The effects of polycyclic aromatic hydrocarbon exposure on the fertilization and larval development of the Pacific oyster, Crassostrea gigas.

ABSTRACT To investigate the polycyclic aromatic hydrocarbon (PAH) effect on fertilization of the Pacific oyster, Crassostrea gigas, we exposed the adult brood stocks to PAHs (200 ppb) for 30 days as the treated (E group) with a nonexposed group as the control (C group). In addition, each subset of the gametes from both groups was further exposed to graded level of PAHs (0, 50, 100, and 200 ppb). Motile sperm and linearity were significantly affected by parental exposure, whereas straight line velocity (VSL) and curvilinear velocity (VCL) were affected by both of parental and larval exposure to PAHs. Fertilization and larval development were also affected by parental exposure. Our result suggests that fertilization capability and larval development are adversely affected by parental exposure whereas sperm movement was more vulnerable to larval exposure. In the brood stock contaminated by PAHs, larval development was also vulnerable to larval exposure to PAHs.

KEY WORDS: Crassostrea gigas, fertilization, larval development, polycyclic aromatic hydrocarbons

INTRODUCTION

Organic contamination has been a great threat to coastal oyster farming. These pollutants can be accumulated in tissues and impair energy reserve for the physiologic function as well as for reproduction (Encomio & Chu 2000). Among the organic contaminant, polycyclic aromatic hydrocarbons (PAHs) are major pollutants of the estuarine and coastal environment, and have the characteristics of persistent, carcinogenic, and antiestrogenic effects on marine organisms (Anderson et al. 1996, Monteiro et al. 2000b).

In general, PAHs do not show high acute toxicity to aquatic animals because of their water solubility limits but deposit in lipid reserve organs because of their lipophilic characteristic. The uptake and storage of PAHs in depot lipids, such as energy reserves or gonadal reserves, may be retained until lipid reserves are mobilized for nutritional or reproductive needs (Neff 1979). Chronic exposure or UV light interaction with PAHs can cause a greater hazard to aquatic organisms (Seagrave & Burchiel 2000, Lyons et al. 2002).

The high deposit of organic compound in reproductive tissue may have profound implications for reproduction and subsequent embryo development (Strandberg et al. 1998). Au et al. (2000) has reported that pollution may impair the reproductive success of the adult by decreasing the quality and/or quantity of the gametes, which may in turn affect the fertilization success, embryo development, and larval viability.

PAHs are also known to have a deleterious effect on the vitellogenesis of fish (Janssen et al. 1995, Nicolas 1999), the process through which maturing oocytes in the ovary accumulate yolk. The reported effects include the alteration in circulating hormones and plasma vitellogenin, estrogenic and antiestorgenic effects, retardation of oocyte maturation, and the reduction of reproductive success in fish (Nicolas 1999). Oviparous invertebrates can synthesize the vitellin within the ovary (Li et al. 1998, Won et al. 1999, Eckelbarger & Davis 1996). Although their endocrine regulations are still unknown, sexual steroids have been found in bivalves (Mori et al. 1969, Li et al. 1998). It is possible that the hormone homeostasis for regulating the reproductive cycle can be disrupted by pollutants (Gagne et al. 2001). Therefore a possible implication can be established that PAHs adversely affects the reproductive cycle and further larval development.

In this study, we evaluated the effect of PAHs on sperm activities, fertilization, and larval development of the Pacific oyster, Crassostrea gigas via parental and larval exposure.

MATERIALS AND METHODS

Experimental Animals

Conditioned C. gigas brood stock was taken from a relative pristine oyster farm around Tongyeong, Korea in May 2003 (Fig. 1). After conditioning for 2 days without feed, the oysters were allocated to four tanks and raised in flow through tanks with an introduction of PAH cocktail at 200 ppb (E groups) to test the effect of parental PAH exposure and a non-exposed group as the control (C groups) for 30 days. Control groups were introduced the same amount of acetone with E groups. Raised oysters were fed on equivalently mixed marine microalgae during the experiment for ripening: Chaetoceros simplex, C. gracilis, Isochrysis galbana, and Tetraselmis tetrathele. After confirmation of being fully ripened, the gametes from each group were taken for further experiment with 4 graded PAHs levels (0, 50, 100, and 200 ppb) at each group, therefore, the experimental groups included 8 groups.

[FIGURE 1 OMITTED]

The stock solution of the PAHs cocktail was prepared by the equivalent amount of 10 species PAHs with acetone; Acenaphthene, Acenaphthylene, Benz(a)anthracene, Benzo(a)pyrene, Chrysene, Dibenz(a,h)anthracene, Fluoranthene, Fluorene, Phenanthrene, and Pyrene. These were obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan).

Sperm Movements

Sperm for the test was removed with a clean Pasteur pipette, which was inserted beneath the epidermis of the gonad. Around 2 cm of sperm in the narrow end of Pasteur pipette was transferred into 5-10 mL of seawater with a gentle suck and blow. After checking for cross contamination, the sperm was tested further.

The motility of the sperm in the eight experimental groups was recorded. Collected sperm was diluted to graded level of PAHs (0, 50, 100, and 200 ppb) with filtered seawater (density ca. x [10.sup.7] [ml.sup.-1]). After a 10-min incubation, subsamples (50 [micro]L) were taken at 1-, 30-, 60-, 150-min intervals and mounted on a Neubauer slide. Two microscopic fields were examined and recorded for 6 sec using a light microscope (Olympus, BX-50, 10 x 40 magnification) with a computer controlled CCD camera (JVC digital camera KY-F1030). The following parameters of individual sperm were assessed using a computer based image analysis program (ImagePro, Media Cybermetics, Inc.): the percent of motile sperm; curvilinear velocity (VCL) ([micro][m.sup.-1]), the time-average velocity of the sperm head along its actual trajectory; straight line velocity (VSL) ([micro][m.sup.-1]), time-average velocity of the sperm head along a straight line between its first detected position and its last position; Linearity, VSL/VCL.

Fertilization and Larval Development

The female gonad was stripped (Thian 1991) and submerged in 1 L of filtered seawater (through 0.2-[micro]m Whatman filters) and squeezed gently to liberate eggs. The eggs were washed through a combination of both 80 and 40 wm screens and rinsed 4 times with filtered seawater. Eggs from C and E groups were allocated at a density of approximately 4,000 eggs/mL to 8 tanks of duplicate 4 subgroups with graded PAH (0, 50, 100, and 200 ppb) and retained them for 30 min at 20[degrees] to allow them to swell. Cross-contamination was carefully checked under a microscope before they were allowed to be tested.

To test the fertilization capability, sperm and egg were mixed with the ratio of no more than 10 sperms surrounding each egg to prevent multiple fertilization. The gametes were deposited in separate vessels containing stock filtered seawater with graded PAHs concentration (0, 50, 100, and 200 ppb for both C and E groups). The mixture had been left for an hour for fertilization and then, once fertilization had occurred, excessive sperm was immediately removed by sieving the gamete suspension through a 40-[micro]m screen and rinsed twice with filtered seawater. One milliliter of the embryo suspension was subsampled by a clean Pasteur pipette before introducing it into the larvae tank. The subsample was introduced into the same amount of 20% buffered formalin fixative for further microscopic examination.

For the larval development test, fertilized eggs were stocked in 5-L plastic tanks with graded PAHs levels (0, 50, 100, and 200 ppb) at a density of 100 embryos/mL at 20[degrees]C. After 48 h, D-shaped larvae were measured under a light microscope (Olympus BX-50).

Statistics

Mean and standard deviation were calculated from the duplicate. Statistical analysis was performed with SPSS statistics package (SPSS Inc.). For the group comparisons, a 1-way analysis of variance (ANOVA) with eight experimental groups was calculated. Post-test were performed by Tukey-HSD with the eight groups at the 95% level.

RESULTS

Sperm Movements

The results of sperm movements were represented in Figure 2. Sperm motility ranged from 30.7% to 63.3% in C groups and 20.5% to 64.9% in E groups with significant difference along with parental exposure (P < 0.05). The motility of CO and C50 were relatively constant during the recording. However, in the other groups, the motility significantly decreased at the end of the recording. In the C groups, the motility of CO was significantly higher than other larval exposed group of control (C50, C100, and C200) and a larval exposed level dependent decrease was observed at the end of the recording (P < 0.05). On the other hand, in the E groups, a larval exposed level dependent decrease was not observed with the lowest at E50 and E0 was not always higher than any other exposed groups (P < 0.05).

[FIGURE 2 OMITTED]

VCL and VSL decreased from the beginning of recording in all groups. The activities in CO and E0 were always higher than any of the other groups in both VCL and VSL (P < 0.05). Although exposed level dependent correlations were not observed, the sperm activities were highly affected by larval exposure to PAHs (P < 0.05). Linearity of the C groups was not decreased during the recording with fluctuation between 0.6 and 0.9, those of the E group, however, were significantly decreased from 0.8-0.9 to 0.40.5 at the end of recording (P < 0.05).

Fertilization and Larval Development

The results of fertilization and D-shaped larval development were represented in Figure 3. The fertilization of the E group at 1 h was significantly lower than that of the C groups (P < 0.05): 51.1% to 68.6% in the C groups, whereas, 7.4% to 26.4% in the E groups. Larval exposed level dependent fertilization decrease was observed in the E groups with [r.sup.2] = 0.971 but not in the C groups ([r.sup.2] = 0.771) (P < 0.05).

[FIGURE 3 OMITTED]

The percent of D-shaped larvae development at 48 h in the C groups ranged from 95.3% to 96.9%, whereas in the E groups ranges were from 57.1% to 68.3%. The percent of D-shaped larvae in the E groups was significantly decreased with an average of 30% to 40% compared with the C groups (95.3% to 96.9%, P < 0.05). For larval exposure, a significant dose-dependent effect was observed in the E group although not being in the C groups (P < 0.05).

DISCUSSION

In vitro screening method using animal spermatozoa has developed rapidly as a simple and valid model for toxicity test and it is highly applied to assess the toxicity of compounds for humans (Petzoldt et al. 1985, Bavister & Andrews 1988, Slott et al. 1993), fish (Kime et al. 1996, Rurangwa et al. 1998), and sea urchins (Dinnel et al. 1989, Geffard et al. 2001, Au et al. 2000). Several authors demonstrated that the sperm activities of marine invertebrate are highly affected by toxicants (Hamoutene et al. 2000, Au et al. 2000, Bellas et al. 2001, Geffard et al. 2001). Although Geffard et al. (2001) suggested that the sea urchin was a more effective tool for spermiotoxicity than spermatozoa of Crassostrea gigas for highly contaminated sediment with heavy metals and PAHs, the pacific oyster is one of the useful tools for spermiotoxicity in marine environmental biomonitoring.

In this study, the movements of the sperm were highly affected by both parental and larval exposure to PAHs. Laval exposure to PAHs affected the viability of sperm with a significant decrease of motility at the end of recording, and parental exposure on the movements with a significant decrease of linearity at the end of recording at only E groups. This was inconsistent with Rurangwa et al. (2002) who reported that TBT exposed fish spermatozoa did not show any difference in linearity of sperm movement. The effects of larval exposed level on the linearity were negligible (P < 0.05). Ellis et al. (1993) demonstrated that eggs and sperm contain significantly more PAHs than somatic tissues in Crassostrea virginica from the survey of Galveston Bay, and oysters may lose up to 50% of their body burden of certain PAHs in a single spawn. Gonad, therefore, is one of the most impacted organs in oysters. In human semen studies, ambient pollution can affect on the semen quality with DNA adducts, morphologic alteration, and decreased motile sperm (Sram et al., 1999). It is evident that many toxicants can affect the ATP content (Vega et al. 1996, Rurangwa et al. 2002). Also, PAHs have been known to affect the cellular-calcium homeostasis that have an influence on cellular signal pathway and to deplete the cellular ATP content (Seagrave & Burchiel 2000). Sperm motility depends mainly on the endogenous ATP level before activation (Perchec et al. 1995) and is closely associated with sperm morphology (Woods & Garside 1996). Therefore the decreased mobility and linearity in parentally exposed groups might be caused by a morphologic alteration and depleted ATP content in the sperm.

The fertilization capability at 1 h showed significant difference between the C and E groups (P < 0.05). Our result was relatively lower than other studies (Nice et al. 2000, Lyons et al. 2002) but higher than that of Wintermyer & Cooper (2000). After 48 h, however, the D-shaped larvae in the C groups reached 95.3% to 96.9% similar to those of other reports with strip spawning (Nice et al. 2000, Lyons et al. 2002), but 57.1% to 68.3% of D-shaped larvae in E groups were significantly lower than the C groups. This implicates that parental exposure to PAHs can adversely affect the fertilization of eggs as well as the larval development.

Some of PAHs are known for their antiestrogenic effect on fish (Nicolas 1999, Monteiro et al. 2000a, Monteiro et al. 2000b, Navas & Segner 2000). Gauthier-Clerc et al. (2002) suggested that a delayed gametogenesis of Mya arenaria in the Saguenay fjord (Canada), which is well known for its multiple contaminations (PAHs, metals and so on), is caused by persistent dysfunction of a vitellogenic process due to the exposure to antiestorgenic contaminants.

In this study fertilization and larval development in the C group were not decreased by larval exposure, whereas, in the E group, they were significantly decreased by larval exposure. This implicates that larvae from contaminated broodstocks can be more vulnerable to contamination when the gamete or larvae is liberated to surrounding waters.

In conclusion, exposure to PAHs of adult brood stock can cause a breakdown in successive reproduction with decreased sperm motility and larval development. When larvae are exposed to PAHs, furthermore, they are more vulnerable if the brood stock were previously exposed to PAHs.

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WOO-GEON JEONG AND SANG-MAN CHO *

Institute of Marine Industry & Division of Marine Bioscience, College of Marine Science, Gyeongsang National University, 445, Inpyeong, Tongyeong, 650-160, Korea

* Corresponding author. E-mail: [e-mail]