Altered challenge response in whitefish subchronically exposed in areas polluted by bleached kraft mill effluents

Exposure to impaired water quality, as in bleached kraft mill effluents (BKMEs), has recently been demonstrated to impair the ability of fish to elicit an acute stress response. Acute stress caused by catching is, in most field studies, an unavoidable incident that may markedly affect physiological functions. Consistently, dissimilar stress responses to catching procedures in exposed and reference animals may lead to altered results. In this study, juvenile whitefish (Coregonus levaretus) were caged for 30 days in two reference areas and three areas affected by different BKMEs, and the immediate effects of low-level handling on physiological functions in four periods within an hour of the start of the disturbance were clarified. Both primary and secondary stress responses were evident and consistent in fish caged in the two reference areas. Instead, despite the strong cortisol response in fish caged in polluted areas, the secondary stress response was weak or altered with respect to all other measured parameters excluding blood glucose in two of the three polluted areas. Thus, exposed fish may, when compared with reference fish, give different results depending on the exact period from the onset of the catching disturbance to sampling, and therefore, the results may be masked or totally inverted. Because the impacts of acute stress on physiological parameters are immediate and, in exposed areas after low-level handling, dissimilar in nature, this study points to the necessity for time-dependent standardization in field studies whenever sampling is delayed.     

Effects of pulp and paper mill effluent (BKME) on physiology and biochemistry of the roach (Rutilus rutilus L.)

The effects of bleached kraft pulp and paper mill effluent (BKME) on the roach (Rutilus rutilus L.) were studied under experimental and natural field conditions. In the acute experiment (72 h exposure to the concentrated BKME), the roach suffered from a general stress syndrome, characterized by a significant increase of cortisol and blood glucose, as well as a significant decrease of leucocrit and total plasma protein. In three weeks' exposure in a polluted and an unpolluted lake and in fish caught from the same lakes, the more specific effects of BKME treatments appeared. During the three weeks' exposure, slight hyperglycaemia as well as a decrease in a transaminase activity (GPT) and increase in the plasma total protein concentration of the fish occurred in the polluted lake. Fish caught from the polluted lake exhibited lower values of haematocrit, transaminases (GOT and GPT), and calcium concentration plus a higher chloride concentration in the plasma than in the unpolluted lake. The differential leukocyte counts also showed slight differences: Fewer lymphocytes and more granulocytes were found in roach from polluted waters. The morphology of the red blood cells in the roach from polluted lakes had more elongate erythrocytes with a longer major axis and a shorter minor axis than in fish from the polluted lake. The possibilities of determining the origin of fish based on their erythrocyte morphology is discussed.     

Capture and Confinement Stress in White Sucker Exposed to Bleached Kraft Pulp Mill Effluent

This study evaluates the effects of handling and confinement stress and a 3-day recovery period on a number of biochemical parameters used to monitor exposure of fish to bleached kraft mill effluent (BKME). Plasma was collected at four times of the day from male and female white sucker subjected to four levels of handling stress during their spawning migration at a BKME and a reference site. Indicators of a general response to stress (plasma cortisol, glucose, lactate, and total protein) and of reproductive fitness (plasma testosterone, 11-ketotestosterone, and 17β-estradiol) were measured. With the exception of 17β-estradiol in females, all of the parameters measured varied with time of day and stress level. The general stress indicators ranged from being highly to marginally responsive to handling and confinement stress and were inconsistent in terms of indicating a site difference. The reproductive steroids were moderately responsive to stress and gave the most consistent site difference with testosterone and 11-ketotestosterone levels reduced in BKME-exposed fish under low levels of stress. There was limited evidence for recovery from capture, handling, and transport in the spring for plasma cortisol, lactate, and 17β-estradiol at the BKME site, plasma glucose at the reference site, and plasma testosterone at both sites. Fish were also more responsive to an additional acute stress on Day 1 and Day 3 of recovery compared to that on Day 0. This study emphasizes the need for standardized methods in field collections and sampling with the least amount of stress possible, and suggests that holding white sucker for 1 or 3 days does not allow them to recover from the stress of capture.     

Oxidative Stress and Genotoxicity Biomarker Responses in Grey Mullet (<Emphasis Type="Italic">Mugil cephalus</Emphasis>) From a Polluted Environment in Saronikos Gulf,Greece

This study aimed to assess oxidative stress and genotoxicity biomarkers in grey mullet Mugil cephalus from a site receiving discharges from industrial and harbor activities and a reference site in Saronikos Gulf, Greece. Cellular antioxidant defenses, i.e., antioxidant enzyme activities (catalase, selenium [Se]-dependent glutathione peroxidase, glutathione-S-transferase) and oxidative damage, i.e., lipid peroxidation (measured as thiobarbituric acid reactive substances) were used as biomarkers of oxidative stress. The micronucleus test was used for evaluation of chromosome damage, and the frequency of cells with double nuclei was used as an indication of damage to cell divisions. Antioxidant enzyme activities were lower in fish from the polluted site compared with fish from the reference site, suggesting deficiency of the antioxidant system to compensate for oxidative stress. This is further supported by the higher lipid peroxidation levels in fish from the polluted site, which represent oxidative damage. Micronuclei and double-nuclei frequencies were higher in fish from the polluted site compared with fish from the reference site, indicating genotoxic effects. Correlations between biomarkers suggest that observed effects were due to contaminants exhibiting oxidative stress potential that can also induce genotoxicity. Results suggest that the selected biomarkers in M. cephalus are useful for the assessment of pollution impacts in coastal environments influenced by multiple pollution sources.     

Polycyclic Aromatic Hydrocarbon Metabolites in the Bile of a Territorial Benthic Fish, Oyster Toadfish (Opsanus tau) from the Elizabeth River, Virginia

Reverse-phase high-performance liquid chromatographic analyses of bile of a territorial benthic fish, oyster toadfish (Opsanus tau), indicated that fish from reference stations in the York and Elizabeth Rivers, Virginia, contained lower concentrations of polycyclic aromatic hydrocarbon (PAH) metabolites than fish from polluted stations. PAH metabolite levels in the bile of fish from mildly polluted stations were 7 to 10 times greater than those from reference stations. PAH metabolite levels in fish from a moderately polluted station and a highly polluted station were, respectively, 20 and 50 times greater than those from reference stations. Differential patterns of five major PAH metabolites in fish from the same station suggested individual variability in metabolic pathways possibly further convoluted by the differential inductions or suppressions of hepatic mixed-function oxygenase isozyme systems under various natural or anthropogenic habitat parameters. PAH metabolite levels in the bile of oyster toadfish correlated well with the gradient of PAH contamination in the Elizabeth River sediments. High levels of biliary PAH metabolites were not detected in muskrats (Ondatra zibethicus) collected along the polluted sections of the Elizabeth River, probably due to their primarily herbivorous nature. Assuming that the hepatobiliary system and the gastrointestinal tract are the major routes of biotransformation and excretion of PAHs in the muskrats, the contaminated diet appears to be a more important routes of exposure of muskrats (and possibly oyster toadfish) to PAHs than the transdermal transfer. Occurrence permitting, we propose oyster toadfish as a useful biological sentinel for early detection of estuarine PAH pollution. Received: 27 January 2001/Accepted: 1 July 2001     

Effects of Methylmercury on Ontogeny of Prey Capture Ability and Growth in Three Populations of Larval Fundulus heteroclitus

We used three populations of mummichogs (Fundulus heteroclitus), one from a polluted site (Piles Creek [PC], New Jersey) and two from cleaner sites (Tuckerton [TK], New Jersey, and East Hampton [EH], New York), to study (1) whether embryonic, embryonic plus larval, or larval exposure to methylmercury (MeHg) altered larval prey capture ability and growth; and (2) whether there were differences in tolerance to MeHg-induced behavioral changes among the three populations. Eggs and sperm were obtained from mummichogs captured in the field, and their embryos and larvae were kept in clean sea water or MeHg solution (5, 10 μg/L). Larvae were then tested regularly for prey capture rates and prey capture efficiencies, and their lengths were measured. Embryonic exposure to MeHg induced transitory and recoverable impairments in larval prey capture ability, whereas larval exposure alone was relatively ineffective. When both embryos and larvae were treated, larval prey capture ability was affected at a lower concentration and a wider range of larval ages. In terms of growth and prey capture ability, response of larvae to embryonic or larval or exposure to both stages to MeHg varied with populations. TK fish were the most tolerant with respect to behavioral changes but were the most sensitive to MeHg in reduction of growth. EH fish were the most sensitive whenever embryos were treated, and PC fish were the most vulnerable after larval exposure. The population differences in response to MeHg intoxication may be due to pollution related factors or differences in behavioral-related genetic factors. Received: 23 February 2000/Accepted: 21 January 2001     

Heavy Metals in the Nase, Chondrostoma nasus (L. 1758), and Its Intestinal Parasite Caryophyllaeus laticeps (Pallas 1781) from Austrian Rivers: Bioindicative Aspects

Tissue samples from 121 adult specimens of the predominantly herbivorous fish species nase, Chondrostoma nasus (L. 1758), from five river sites in Austria were analyzed for their metal content. Sediments and water samples of the sites show different levels of metal load, with only one site considered being polluted with metals. The concentrations of cadmium, copper, lead, and zinc in the tissue of the gills, muscle, intestine, and liver of the fish were determined by inductively coupled plasma–optical emission spectrometry (ICP-OES). As in one of the unpolluted and the polluted river site, a well-established population of the tapeworm Caryophyllaeus laticeps (Pallas, 1781) occurred in the intestine of the fish; pooled samples of this worm were analysed as well. Both the nase and C. laticeps show bioindicative ability for metal pollution in rivers. The results allow a more differentiated characterization of the rivers than the sediment analyses. Cadmium is found mainly in the liver, with maximum concentrations of 1.57 μg/g dry weight from unpolluted sites and 5.58 μg/g from the polluted site. The highest concentrations of copper are also found in the liver, with values between 25 and 333 μg/g. A significantly elevated concentration of Cu in the intestine from an “unpolluted” site (mean: 24.06 μg/g) indicates an acute pollution in this area at the time of sampling. Lead was found mainly in the intestine and liver in concentrations between 0.09 and 4.05 μg/g and 0.26 and 1.94 μg/g, respectively. In the samples from the polluted site, it also could be detected in the gills (mean: 1.38 μg/g). The parasite C. laticeps shows different capacities for metal accumulation: Although the concentrations of Cu were significantly lower compared to the values of the fish liver, cadmium was detected in concentrations up to 5.1 times higher. Lead and zinc concentrations were found to be up to 9.7 and 3.0 times higher in the tapeworm compared to the fish liver, respectively.     

Assessment of tolerant sunfish populations (Lepomis sp.) inhabiting selenium-laden coal ash effluents. 3. Serum chemistry and fish health indicators

Sunfish were collected from fly ash discharge-receiving streams to assess the possible effects of exposure to elevated selenium. Concentrations of selenium, copper, and arsenic were statistically higher in fish tissue (liver) samples from effluent-exposed fish than in reference fish. Several biomarkers were indicative of metal exposure and effect. Plasma protein levels and cholesterol levels were significantly lower in exposed fish, indicating nutritional stress. Ion levels (i.e., K) increased with exposure to ash pond metals, indicating possible gill damage. Fish from the receiving streams also had increased serum glucose and osmolality indicating possible acute stress due to sampling. Fish health assessments revealed a lower incidence of fin erosion, kidney discoloration, urolithiasis or nephrocalcinosis, liver discoloration, and parasites in exposed fish and a higher incidence of skin, eye, and gill aberrations. Condition factors of exposed fish were correlated with biomarker response and were the same as or lower than those of reference fish, but not related to selenium levels. Although several serum biochemical indicators differed between the ash pond-receiving stream and reference sites, pollutant exposure was apparently not sufficient to cause functional damage to critical organ systems.     

Assessment of Tolerant Sunfish Populations (Lepomis sp.) Inhabiting Selenium-Laden Coal Ash Effluents: 3. Serum Chemistry and Fish Health Indicators

Sunfish were collected from fly ash discharge-receiving streams to assess the possible effects of exposure to elevated selenium. Concentrations of selenium, copper, and arsenic were statistically higher in fish tissue (liver) samples from effluent-exposed fish than in reference fish. Several biomarkers were indicative of metal exposure and effect. Plasma protein levels and cholesterol levels were significantly lower in exposed fish, indicating nutritional stress. Ion levels (i.e., K) increased with exposure to ash pond metals, indicating possible gill damage. Fish from the receiving streams also had increased serum glucose and osmolality indicating possible acute stress due to sampling. Fish health assessments revealed a lower incidence of fin erosion, kidney discoloration, urolithiasis or nephrocalcinosis, liver discoloration, and parasites in exposed fish and a higher incidence of skin, eye, and gill aberrations. Condition factors of exposed fish were correlated with biomarker response and were the same as or lower than those of reference fish, but not related to selenium levels. Although several serum biochemical indicators differed between the ash pond-receiving stream and reference sites, pollutant exposure was apparently not sufficient to cause functional damage to critical organ systems.     

Fish thyroidal and stress responses in contamination monitoring--an integrated biomarker approach

Xenobiotics may affect fish endocrine system, making endocrine responses relevant in pollution monitoring. Five critical sites in Ria de Aveiro (Portugal), having different contamination profiles, were assessed in comparison to a reference site (Torreira), focusing on Liza aurata stress (plasma cortisol, glucose and lactate) and thyroidal (plasma thyroid stimulating hormone—TSH, thyroxine—T4 and triiodothyronine—T3) responses. L. aurata at Vagos (PAHs contaminated) and Laranjo (mercury contaminated), respectively, displayed, low cortisol and high glucose as well as lactate levels. Considering thyroidal responses, high TSH levels were found at Laranjo; low T4 levels were found at Barra (subject to naval traffic) and low T3 levels were found at Rio Novo do Principe (near a former bleached kraft pulp mill effluent), Laranjo and Vagos. The integrated biomarker index ranked impacted sites as: Laranjo>Vagos>Gafanha>Rio Novo do Principe>Barra>Torreira. Endocrine responses reflected xenobiotics exposure suggesting environmental risk to fish at Ria de Aveiro. However no clear relation was found between stress and thyroidal responses.     

A Study of the Lethal and Sublethal Toxicity of Polyphase P-100, an Antisapstain Fungicide Containing 3-Iodo-2-Propynyl Butyl Carbamate (IPBC), on Fish and Aquatic Invertebrates

The acute toxicity of Polyphase P-100, an antisapstain wood preservative that contains 97% 3-iodo-2-propynyl butyl carbamate (IPBC), was determined for three species of fish (coho salmon, rainbow trout, and starry flounder) and three species of aquatic invertebrates (Daphnia magna, Hyalella azteca, and Neomysis mercedis). The 96-h LC₅₀ values for the various fish species exposed to Polyphase P-100 ranged from 95 ppb for coho smolts (Oncorhynchus kisutch) to 370 ppm for juvenile starry flounder (Platichthys stellatus). The sensitivity of coho to Polyphase P-100 was altered by their developmental stage. Coho embryos were six to nine times more tolerant of Polyphase P-100 than coho alevins, which were twice as tolerant as coho smolts. The 48-h LC₅₀ values for the invertebrates D. magna, H. azteca, and N. mercedis were 40 ppb, 500 ppb, and 2,920 ppb, respectively. In addition to a wider range of sensitivity to Polyphase P-100 compared with the fish species, the invertebrate species were characterized by a shallower concentration-response. In acute, 24-h sublethal tests with juvenile starry flounder and rainbow trout, there was no primary or secondary stress response (changes in hematocrit, leucocrit, hemoglobin concentration, plasma lactate concentration, and plasma cortisol concentration) at concentrations up to 50% of the 96-h LC₅₀ value. The acute toxicity of a 1:8 mixture of Polyphase P-100 and Bardac 2280 (another antisapstain compound that contains didecyldimethylammonium chloride [DDAC] as the active ingredient) was close to additive for fish, but not for invertebrate species. The acute toxicity of the mixture was seven to eight times more than additive for H. azteca, but two to three times less than additive for D. magna. Some sublethal stress responses were revealed with the mixture that were not observed with the test chemicals alone. Received: 10 October 1997/Accepted: 28 March 1998     

Comparison of the Heavy Metals,Copper, Iron,Magnesium, Nickel,and Zinc Between Muscle and Gills of Four Benthic Fish Species from Shif Island (Iran)

Concentrations of copper (Cu), iron (Fe), manganese (Mn), nickel (Ni) and zinc (Zn) in muscle and gills of four Benthic fish species (Brachirus orientalis, Argyrops spinifer, Platycephalus indicus and Sillago sihama) from Shif Island of Iran were analyzed using flame atomic absorption spectrometry. The heavy metal levels observed in tissues varied for Cu: 2.15–820.98, Fe: 24.46–533.26, Mn: 1.80–53.17, Ni: 1.86–65.54 and Zn: 28.77–375.79 mg/kg. Significant differences were found among muscle and gill of the four fish species. Heavy metals concentration were significantly (p < 0.05) higher in the gill than in the muscles of all fish species studied. The metal levels in benthic fish species should be continuously monitored in polluted areas since benthic fish indicated a tendency to bioaccumulate pollutants in their tissues from polluted water. The indices of Estimated Daily Intake, Target Hazard Quotient and Hazard Index suggests no risk while carcinogenic risk for Ni showed unacceptable carcinogenic risk.

     

Oxidative stress and endocrine endpoints in white sucker (Catostomus commersoni) from a river impacted by agricultural chemicals

The effects of agricultural chemicals on cortisol secretion, antioxidants, and lipid peroxidation were investigated in hepatic and adrenal tissue of white sucker (Catostomus commersoni) from a river (Yamaska) that drains an agricultural region in Québec (Canada). Plasma cholinesterase (ChE) activity, used as a biomarker of exposure to pesticides, was elevated in fish from the reference site compared to fish from the contaminated sites. Plasma concentrations of cortisol and thyroid hormones (T3 and T4) were higher in fish from the reference site compared to contaminated sites; reduced glutathione (GSH) levels, catalase (CAT), and glutathione peroxidase (GPx) activities were higher and lipid peroxidation (LPO) was lower. Levels of antioxidants (CAT, Gpx, and GSH) were higher (10-90%) and LPO levels were lower (50%) in the liver than in the adrenal tissue. The present in situ study provided evidence that antioxidants, lipid peroxidation, and plasma hormones were altered in fish sampled in areas impacted by agricultural chemicals. Endocrine-disrupting effects were associated with oxidative stress. The results suggest that antioxidants and lipid peroxidation could be used as markers of contaminant exposure in fish.     

Effects of Acute Handling Stress on Whitefish Coregonus lavaretus After Prolonged Exposure to Biologically Treated and Untreated Bleached Kraft Mill Effluent

Exposure of fish to water of impaired quality has been shown to disrupt the function of the hypothalamo-pituitary-interrenal (HPI) axis and alter the interpretation of data from field studies due to the varying effects of handling and delayed sampling on exposed and reference animals. In the present study, juvenile whitefish, Coregonus lavaretus, were exposed for 6 weeks to diluted (4–8%) untreated and biologically treated bleached kraft mill effluent (BKME) and their response to acute handling was investigated. Liver microsomal EROD activity and glycogen phosphorylase (GPase) activity, in addition to gill Na⁺-K⁺-ATPase activity, and blood hemoglobin and hematocrit levels were increased in whitefish exposed for 6 weeks to untreated BKME, whereas those exposed to treated BKME exhibited increased blood hemoglobin and red blood cell K⁺ concentrations. Both handling procedures, exposure to a shallow water (10 cm, 5 min) and to an air challenge (10 s air/10 s water/30 s air/10 s water/10 s air), resulted in acute physiological stress, as recorded after 5-, 60-, and 120-min recovery periods. Following air exposure, the levels of plasma cortisol, blood glucose, hemoglobin, and hematocrit as well as the liver GPase activity were increased, and liver glycogen concentration decreased in control fish. These responses were attenuated in fish exposed to untreated or treated BKME. Plasma estradiol and testosterone levels were not affected by the BKME exposures or by the air challenge. Handling also resulted in attenuated EROD induction in fish exposed to untreated BKME. According to the present findings, the sensitivity of some widely used cellular and physiological variables may be improved by time-dependent standardization when interpreting data obtained following delayed sampling. Received: 3 July 2000/Accepted: 26 January 2001     

Anomalies in sculpins (Myoxocephalus spp.) sampled near a pulp and paper mill

This study investigated anomalies in two species of benthic sculpins (Myoxocephalus octodecemspinosus and M. scorpius) inhabiting a marine inlet in Newfoundland, Canada, where effluent from a pulp and paper mill is discharged and from two reference sites. Condition factor of both species of sculpins from the polluted and a reference site was similar. Some differences in parasite fauna were observed. Tissues from reference fish, obtained from two uncontaminated sites for comparison, were normal. Impaired vision, fin and tail necrosis, hyperplasia of the gill epithelium, depletion of energy reserves in the liver, and multifocal hemosiderosis in the spleen were observed primarily in M. octodecemspinosus captured near the paper mill. These stress-associated lesions, probably caused by undetermined xenobiotics, appear to be associated with the sediment.     

CYP1A Expression in Caged Rainbow Trout Discriminates Among Sites with Various Degrees of Polychlorinated Biphenyl Contamination

It has become increasingly apparent that resident fish can develop resistance to chemicals in their environment, thus compromising their usefulness as sentinels of site-specific pollution. By using a stream system whose resident fish appear to have developed pollutant resistance (Brammell et al., Mar Environ Res 58:251–255, 2005), we tested the hypothesis that the pollutant-inducible biomarker, cytochrome P4501A (CYP1A), as measured in field-caged juvenile rainbow trout (Oncorhynchus mykiss), would reflect relative pollution differences between reference and polychlorinated biphenyl (PCB)-contaminated sites. Trout were caged in the Town Branch/Mud River system (Logan County, KY), a stream system undergoing remediation for PCBs. Fish were held in remediated (Town Branch), unremeditated (Mud River), and reference sites for 2 weeks during spring 2002. At the end of this period, gill and hepatic CYP1A expression were measured. To evaluate the relative PCB exposure of caged trout and provide a reference point against which to calibrate CYP1A response, PCB levels were quantified in sediments from each site. Hepatic CYP1A expression in caged trout clearly detected the presence of PCBs in the Town Branch/Mud River stream system. Sediment PCB levels and hepatic CYP1A expression in caged trout produced identical pollution rankings for the study sites. Gill CYP1A expression, although suggestive of site differences, was not statistically different among sites. Unlike resident fish, which failed to show site differences in hepatic CYP1A expression in this waterway (Brammell et al. 2005), caged fish proved to be a sensitive discriminator of relative PCB contamination in this system. In summary, we determined that CYP1A expression in caged fish reflected relative in situ pollutant exposure. The exposure paradigm confirmed that 2 weeks was a sufficient caging period for evaluating CYP1A response in this species at these temperatures (13–19°C). In addition, these studies demonstrate that tissue-specific CYP1A expression can provide insights into likely routes of exposure. We conclude that CYP1A expression in caged trout is a reliable and inexpensive first-pass determination of relative environmental pollutant exposure and bioavailability in aqueous systems.     

Supplementation with macular carotenoids reduces psychological stress,serum cortisol,and sub-optimal symptoms of physical and emotional health in young adults

Purpose: Oxidative stress and systemic inflammation are the root cause of several deleterious effects of chronic psychological stress. We hypothesize that the antioxidant and anti-inflammatory capabilities of the macular carotenoids (MCs) lutein, zeaxanthin, and meso-zeaxanthin could, via daily supplementation, provide a dietary means of benefit.

Methods: A total of 59 young healthy subjects participated in a 12-month, double-blind, placebo-controlled trial to evaluate the effects of MC supplementation on blood cortisol, psychological stress ratings, behavioural measures of mood, and symptoms of sub-optimal health. Subjects were randomly assigned to one of three groups: placebo, 13?mg, or 27?mg / day total MCs. All parameters were assessed at baseline, 6 months, and 12 months. Serum MCs were determined via HPLC, serum cortisol via ELISA, and macular pigment optical density (MPOD) via customized heterochromatic flicker photometry. Behavioural data were obtained via questionnaire.

Results: Significant baseline correlations were found between MPOD and Beck anxiety scores (r?=??0.28; P?=?0.032), MPOD and Brief Symptom Inventory scores (r?=?0.27; P?=?0.037), and serum cortisol and psychological stress scores (r?=?0.46; P?P?Conclusions: Supplementation with the MCs significantly reduces stress, cortisol, and symptoms of sub-optimal emotional and physical health. Determining the basis for these effects, whether systemic or a more central (i.e. brain) is a question that warrants further study.     

Attenuated carbohydrate and gill Na+, K+ -ATPase stress responses in whitefish caged near bleached kraft mill discharges.

Exposure to biologically treated bleached kraft mill effluent (BKME) is demonstrated to greatly modify the acute physiological stress response in fish and, accordingly, to lead to inconsistencies in data interpretation due to dissimilar effects of handling procedures on reference and exposed fish. To consider this phenomenon, juvenile whitefish (Coregonus lavaretus) were caged for 30 days in four reference sites and in three areas influenced by different BKME discharges. After exposure, fish were subjected to the impacts of low-level handling by raising the cages to the water surface, serially handnetting the fish, and transferring ( approximately 10 min) the submerged cages to the research vessel. The data on physiological variables were pooled within each area to three groups according to the periods (<4 min, 10-20 min, 21-40 min) from the onset of handling to the sampling of individual fish. BKME-exposed whitefish sampled during the first period exhibited lower plasma cortisol and blood lactate levels and higher red blood cell Na+ concentrations than reference fish sampled during the same period. In reference whitefish, along with increased plasma cortisol and blood lactate levels, gill ATPase activity as well as liver glycogen and blood glucose levels were markedly affected by the handling procedure, while the latter three responses were strongly attenuated in exposed whitefish. Red blood cell Na+ and K+ and blood hemoglobin responses vanished in only one mill area. These findings, in accordance with similar results of earlier studies dealing with the same species, point to the need for this kind of time-dependent approach to achieve reliable and comparable outcomes in field experiments and encourage work on determining the importance of functional deviations noted in fish exposed to anthropogenically modified water qualities.     

Exposure to Water from the Lower Rhine Induces a Stress Response in the Rainbow Trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

The water quality of the river Rhine has improved in recent years and populations of salmonids are increasing. Nevertheless at present, the water from the lower Rhine still contains a complex mixture of low levels of many pollutants and it is not known whether exposure to such water is stressful to salmonid fish. For 31 days we continuously exposed the trout Oncorhynchus mykiss to water from the lower Rhine in the Netherlands and measured a variety of physiological, biochemical, and histological parameters, including the stress parameters cortisol and glucose. Exposure to Rhine water significantly increased cortisol and glucose after 3 h. At 21 and 31 days, cortisol was lower in exposed fish, indicating inhibition or exhaustion of the hypothalamic-pituitary-interrenal (HPI) axis. Electron microscopical analysis of the skin and gill epithelia revealed stressor-related effects that reflected disruption of the skin epithelium, the interface between the fish and the environment. This had little influence on hydromineral balance, as neither gill Na⁺/K⁺-ATPase activity nor plasma Na⁺ and Cl^– were altered, although intestine- and kidney-specific Na⁺/K⁺-ATPase activities were affected. Analysis of heavy metal concentrations in the liver, kidney, and intestine indicated no bioaccumulation. Immunostimulation was reflected by increased respiratory burst activity of the head kidney leukocytes. From 7 days onwards, the body weight of the Rhine water fish was significantly lower than that of control fish. Overall, the data show that acute exposure to present day water from the lower Rhine induced a stress response in the fish that, during chronic exposure, was followed by impairment of the HPI axis, reduced growth, and prolonged immunostimulation.     

A Biomonitoring Study Assessing the Residual Biological Effects of Pollution Caused by the HAVEN Wreck on Marine Organisms in the Ligurian Sea (Italy)

Residual biological effects of the 1991 HAVEN oil spill off the Ligurian (Arenzano) coast were assessed in this study. Samples of the fish species Boops boops, Mullus barbatus, and Uranoscupus scaber were collected from two polluted sites near the HAVEN wreck and from an uncontaminated area. In addition to this, mussels were caged along the coast affected by the HAVEN disaster. The physiological status of fish and mussels was assessed using a battery of stress and exposure biomarkers. The PAH content of mussel and fish tissues was also analyzed. Significant biological responses were observed in lysosomal membrane stability, neutral lipid and lipofuscin accumulation and micronucleus frequency for mussels caged at two sites close to the HAVEN wreck. Chemical analyses indicated, however, that these effects are not caused by aromatic hydrocarbons. For this reason, we suggest that the aftermath of the HAVEN disaster contributes very little to coastal ecosystem pollution. This was also confirmed by the few biological effects observed in fish specimens (Boops boops) collected from surface waters. Nevertheless, it is important to point out that benthic fish displayed a stress syndrome potentially caused by aromatic hydrocarbons released from the oil tanker, as witnessed by an enhanced EROD activity and increased lipofuscin and neutral lipid lysosomal contents.