Exclusion of tidal influence on ambient sound measurements.

Growing concern about the impacts of anthropogenic noise on marine life has led to a global increase in the number of acoustic monitoring programmes aiming to quantify underwater soundscapes. However, low-frequency measurements in coastal sites may be affected by flow noise that is not actually present in the environment, but is caused by tidal flow turbulence around the hydrophone. At present, there is no standard way of removing this contaminating noise. This study presents an approach to exclude tidal influences (flow noise and other tidal-related acoustic self-noise) on ambient sound measurements, using data recorded at ten Scottish coastal sites between 2013 and 2017, and with a focus on the 63 and 125 Hz 1/3-octave bands. The annual ambient sound pressure levels (SPL) of the full and "tidal influence excluded" datasets of the three most tidally affected sites were compared against hypothetical noise thresholds. For the 63 Hz 1/3-octave band, results revealed: Site-specific patterns in the amount of data excluded (28.2%-89.2%), decreases in SPL (0.7-8.5 dB), and differences in the percentage of time that noise thresholds were exceeded. The described approach may serve as a standardised way of excluding tidal influence on soundscape descriptors.

Integrated survey methodologies provide process-driven framework for marine renewable energy environmental impact assessment.

Environmental interactions of marine renewable energy developments vary from fine-scale direct (e.g. potential collision) to indirect wide-scale hydrodynamic changes altering oceanographic features. Current UK Environmental Impact Assessment (EIA) and associated Habitats Regulations Appraisal (HRA) guidelines have limited focus on underlying processes affecting distribution and movements (hence vulnerability) of top predators. This study integrates multi-trophic ship survey (active acoustics and observer data) with an upward-facing seabed platform and 3-dimensional hydrodynamic model as a process-driven framework to investigate predator-prey linkages between seabirds and fish schools. Observer-only data highlighted the need to measure physical drivers of variance in species abundances and distributions. Active acoustics indicated that in situ (preferable to modelled) data were needed to identify temporal changes in hydrodynamics to predict prey and consequently top predator presence. Revising methods to identify key habitats and environmental covariates within current regulatory frameworks will enable more robust and transferable EIA and HRA processes and outputs, and at larger scales for cumulative and strategic-level assessments, enabling future modelling of ecosystem impacts from both climate change and renewable energy extraction.

Reduced Lepeophtheirus salmonis larval abundance in a sea loch on the west coast of Scotland between 2002 and 2006.

A survey for planktonic sea louse larvae was carried out in Loch Shieldaig, Scotland, between 2002 and 2006, and spanned 2 successive production cycles (Cycles 1 and 2) at a local Atlantic salmon Salmo salar L. farm. The vast majority of the caligid copepodids recovered were Lepeophtheirus salmonis; however, the methodology was unable to determine the species of the caligid nauplii. Greatest densities of nauplii were found at the sampling station adjacent to the salmon farm, and larval densities were low during the fallow period of both cycles. Peaks in nauplius densities occurred around the same time in the 2 cycles, but the peaks were significantly lower during Cycle 2 than Cycle 1. Lepeophtheirus salmonis copepodid densities varied temporally, but not spatially. During most of Cycle 2, copepodid densities were significantly lower than those recovered during Cycle 1. Numbers of gravid L. salmonis at the local salmon farm correlated significantly with densities of louse nauplii and L. salmonis copepodids in the water at time lags of 0 and 1 wk, and 1 and 2 wk, respectively. This survey demonstrated a reduction in densities of L. salmonis larvae in the plankton (an indication of L. salmonis infectious pressure) between the 2 cycles and indicated that the farm was an important source of L. salmonis larvae. The application of anti-louse treatments using emamectin benzoate reduced the numbers of gravid L. salmonis at the farm, and this was the main factor influencing the apparent reduction in L. salmonis infectious pressure in the loch between cycles.

Offshore pipeline decommissioning: Scale and context.

The spatial extent of human activities must be understood for consistent and proportionate regulation, and effective marine planning. Redundant offshore pipelines can be removed or left in situ, but data on the footprint of these options are not readily available. The extents of three North Sea in situ decommissioning scenarios are presented. Leaving pipelines in situ would occupy <0.01% (12.3 km2) of UK waters, and this was similar to, or smaller than, other regulated activities (e.g. aggregate extraction). Adding armouring to large pipelines occupied up to 95 km2, while creating fisheries exclusion zones occupied up to 1119 km2. Removal of pipelines >30″ would be required to regain 50% or more of the seabed currently occupied. At present, the technology to remove pipelines >16″ safely and cost-efficiently is untested for large-scale decommissioning projects. The summaries presented inform the debate over the significance of decommissioning, and the regional consequences of different options.

Bioindicators and reproductive effects of prolonged 17beta-oestradiol exposure in a marine fish, the sand goby (Pomatoschistus minutus).

The effects of 17beta-oestradiol (E2) on mortality, growth rates, sexual maturation, hepatic vitellogenin (VTG) mRNA expression and reproductive success were investigated during an 8-month, water-borne exposure of a marine fish, the sand goby (Pomatoschistus minutus). Indicators of oestrogenic exposure were investigated as predictors of population-level reproductive success. E2 exposure concentrations were <5 (below limit of detection), 16+/-3, 97+/-20 and 669+/-151 ng l(-1) (bootstrap means and standard errors). The carrier solvent (<20 microl l(-1) propan-2-ol) significantly reduced the rate of egg production compared to untreated fish, but did not significantly affect male VTG mRNA expression, brood size, or the other studied parameters. Fish exposed to 16 ng l(-1) E2 showed few adverse effects compared with solvent only-exposed fish. Exposure to 97 ng l(-1) E2 significantly inhibited male sexual maturation, induced male VTG mRNA expression and delayed spawning. The 97 ng l(-1) E2 exposed population also produced fertile eggs at a significantly slower rate than solvent controls; however, brood size, fertility and overall reproductive success were not significantly affected. Exposure to 669 ng l(-1) E2 significantly increased mortality, adversely affected haematological parameters and caused an almost total lack of reproductive activity, with both sexes failing to mature. Reproductive failure following exposure to 669 ng l(-1) E2 was evident in both sexes when crossed with untreated animals. This work indicates that marine fish are similarly as sensitive to oestrogenic exposure as freshwater fish, that exposure biomarkers such as VTG are more sensitive to exposure than are reproductive effects, and that the use of carrier solvents in long-term reproductive studies should be avoided.

Taking movement data to new depths: Inferring prey availability and patch profitability from seabird foraging behavior.

Detailed information acquired using tracking technology has the potential to provide accurate pictures of the types of movements and behaviors performed by animals. To date, such data have not been widely exploited to provide inferred information about the foraging habitat. We collected data using multiple sensors (GPS, time depth recorders, and accelerometers) from two species of diving seabirds, razorbills (Alca torda, N = 5, from Fair Isle, UK) and common guillemots (Uria aalge, N = 2 from Fair Isle and N = 2 from Colonsay, UK). We used a clustering algorithm to identify pursuit and catching events and the time spent pursuing and catching underwater, which we then used as indicators for inferring prey encounters throughout the water column and responses to changes in prey availability of the areas visited at two levels: individual dives and groups of dives. For each individual dive (N = 661 for guillemots, 6214 for razorbills), we modeled the number of pursuit and catching events, in relation to dive depth, duration, and type of dive performed (benthic vs. pelagic). For groups of dives (N = 58 for guillemots, 156 for razorbills), we modeled the total time spent pursuing and catching in relation to time spent underwater. Razorbills performed only pelagic dives, most likely exploiting prey available at shallow depths as indicated by the vertical distribution of pursuit and catching events. In contrast, guillemots were more flexible in their behavior, switching between benthic and pelagic dives. Capture attempt rates indicated that they were exploiting deep prey aggregations. The study highlights how novel analysis of movement data can give new insights into how animals exploit food patches, offering a unique opportunity to comprehend the behavioral ecology behind different movement patterns and understand how animals might respond to changes in prey distributions.

Integrated indicator framework and methodology for monitoring and assessment of hazardous substances and their effects in the marine environment.

Many maritime countries in Europe have implemented marine environmental monitoring programmes which include the measurement of chemical contaminants and related biological effects. How best to integrate data obtained in these two types of monitoring into meaningful assessments has been the subject of recent efforts by the International Council for Exploration of the Sea (ICES) Expert Groups. Work within these groups has concentrated on defining a core set of chemical and biological endpoints that can be used across maritime areas, defining confounding factors, supporting parameters and protocols for measurement. The framework comprised markers for concentrations of, exposure to and effects from, contaminants. Most importantly, assessment criteria for biological effect measurements have been set and the framework suggests how these measurements can be used in an integrated manner alongside contaminant measurements in biota, sediments and potentially water. Output from this process resulted in OSPAR Commission (www.ospar.org) guidelines that were adopted in 2012 on a trial basis for a period of 3 years. The developed assessment framework can furthermore provide a suitable approach for the assessment of Good Environmental Status (GES) for Descriptor 8 of the European Union (EU) Marine Strategy Framework Directive (MSFD).

Vitellogenin in the blood plasma of male cod (Gadus morhua): a sign of oestrogenic endocrine disruption in the open sea?

An ELISA for cod vitellogenin (VTG) has been set up using cod lipovitellin for plate coating and standardisation. The assay has been applied to plasma samples collected from male and female cod caught in three distinct areas around the UK, three areas off the Norwegian coast and also to cod reared initially at an aquaculture site and subsequently maintained at a research station. The aim of the study was to determine whether there were any signs of oestrogenic endocrine disruption in a fish species living offshore. VTG induction was found in male cod caught in the North Sea, the Shetland Box area, in Oslofjord and also in cultivated fish. There was a strong relationship between concentrations of VTG and fish size. There was no evidence that the presence of VTG in the plasma of males is a natural part of their life cycle. On the other hand, the size of fish at which these elevated VTG concentrations appear (ca. 5 kg) is about the size that cod change from feeding primarily on benthic invertebrates to mainly other fish, both benthic and pelagic. The possibility is suggested that large cod pick up oestrogenic endocrine disrupters through the food chain.

Can industrial pollution cause intersexuality in the amphipod, Echinogammarus marinus?

Following recent studies, in which intersex and/or reproductive abnormalities have been observed in a number of crustaceans in association with pollution, the prevalence of intersexuality in the amphipod, Echinogammarus marinus was monitored at sites receiving industrial discharges for one year in 2002/2003. Based upon reports of intersexuality occurring in amphipods due to feminising microsporidian parasites, the occurrence, and role of microsporidian parasites in causing intersex was investigated through histological examination. Results demonstrate a significantly higher prevalence of intersex organisms was found at sites receiving industrial discharges throughout the year when compared to a reference site, with the phenotype of intersex (intersex male and intersex female) varying in its dominance between impacted sites. Intersex specimens were significantly more likely to be infected with microsporidian parasites at sites receiving discharges than reference sites, however relatively few specimens (normal or intersex) were infected at reference sites suggesting parasitism is not the only cause of intersex. The direct/indirect role of pollution in the observed intersexuality is discussed.

The use of an unsupervised learning approach for characterizing latent behaviors in accelerometer data.

The recent increase in data accuracy from high resolution accelerometers offers substantial potential for improved understanding and prediction of animal movements. However, current approaches used for analysing these multivariable datasets typically require existing knowledge of the behaviors of the animals to inform the behavioral classification process. These methods are thus not well-suited for the many cases where limited knowledge of the different behaviors performed exist. Here, we introduce the use of an unsupervised learning algorithm. To illustrate the method's capability we analyse data collected using a combination of GPS and Accelerometers on two seabird species: razorbills (Alca torda) and common guillemots (Uria aalge). We applied the unsupervised learning algorithm Expectation Maximization to characterize latent behavioral states both above and below water at both individual and group level. The application of this flexible approach yielded significant new insights into the foraging strategies of the two study species, both above and below the surface of the water. In addition to general behavioral modes such as flying, floating, as well as descending and ascending phases within the water column, this approach allowed an exploration of previously unstudied and important behaviors such as searching and prey chasing/capture events. We propose that this unsupervised learning approach provides an ideal tool for the systematic analysis of such complex multivariable movement data that are increasingly being obtained with accelerometer tags across species. In particular, we recommend its application in cases where we have limited current knowledge of the behaviors performed and existing supervised learning approaches may have limited utility.

Use of mathematical modeling to study copper metabolism in humans.

BACKGROUND: An improved understanding of copper metabolism is needed to derive more precise estimates of dietary requirements. OBJECTIVES: The objectives were to validate a method for estimating endogenous losses of copper, test whether a simple model can predict true absorption from the plasma appearance of labeled copper, and develop a compartmental model for copper metabolism by using stable isotopes. DESIGN: A stable isotope of copper was intravenously administered to 6 men, and fecal samples were collected for 14 d. Four weeks later the study was repeated, but with an oral dose, and blood samples were collected for 7 d and fecal samples for 14 d. RESULTS: There was no significant difference (P = 0.48) in the estimated endogenous loss of copper calculated by using either the excreted intravenous dose (x +/- SD: 32 +/- 5%) or the absorbed and excreted oral dose (35 +/- 2%). A simple mathematical model fitted to plasma isotope appearance data estimated true absorption to be 8 +/- 2% compared with 48-49% measured by fecal monitoring. A more complicated compartmental model predicted that, when newly absorbed copper first enters the blood, 74% is removed by the liver and 99% is bound to ceruloplasmin in the plasma. The exchangeable pool of copper was estimated to be 43 +/- 30 mg. Daily endogenous losses were predicted to be 2.4 mg. CONCLUSIONS: The results showed that fecal monitoring is the only method that can reliably measure labeled copper absorption, and it is not necessary to administer an intravenous dose of copper to estimate endogenous losses. The compartmental model provides new insights into human copper metabolism.

A new perspective on how humans assess their surroundings; derivation of head orientation and its role in 'framing' the environment.

Understanding the way humans inform themselves about their environment is pivotal in helping explain our susceptibility to stimuli and how this modulates behaviour and movement patterns. We present a new device, the Human Interfaced Personal Observation Platform (HIPOP), which is a head-mounted (typically on a hat) unit that logs magnetometry and accelerometry data at high rates and, following appropriate calibration, can be used to determine the heading and pitch of the wearer's head. We used this device on participants visiting a botanical garden and noted that although head pitch ranged between -80° and 60°, 25% confidence limits were restricted to an arc of about 25° with a tendency for the head to be pitched down (mean head pitch ranged between -43° and 0°). Mean rates of change of head pitch varied between -0.00187°/0.1 s and 0.00187°/0.1 s, markedly slower than rates of change of head heading which varied between -0.3141°/0.1 s and 0.01263°/0.1 s although frequency distributions of both parameters showed them to be symmetrical and monomodal. Overall, there was considerable variation in both head pitch and head heading, which highlighted the role that head orientation might play in exposing people to certain features of the environment. Thus, when used in tandem with accurate position-determining systems, the HIPOP can be used to determine how the head is orientated relative to gravity and geographic North and in relation to geographic position, presenting data on how the environment is being 'framed' by people in relation to environmental content.

Temporal changes in gene expression in the liver of male plaice (Pleuronectes platessa) in response to exposure to ethynyl oestradiol analysed by macroarray and Real-Time PCR.

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially-expressed in liver from male plaice (Pleuronectes platessa) exposed to ethynyl oestradiol (EE2). BLAST analysis and alignments of the clones with database sequence suggested at least three vitellogenin (VTG) genes and three zona radiata protein (ZRP) genes were represented. Clones with unique sequence (62 up-, 13 down-regulated) were arrayed as probes on nylon membranes to investigate temporal expression of oestrogen-responsive genes in experimental animals. Arrays were hybridised with radiolabelled cDNAs prepared from hepatic mRNA from animals treated with EE2 for various times upto 21 days and from treated animals transferred to clean water for upto a further 31 days. By day 21 of treatment 11 out of 17 probes from unidentified genes, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other gene sequences were induced by EE2 exposure. Of the down-regulated sequences, only three showed significant, decreased expression and these encode cytochrome b and two with cryptic functions. Based on the pattern of temporal response the up-regulated probes fell into two classes. Pattern A reached maximum expression by day 16 of exposure and then declined prior to removal of EE2 at 21 days. Pattern B genes reached maximal expression between day 16 and 22, declining only after removal of EE2. Independent investigation of the expression patterns of selected probes using quantitative Real-Time PCR reproduced the distinctive patterns. The results indicate a previously unrecognised mechanism for oestrogenic toxicity in which there is a selective down-regulation of some egg proteins, potentially diminishing the quality of eggs and this may contribute to reproductive failure described elsewhere.

Effects of sewage effluent and ethynyl oestradiol upon molecular markers of oestrogenic exposure, maturation and reproductive success in the sand goby (Pomatoschistus minutus, Pallas).

Male fish in several UK estuaries are known to be exposed to oestrogenic contamination, and whilst a limited number of studies have shown that exposure to oestrogens can reduce the reproductive success of fish, the impact of environmentally relevant exposures is less clear. The aim of this study was, therefore, to investigate the effects of exposure to environmentally realistic concentrations of a sewage effluent and the synthetic oestrogen 17alpha-ethynyl oestradiol (EE(2)) upon the reproductive success of a marine fish. Sand goby (Pomatoschistus minutus) were exposed for 7 months to EE(2) or a sewage effluent containing known xeno-oestrogens (alkylphenol polyethoxylates) and bred using within treatment crosses. Nominal exposure concentrations were 6 ng l(-1) EE(2), 0.3 or 0.03% v/v sewage effluent. At the end of the breeding trials, expression of hepatic zona radiata protein (Zrp) and vitellogenin (Vtg) mRNA were determined using two recently developed cDNA probes. Exposure to 6 ng l(-1) EE(2) induced Zrp and Vtg mRNA expression in male and female sand goby, impaired male maturation and reproductive behaviour, reduced female fecundity and reduced egg fertility. As a consequence, fertile egg production of the EE(2)-exposed population was reduced by 90%. Exposure to sewage effluent (0.3% v/v) increased adult mortality and female Zrp and Vtg mRNA expression, but did not induce male vitellogenesis. Exposure to EE(2) and 0.3% v/v sewage effluent impaired development of the male urogenital papilla. Fish exposed to 0.03% v/v sewage effluent produced more fertile eggs than those exposed to 0.3% effluent, or those receiving no effluent. It is concluded that male vitellogenesis in an oestrogenically exposed population may be accompanied by reduced reproductive success, but that it may not be indicative of altered reproductive output in a population exposed to an industrial sewage effluent.

Localised remobilization of metals in a marine sediment.

Trace metals and Fe and Mn were measured at vertical spatial resolutions of 2.5 and 5 mm in the top 35 cm of the profundal sediment of a Scottish sea-loch using DGT (diffusive gradients in thin films) technique. DGT probes lower adjacent metal concentrations in pore waters and induce a flux of metal from the solid phase to porewater. The concentrations of metals in porewaters at the interface of the probe were measured during its deployment in a box core. These measurements reflect porewater concentrations of metals and their rates of resupply from the local solid phase of a very small volume (25 microl) of sediment. There was pronounced horizontal and vertical structure in the interfacial concentrations. Horizontal variations were shown by results from adjacent DGT assemblies being markedly different in detail, while vertical structure was measured directly by the DGT-depth profiles. Iron and Mn varied systematically with depth, with both broad and detailed features of Co aligning with those of Mn. There was, however, evidence of additional localised sources of Co that were apparently unrelated to the redox behaviour that Mn typifies, but associated with the remobilization of Ni, possibly from mineral dissolution. Arsenic(III) was remobilized in well-defined zones. Detailed correspondence of As(II) with some Fe features suggest that its release is mechanistically-related to iron oxide dissolution, but the 3 orders of magnitude higher concentrations of Fe may sometimes obscure the association. These results demonstrate that, within sediments, metals may be released in discrete locations that are not measured by conventional porewater sampling techniques due to their horizontal averaging.

Horizontal and vertical distribution of organic contaminants in deep-sea fish species.

Polychlorobiphenyls (PCBs) and pesticides are persistent, semivolatile contaminants whose global circulation is now well established. One of their ultimate sink is the bio- and geo-phases in the deep-sea. We have investigated the distribution of selected PCBs and pesticides with depth and longitude in various deep-sea fish, namely black scabbardfish (Aphanopus carbo), orange roughy (Hoplostethus atlanticus), roundnose grenadier (Coryphaenoides rupestris) and Bathysaurus ferox from Madeira, Sesimbra, the Meriadzec Terrace and Rockall Trough in the North Eastern Atlantic at 1000 m depth, and at 2000 m depth where available. In most species, males presented higher levels of contamination than females. This pattern was probably due to the females eliminating contaminants through egg production. Roundnose grenadier presented higher levels of contamination (mainly p,p'-DDE, CB 153, 138, 180, sigmaCB(24) and p,p'-DDT) in fish caught at 2000 m than at 1000 m depth. Similarly, B. ferox, which is found deeper than the other species studied, showed much higher levels of most CBs and pesticides (lipid normalised) than the other species. Concentrations were up to 10 times higher, and showed differences between water masses.

Effects of prolonged exposure to 4-tert-octylphenol on toxicity and indices of oestrogenic exposure in the sand goby (Pomatoschistus minutus, Pallas).

Alkylphenolic compounds are present in estuarine and marine environments. They are moderately bioaccumulative, and oestrogenic to fish following three week, in vivo, exposures. The effects of prolonged exposure are, as yet, unclear. Sand goby (Pomatoschistus minutus) were exposed to measured concentrations of 4-tert-octylphenol (OP) in a 28-day, dose-ranging study, and a six month temporal response study. Following 28-days exposure to measured 31+/-6 or 101+/-47 microg l(-1) OP (mean+/-SD), immature male sand goby showed elevated vitellogenin (VTG) mRNA expression. Plasma alkali-labile phosphate concentrations were elevated in both sexes and were higher in females than males. Although measured OP concentrations were variable, they were significantly different for each treatment and a range of concentration-dependent effects were observed. The LC(50) for chronic OP exposure (8 weeks) was 29+/-6 microg l(-1), and both sexes demonstrated concentration and duration dependent increases in VTG mRNA expression. Exposure to 28+/-20 microg l(-1) OP (median+/-95% CI) for 6 months inhibited development of male nuptial colouration and sperm duct glands. These findings are discussed in relation to environmental concentrations of alkylphenols and ecological concerns.

Identification of transcriptional effects of ethynyl oestradiol in male plaice (Pleuronectes platessa) by suppression subtractive hybridisation and a nylon macroarray.

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.

Cypermethrin induces glutathione S-transferase activity in the shore crab, Carcinus maenas.

Cypermethrin is a synthetic pyrethroid that is particularly toxic to crustacea. It is therefore applied as a chemotherapeutant in the salmonid aquaculture industry for the treatment of sea lice infestations. After use, cypermethrin is released directly into the marine environment, to be diluted by fresh seawater. The shore crab, Carcinus maenas is found in the vicinity of fish farms, and may come into contact with released cypermethrin. The detoxification enzyme, glutathione S-transferase (GST) has been implicated in cypermethrin metabolism in terrestrial arthropods, but this has not yet been demonstrated in crustacea. In this paper we investigate the response of GST activity in Carcinus to cypermethrin exposure, and also the time course of the induction process. GST activity is significantly increased in Carcinus exposed to nominal concentrations of 50 and 500 ng/l of water-borne cypermethrin. Carcinus demonstrate a significant elevation in GST activity following intra-cephalothoracic injection with 10 ng of cypermethrin. GST activity returns to basal levels after 36 h. The potential application of GST activity in Carcinus as a biomarker of cypermethrin exposure is discussed.

Characterisation of choline esterases and their tissue and subcellular distribution in mussel (Mytilus edulis).

Acetylcholinesterase in mussel is potentially a useful biomarker of exposure to organophosphates (OP) in the marine environment. This study looked at cholinesterase activity in subcellular fractions of various tissues from the common mussel, Mytilus edulis. Measurement of enzyme rates demonstrated that although highest specific activity was found in foot 'mitochondrial' fraction, recovery of activity was very low. Gill 'microsomal' fraction had the second highest specific activity with a useful level of recovery and therefore was the most suitable tissue fraction for biomarker applications. Comparative studies of alternative alkylthiocholine substrates and competitive inhibitors suggest there is a single cholinesterase enzyme type present in this fraction. Inhibition of alkylcholine hydrolysis by BW284C51, specific to acetylcholinesterase in vertebrates, showed that cholinesterase activity in gill 'microsomal' fraction is inhibited by this compound but to a lesser extent than in vertebrate AChE. Inhibition of cholinesterase activity by azamethiphos in gill 'microsomal' fraction gave an IC50 of approximately 100 microM and showed both time and concentration dependence. However this indicates a lower potency compared to other animals and it is debatable whether mussel cholinesterase activity is useful as a biomarker of exposure in the field.