenesis, with previtellogenic and vitellogenic oocytes and only some atresic follicles (Fig. 3D).

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Figure 2. Hematoxylin and eosin–stained sections of the ovary of the hermit crab Clibanarius vittatus, without tributyltin (TBT). (A) General view of the ovary, with previtellogenic and vitellogenic oocytes and absence of atretic follicles. (BD) Details of normal oocyte development from control females, showing the normal vitellogenesis process. pvo = revitellogenic; vo = vitellogenic oocytes. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com.]

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thumbnail image

Figure 3. Hematoxylin and eosin–stained sections of the ovary of the hermit crab Clibanarius vittatus, treated with tributyltin (TBT). (A) General view of the ovary showing vitellogenic oocytes with an irregular membrane, degenerated oocytes, and large numbers of atresic follicles characterized by a space surrounded by follicle cells. (B, C) Detail of some atresic follicles and oocytes with an irregular membrane. (D) Functional ovary with previtellogenic oocytes and vitellogenic oocytes. Note the presence of atresic follicles among the oocytes. (E–H) Sequence of oocyte degeneration; observe the vitellogenic oocytes with a relatively regular membrane (E), degenerate oocytes with an irregular and broken membrane (F) that releases yolk granules (G), resulting in the atresic follicles (H). Atresic follicles (black arrows), yolk granules (white arrows). pvo = previtellogenic oocytes; vo = vitellogenic oocytes; do = degenerated oocytes. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com.]

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Males maintained with and without TBT showed normal spermatogenesis with sperm production in the testis, and spermatophore formation in the vas deferens (Fig. 4A–H). No influence of TBT contamination on sperm size was detected (males with TBT x = 14.72, and males without TBT x = 14.70 µm; t = 0.1744; df = 198; p = 0.8618).

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Figure 4. Histological sections of hematoxylin and eosin–stained male reproductive system of the hermit crab Clibanarius vittatus. (A–D) Males without tributyltin (TBT). (A) Testis showing normal characteristics with germinative cells and mature sperm. (B) Seminiferous duct filled with spermatozoa. (C) Normal ribbon of spermatophores surrounded with seminal fluid within the distal vas deferens. (D) Detail of a normal spermatophore filled with mature sperm. (EH) Males treated with TBT. (E) General view of testis displaying normal spermatogenesis with germinative cells and mature sperm. (F) Detail of seminiferous duct and sperm. (G) Normal ribbon of spermatophores in the distal vas deferens. (H) Detail of normal spermatophores with sperm from TBT-treated males. Seminiferous duct (black arrow), distal vas deferens (white arrow). gc = male germinative cells; sz = mature sperm; st = spermatophores. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com.]

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Individuals treated with TBT assimilated this pollutant, and the animals maintained without TBT showed no trace of this substance in their tissues. These results agree with the model of assimilation and rapid (within a few weeks) depuration demonstrated for C. vittatus23. The TBT effect was observed only in females, which showed disorganization of the ovaries and absorption of the vitellogenic oocytes that caused ovary atrophy. However, the degenerative effect of TBT is apparently related to the individual characteristics of each female. Of the five females analyzed histologically, not all showed alterations in the ovary. Two females showed normal oogenesis, with previtellogenic and vitellogenic oocytes and some atresic follicles. This process of atresia was characterized by the participation of hemocytes, as reported for other crustacean species 27. Males treated with TBT showed normal spermatogenesis with no anatomical changes.

Effects on the biota reproduction caused by TBT contamination were reported more than 20 years ago, when Thain 28 and Alzieu 29 recorded effects on growth, reproduction, and survival of oysters. In the bivalve Scrobicularia plana (da Costa, 1778), Ruiz et al. 30 observed that TBT was toxic to embryonic development, whereas in the hermaphroditic snail Lymnaea stagnalis (Linnaeus, 1758), Czech et al. 31 detected effects on the egg production and hatching rate. Similar to the present study, effects of TBT on female ovarian development were observed by Jacobson et al. 32 in the amphipod Monoporeia affinis (Lindström, 1855). In the study by Jacobson et al. 32, females of M. affinis treated with TBT showed a significantly higher proportion of dead oocytes, which was related to TBT contamination or to an increase in parasite prevalence. In the present study, no parasites were observed in the anatomical analysis, so that only the effect of TBT is being considered.

The toxic effect of TBT in hermit crab females led to fertility reduction through ovarian disorganization or atrophy, which may result in a decrease in the hermit crab population over the long term. This result seems similar to observations in gastropod mollusks in which ovarian atrophy and sterility were also demonstrated 12, 33, 34.

In crustaceans, sex determination is controlled by the androgenic gland 35, 36, which produces an insulin-like peptide hormone that controls the development of primary and secondary male sex characteristics 37–40. This may explain why the imposition of the male sex characters in hermit crab females was not observed, unlike gastropod mollusks. In these organisms, TBT acts as an endocrine disruptor and leads to the imposition of male sex organs (penis or vas deferens and prostate tissue) in female gastropods 41 because of aromatase competition, disrupted testosterone conjugation, neuropeptide interaction, and interference with receptors 42–44.

As mentioned, TBT use in antifouling paints was banned in many parts of the world 2, 16, 20, 21, but the presence of high concentrations of butyltin compounds in the environment is still recorded in different locations 19, 45. This may explain the continuing occurrence of imposex development in gastropods 15, 46 as well as other effects on the biota, such as those demonstrated here.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

With the experimental design of the present study, we can conclude that TBT has a toxic effect on the female reproductive system of the hermit crab C. vittatus, which showed ovarian disorganization and atrophy. This impact may result in reductions of fertility and embryo success, and even population decreases in the long term.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

The authors thank the Foundation for Research Support of the State of São Paulo, F. J. Zara (proc. 2005/04707-5; Alexander Turra proc. 2006/57007-3,) for financial support, the National Council of Scientific and Technological Development (proc. 301240/2006-0; proc. 308215/2010-9), and Foundation for Research Support of the State of São Paulo (proc. 2006/61589-8) for scholarships to A. Turra, F. J. Zara, and B. S. Sant'Anna, respectively. Thanks to Oceanographer R. Haruo Ota and graduate students S. Cardoso de Souza and D. Corsino Sandron for technical support, and J. Reid (JWR Associates) for editing the English text.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES
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