Evaluation of the lethal and sub-lethal toxicity and potential endocrine disrupting effect of nonylphenol on the zebra mussel (Dreissena polymorpha)

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Abstract

Nonylphenol (NP) is commonly found in surface waters nearby municipal wastewater treatment plants and was shown to have endocrine disrupting effects in aquatic organisms. The purpose of this study was to investigate the toxicity and potential endocrine disrupting effects of NP on the freshwater zebra mussel (Dreissena polymorpha). Toxicity assessment yielded LC50 values of 3.68, 2.19 and 1.62 mg L 1 after 15, 35 and 50 days of exposure, respectively. LC10 values of 1.6, 1.11 and 0.68 mg L 1 were respectively obtained for similar exposure periods. At concentrations > 5 mg L 1, mortality effects were significant, as were those relating to attachment and siphon extension (indicating filtration), both general indicators of health. Endocrine disruption effects were investigated after a prolonged exposure (112 d) to 5 and 500 μg L 1 NP by measuring Vitellin (Vn)-like protein levels using the alkali-labile phosphate (ALP) assay and gel electrophoresis (GE). An increase in ALP levels was observed in both male and female mussels, although only marginal owing to a significant decrease in the mussels' health indicated by its condition, during the experiment. These levels, however, increased proportionally with NP concentration. Using solid phase thin-layer chromatography, we confirmed increased levels of the steroid cholesterol and evidence of NP uptake. Cholesterol levels in gonad tissue proved to be a more responsive biomarker of exposure to NP than levels of ALP. Further implications relating to the occurrence of endocrine disruption in the zebra mussel are discussed.

Introduction

Nonylphenol (NP) is one of a group of chemicals known as oestrogen mimicking compounds or xeno-oestrogens. These chemicals interfere with the action of an animal's endogenous hormones by binding to the oestrogen receptor and eliciting a biological response resulting in endocrine disruption (Jobling et al., 1996, Giesy et al., 2000). NP is formed by the breakdown of alkylphenol ethoxylates, non-ionic surfactants used in the formulation of detergents, pesticides and emulsion paints and is commonly released into the environment mainly via sewage effluent (for a review, see Montgomery-Brown and Reinhard, 2003). This compound is generally found at concentrations < 0.1 μg L 1 in municipal effluent and in environments polluted by such discharges (Blackburn and Waldock, 1995), but has been found at concentrations up to hundreds of micrograms per liter, high enough to elicit a biological endocrine disrupting response (Bennie, 1999).

The endocrine disrupting effects of these non-ionic surfactants in the aquatic environment are particularly well documented in studies conducted with teleost fish species. Exposures to sewage discharges containing alkylphenols including NP resulted in the stimulation of the synthesis of the female egg yolk precursor protein vitellogenin (Vtg) in male fish and the inhibition of testicular growth (Jobling et al., 1996, Ashfield et al., 1998, Giesy et al., 2000). A significant elevation of Vtg levels in male fish is now recognised as a highly specific biomarker of exposure to xeno-estrogens. Direct exposure of fish to NP in laboratory tests has resulted in significant effects on the growth and ovosomatic index, Vtg induction, inhibition of testicular growth, intersex and a reduction in somatic growth and the gonado-somatic index (for a summary, see Vos et al., 2000). In another study with the eelpout Zoarces viviparus, nonylphenol was able to increase vitellogenins in pregnant females as well as in the embryos (Korsgaard and Pedersen, 1998). Thus, evidence of endocrine disruption (ED) has been extensively documented and is presently considered a potential threat for the reproductive efficiency of fish populations. NP was also shown to decrease muscarinic acetyl cholinergic receptors in the brain of cold-water salmonids species as with copper, carbaryl and permethrin (Jones et al., 1998).

Despite their obvious ecological importance, relatively little ED research has been carried out on invertebrates, with the exception of imposex (the imposition of the male sex organs including a penis and vas deferens on female reproductive organs) in gastropod molluscs exposed to organotin compounds (for a review, see Matthiessen and Gibbs, 1998). However, some evidence of suspected ED was found in the ovotestes in male lobster in feral populations exposed to sewage effluent (Sangalang and Jones, 1997) and the stimulation of secondary sexual characteristics in Daphnia exposed to diethylstilbestrol (Olmstead and LeBlanc, 2000). Exposure to NP has resulted in accumulation of endogenous testosterone in Daphnia magna (Baldwin and LeBlanc, 1994, Baldwin et al., 1997), reduced fecundity in adult female Daphnia (Shurin and Dodson, 1997) and increased levels of cyprid major protein in barnacle (Balanus amphitrite) larvae (Billinghurst et al., 2000). Recent controversy has emerged regarding what constitutes an endocrine disrupting effect in invertebrates. Although the different effects described above were observed after exposure to EDCs, these effects could be normal toxicological responses (i.e., related to narcosis), not necessarily receptor-mediated.

Although relatively little is known about the endocrine system of invertebrates, the use of hormones to control and co-ordinate biochemical, physiological and behavioural processes is common to all invertebrate taxa (DeFur et al., 1999). Most invertebrate species studied have vertebrate-type hormones (testosterone and estrogen) in measurable quantities (Baldwin and LeBlanc, 1994, DeLongcamp et al., 1974, Reis-Henriques et al., 1990) and an endocrine pathway that is theoretically susceptible to disruption (DeFur et al., 1999). In bivalves, vitellogenesis occurs in the gonad where the oocytes are believed to produce yolk proteins autosynthetically (Pipe, 1987) through a process induced and regulated by estrogens (Li et al., 1998). This process appears to be susceptible to ED as bivalves reportedly respond to exposure to estrogens and xeno-estrogens in a manner similar to fish, by increasing levels of vitellogenin (Vtg) or vitellin (Vn)-like proteins (major protein found in oocytes of invertebrates synthesised from vitellogenin). This response has been observed in both male and female clams (Blaise et al., 1999), mussels (Gagné et al., 2001a) and oysters (Li et al., 1998) exposed to estradiol, municipal effluent and NP, respectively, and more recently in zebra mussels exposed to tertiary treated municipal effluent (Quinn et al., 2004).

As with the ED effect, most research regarding the toxicity of NP has also focused on vertebrates, particularly fish, with LC50 values reported between 17 and 3000 μg L 1 (for a review, see Servos, 1999). Median lethal effect concentrations of NP have also been calculated for several invertebrate species (Table 1). Fish seem to be more susceptible to the lethal effects of NP than crustaceans, while bivalves were considerably more resistant with recorded LC50 values ranging between 500 and 5000 μg L 1 (Servos, 1999). Sublethal effects including a reduction in taxon richness of zooplankton exposed to > 30 μg L 1 NP (Severin et al., 2003) and a significant decrease in clearance rate and scope for growth in the clam Tapes philippinarum at 100 and 200 μg L 1 (Marcial et al., 2003) have also been reported. The freshwater bivalve Dreissena polymorpha, commonly known as the zebra mussel, is largely regarded as a pest species. It is found biofouling hard substrates in Europe and in North America where it was recently introduced. Its high filtration rate, ability to accumulate toxicants and widespread distribution and abundance has made the zebra mussel a valuable bio-indicator species (Lafontaine et al., 2000). It has even been proposed as the counterpart of the blue mussel Mytilus edulis in mussel watch programmes for freshwater environments (Mersch et al., 1992). Zebra mussels have been used in both passive and active bio-monitoring for heavy metals (Lecoeur et al., 2004) and organic contaminants (Dauberschmidt et al., 1997) and are regarded as sentinel organisms for monitoring environmental quality.

In the present study, the effects of exposure to NP were investigated on the zebra mussel. We initially established its LC50 and assessed its sub-lethal effects on attachment and siphon extension (indicating feeding). Afterwards, environmentally relevant (5 μg L 1) and elevated (500 μg L 1) concentrations of NP were employed to investigate the possible ED effects of NP on the zebra mussel. In this second exposure, (1) the influence of NP on levels of Vn-like proteins in both male and female mussels using an indirect alkali-labile phosphate (ALP) method and gel electrophoresis (GE) was appraised; (2) uptake and bioaccumulation of NP and its effect on cholesterol levels in the mussels were measured using high-performance thin-layer chromatography (HPTLC); (3) condition indices were determined to measure the overall impact of exposure to NP on zebra mussels. Moreover, the reproductive period in bivalves is purported as the life cycle stage that could be particularly sensitive to endocrine disturbance (Mori et al., 1969). For this reason, a prolonged exposure to NP (112 days) was undertaken between mid-January and mid-May to coincide with the onset of early gametogenesis (i.e., the development of spermatocytes or ovocytes in pre-vitellogenesis).

Section snippets

NP test solution

Working solutions were prepared by dissolving NP (4-n-nonylphenol, 98+% purity Lancaster Synthesis Ltd., U.K.) in the carrier solvent ethyl acetate (99.9%, Romia Chemicals). For lethality experiments, the nominal NP concentrations used were 0.1, 1.0, 5.0, 10.0 mg L 1 NP. For the second set of experiments, hereafter referred to as the ED exposure, concentrations of 5 and 500 μg L 1 NP were employed. A control tank and solvent control tank were added in each experiment. In both experiments, 250

Lethal effects

Zebra mussels showed chronic responses after exposure to NP with significant effects on mortality, attachment and siphon extension at the highest concentrations of 5 and 10 mg L 1 (Table 2). After 15 days of exposure, an LC50 of 3.68 mg L 1 was calculated, falling to 2.19 and 1.62 mg L 1 after 35 and 50 days of exposure, respectively (Fig. 1). The LC10 for the same time periods were 1.6 (2.3-fold lower than the LC50), 1.11 (2-fold lower than the LC50) and 0.68 (2.4-fold lower than the LC50) mg L

Discussion

The NP LC50 for the zebra mussel D. polymorpha was somewhat high (3.68 and 2.19 mg L 1 for 15 and 35 days exposure, respectively) when compared to LC50 values for other invertebrate species (Table 1). The marine blue mussel M. edulis, often used in comparative studies with the zebra mussel, yielded lower LC50s measuring 3.0, 0.5 and 0.14 mg L 1 (nominal concentration) after 4 days, 15 days and 35 days of exposure, respectively (Granmo et al., 1989). Based on previous experiments, it would

Conclusions

Although zebra mussels appear to be less sensitive than other species to the toxic effects of NP, an LC10 threshold level concentration of 0.68 mg L 1 was established. When taking NP degradation and a factor of 10 used in risk management into account, this threshold approaches the maximum levels of NP reported in the environment. Although not acutely toxic to zebra mussels, NP is likely to cause sub-lethal effects, thereby reducing the fitness of mussel populations in the wild. It is possible

Acknowledgements

The first author thanks Peter Stafford (Trinity College Dublin) for his help in setting up the in vivo experiments.

References (55)

  • F. Gagné et al.

    Estrogenic properties of municipal and industrial waste waters evaluated with a rapid and sensitive chemoluminescent in situ hybridization assay (CISH) in rainbow trout hepatocytes

    Aquat. Toxicol.

    (1998)
  • F. Gagné et al.

    Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata

    Comp. Biochem. Physiol. C

    (2001)
  • F. Gagné et al.

    Evidence of coprostanol estrogenicity to the freshwater mussel Elliptio complanata

    Environ. Pollut.

    (2001)
  • F. Gagné et al.

    Alteration of the biochemical properties of female gonads and vitellins in the clam Mya arenaria at contaminated sites in the Saguenay fjord

    Mar. Environ. Res.

    (2002)
  • L. Giamberini et al.

    The pericardial glands of the zebra mussel: ultrastructure and implication in lead detoxification process

    Biol. Cell

    (1996)
  • A. Granmo et al.

    Lethal and sublethal toxicity of 4-nonylphenol to the common mussel (Mytilus edulis L.)

    Environ. Pollut.

    (1989)
  • S.B. Jones et al.

    Effects of carbaryl, permethrin, 4-nonylphenol and copper on muscarinic cholinergic receptors in brain of surrogate and listed fish species

    Comp. Biochem. Physiol.

    (1998)
  • B. Korsgaard et al.

    Vitellogenin in Zoarces viviparus: purification, quantification by ELISA and induction by estradiol-17 beta and 4-nonylphenol

    Comp. Biochem. Physiol.

    (1998)
  • S. Lecoeur et al.

    Evaluation of metallothionein as a biomarker of single and combined Cd/Cu exposure in Dreissena polymorpha

    Environ. Res.

    (2004)
  • V. Matozzo et al.

    Evaluation of 4-nonylphenol toxicity in the clam Tapes philippinarum

    Environ. Res.

    (2003)
  • D.W. McLeese et al.

    Lethality of aminocarb and the components of the animocarb formulation to juvenile Atlantic salmon, marine invertebrates and a freshwater clam

    Chemosphere

    (1980)
  • B. Quinn et al.

    The endocrine disrupting effect of municipal effluent on the zebra mussel (Dreissena polymorpha)

    Aquat. Toxicol.

    (2004)
  • M.A. Reis-Henriques et al.

    Studies of endogenous steroids from the marine mollusc Mytilus edulis by gas chromatography and mass spectrometry

    Comp. Biochem. Physiol.

    (1990)
  • M.G. Stanton

    Colorimetric determination of inorganic phosphate in the presence of biological material and adenosine triphosphate

    Anal. Biochem.

    (1968)
  • L.A. Ashfield et al.

    Exposure of female juvenile rainbow trout to alkylphenolic compounds results in modifications to growth and ovosomatic index

    Environ. Toxicol. Chem.

    (1998)
  • W.S. Baldwin et al.

    Metabolic androgenization of female Daphnia magna by the xenoestrogen 4-nonylphenol

    Environ. Toxicol. Chem.

    (1997)
  • D.T. Bennie

    Review of the environmental occurrence of alkylphenols and alkylphenol ethoxylates

    Water Qual. Res. J. Can.

    (1999)
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