Assessing the impact of diclofenac, ibuprofen and sildenafil citrate (Viagra®) on the fertilisation biology of broadcast spawning marine invertebrates.

Exposure to synthetic chemicals is a key environmental challenge faced by aquatic organisms. The time and dose effects of the pharmaceuticals diclofenac, ibuprofen, and sildenafil citrate on sperm motility and successful fertilisation are studied using the echinoderms, Asterias rubens and Psammechinus miliaris, and the polychaete worm Arenicola marina, all important components of the marine benthos. Motility was reduced for all species when exposed to diclofenac concentrations ≥0.1 µg/L. Exposure to ≥1.0 µg/L of ibuprofen affected only P. miliaris gametes and fertilisation success of A. marina. A. rubens and P. miliaris sperm increased in both percentage motility and swimming velocity when exposed to sildenafil citrate at concentrations ≥18 and ≥ 50 ng/L, respectively. Pre-incubation of sperm with sildenafil citrate significantly increased fertilisation success in A. rubens and P. miliaris but not in A. marina. Pre-incubated A. rubens oocytes fertilised successfully in ibuprofen. According to EU Directive 93/67/EEC, diclofenac is classified as a very toxic substance to gametes of A. rubens, P. miliaris, and A. marina (EC50 = 100-1000 µg/L) while ibuprofen is classified as very toxic to gametes of P. miliaris but non-toxic to gametes of A. marina (EC50 > 10,000 µg/L). The present study indicates that diclofenac exposure may have negative impacts on invertebrate reproductive success, whereas ibuprofen potentially may compromise P. miliaris reproduction. This study provides a valuable insight into the mechanisms that allow marine invertebrates to survive and reproduce in contaminated and changing habitats.

Response of copepods to elevated pCO2 and environmental copper as co-stressors--a multigenerational study.

We examined the impacts of ocean acidification and copper as co-stressors on the reproduction and population level responses of the benthic copepod Tisbe battagliai across two generations. Naupliar production, growth, and cuticle elemental composition were determined for four pH values: 8.06 (control); 7.95; 7.82; 7.67, with copper addition to concentrations equivalent to those in benthic pore waters. An additive synergistic effect was observed; the decline in naupliar production was greater with added copper at decreasing pH than for decreasing pH alone. Naupliar production modelled for the two generations revealed a negative synergistic impact between ocean acidification and environmentally relevant copper concentrations. Conversely, copper addition enhanced copepod growth, with larger copepods produced at each pH compared to the impact of pH alone. Copepod digests revealed significantly reduced cuticle concentrations of sulphur, phosphorus and calcium under decreasing pH; further, copper uptake increased to toxic levels that lead to reduced naupliar production. These data suggest that ocean acidification will enhance copper bioavailability, resulting in larger, but less fecund individuals that may have an overall detrimental outcome for copepod populations.

The relevance of marine chemical ecology to plankton and ecosystem function: an emerging field.

Marine chemical ecology comprises the study of the production and interaction of bioactive molecules affecting organism behavior and function. Here we focus on bioactive compounds and interactions associated with phytoplankton, particularly bloom-forming diatoms, prymnesiophytes and dinoflagellates. Planktonic bioactive metabolites are structurally and functionally diverse and some may have multiple simultaneous functions including roles in chemical defense (antipredator, allelopathic and antibacterial compounds), and/or cell-to-cell signaling (e.g., polyunsaturated aldehydes (PUAs) of diatoms). Among inducible chemical defenses in response to grazing, there is high species-specific variability in the effects on grazers, ranging from severe physical incapacitation and/or death to no apparent physiological response, depending on predator susceptibility and detoxification capability. Most bioactive compounds are present in very low concentrations, in both the producing organism and the surrounding aqueous medium. Furthermore, bioactivity may be subject to synergistic interactions with other natural and anthropogenic environmental toxicants. Most, if not all phycotoxins are classic secondary metabolites, but many other bioactive metabolites are simple molecules derived from primary metabolism (e.g., PUAs in diatoms, dimethylsulfoniopropionate (DMSP) in prymnesiophytes). Producing cells do not seem to suffer physiological impact due to their synthesis. Functional genome sequence data and gene expression analysis will provide insights into regulatory and metabolic pathways in producer organisms, as well as identification of mechanisms of action in target organisms. Understanding chemical ecological responses to environmental triggers and chemically-mediated species interactions will help define crucial chemical and molecular processes that help maintain biodiversity and ecosystem functionality.

Exposure to copper and a cytotoxic polyunsaturated aldehyde induces reproductive failure in the marine polychaete Nereis virens (Sars).

A number of metabolites from microalgae, including polyunsaturated aldehydes (PUAs), have been implicated as inducers of reproductive failure in aquatic invertebrates. Current work describes the impacts of the model PUA 2E, 4E-decadienal and copper sulphate applied in isolation and combination on the reproductive performance of the infaunal polychaete, Nereis virens (Sars). The reproductive and life cycle parameters investigated were; fertilisation success, larval survival, sperm motility (percent motility and curvilinear velocity) and sperm DNA damage. Exposure to decadienal and copper sulphate in isolation resulted in dose- and time-dependent reductions for each evaluated endpoint. Fertilisation success was heavily impacted at concentrations of up to 10µM for both compounds. Copper sulphate was more toxic in larval survival assays. Sperm motility impacts, although variable, exhibited rapid onset with pronounced reductions in sperm swimming performance observed within 3min of exposure. The extent of DNA damage was dose-dependent, and in the case of decadienal, rapid in onset. Dual compound exposures resulted in enhanced overall toxicity in all assays. Logistic regression analysis of fertilisation and larval survival assays showed significant synergistic interactions between decadienal and copper sulphate; an increase in concentration of either compound resulted in enhanced toxicity of the other. Longer exposure durations during larval survival assays demonstrated a further increase in both toxicity and synergism. The results indicate that the effects of additional environmental stressors must be considered when attempting to extrapolate laboratory-derived single compound exposures to field situations.

Effects of 4-nonylphenol on the endocrine system of the shore crab, Carcinus maenas.

There is a considerable body of evidence to suggest that many anthropogenic chemicals, most notably xeno-estrogens, are able to disrupt the endocrine system of vertebrates. There have been few comparable studies on the effects of exposure to these chemicals that may serve as biomarkers of endocrine disruption in aquatic invertebrate species. In addition, the evidence available is complex, conflicting, and far from conclusive. The present study aimed to investigate the impact of the xeno-estrogen 4-nonylphenol (4-NP, nominal concentrations 10-100 microg L(-1)) on the regulation and functioning of the endocrine system of the shore crab Carcinus maenas. It also set out to establish whether 4-NP are causing the effects (i.e., changes of exoskeletons including secondary sexual characteristics, pheromonally mediated behavior and ecdysone levels, and the presence of vt in the male hepatopancreas) found recently in wild shore crabs (Lye et al.,2005). The study utilizes morphological (e.g., gonadosomatic and hepatosomatic indices) and hormonal (ecdysteroid moulting hormone levels and the induction of female specific proteins, vitellins) biomarkers using radioimmunoassay and an indirect enzyme linked immunosorbent assay applied to the soluble protein fraction of adult male hepatopancreatic homogenates. Exposure of C. maenas to an effective concentration as low as 1.5 microg L(-1) 4-NP resulted in a reduced testis weight, increased liver weight, and altered levels of ecdysone equivalents compared to controls. Induction of vitellin-like proteins was absent in all samples tested. The ecological implications and the possible mechanisms for the action of 4-NP on the response of the shore crab to xeno-estrogen exposure are discussed.

Toxicity of algal-derived aldehydes to two invertebrate species: do heavy metal pollutants have a synergistic effect?

The recent discovery of the production of anti-proliferative aldehydes in a variety of microalgal species has lead to considerable investigation into the effects of these toxins on aquatic invertebrates. Studies have, however, rarely considered the impact pollutants may have on grazer responses to algal toxins. In this study, the acute toxicities of five aldehydes to the rotifer Brachionus plicatilis and nauplii of the brine shrimp Artemia salina are examined using immersion assays. In addition, the effect of a representative of these aldehydes in the presence of sub-lethal levels of heavy metals was examined. B. plicatilis generally showed greater sensitivity to the aldehydes than A. salina. The polyunsaturated 2-trans,4-trans-decadienal was the most toxic to both species having 24h LD(50) values of 7 and 20 microM for B. plicatilis and A. salina, respectively. The remaining aldehydes had different orders of toxicity for the two species with a stronger relationship observed between mortality and aldehyde carbon-chain length for A. salina whereas B. plicatilis mortality showed a stronger dependence on the presence of carbon-carbon double bonds in the aldehydes. The presence of 1 microM of copper sulphate in solutions of decadienal resulted in the reduction of the 24h LD(50) of decadienal by approximately a third for both species. 1 microM of copper chloride in solutions of decadienal reduced the 24h LD(50) of decadienal to A. salina nauplii by approximately 11% and 1 microM zinc sulphate caused a reduction of only 3%. Pre-exposure of the organisms to 1 microM copper sulphate had no significant impact on their subsequent mortality in decadienal. The ecological implications and the possible mechanisms for the action of copper sulphate on the response of organisms to decadienal are discussed.

Exposure to 2,4-decadienal negatively impacts upon marine invertebrate larval fitness.

Diatoms liberate volatile, biologically active unsaturated aldehydes following cell damage, which negatively impact upon invertebrate reproductive processes such as fertilization, embryogenesis and larval survival. 2,4-Decadienal is frequently identified among the aldehydes produced and is one of the more biologically active. The majority of studies which have examined the toxic effects of diatom aldehydes to invertebrate reproduction have scored egg production and/or hatching success as indicators of biological impacts. There are very few studies which have dealt specifically with the impacts of diatom-derived aldehydes on larval fitness. Larval stages of the polychaetes Arenicola marina and Nereis virens and the echinoderms Asterias rubens and Psammechinus miliaris exposed to 2,4-decadienal at sub 1 microg ml(-1) concentrations suffered reduced survival over the incubation period (day 1-8 post fertilization) with detectable differences for the polychates at a concentration of 0.005 and 0.01-0.1 microg ml(-1) for the echinoderms. Susceptibility of larval N. virens was investigated using stage specific 24 h exposures at 2,4-decadienal concentrations up to 1.5 microg ml(-1). A clear stage specific effect was found, with earlier larval stages most vulnerable. Nectochaete larvae (9-10 d) showed no reduction in survival at the concentrations assayed. Fluctuating asymmetry (FA), defined as random deviations from perfect bilateral symmetry, was used to analyse fitness of larval P. miliaris exposed to 2,4-decadienal at concentrations of 0.1, 0.5 and 1 microg ml(-1). The degree and frequency of asymmetrical development increased with increasing 2,4-decadienal concentration. Equally, as FA increased larval survival decreased. These results provide further support for the teratogenic nature of 2,4-decadienal and its negative impact on invertebrate larval fitness.

CDK1/cyclin B regulation during oocyte maturation in two closely related lugworm species, Arenicola marina and Arenicola defodiens.

The molecular mechanisms underlying oocyte maturation in the annelid polychaetes Arenicola marina and Arenicola defodiens were investigated. In both species, a hitherto unidentified hormone triggers synchronous and rapid transition from prophase to metaphase, a maturation process which can be easily reproduced in vitro. Activation of a roscovitine- and olomoucine-sensitive M-phase-specific histone, H1 kinase, occurs during oocyte maturation. Using affinity chromatography on immobilized p9CKShs1, we purified CDK1 and cyclin B from oocyte extracts prepared from both phases and both species. In prophase, CDK1 is present both as an inactive, but Thr161-phosphorylated monomer, and as an inactive (Tyr15-phosphorylated) heterodimer with cyclin B. Prophase to metaphase transition is associated with complete tyrosine dephosphorylation of the cyclin B-associated CDK1, with phosphorylation of cyclin B, and with dramatic activation of the kinase activity of the CDK1/cyclin B complex. We propose that Arenicola oocytes may provide an ideal model system to investigate the acquisition of the ability of oocytes to be fertilized that occurs as oocyte shift from prophase to metaphase, an important physiological event, probably regulated by active CDK1/cyclin B.

The use of a brine shrimp (Artemia salina) bioassay to assess the toxicity of diatom extracts and short chain aldehydes.

Water soluble algal extracts, the aldehydes 2E,4E-decadienal, decanal, undecanal and the fatty acid eicosapentaenoic acid (EPA) were assayed for toxicity to hatching success and larval mortality of the brine shrimp Artemia salina. Both crude cellular extracts of the diatoms Skeletonema costatum and Nitzschia commutata and the diatom-derived short chain aldehyde decadienal were found to inhibit hatching success of A. salina cysts in a dose-dependent manner. Decadienal also significantly affected larval mortality rates in 24 and 72 h exposure incubations. The Artemia hatching success assay was the least sensitive of the three (EC50=3.94 microg ml(-1)). A greater sensitivity was observed for the 72 h compared with the 24 h exposure trials (EC50 for 24h=2.14, 72 h=0.023 microg ml(-1)). Decanal did not significantly affect survival or hatching success at the concentrations tested. Undecanal and EPA showed a limited toxic effect in naupliar mortality trials. We suggest that 72 h Artemia exposure trials represent an acceptable bioassay for diatom toxicity where alternative bioassays are unavailable.

Inhibition of embryonic development and fertilization in broadcast spawning marine invertebrates by water soluble diatom extracts and the diatom toxin 2-trans,4-trans decadienal.

Water soluble diatom extracts and the diatom aldehyde 2-trans,4-trans decadienal were assayed on the gametes and embryos of the broadcast spawning polychaetes Arenicola marina and Nereis virens and the echinoderms Asterias rubens and Psammechinus miliaris. Both crude cellular extracts and purified aldehyde were found to inhibit fertilization, embryogenesis and hatching success in a dose dependent manner. Intact diatom cells had no discernable effect on fertilization or development. Extracts of Skeletonema costatum were generally more effective than Nitzschia commutata in inhibiting development and fertilization. There was considerable interspecific variation in terms of toxin sensitivity. The polychaetes were more sensitive to the effects than the echinoderms. Within the polychaetes A. marina was the more tolerant in terms of developmental competence but N. virens had a higher fertilization rate. Echinoid embryos were more tolerant than asteroid embryos. This is the first study to present data on the inhibition of fertilization success by diatom extracts and aldehydes. Our observations are discussed in relation to temporal patterns in spawning and possible adaptive mechanisms to avoid diatom toxicity.