Identification of a gene set to evaluate the potential effects of loud sounds from seismic surveys on the ears of fishes: a study with Salmo salar.

Functional genomic studies were carried out on the inner ear of Atlantic salmon Salmo salar following exposure to a seismic airgun. Microarray analyses revealed 79 unique transcripts (passing background threshold), with 42 reproducibly up-regulated and 37 reproducibly down-regulated in exposed v. control fish. Regarding the potential effects on cellular energetics and cellular respiration, altered transcripts included those with roles in oxygen transport, the glycolytic pathway, the Krebs cycle and the electron transport chain. Of these, a number of transcripts encoding haemoglobins that are important in oxygen transport were up-regulated and among the most highly expressed. Up-regulation of transcripts encoding nicotinamide riboside kinase 2, which is also important in energy production and linked to nerve cell damage, points to evidence of neuronal damage in the ear following noise exposure. Transcripts related to protein modification or degradation also indicated potential damaging effects of sound on ear tissues. Notable in this regard were transcripts associated with the proteasome-ubiquitin pathway, which is involved in protein degradation, with the transcript encoding ubiquitin family domain-containing protein 1 displaying the highest response to exposure. The differential expression of transcripts observed for some immune responses could potentially be linked to the rupture of cell membranes. Meanwhile, the altered expression of transcripts for cytoskeletal proteins that contribute to the structural integrity of the inner ear could point to repair or regeneration of ear tissues including auditory hair cells. Regarding potential effects on hormones and vitamins, the protein carrier for thyroxine and retinol (vitamin A), namely transthyretin, was altered at the transcript expression level and it has been suggested from studies in mammalian systems that retinoic acid may play a role in the regeneration of damaged hair cells. The microarray experiment identified the transcript encoding growth hormone I as up-regulated by loud sound, supporting previous evidence linking growth hormone to hair cell regeneration in fishes. Quantitative (q) reverse transcription (RT) polymerase chain reaction (qRT-PCR) analyses confirmed dysregulation of some microarray-identified transcripts and in some cases revealed a high level of biological variability in the exposed group. These results support the potential utility of molecular biomarkers to evaluate the effect of seismic surveys on fishes with studies on the ears being placed in a priority category for development of exposure-response relationships. Knowledge of such relationships is necessary for addressing the question of potential size of injury zones.

Effects of tebufenozide on some aspects of lake trout (Salvelinus namaycush) immune response.

Tebufenozide is a nonsteroid ecdysone agonist that causes premature and incomplete molting in Lepidoptera and is used on crops and in forest spray programs. Lake trout (Salvelinus namaycush) were exposed to one pulse of 0.25 ppm tebufenozide every 3 days (12 pulses in total). Cell ratios and respiratory burst responses of circulating white blood cells (WBCs) as well as head-kidney (HK) WBCs were investigated by using cell observation, Nitro-Blue Tetrazolium tests, and flow cytometry. Endpoints studied suggest a difference in sensitivity between HK and circulating blood cells to tebufenozide and show a stimulation of fish cell function and changes in percentages of cell types. Responses are not associated with a strong stress response as highlighted by the absence of effect on cortisol and blood protein levels. These results and tebufenozide persistence in water warrant further studies on pesticide impact on fish immunity when used on crops or in forest spray programs near lakes and streams.

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons.

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.

Some factors influencing EROD activity in winter flounder (Pleuronectes americanus) exposed to effluent from a pulp and paper mill.

EROD (ethoxyresorufin-O-deethylase) activity was determined in winter flounder, a sediment-inhabiting and non-migratory fish species, living near a pulp and paper mill in Newfoundland in relation to temperature, gender, sexual maturity, and lesions in the liver. Samples of liver were taken from fish captured by SCUBA divers at 0 degrees C, 5 degrees C and 16 degrees C. Enzymic activity was detected in fish living only above 0 degrees C. Adult males and juvenile fish had higher levels of EROD activity than prespawning females at 5 degrees C. Macrophage aggregates only or occurring simultaneously with bile ductule hyperplasia and clear cell foci in the liver, did not impair EROD activity but necrosis had a negative effect. Results from this study indicate the importance of water temperature, gender, sexual maturity and liver pathology in assessing EROD activity of fish in biomonitoring programs.

Are metal mining effluent regulations adequate: identification of a novel bleached fish syndrome in association with iron-ore mining effluents in Labrador, Newfoundland.

Water quality guidelines for industrial effluents are in place in many countries but they have generally evolved within a limited ecotoxicological framework. Effluents from iron-ore mines have traditionally been viewed by regulatory bodies as posing little or no risk to the aquatic environment. However, it was recently reported that lake trout taken from a large iron-ore contaminated Lake in Labrador (Wabush Lake) had elevated levels of DNA oxidative damage and were markedly depleted in levels of vitamin A (Payne et al., 1998) in comparison with fish from a Lake (Shabogamo Lake) receiving lesser levels of effluents. Through further observations, it has now been established that the lake trout in Wabush Lake are commonly affected with a marked skin bleaching syndrome in comparison with fish in Shabogamo Lake and a nearby Lake (Ashuanipi) which does not receive effluents. To the authors' knowledge such a syndrome which is characterized by marked reduction in skin pigmentation and overall increase in skin whitening has not been reported before in any fish population in association with contamination. Preliminary information for liver histopathological and blood cell differences have also been obtained in fish in Wabush Lake in comparison with Ashuanipi Lake. It has also been observed through studies on phosphatidyl liposomes that iron-ore leachate contains redox-active material (iron but possibly other transition metals) that has considerable potential for causing oxidative damage to cellular constituents. Using the weight of evidence approach it is indicated that iron-ore effluents may pose more of a risk to the aquatic environment than traditionally considered by regulatory agencies.

Note: evaluation of selective media for the enumeration of Bifidobacterium sp. in milk.

Pure cultures of three species of bifidobacteria (Bifidobacterium longum, Bif. adolescentis and Bif. bifidum), Lactobacillus acidophilus and a mixed culture of Lact. delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus were each enumerated on two differential media and six selective media for the enumeration of bifidobacteria. The appearance of the colonies on the differential media was as expected but when mixed cultures were present, it proved extremely difficult to distinguish one species from another. Of the selective media, AMC, RMS, NPNL and BL-OG performed well in that they gave good recoveries of bifidobacteria and were inhibitory to the growth of Lact. delbrueckii subsp. bulgaricus, Strep. salivarius subsp. thermophilus and Lact. acidophilus. However, of these four media, AMC was most convenient as it is based on a commercially available medium, whereas the others must be made up from individual constituents. The AMC agar is thus a good choice for the routine enumeration of bifidobacteria from mixed cultures.

Polycyclic aromatic hydrocarbon-DNA adducts in beluga whales from the Arctic.

The Arctic is still relatively pristine in nature, but it is also vulnerable to pollution because contaminants originating from midlatitudes are transported to the Arctic by atmospheric processes, ocean currents, and rivers (Muir et al., 1992). Recognition of this fact of Arctic vulnerability has resulted in a Declaration on the Protection of the Arctic Environment by eight Arctic countries. A manifest aim of this declaration is to develop an Arctic Monitoring and Assessment Program. We report here on the presence of measurable levels of polycyclic aromatic hydrocarbon-DNA adducts, including relatively high levels in Arctic beluga (Delphinapterus leucas). These results lend support to the value of developing biological assessment programs for Arctic wildlife.

Effect of toxaphene on reproduction of fish.

The effect of Toxaphene on fish reproduction was investigated in sexually mature female zebrafish fed for two weeks with food contaminated with three different concentrations of the Pesticide (0.02, 0.23 and 2.2 micrograms/g fish/day). No overt differences were observed in reproductive success as assessed by examination of (a) total number of eggs spawned by each female, (b) percentage of fertilized eggs 24 hours after fertilization, (c) percentage of embryo mortality 72 hours after fertilization and (d) percentage of hatching 72 hours after fertilization. By contrast Toxaphene produced distinct effects of a dose response nature on oviposition. This observation is not only of interest with respect to Toxaphene, but also points to the importance of evaluating other pesticides for their effect on the oviposition of feral species that might be even more sensitive than zebrafish. Toxicity of Toxaphene was manifested both in the parent fish (skin discoloration, subcutaneous hemorrhages, particularly around the gill areas and backbones curved in the vertical plane) and in hatching embryos (half hatched).

A multidisciplinary approach using several biomarkers, including a parasite, as indicators of pollution: a case history from a paper mill in Newfoundland.

Several biomarkers are currently used to determine chronic exposure of fish to environmental contaminants. In studies to assess the effects of pulp mill effluent on winter flounder (Pleuronectes americanus), body and hepatosomatic indices (HSIs), histopathology, levels of hepatic detoxifying enzymes, mixed function oxygenases (MFO), muscle acetylcholinesterase (Ache) and selected parasite abundance were compared in samples taken near a paper mill (sulphite-bleaching) and a number of reference sites. Our studies revealed a number of significant (P < 0.05) differences between samples taken near the paper mill and reference sites. Flounder captured at two locations near the mill were greater in length and weight, had elevated HSIs and more lesions in the liver and spleen while an acanthocephalan, Echinorhynchus gadi, located in the digestive tract, was lower than at more distant sites. Activity of MFO and Ache were similar at most sites and might be a reflection of the fish's foraging behaviour and consequently intermingling of populations from different locations during summer. These results support the view that several biomarkers should be utilised when assessing the health of fish living in habitats degraded by pollutants.

Bioaccumulation of aromatic hydrocarbons from sediments: a dose-response study with flounder (Pseudopleuronectes americanus).

Pseudopleuronectes americanus were chronically exposed to Hibernia crude oil in sediments, for 4 months. Oil was added to sediments at five concentrations between 0.09 and 4.5 mg/g (dry weight) and was 0.10-0.90 mg/g, at the termination of the exposure. Bioaccumulation measured in terms of fluorescence or in terms of the concentration of specific aromatic targets, increased with dosage. Accumulation of hydrocarbons was observed in muscle tissue (0.22 microgram/g, dry weight), when concentration of the sum of 27 polycyclic aromatic compounds (PAC) in sediments was of 0.65 microgram/g (E-50), at the end of the 4-month period. Of the 27 parental and alkylated polycyclic aromatic compounds analyzed, alkylated naphthalenes predominated in muscle (90-100%) and in sediments (30-60%). Bioaccumulation factors were derived for 13 compounds detected in muscle, at the three higher exposures. Liver concentrations (fluorescence) were higher than in muscle, but did not display a noticeable dose-response. Several alkylbenzenes, a C-2 biphenyl and C-4 acenaphthene were also detected in muscle extracts. The development of dose-response relationships for polycyclic aromatic hydrocarbons (PAH) present in sediment, in relation to bioaccumulation in flatfish, is of major interest for evaluating the environmental effects of oil contamination.

Polycyclic aromatic compounds in cod (Gadus morhua) from the Northwest Atlantic and St. Lawrence Estuary.

There is limited information available on levels of polycyclic aromatic hydrocarbons (PAH) in major fish populations including populations from the Northwest Atlantic. The cod (Gadus morhua) stocks off eastern Canada form the basis of one of the world's most important fisheries. Muscle tissues of cod collected from Northwest Atlantic Fisheries Organisation (NAFO) management Divisions 2J, 3K and 3Ps off Newfoundland and Labrador, as well as three contaminated sites in the Gulf of St. Lawrence were analyzed for total polycyclic aromatic (PAC) by fluorimetry. Concentrations were determined in terms of crude oil and chrysene equivalents in line with recommendations of the International Oceanographic Commission. Overall, relatively low concentrations of PAC were found, the highest values generally being below 0.1 microgram/g (dry weight) in terms of chrysene equivalents. Similarly, only trace levels of a few PAH were detected in composite samples analyzed by gas chromatography-mass spectrometry (GC-MS). It is of interest that the highest levels of PAC were found in fish from NAFO Division 3K, while concentrations in fish from the two contiguous zones, 2J and 3Ps, as well as the Gulf of St. Lawrence, were similar. Division 3K is a major fishing zone and it is important to determine if trawler fleets are important sources of hydrocarbons derived from fossil fuels, in this and similar fishing areas of the world's oceans.

Polycyclic aromatic compounds in Northwest Atlantic COD (Gadus morhua).

Twenty-seven polycyclic aromatic compounds (PAC) including polycyclic aromatic hydrocarbons (PAH) and polycyclic aromatic sulphur heterocycles were analysed in muscle, liver and ovaries of cod (Gadus morhua) from the Northwest Atlantic. These PAC include the 16 PAH priority pollutants (EPA recommendation), alkylated naphthalenes, phenanthrene-anthracenes as well as dibenzothiophenes (DBT). Aromatics were undetectable in muscle. One of the analysed PAC was detected in one ovary and six in another ovary sample. Liver samples contained between 1 and 9 PAC. Of the parental PAH, only acenapthene (18 ng/g, dry weight), fluorene (28 ng/g) and chrysene (22 ng/g) were detected once each in two liver samples, while fluorene (72 ng/g) was detected once in one of the ovaries. C-2 DBT was the major component in ovaries and liver (8-86 ng/g), while C-3 and C-4 alkylated phenanthrene-anthracene (12-78 ng/g) were the next major components detected in the liver samples. This is the first detailed study of PAH and PAC in finfish from the Northwest Atlantic.

Organochlorines including polychlorinated biphenyls in muscle, liver, and ovaries of cod, Gadus morhua.

Twenty-three specific organochlorine contaminants and polychlorinated biphenyls (PCBs), measured as three Aroclor standards were analyzed in muscle, liver, and ovaries of cod, Gadus morhua, collected in the Northwest Atlantic. In general, contaminants were undetectable in muscle tissue, while concentrations were 10 times lower in ovaries than liver (wet weight). Comparison of results to other locations indicated a similarity between the ratio of the concentrations of p,p'-DDE, p,p'-DDD and p,p'-DDT, in liver of cod from the northern North Sea and from the Northwest Atlantic, although with lower levels in the present study. The ratio of alpha-HCH and gamma-HCH was between that of the central and northern North Sea. Similar ratios tend to indicate similar residence times in the atmosphere, from source to sampling area. Comparison of sigma PCB and sigma DDT in the liver of cod from various geographical locations showed the following general trend in concentrations: Arctic, Northwest Atlantic, West Atlantic, Norway < North Baltic, Nova Scotia, North Sea < South Baltic. It was observed that if the liver concentration of one compound was low (high), there was a tendency for all compounds to be low (high). Cluster analysis of organochlorines in liver pointed to the presence of four basic clusters, which could reflect similar physical chemical properties within a group. Concentrations of organochlorines in ovaries were below levels expected to affect egg and larval viability.