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SummaryThe indiscriminate disposal of plastic to the environment is of concern. Microscopic plastic litter (

Small plastic detritus, termed "microplastics", are a widespread and ubiquitous contaminant of marine ecosystems across the globe. Ingestion of microplastics by marine biota, including mussels, worms, fish, and seabirds, has been widely reported, but despite their vital ecological role in marine food-webs, the impact of microplastics on zooplankton remains under-researched. Here, we show that microplastics are ingested by, and may impact upon, zooplankton. We used bioimaging techniques to document ingestion, egestion, and adherence of microplastics in a range of zooplankton common to the northeast Atlantic, and employed feeding rate studies to determine the impact of plastic detritus on algal ingestion rates in copepods. Using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy we identified that thirteen zooplankton taxa had the capacity to ingest 1.7-30.6 μm polystyrene beads, with uptake varying by taxa, life-stage and bead-size. Post-ingestion, copepods egested faecal pellets laden with microplastics. We further observed microplastics adhered to the external carapace and appendages of exposed zooplankton. Exposure of the copepod Centropages typicus to natural assemblages of algae with and without microplastics showed that 7.3 μm microplastics (4000 mL(-1)) significantly decreased algal feeding. Our findings imply that marine microplastic debris can negatively impact upon zooplankton function and health.

Low level chronic exposure to toxicants is associated with a range of adverse health effects. Understanding the various factors that influence the chemical burden of an individual is of critical importance to public health strategies. We investigated the relationships between socioeconomic status (SES) and bio-monitored chemical concentration in five cross-sectional waves of the U.S. National Health and Nutrition Examination Survey (NHANES).We utilised adjusted linear regression models to investigate the association between 179 toxicants and the poverty income ratio (PIR) for five NHANES waves. We then selected a subset of chemicals associated with PIR in 3 or more NHANES waves and investigated potential mediating factors using structural equation modelling.PIR was associated with 18 chemicals in 3 or more NHANES waves. Higher SES individuals had higher burdens of serum and urinary mercury, arsenic, caesium, thallium, perfluorooctanoic acid, perfluorononanoic acid, mono(carboxyoctyl) phthalate and benzophenone-3. Inverse associations were noted between PIR and serum and urinary lead and cadmium, antimony, bisphenol A and three phthalates (mono-benzyl, mono-isobutyl, mono-n-butyl). Key mediators included fish and shellfish consumption for the PIR, mercury, arsenic, thallium and perfluorononanoic acid associations. Sunscreen use was an important mediator in the benzophenone-3/PIR relationship. The association between PIR and cadmium or lead was partially mediated by smoking, occupation and diet.These results provide a comprehensive analysis of exposure patterns as a function of socioeconomic status in US adults, providing important information to guide future public health remediation measures to decrease toxicant and disease burdens within society. Keywords: Chemical toxicants, Socioeconomic status, Environment, NHANES

The third author's name was spelled incorrectly. The correct name is: Nicholas J. Osborne. The correct citation is: Melzer D, Gates P, Osborne NJ, Henley WE, Cipelli R, et al. (2012) Urinary Bisphenol A Concentration and Angiography-Defined Coronary Artery Stenosis. PLoS ONE 7(8): e43378. doi:10.1371/journal.pone.0043378

Zinc oxide nanoparticles (ZnO NPs) are widely used in commercial products and knowledge of their environmental fate is a priority for ecological protection. Here we synthesized model ZnO NPs that were made from and thus labeled with the stable isotope (68)Zn and this enables highly sensitive and selective detection of labeled components against high natural Zn background levels. We combine high precision stable isotope measurements and novel bioimaging techniques to characterize parallel water-borne exposures of the common mudshrimp Corophium volutator to (68)ZnO NPs, bulk (68)ZnO, and soluble (68)ZnCl(2) in the presence of sediment. C. volutator is an important component of coastal ecosystems where river-borne NPs will accumulate and is used on a routine basis for toxicity assessments. Our results demonstrate that ionic Zn from ZnO NPs is bioavailable to C. volutator and that Zn uptake is active. Bioavailability appears to be governed primarily by the dissolved Zn content of the water, whereby Zn uptake occurs via the aqueous phase and/or the ingestion of sediment particles with adsorbed Zn from dissolution of ZnO particles. The high sorption capacity of sediments for Zn thus enhances the potential for trophic transfer of Zn derived from readily soluble ZnO NPs. The uncertainties of our isotopic data are too large, however, to conclusively rule out any additional direct uptake route of ZnO NPs by C. volutator.

Background— The endocrine-disrupting chemical bisphenol A (BPA) is widely used in food and beverage packaging. Higher urinary BPA concentrations were cross-sectionally associated with heart disease in National Health and Nutrition Examination Survey (NHANES) 2003–2004 and NHANES 2005–2006, independent of traditional risk factors. Methods and Results— We included 758 incident coronary artery disease (CAD) cases and 861 controls followed for 10.8 years from the European Prospective Investigation of Cancer–Norfolk UK. Respondents aged 40 to 74 years and free of CAD, stroke, or diabetes mellitus provided baseline spot urine samples. Urinary BPA concentrations (median value, 1.3 ng/mL) were low. Per-SD (4.56 ng/mL) increases in urinary BPA concentration were associated with incident CAD in age-, sex-, and urinary creatinine–adjusted models (n=1919; odds ratio=1.13; 95% confidence interval, 1.02–1.24; P =0.017). With CAD risk factor adjustment (including education, occupational social class, body mass index category, systolic blood pressure, lipid concentrations, and exercise), the estimate was similar but narrowly missed 2-sided significance (n=1744; odds ratio=1.11; 95% confidence interval, 1.00–1.23; P =0.058). Sensitivity analyses with the fully adjusted model, excluding those with early CAD (30, or abnormal renal function or with additional adjustment for vitamin C, C-reactive protein, or alcohol consumption, all produced similar estimates, and all showed associations at P ≤0.05. Conclusions— Associations between higher BPA exposure (reflected in higher urinary concentrations) and incident CAD during >10 years of follow-up showed trends similar to previously reported cross-sectional findings in the more highly exposed NHANES respondents. Further work is needed to accurately estimate the prospective exposure-response curve and to establish the underlying mechanisms.

Naphthenic acids (NAs) are considered to be a major toxic component of oil sands process-affected waters (OSPW) and are also widely used for industrial processes. The effects of previously identified NAs (54 in total), together with six alkylphenols, were modelled for a range of environmental and human toxicity related endpoints using ADMET predictor™ software. In addition to the models, experimental CALUX® assays were performed on seven tricyclic diamondoid acids. Most of the NAs modelled were predicted to have lethal median concentrations (LC 50 ) > 100 μM for the three aquatic species modelled. Polycyclic acids containing a single aromatic ring were predicted to be the most toxic to fathead minnows with LC 50 s typically ca 1 μM. Some of these compounds were also predicted to be the most carcinogenic (based on rat and mouse models), possess human estrogenic and androgenic activity and potentially disrupt reproductive processes. Some aliphatic pentacyclic acids also were predicted to exhibit androgenic activity and, uniquely amongst the compounds tested, act as substrates for the cytochrome P450 enzyme CYP3A4. Consistent with the models' predictions for the tricyclic acids, no estrogenic or androgenic activity was detected by ER/AR CALUX®. Further experimental validation of the predictions should now be performed for the compounds highlighted by the models (e.g. priority should perhaps be focused on the polycyclic monoaromatic acids and the aliphatic pentacyclic acids). If shown to be accurate, these compounds can then be targeted for toxicity reduction remediation efforts.

Since the mass production of plastics began in the 1940s, microplastic contamination of the marine environment has been a growing problem. Here, a review of the literature has been conducted with the following objectives: (1) to summarise the properties, nomenclature and sources of microplastics; (2) to discuss the routes by which microplastics enter the marine environment; (3) to evaluate the methods by which microplastics are detected in the marine environment; (4) to assess spatial and temporal trends of microplastic abundance; and (5) to discuss the environmental impact of microplastics. Microplastics are both abundant and widespread within the marine environment, found in their highest concentrations along coastlines and within mid-ocean gyres. Ingestion of microplastics has been demonstrated in a range of marine organisms, a process which may facilitate the transfer of chemical additives or hydrophobic waterborne pollutants to biota. We conclude by highlighting key future research areas for scientists and policymakers.

A full assessment of the impact of oil and chemical spills at sea requires the identification of both the polluting chemicals and the biological effects they cause. Here, a combination of chemical fingerprinting of surface oils, tissue residue analysis, and biological effects measures was used to explore the relationship between spilled oil and biological impact following the grounding of the MSC Napoli container ship in Lyme Bay, England in January 2007. Initially, oil contamination remained restricted to a surface slick in the vicinity of the wreck, and there was no chemical evidence to link biological impairment of animals (the common limpet, Patella vulgata) on the shore adjacent to the oil spill. Secondary oil contamination associated with salvage activities in July 2007 was also assessed. Chemical analyses of aliphatic hydrocarbons and terpanes in shell swabs taken from limpet shells provided an unequivocal match with the fuel oil carried by the ship. Corresponding chemical analysis of limpet tissues revealed increased concentrations of polycyclic aromatic hydrocarbons (PAHs) dominated by phenanthrene and C1 to C3 phenanthrenes with smaller contributions from heavier molecular weight PAHs. Concurrent ecotoxicological tests indicated impairment of cellular viability (p < 0.001), reduced immune function (p < 0.001), and damage to DNA (Comet assay, p < 0.001) in these animals, whereas antioxidant defenses were elevated relative to un-oiled animals. These results illustrate the value of combining biological monitoring with chemical fingerprinting for the rapid identification of spilled oils and their sublethal impacts on biota in situ.

Background Bisphenol A (BPA) is a high production volume chemical widely used in packaging for food and beverages. Numerous studies have demonstrated that BPA can alter endocrine function in animals, yet human studies remain limited. Objective We estimated daily excretion of BPA among adults and examined hypothesized associations with serum estrogen and testosterone concentrations. Methods We conducted cross-sectional analyses using data from the InCHIANTI Study, a prospective population-based study of Italian adults. Our study included 715 adults between 20 and 74 years old. BPA concentrations were measured by liquid chromatography–mass spectrometry in 24-hr urine samples. The main outcome measures were serum concentrations of total testosterone and 17β-estradiol. Results Geometric mean urinary BPA concentration was 3.59 ng/mL [95% confidence interval (CI), 3.42–3.77 ng/mL], and mean excretion was 5.63 μg/day (5th population percentile, 2.1 μg/day; 95th percentile, 16.4 μg/day). We found higher excretion rates among men, younger respondents, and those with increasing waist circumference (p = 0.013) and weight (p = 0.003). Higher daily BPA excretion was associated with higher total testosterone concentrations in men, in models adjusted for age and study site (p = 0.044), and in models additionally adjusted for smoking, measures of obesity, and urinary creatinine concentrations (β = 0.046; 95% CI, 0.015–0.076; p = 0.004). We found no associations with the other serum measures. We also found no associations with the primary outcomes among women, but we did find an association between BPA and SHBG concentrations in the 60 premenopausal women. Conclusion Higher BPA exposure may be associated with endocrine changes in men. The mechanisms involved in the observed cross-sectional association with total testosterone concentrations need to be clarified.

The human population generates vast quantities of waste material. Macro (1 mm) and microscopic (1 mm) fragments of plastic debris represent a substantial contamination problem. Here, we test hypotheses about the influence of wind and depositional regime on spatial patterns of micro- and macro-plastic debris within the Tamar Estuary, UK. Debris was identified to the type of polymer using Fourier-transform infrared spectroscopy (FT-IR) and categorized according to density. In terms of abundance, microplastic accounted for 65% of debris recorded and mainly comprised polyvinylchloride, polyester, and polyamide. Generally, there were greater quantities of plastic at downwind sites. For macroplastic, there were clear patterns of distribution for less dense items, while for microplastic debris, clear patterns were for denser material. Small particles of sediment and plastic are both likely to settle slowly from the water-column and are likely to be transported by the flow of water and be deposited in areas where the movements of water are slower. There was, however, no relationship between the abundance of microplastic and the proportion of clay in sediments from the strandline. These results illustrate how FT-IR spectroscopy can be used to identify the different types of plastic and in this case was used to indicate spatial patterns, demonstrating habitats that are downwind acting as potential sinks for the accumulation of debris.

Environmentally persistent aromatic hydrocarbons known as unresolved complex mixtures (UCMs) derived from crude oil can be accumulated by, and elicit toxicological responses in, marine organisms (e.g. mussels, Mytilus edulis ). Comprehensive two-dimensional gas chromatography time-of-flight mass-spectrometry (GC × GC–ToF-MS) previously revealed that these UCMs included highly branched alkylated aromatic hydrocarbons. Here, the effects of biodegradation on the toxicity and chemical composition of an aromatic UCM hydrocarbon fraction isolated from Tia Juana Pesado (TJP) crude oil were examined. 48 h exposure of mussels to the aromatic hydrocarbon fraction (F2) resulted in tissue concentrations of 900 µg g − 1 (dry wt.) and ∼ 45% decrease in clearance rate. Over 90% of the hydrocarbon burden corresponded to an UCM. Following a 5 day recovery period, GC × GC–ToF-MS analysis of the tissues indicated depuration of most accumulated hydrocarbons and clearance rates returned to those observed in controls. To assess the potential of biodegradation to reduce UCM toxicity, TJP F2 was exposed to bacteria isolated from Whitley Bay, UK, for 46 days. Mussels exposed to the undegraded TJP F2 from the abiotic control exhibited a reduction in clearance rate comparable with values for the pure crude oil TJP F2. Clearance rates of mussels exposed to biodegraded TJP F2 were statistically similar to seawater controls, suggesting biodegradation had reduced the TJP F2 toxicity. GC × GC–ToF-MS analysis revealed the same compound groups in the tissue of mussels exposed to pure TJP F2, undegraded TJP F2 and biodegraded TJP F2 samples; however > 300 fewer compounds were observed in the biodegraded (954 compounds) compared to the undegraded TJP F2 (1261). The compound distributions were markedly different, possibly accounting for the decrease in toxicity. Extraction and analysis of pelleted bacterial cell material revealed that a significant proportion of the TJP F2 had adsorbed onto the cells. Thus extreme care must be taken in interpreting biodegradation data from recalcitrant UCM hydrocarbons.

The utility of some biomarkers in environmental monitoring may be limited due to the lack of knowledge that exists on how they respond to extrinsic abiotic and intrinsic biotic factors. During the present study we investigated the seasonal responses of three biomarkers, Neutral Red Retention, clearance/filtration rate and heart rate in the common blue mussel Mytilus edulis located in the Exe Estuary, UK during September 2006-September 2007. During the current study, a significant decrease in feeding rate was observed in mussels during June, July and August 2007, coinciding with the period following spawning when the mussels lay down nutrient reserves. Heart rate also increased between April and September 2007 and corresponded with times when mussels were spawning and laying down nutrient reserves. By integrating the individual biomarker responses into a Biomarker Response Index (BRI) we were able to identify times of the year when environmental impact was highest and hence when the timing of monitoring programmes using biomarkers should be carried out. For many years the lack of knowledge of normal physiological ranges of biomarkers has impeded their applied use, however by integrating biomarker responses into the BRI and creating an index of health, we have shown that we can limit the natural variability of individual responses; and thus we are better able to make informed judgements on the overall health status of these populations of mussels.

BackgroundBisphenol A (BPA) is a high production volume chemical widely used in food and drinks packaging. Associations have previously been reported between urinary BPA concentrations and heart disease, diabetes and liver enzymes in adult participants of the National Health and Nutrition Examination Survey (NHANES) 2003/04. We aimed to estimate associations between urinary BPA concentrations and health measures in NHANES 2005/06 and in data pooled across collection years.Methodology and findingsA cross-sectional analysis of NHANES: subjects were n = 1455 (2003/04) and n = 1493 (2005/06) adults aged 18-74 years, representative of the general adult population of the United States. Regression models were adjusted for age, sex, race/ethnicity, education, income, smoking, BMI, waist circumference, and urinary creatinine concentration. Main outcomes were reported diagnoses of heart attack, coronary heart disease, angina and diabetes and serum liver enzyme levels. Urinary BPA concentrations in 2005/06 (geometric mean 1.79 ng/ml, 95% CI: 1.64 to 1.96) were lower than in 2003/04 (2.49 ng/ml, CI: 2.20 to 2.83, difference p-value = 0.00002). Higher BPA concentrations were associated with coronary heart disease in 2005/06 (OR per z-score increase in BPA = 1.33, 95%CI: 1.01 to 1.75, p = 0.043) and in pooled data (OR = 1.42, CI: 1.17 to 1.72, p = 0.001). Associations with diabetes did not reach significance in 2005/06, but pooled estimates remained significant (OR = 1.24, CI: 1.10 to 1.40, p = 0.001). There was no overall association with gamma glutamyl transferase concentrations, but pooled associations with alkaline phosphatase and lactate dehydrogenase remained significant.ConclusionsHigher BPA exposure, reflected in higher urinary concentrations of BPA, is consistently associated with reported heart disease in the general adult population of the USA. Studies to clarify the mechanisms of these associations are urgently needed.

Background Perfluorooctanoic acid (PFOA, also known as C8) and perfluorooctane sulfonate (PFOS) are stable compounds with many industrial and consumer uses. Their persistence in the environment plus toxicity in animal models has raised concern over low-level chronic exposure effects on human health. Objectives We estimated associations between serum PFOA and PFOS concentrations and thyroid disease prevalence in representative samples of the U.S. general population. Methods Analyses of PFOA/PFOS versus disease status in the National Health and Nutrition Examination Survey (NHANES) for 1999–2000, 2003–2004, and 2005–2006 included 3,974 adults with measured concentrations for perfluorinated chemicals. Regression models were adjusted for age, sex, race/ethnicity, education, smoking status, body mass index, and alcohol intake. Results The NHANES-weighted prevalence of reporting any thyroid disease was 16.18% (n = 292) in women and 3.06% (n = 69) in men; prevalence of current thyroid disease with related medication was 9.89% (n = 163) in women and 1.88% (n = 46) in men. In fully adjusted logistic models, women with PFOA ≥ 5.7 ng/mL [fourth (highest) population quartile] were more likely to report current treated thyroid disease [odds ratio (OR) = 2.24; 95% confidence interval (CI), 1.38–3.65; p = 0.002] compared with PFOA ≤ 4.0 ng/mL (quartiles 1 and 2); we found a near significant similar trend in men (OR = 2.12; 95% CI, 0.93–4.82; p = 0.073). For PFOS, in men we found a similar association for those with PFOS ≥ 36.8 ng/mL (quartile 4) versus ≤ 25.5 ng/mL (quartiles 1 and 2: OR for treated disease = 2.68; 95% CI, 1.03–6.98; p = 0.043); in women this association was not significant. Conclusions Higher concentrations of serum PFOA and PFOS are associated with current thyroid disease in the U.S. general adult population. More work is needed to establish the mechanisms involved and to exclude confounding and pharmacokinetic explanations.

Pathogen-associated molecular patterns (PAMPs) enable recognition of structures present in microorganisms such as lipopolysaccharides (LPS). LPS are an essential constituent of the outer membrane of Gram-negative bacteria, stimulating the innate immune system of invertebrates. Here, LPS from Escherichia coli (055:B5) were used to investigate the functional immune response of Pecten maximus after stimulation with a PAMP and to determine the combined effect of a phenanthrene exposure and LPS challenge. Organisms were exposed to 200 mug l(-1) phenanthrene and after 7 d were injected with either physiological saline (injection controls) or LPS solution, and returned to their respective exposure tanks. Haemolymph was sampled from the scallops 48 h post-injection and immune function was assessed using a combination of cellular biological responses. The LPS challenge significantly altered the immune response in P. maximus with increased cell counts and phagocytic activity. An immunosuppressive effect of phenanthrene was also observed in this study; however, exposure to phenanthrene did not significantly impair the organism's ability to respond to a PAMP challenge. The overall level of phagocytosis and cytotoxic capability following the LPS challenge was lower in phenanthrene exposed scallops and may have consequences for disease resistance in this commercially-exploited species.

The trophic transfer of monoaromatic hydrocarbons to predatory organisms feeding upon contaminated marine animals is not well reported within the scientific literature. Branched alkylbenzenes (BABs) unresolved by gas chromatography have been reported to be principal toxic components of bioaccumulated hydrocarbons in the tissues of some wild mussel, Mytilus edulis, populations with poor health status. Mussels, M. edulis, contaminated with a commercial mixture of BABs were fed to shore crabs, Carcinus maenas, for 7 d, and effects upon the behavior, heart rate, hemolymph cellular viability, and immune response of the crabs were assessed. Accumulation of BABs in crab midgut gland tissue was quantified by gas chromatography–mass spectrometry, and the presence of BABs in crab urine was detected spectrophotometrically using ultraviolet fluorescence spectroscopy. Analysis of crab tissues and urine revealed a proportion of the BABs was transferred from the mussel tissues to the crabs, but the majority was not present 3 d after consumption of the mussels and may have been metabolized, excreted, or both. The results do not support the hypothesis that BABs are likely to be biomagnified, at least by crabs, in the marine environment. Alterations to measured cellular and physiological responses of crabs fed BAB-exposed mussels were not significant. Consumption of contaminated mussels was shown to cause highly significant abnormal behavior that, in the wild, may affect the feeding ability of crabs and make them more vulnerable to predation.

Chemicals with the potential to damage DNA are increasingly present in the marine environment; yet our understanding of the long-term consequences of DNA damage for populations remains limited. We explore the impact of paternal genotoxin exposure on the reproductive biology of two ecologically important free-spawning marine invertebrates: the polychaete Arenicola marina and the mussel Mytilus edulis. Males were exposed in vivo for 72 h to methyl methanesulfonate and benzo(a)pyrene and the impact on somatic cells and sperm assessed using the Comet assay. A strong correlation between DNA damage in somatic cells and sperm was observed after 24 h exposure (P0.001). Recovery in sperm was significantly lower than in coelomocytes after 72 h. The fertilization success of DNA-damaged sperm was unaffected, but a significant percentage of embryos derived from sperm with induced DNA damage exhibited severe developmental abnormalities within 24 h of fertilization with potential long-term consequences for population success. Further research is required to determine the mechanism by which paternal DNA damage causes disruption of development at this early stage.

The population of Nereis diversicolor inhabiting the upper reaches of Restronguet Creek, Cornwall, UK is highly resistant to acute zinc and copper toxicity. Here we employ bioenergetic accounting and fecundity counts to demonstrate the energetic costs associated with this phenomenon in terms of the worms’ allocation of metabolic resources and reproductive output [Pr]. Metal-resistant animals exhibited a scope for growth that was 46–62% less than that of animals from two non-resistant reference populations, corresponding to a mean metabolic cost of 1.31 mJ h−1 mg DW−1. The resistant population also contained 13% less lipid than animals from the reference populations and 73–81% less carbohydrates. Consequently, mass-specific fecundity was reduced in resistant animals by 39–45%, although material investment in individual gametes did not appear to vary. This demonstrates fitness costs associated with metal resistance in this ecologically important polychaete and adds to our understanding of phenotypic trade-offs associated with resistance.

The discharge of oil well produced water (PW) provides a constant source of contaminants to the marine environment including polycyclic aromatic hydrocarbons, alkylated phenols, metals and production chemicals. High concentrations of PW cause adverse effects to exposed biota, including reduced survival, growth and reproduction. Here we explore the effects of PW on immune function in the blue mussel, Mytilus edulis. Mussels were exposed for 21 days to sublethal PW concentrations (0.125-0.5%) and cellular parameters were measured. Cell viability, phagocytosis and cytotoxicity were inhibited after exposure to 0.25% and 0.5% PW, whilst the 0.125% PW treatment produced significant increases in these biomarker responses. This biphasic response was only observed after 7 days exposure; longer exposure periods led to a reduction in immune parameters. Results indicate that PW concentrations close to the discharge point cause modulation to cellular immunity. The implications for longer-term disease resistance are discussed.

A series of European Marine Sites has been designated as Special Areas of Conservation (SAC) in England. The aim of this study was to develop a practical methodology to assess the condition of SACs by applying a suite of biomarkers. Biomarkers were applied to the blue mussel Mytilus edulis and the shore crab Carcinus maenas from the Fal and Helford SAC (Cornwall). Individual biomarkers provided useful diagnostic information on the activity of certain classes of contaminants and an integrated Biomarker Response Index (BRI) was used to achieve a more holistic understanding of the condition of the SAC. The BRI indicated that the general health of both organisms was impacted in the upper part of the SAC (Fal Estuary) which correlated well with known chemical hotspots and sources of contamination. The BRI allows a pragmatic way to prioritise SAC sites that may require further investigative studies.

With the current expansion of offshore oil activities in Arctic regions, there is an urgent need to establish the potential effects of oil-related compounds on Arctic organisms. As susceptibility to growth, disease and survival is determined partly by the condition of an organism's immune system, measurement of endpoints linked to the latter system provide important early warning signals of the sub-lethal effects of exposure to contaminants. This study assessed the impact of dispersed oil exposure on immune endpoints in the Arctic Scallop Chlamys islandica, using a combination of cellular and humoral biological responses. Laboratory exposures of C. islandica to sub-lethal dispersed oil concentrations (0.06 and 0.25 mg l(-1)) were conducted over 15 days, followed by a 7-day recovery period in clean, filtered seawater. Cellular endpoints were significantly altered following dispersed oil exposure: haemocyte counts (P

Plastics debris is accumulating in the environment and is fragmenting into smaller pieces; as it does, the potential for ingestion by animals increases. The consequences of macroplastic debris for wildlife are well documented, however the impacts of microplastic (< 1 mm) are poorly understood. The mussel, Mytilus edulis, was used to investigate ingestion, translocation, and accumulation of this debris. Initial experiments showed that upon ingestion, microplastic accumulated in the gut. Mussels were subsequently exposed to treatments containing seawater and microplastic (3.0 or 9.6 microm). After transfer to clean conditions, microplastic was tracked in the hemolymph. Particles translocated from the gut to the circulatory system within 3 days and persisted for over 48 days. Abundance of microplastic was greatest after 12 days and declined thereafter. Smaller particles were more abundant than larger particles and our data indicate as plastic fragments into smaller particles, the potential for accumulation in the tissues of an organism increases. The short-term pulse exposure used here did not result in significant biological effects. However, plastics are exceedingly durable and so further work using a wider range of organisms, polymers, and periods of exposure will be required to establish the biological consequences of this debris.

Marine and coastal sediments can accumulate substantial concentrations of metals and hydrocarbons, yet the consequences of this contamination for exposed biota in situ can be difficult to establish. Here, we examine the hypothesis that exposure to contaminated sediments can lead to detrimental effects in sediment-dwelling species. The combination of chemical and biological assessment allows the identification of the impact of chemical contamination, and their use as assessment tools is becoming increasingly important.The study was applied to marine sediments from the Bay of Algeciras (S Spain) impacted by multiple, low-level contaminant inputs, and the Galician Coast (NW Spain), historically impacted by an oil spill (Prestige 2002), with two reference sites selected in UK and Spain. The common lugworm Arenicola marina was exposed in the laboratory for 14 days to the marine sediments, and a suite of biomarkers of sublethal toxicity was combined with analytical chemistry to test for relationships between sediment contamination and effect.Moderate to strong correlations between organics, metals, and biological responses were observed, with DNA damage as measured using the Comet assay forming the largest contribution toward the observed differences (p0.05). The responses of worms from sites experiencing different contamination loads were clearly distinguishable.We show how a combination of multibiomarkers with analytical chemistry can be used to investigate the toxicity of marine sediments, enabling the differentiation of sites showing different types of contamination. There are clear relationships in sublethal assays that can be related to the putative mode of toxicity of the contaminants.The use of A. marina in this way provides a sensitive, holistic approach to sediment toxicity assessment, enabling comparisons between oil-polluted sites to be quantified.These tools provide a relatively simple, rapid, and economic way to test the environmental status of oil-contaminated sediment.

Accidental pollution incidents are common in the marine environment and are often caused by oil-related activities. Here the potential of such an incident to disrupt reproduction in two polychaete species is investigated, using an environmentally relevant preparation of weathered Forties crude oil, i.e. the water accommodated fraction (WAF). Oocytes were collected and exposed to three concentrations of WAF for 1h prior to the addition of sperm, so that fertilization took place under exposure conditions. Fertilization success was significantly reduced in both species by an exposure to WAF concentrations equivalent to 0.38 mgL(-1) PAHs, to just 26.8% in Arenicola marina compared to 76% in Nereis virens. The effects of WAF exposure on fertilization were greatly enhanced at lower sperm concentrations in N. virens, with a complete lack of fertilization reactions observed at sperm concentrations of 10(3)sperm per mL. We therefore suggest a mechanism of toxicity related to sperm swimming behaviour, resulting in reduced sperm:egg collision rates. WAF was found to reduce post-fertilization development rates and have teratogenic effects on early embryonic stages in both species, which exhibited abnormal cleavage patterns and high levels of fluctuating asymmetry. These results illustrate how the presence of crude oil in its soluble form in seawater at the time of a spawning event for either A. marina or N. virens could impact on fertilization success with implications for the fertilization ecology of these free spawning marine invertebrates.

The marine environment is becoming increasingly contaminated by environmental pollutants with the potential to damage DNA, with marine sediments acting as a sink for many of these contaminants. Understanding genotoxic responses in sediment-dwelling marine organisms, such as polychaetes, is therefore of increasing importance. This study is an exploration of species-specific and cell-specific differences in cell sensitivities to DNA-damaging agents in polychaete worms, aimed at increasing fundamental knowledge of their responses to genotoxic damage. The sensitivities of coelomocytes from three polychaetes species of high ecological relevance, i.e. the lugworm Arenicola marina, the harbour ragworm Nereis diversicolor and the king ragworm Nereis virens to genotoxic damage are compared, and differences in sensitivities of their different coelomic cell types determined by use of the comet assay. A. marina was found to be the most sensitive to genotoxic damage induced by the direct-acting mutagen methyl methanesulfonate (MMS), and showed dose-dependent responses to MMS and the polycyclic aromatic hydrocarbon benzo(a)pyrene. Significant differences in sensitivity were also measured for the different types of coelomocyte. Eleocytes were more sensitive to induction of DNA damage than amoebocytes in both N. virens and N. diversicolor. Spermatozoa from A. marina showed significant DNA damage following in vitro exposure to MMS, but were less sensitive to DNA damage than coelomocytes. This investigation has clearly demonstrated that different cell types within the same species and different species within the polychaetes show significantly different responses to genotoxic insult. These findings are discussed in terms of the relationship between cell function and sensitivity and their implications for the use of polychaetes in environmental genotoxicity studies.

There is heightened concern worldwide about the impacts of endocrine-disrupting chemicals present in the aquatic environment that can alter physiological function in wildlife and humans (reviewed by Tyler et al. 1998). Effluents from wastewater treatment works (WwTWs) are a major point source of endocrine-disrupting chemicals into watercourses, and exposure has been associated with a range of reproductive impacts, particularly in fish, including the induction of intersex (Jobling et al. 1998), lowered hormone levels (Folmar et al. 1996), and reduced gamete production and fertilization capability (Jobling et al. 2002a, 2002b). In addition to effects on reproductive development and function, exposure to WwTW effluents has been associated with wider adverse health effects, including genotoxic damage (Gravato and Santos 2003; Liney et al. 2006), immunosuppression (Hoeger et al. 2005; Liney et al. 2006; Price et al. 1997), altered activity of hepatic phase I/phase II biotransformation enzymes (Gagne et al. 2006; Gravato and Santos 2003; Hoeger et al. 2005), nephrotoxicity (Liney et al. 2006), and reduced growth/condition (Smolders et al. 2002). Moreover, some of these effects have been shown to occur at effluent concentrations considerably lower than those that cause feminization of the reproductive system (Liney et al. 2006). The potential for WwTW effluents to affect health in these ways is of significant concern. For example, immune suppression can lead to higher disease susceptibility; altered metabolic capability can lead to toxic accumulation of pollutants or production of reactive metabolites; and damage to DNA can result in embryo mortality, developmental abnormalities, and/or cancer. There is now considerable evidence from both field and laboratory studies that the reproductive effects of WwTW effluents result from exposure to environmental estrogens present in effluents, in particular natural and synthetic steroidal estrogens [e.g., estrone, 17β-estradiol (E2), 17α-ethinyl-estradiol (EE2) (Desbrow et al. 1998)]. Studies in mammalian systems have shown that some estrogens (and/or their metabolites) also have, for example, genotoxic properties (reviewed by Roy and Liehr 1999) and immunotoxic properties (Ahmed 2000). However, the possible role(s) of steroidal estrogens as causative agents of such wider health effects associated with exposure to WwTW effluents has not been established. Furthermore, essentially nothing known health effects of steroidal contained in complex environmental mixtures, such as effluent discharges, compared with their effects when exposed as individual chemicals. This is a significant information gap given the potential for interactive biological effects of estrogens with other environmental chemical present in mixtures. In this study, we investigated the contribution of steroidal estrogens to a suite of adverse sublethal health effects induced by exposure to WwTW effluents via two sets of experiments with a model fish species (fathead minnow; Pimephales promelas). The work included a comparative analysis on the biological responses induced by the steroidal estrogen EE2, as a single chemical and as part of a complex environmental mixture (spiked into effluent). Health effects were determined using a battery of end points indicative of growth effects [length, weight, condition factor, and hepatic expression of the gene for the somatotropic hormone insulin-like growth factor-1 (igf1)], genotoxicity (comet assay), immunotoxicity [differential white blood cell (WBC) count and phagocytotic activity of liver cells], and metabolic responses [hepatic ethoxy-resorufin-O-deethylase (EROD) activity and cytochrome P450 1a (cyp1a) and 3a (cyp3a) gene expression, as indicators of phase I bio-transformation, and glutathione S-transferase (gst) gene expression, as an indicator of phase II biotransformation]. These responses were measured alongside endocrine (feminized) responses [gonadal growth, development of secondary sex characters, the female yolk protein precursor vitellogenin (VTG), and sex steroid receptor gene expression].

Environmental context Engineered nanomaterials are increasingly being used and their release to the aquatic environment poses potential risk. We review the research on transformations of engineered nanomaterial in the aquatic sediment environments, and consider the implications of their release. The key factors defining the fate of engineered nanomaterials in aqueous and sediment systems are identified. Abstract Inorganic nanoparticles are at risk of release into the aquatic environment owing to their function, use and methods of disposal. Aquatic sediments are predicted to be a large potential sink for such engineered nanomaterial (ENM) emissions. On entering water bodies, ENMs undergo a range of transformations dependent on the physicochemical nature of the immediate environment, as they pass from the surface waters to sediments and into sediment-dwelling organisms. This review assesses the current state of research on transformations of metal-based ENMs in the aquatic environment, and considers the implications of these transformations for the fate and persistence of ENMs and their bioavailability to organisms within the benthos. We identify the following factors of key importance in the fate pathways of ENMs in aqueous systems: (1) extracellular polymeric substances, prevalent in many aquatic systems, create the potential for temporal fluxes of ENMs to the benthos, currently unaccounted for in predictive models. (2) Weak secondary deposition onto sediment grains may dominate sediment–ENM interactions for larger aggregates >500nm, potentially granting dynamic long-term mobility of ENMs within sediments. (3) Sulfurisation, aggregation and reduction in the presence of humic acid is likely to limit the presence of dissolved ions from soluble ENMs within sediments. (4) Key benthic species are identified based on their ecosystem functionality and potential for ENM exposure. On the basis of these findings, we recommend future research areas which will support prospective risk assessment by enhancing our knowledge of the transformations ENMs undergo and the likely effects these will have.

Marine pollution is a major threat to human and environmental health. Given the complexity of function of marine and coastal ecosystems, it is unlikely that a balanced view of the nature and extent of risk will easily be achieved if human and environmental risk assessments continue to be conducted in isolation. Here, the integration of assessment protocols is advocated as a holistic means of improving risk management. Biomarkers can provide the common conceptual framework and measurable endpoints necessary for successful integration. Examples are given of the ways in which suites of biomarkers encompassing molecular change, cellular pathology and physiological impairment can be developed and adapted for human and ecological scenarios. By placing a greater emphasis on the health status of impacted biota, it is more likely that risk assessment will develop the efficiency, reliability and predictive power to adapt to the unforeseen environmental threats that are an inevitable consequence of human development and global change.

The European Union has adopted several environmental directives, strategies, recommendations, and agreements that require a shift from local- or regional-based regulations to more ecosystem-based, holistic environmental management. Over the next decade, environmental management in Europe is likely to focus more on biological and ecological conditions rather than physical and chemical conditions, with ecosystem health at the center of regulation and management decision making. Successful implementation of this new ecosystem management and strategic assessment process in Europe will require the integration of regulatory and technical information and extensive collaboration from among European Union member countries, between agencies, and across disciplines to an unprecedented degree. It will also require extensive efforts to adapt current systems of environmental assessment and management to the basin and ecosystem level, across media and habitats, and considering a much broader set of impacts on ecosystem status than is currently addressed in most risk assessments. This will require the understanding, integration, and communication of economic, ecological, hydrological, and other processes across many spatial and temporal scales. This article discusses these challenges and describes some of the research initiatives that will help achieve integrated ecosystem management in Europe.

Our previous studies have demonstrated that tributyltin (TBT) is genotoxic to the early life stages of marine mussels and worms. Here, the toxicity of TBT to adult organisms was determined using a suite of biomarkers designed to detect cytotoxic, immunotoxic and genotoxic effects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days resulted in a statistically significant decrease in cell viability at concentrations of 0.5 μg/l and above. TBT had no effect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically significant increase in DNA damage detected using the comet and micronucleus assays between the controls and 0.5, 1 and 5 μg/l of TBTO (P > 0.0005). Furthermore there was a strong correlation between DNA strand breaks (comet assay) and formation of micronuclei (P = 0.0005; R2 = 61.5%). Possible mechanisms by which TBT could damage DNA either directly or indirectly are discussed including the possibility that TBT is genotoxic due to its ability to disrupt calcium homeostasis.

The prodromal phase of type 1 diabetes is characterised by the appearance of multiple islet-cell related autoantibodies (Aab). The major target antigens are islet-cell antigen, glutamic acid decarboxylase (GAD), protein-tyrosine phosphatase-2 (IA-2) and insulin. Insulin autoantibodies (IAA), in contrast to the other autoimmune markers, are the only β-cell specific antibodies. There is general consensus that the presence of multiple Aab (≥ 3) is associated with a high risk of developing diabetes, where the presence of a single islet-cell-related Aab has usually a low predictive value. The most commonly used assay format for the detection of Aab to GAD, IA-2 and insulin is the fluid-phase radiobinding assay. The RBA does not identify or measure Aab, but merely detects its presence. However, on the basis of molecular studies, disease-specific constructs of GAD and IA-2 have been employed leading to somewhat improved sensitivity and specificity of the RBA. Serological studies have shown epitope restriction of IAA that can differentiate diabetes-related from unrelated IAA, but current assays do not distinguish between disease-predictive and non-predictive IAA or between IAA and insulin antibodies (IA). More recently, phage display technology has been successful in identifying disease-specific anti-idiotopes of insulin. In addition, phage display has facilitated the in vitro production of antibodies with high affinity. Identification of disease-specific anti-idiotopes of insulin should enable the production of a high affinity reagent against the same anti-idiotope. Such a development would form the basis of a disease-specific radioimmunoassay able to identify and measure particular idiotypes, rather than merely detect and titrate IAA. Copyright © 2005 John Wiley & Sons, Ltd.

Aims Complete or partial remission can occur in newly diagnosed Type 1 diabetes patients. We created idiotype-specific reagents to explore the idiotypes of insulin antibodies (IA) in a patient in remission, and to compare with a patient who was not. Methods Phage display was used to create a library of phagotopes specific to insulin binding in four sera. Sera from a Type 1 diabetes subject deemed to have undergone remission were taken at diagnosis and again during remission. Sera from a non-remitter were taken at diagnosis and after 3 months on insulin. Phagotopes from the four sera were randomly selected and tested for insulin specificity in a radiobinding assay by using sera from remitters and non-remitters. Results IA-binding phagotope selected from serum during remission displaced insulin binding in all nine IA+ remitters and all 10 IA+ non-remitters. IA-binding phagotope selected from the non-remission patient (3 months after insulin therapy) displaced insulin binding in 8/9 IA+ remitters and 8/10 IA+ non-remitters. The consensus peptide sequences adduced from the phages were identical for both these phagotopes. Phagotopes derived from insulin autoantibody-positive individuals at diagnosis were unable to displace insulin binding in the IA+ sera 3 months later, whether in remission or not. Conclusions We have established the principle of using phage display in the investigation of insulin antibodies during remission in Type 1 diabetes. The immunological characteristics of IA 3 months after the introduction of insulin treatment were different from those at diagnosis of Type 1 diabetes (IAA). Using phage display technology, it was not possible to distinguish insulin antibodies according to remission status.

Insulin autoantibodies (IAA) are present in type 1 diabetes (T1D) and other autoimmune diseases. The differences in the IAA epitopes in various clinical diseases have not been evaluated. We used phage display to select phagotopes specific to IAA from a newly diagnosed T1D child (designated FPP) and from an adult-onset T1D subject with autoimmune polyendocrine syndrome type 2 (APS-II). The phagotopes randomly selected were tested as antiidiotope reagents to displace human radiolabeled insulin in the microfiltration radiobinding assay using IAA sera from T1D subjects and insulin antibody (IA) sera from insulin-treated type 2 diabetes subjects. The DNA of the phagotopes selected from the FPP and APS sera revealed consensus amino acid sequences of GRG and LGKRS, respectively. Phagotope FPP-10 displaced insulin binding in 90% of IAA subjects but not in the IA or the APS subject. Phagotope APS-4 was able to displace insulin binding from the APS subject but not in the IAA or IA subjects. We have demonstrated antiidiotope reagents able to distinguish childhood-onset T1D-associated IAA from adult-onset T1D (APSII-associated IAA) that are different from their specificity for human insulin and from its antiidiotope amino acid sequence. (J Clin Endocrinol Metab 89: 5266 –5270, 2004)

Increasing numbers of industrial, agricultural and natural chemicals are present in sewage effluent and are known to elicit toxic effects in laboratory exposures, but little is known of their combined sub-lethal effect in the field. In this study, a combination of esterase activity and ventilation rate assays was performed to determine the neurological and physiological function of the freshwater crustacean Asellus aquaticus (L.) at sites above and below a sewage treatment works (STW). Cholinesterase and carboxylesterase activities were significantly inhibited (n=8, P

Norwegian coastal waters are subject to PAH contamination from electrochemical industries such as aluminium smelters. Evidence of PAH exposure has been established in fish and bivalves. The present study tests the applicability of a novel crustacean PAH exposure biomarker to a PAH contamination gradient in the field (Karmsund Strait, SW Norway). Fluorescence analysis of urine samples collected from crabs at each site revealed 1-OH pyrene "equivalent" levels (indicative of pyrogenic PAH contamination) decreased with increasing distance from a point source of pyrogenic PAH (a large aluminium works). The assay was shown to be suitable for the detection of PAH exposure in wild crustacean populations, for discriminating between contaminated and clean sites and is also sufficiently sensitive to detect gradients of PAH contamination. The method provides a rapid, inexpensive and non-destructive measure of biologically available PAH in crustaceans.

A field study was carried out to investigate whether polyaromatic hydrocarbon (PAH) contamination in shore crabs (Carcinus maenas) could be measured using fluorescence spectrometry. Crabs were collected as part of an integrated study to assess PAH contamination in Southampton Water, UK. Urine was collected from crabs via a non-destructive technique and analysed using fixed wavelength fluorescence (FF) and synchronous fluorescence spectrophotometry (SFS) in order to determine the presence of PAH metabolites, indicative of PAH exposure. By scanning at wavelength pairs specific to a variety of PAH groups the fluorescence of metabolites was analysed and attributed to the following PAH groups; naphthalenes, pyrenes and benzo[a]pyrene (BAP). Naphthalene-type, pyrene-type and BAP-type metabolites were detected using FF and SFS techniques and two distinct parameters were measured; Peak Intensity and Peak Area. Results showed that crabs obtained near a petroleum refinery, a point source of PAH discharge, exhibited significantly higher PAH metabolites for all PAH groups, than at sites sampled along the estuary (ANOVA, p

Incorporation of ecologically relevant biomarkers into routine environmental management programs has been advocated as a pragmatic means of linking environmental degradation with its causes. Here, suites of biomarkers, devised to measure molecular damage, developmental abnormality and physiological impairment, were combined with chemical analysis to determine exposure to and the effects of pollution at sites within Southampton Water (UK). Test species included a filter feeder, a grazer, and an omnivore to determine the sensitivity of organisms occupying different trophic levels. Linear regression confirmed a significant association between incidence of intersex in Littorina littorea and tributyltin (TBT) concentrations (R2 = 0.954) and between PAH metabolites in Carcinus maenas urine and PAHs in sediments (R2 = 0.754). Principal component analysis revealed a gradient of detrimental impact to biota from the head to the mouth of the estuary, coincident with high sediment concentrations of heavy metals, PAHs, and biocides. Multidimensional scaling identified C. maenas as the organism most sensitive to contamination. Carboxylesterase activity, metallothionein and total haemolymph protein were the most discriminating biomarkers among sites. This holistic approach to environmental assessment is encouraged as it helps to identify the integrated impact of chemical contamination on organisms and to provide a realistic measure of environmental quality.

Vibrio cholerae extracellular proteinases (proteases) have been studied as potential candidate antigens for acellular cholera vaccines. Proteinases from V. cholerae NCTC 10732 were prepared from batch culture either by ammonium sulphate precipitation and G100 Sephadex gel filtration or by isoelectric focusing (IEF). Proteinase activity was at a maximum level after 24 h, coincident with the late exponential phase and early stationary phase. Three major IEF peaks of activity were resolved with specific activities in the range 17.2-195 EU ml(-1 )mg(-1). Sodium dodecyl sulphate-polyacrylamide gel electrophoreses (SDS-PAGE) of these fractions revealed 42, 45, 57 and 75 kDa bands in which proteinase activity was demonstrable. Peptide digest analysis suggested different catalytic specificities for each proteinase fraction. Metalloproteinase and serine proteinase inhibitors, alpha(2)-macroglobulin (alpha(2)-M), the thiol proteinase inhibitor and N-ethylmaleimide inhibited the proteinases. The proteinases nicked Escherichia coli heat-labile toxin to yield catalytically active sub-units, confirmed by the measurement of intrinsic ADP-ribosylation activity. The possible value of these putative V. cholerae antigens in an acellular vaccine is discussed.

Despite a wealth of information on biomarkers, they are not routinely used for regulatory purposes, even though the potential benefits of biomarkers to rationalise complex exposure-response relationships are clear. Biomarkers can be inappropriately applied or misinterpreted, because the fundamental assumptions in exposure-response relations have not been considered. Factors causing temporal and spatial variability in biomarker responses are reviewed. These include numerous geochemical and biotic variables. The variation can be minimised by appropriate study site selection, experimental replication, multivariate epidemiological approaches, normalised controls, and temporal calibration of responses; so that the regulatory use of biomarkers for biomonitoring and tracking pollution events, including chronic or multiple exposures to complex mixtures is possible. We propose and define the characteristics of biomarkers of chronic exposure or effect, which must measure changes in pollution/effect against long-term changes in other general stresses (disease, nutrition, environmental quality), relate to cumulative injury, and remain responsive over months or years. Neuroendocrine, immunological, and histological biomarkers are suggested for chronic pollution. We propose a regulatory framework for biomarkers based on a weight of evidence approach that can integrate biomarkers in risk assessment and long-term monitoring programmes.

The inhibition of acetylcholinesterase (AChE) activity has been used widely as a biomarker of exposure to organophosphorous pesticides (OPs). However, scientific uncertainty about the risk assessment implications of data describing inhibition of cholinesterases in diverse species and tissues has hampered the use of AChE activity as a biomarker of adverse effect. Here, haemolymph AChE activity was combined with biomarkers of cellular integrity, immunotoxicity and physiological status in order to measure exposure to and the effects of the priority pollutant chlorfenvinphos. Laboratory exposures of the blue mussel Mytilus edulis to commercial grade chlorfenvinphos (Sapecron) were conducted over 24, 48 and 96 h. AChE activity in haemolymph of M. edulis was highly variable and bore no relationship to either sublethal effects or lethality over the range 0.003-0.03 mg/l chlorfenvinphos. In comparison, concentration dependent inhibition was evident for each of the remaining biomarkers (phagocytic activity, spontaneous cytotoxicity, neutral red retention time, total haemolymph protein). Mussels at the highest exposure concentration showed visual signs of neurotoxicity (impaired neuromuscular control). Haemocyte phagocytic activity and spontaneous cytotoxicity responses were highly sensitive to chlorfenvinphos with significant modulation evident after 24 h exposure to environmentally realistic concentrations of 0.007 mg/l (P = 0.0003). Thus the immune function and well being of the mussels was significantly impacted in the absence of measurable inhibition of haemolymph AChE.

Carboxylesterases in bivalve molluscs exhibit greater sensitivity to organophosphorous and carbamate pesticides than acetylcholinesterase and are present at higher levels. The aim of the present study was to combine measurement of both acetylcholinesterase and carboxylesterase activities in the marine bivalve Mytilus edulis in order to detect the effects of pesticide exposure. Spectrophotometric assays in microtitreplate format were optimised for use with M. edulis haemolymph and tissue homogenate samples. This permitted the nature and distribution of the enzymes to be determined. One predominant pharmacological form of activity consistent in its patterns of activation and inhibition with acetylcholinesterase was identified in the haemolymph with an apparent K(m) for acetylthiocholineiodide of 1.33 mM. Carboxylesterase activity in the tissues was characterised by its preferential hydrolysis of the substrate analogue phenylthioacetate. Concentration-dependent inhibition of both activities was demonstrated following in vitro incubation with diisopropylfluorophosphate (DFP), paraoxon and eserine in the range 0.1-3.0 mM. When M. edulis (n=10) were exposed for 24 h to concentrations of eserine or paraoxon of 0.05-1.0 mM, the percentage inhibition of acetylcholinesterase was in each case greater than for carboxylesterase and reached statistical significance at lower concentrations. In all exposures, a proportion of carboxylesterase activity was present which remained resistant to inhibition by either organophosphorous or carbamate compounds. The ecotoxicological significance of these findings for the environmental monitoring of pesticide exposure is discussed.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and potentially harmful contaminants of the coastal and marine environment. Studies of their bioavailability, disposition and metabolism in marine organisms are therefore important for environmental monitoring purposes. Detecting PAH compounds in the biological fluids of marine organisms provides a measure of their environmental exposure to PAHs. In the present study, the shore crab Carcinus maenas was exposed to waterborne pyrene for 48h. Urine and haemolymph samples were analysed by direct fluorimetry utilising both fixed wavelength (FF) and synchronous scanning fluorescence (SFS) techniques. Samples from exposed crabs exhibited fluorescence due to 1-OH pyrene equivalents, whilst samples from control crabs did not. Levels of equivalents were exposure dependent. Urine was shown to be a more suitable medium for the analysis of PAH equivalents. In a separate experiment, depuration of pyrene equivalents in urine was monitored over time. Urinary levels reached a maximum 2-4 days after initial exposure and decreased steadily thereafter. No unchanged parent pyrene was detected in samples from exposed crabs. While fluorimetric techniques could discriminate between 1-OH pyrene equivalents and parent pyrene, identification of specific metabolites was only possible with HPLC/F analysis. This revealed crabs had bio-transformed pyrene into 3 major conjugates of 1-OH pyrene, which were excreted in the urine. While such biotransformation of PAH is well documented in fish and several crustaceans, this is the first study to use direct fluorimetry to detect PAH equivalents in exposed crustacean urine. Fluorimetric results correlated well with those obtained by HPLC/F and ELISA techniques. The technique has great potential as a rapid, inexpensive and non-destructive technique for field biomonitoring of PAH exposure in crustaceans.

To fully assess the impact of pollutant releases into the environment, it is necessary to determine both the concentration of chemicals accumulating in biota and the biological effects they give rise to. Owing to time, expertise, and cost constraints, this is, however, rarely achieved. Here, quick, simple to perform, and inexpensive biomarkers and chemical immunoassays were combined in a rapid assessment approach to measure exposure to and effects of organic and metal pollutants on the ribbed mussel (Geukensia demmissa) from New Bedford Harbor, MA. Significant differences in polychlorinated biphenyl (PCB) and polyaromatic hydrocarbon (PAH) tissue residue concentrations were detected among sites using RaPID immunoassay. Selected analyses were verified using GC/MS. No significant differences were observed in metal concentrations (Cu, Cd, Pb, As, Hg, Ni) throughout the area. While causality cannot be attributed, multivariate canonical correlation analysis indicated that PCB and PAH concentrations were strongly associated with the induction of biomarkers of genotoxicity (micronucleus formation), immunotoxicity (spontaneous cytotoxicity), and physiological impairment (heart rate). It is concluded thatthe incorporation of chemical immunoassays with biological monitoring tools into routine management procedures is clearly viable and valuable as a means of identifying toxic impacts of pollutants on biota in situ.

The chemical is found in many plastic food containers, but mounting evidence about its safety is being ignored. What more do regulators need, ask David Melzer and Tamara Galloway