Growth of estuarine fish is associated with the combined concentration of sediment contaminants and shows no adaptation or acclimation to past conditions

We tested whether the growth rates of small benthic fish (Gillichthys mirabilis) in three southern California estuaries corresponded with the local concentrations of contaminants. Fish originating from each estuary were transplanted to cages in each estuary in two reciprocal transplant experiments. The growth rates of caged fish, and the size-distribution of natural populations, showed the same pattern of difference among estuaries. Twelve metals and organic contaminants occurred in bulk sediments at concentrations close to their individual ERL values, and a simple index of their combined concentration (the mean ERL quotient) was inversely correlated to the growth of caged fish. Metals in the water column occurred at lower concentrations, relative to toxicity thresholds, than those in sediments and were unrelated to fish growth. Fish used in the field caging experiments, and other fish held in the laboratory under constant conditions, showed no difference in growth according to their estuary of origin. Fish originating from different estuaries also showed no consistent differences in their tissue burden of organic contaminants. Our results thus suggested no genetic adaptation or physiological acclimation to the past contaminant regime, but revealed a possible association between fish growth rates and the combined concentration of multiple sediment contaminants.     

Evaluation of fixed wavelength fluorescence and synchronous fluorescence spectrophotometry as a biomonitoring tool of environmental contamination

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<0.05). This study highlights the effectiveness of using FF and SFS as a rapid and cost-effective biomonitoring tool for detecting PAH contamination.     

Detecting a field gradient of PAH exposure in decapod crustacea using a novel urinary biomarker

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.     

High matrix interference affecting detection of PAH metabolites in bile of Atlantic hagfish (Myxine glutinosa) used for biomonitoring of deep-water oil production

The characteristic biology and wide distribution of hagfish species makes them relevant for use in pollution biomonitoring at great water depths, particularly in regions where deep-water oil production may take place. The exposure of fish to petrogenic contaminants can normally be detected from the level of polycyclic aromatic hydrocarbon (PAH) metabolites in bile fluid. Some of these metabolites are strong fluorophores, allowing analytical detection by means of simple fluorometric techniques such as fixed wavelength fluorescence (FF) and synchronous fluorescence scanning (SFS). In the present study bile from Atlantic hagfish (Myxine glutinosa) collected in pristine areas (Barents Sea and southwestern Norway) displayed strong bile fluorescence levels, suggesting the presence of PAH contaminants. However, gas-chromatography-mass spectrometry (GC-MS) analyses ruled out PAHs as the origin for this fluorescence signal. Rather, the bile of Myxine contains components resulting in unusually strong background fluorescence interfering at the wavelength pairs used for detection of PAH metabolites. Possible background for the observed matrix interference and implications for detection of PAH metabolites in hagfish is discussed.     

Evidence of uptake, biotransformation and DNA binding of polyaromatic hydrocarbons in Atlantic cod and corkwing wrasse caught in the vicinity of an aluminium works

Feral Atlantic cod (Gadus morhua) and corkwing wrasse (Symphodus melops) were investigated for polyaromatic hydrocarbon (PAH) contamination in the Karmsund strait, western Norway. This strait is highly contaminated with PAHs, and a main source is the chronic release of gas-scrubbing effluents from a local aluminium works. In both species, the level of biliary PAH metabolites and hepatic DNA adducts were higher in fish collected near the aluminium works. Interestingly, a significantly higher level of both biliary PAH metabolites and hepatic DNA adducts was found in corkwing wrasse as compared to cod, indicating a higher potential for genotoxic effects in this species. Hepatic cytochrome P4501A (CYP1A) in cod estimated by ethoxyresorufin-O-deethylase and an immunoassay technique (ELISA), seemed to be weakly induced at the contaminated sites. At the most contaminated site, skin ulcers and fin erosion were detected in about 70 and 45% of the cods, respectively. The data demonstrated that both cod and corkwing wrasse may be suitable target species for PAH pollution monitoring.     

PAH in fish bile detected by fixed wavelength fluorescence

Fixed wavelength fluorescence (FF) for detection of polyaromatic hydrocarbon (PAH) metabolites in fish bile has been studied, and is presented through a laboratory and a field experiment. The method, which can be regarded as a biomarker of exposure to PAH, has proved to be a simple, rapid and cost effective method for monitoring PAH contamination.     

Oxidative damage in rainbow trout caged in a polluted river

Sewage treatment works (STWs) are a common source of chemicals entering into the aquatic environment. In order to assess effects of these effluents on oxidative stress parameters in aquatic organisms, we caged rainbow trout at five sites: upstream, near an STW effluent, and three sites downstream in the river Viskan in western Sweden for 14 days during autumn, 2006. We then measured protein carbonyls in plasma as well as 20S proteosome activity and lipid peroxidation products, i.e. MDA and 4-HNE, in liver samples. Levels of both lipid and protein oxidative damage products were elevated in fish caged near the STW effluent while 20S activity showed no differences. This argues that complex mixtures of chemicals entering into the aquatic environment do have deleterious effects on fish. Additionally, oxidative stress parameters can serve as a biomarker in aquatic organisms.     

Confinement during field studies may jeopardize antioxidant and physiological responses of Nile tilapia to contaminants

This work evaluates the effects of caging, a known confinement stress, in Nile tilapia (Oreochromis niloticus) during an environmental study in Cubatão river, southern Brazil. Caging animals for 7 days, regardless of being at the reference or at a contaminated site, resulted in lower levels of antioxidant-related defenses (glutathione, glutathione S-transferase, glucose-6-phosphate dehydrogenase) in liver and physiological parameters (blood glucose and lactate) as compared with free-swimming animals. Higher hepatic glutathione reductase activity and elevated Hb content could be associated to contaminant exposure. In conclusion, the confinement stress in caged Nile tilapia biochemical and physiological disturbances, acting as a confounding factor in field studies.     

Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla: Seasonal, dose- and time-response variability in field and laboratory conditions

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.     

In vivo formation of (+)-anti-benzo[a]pyrene diol-epoxide-plasma albumin adducts in fish.

Benzo[a]pyrene (BaP), a procarcinogenic polycyclic aromatic hydrocarbon (PAH), is bioactivated to BaP diol-epoxides (BPDEs) that can form adducts with DNA and blood proteins. We report here for the first time the in vivo formation of adducts between BPDE and plasma albumin (Alb) from two fish species experimentally exposed to BaP. Brook trout (Salvelinus fontinalis) received either a single i.p. dose (10 mg/kg) or two separate i.p. doses (25 mg/kg; 7 days apart) of BaP, and blood was collected 2 (single exposure) or 3 (multiple exposure) days post-treatment. Arctic charr (Salvelinus alpinus) received 10 i.p. doses (3 mg/kg; a single dose every 6 days), and blood was collected 2 days after the second, sixth, and 10th injections. BPDE-Alb adducts were measured by an improved HPLC/fluorescence method developed to detect and quantify BaP-tetrols released after acid hydrolysis of adducted Alb. HPLC/fluorescence chromatograms of Alb from BaP-treated fish revealed only BaP-tetrol I-1, thus indicating the formation of adducts exclusively via the (+)-anti-BPDE metabolite. Levels of (+)-anti-BPDE-Alb adduct ranged from 0.68 to 19.6 ng of tetrol I-1 per gram of Alb. Notably, adduct level was not related to BaP dose and there was no accumulation of adducts with repeated exposure, which may indicate a very short half-life (< 2 days) of plasma Alb in fish. The data suggest that BPDE-Alb adducts in fish could be useful as a non-destructive biomarker of recent exposure to bioactivated BaP.     

Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla: Seasonal, dose- and time-response variability in field and laboratory conditions

Induction of cytochrome P4501A in fish is a well-known indicator of exposure to polycyclic aromatic hydrocarbons (PAHs) and determination of PAH metabolites in bile by fixed wavelength fluorescence (FF) or synchronous fluorescence spectroscopy (SFS), has become an useful method in monitoring programs. In this work the relationship between cytochrome P4501A (EROD activity) and levels of biliary PAH metabolites was measured in the European eel Anguilla anguilla, in both field and laboratory conditions: organisms were sampled on a seasonal basis from the Orbetello lagoon (Tuscany) to characterize the natural variability of these biological parameters, while in laboratory eels were intraperitoneally injected with benzo[a]pyrene to investigate temporal and dose-dependent induction patterns. Results showed that induction of cytochrome P450 and accumulation of PAHs metabolites in bile are not necessarily correlated either in field, or in laboratory investigations; different seasonal changes were measured in natural conditions and slight variations in dose and time response patterns were also obtained in laboratory exposures.     

Biomarker induction in tropical fish species on the Northwest Shelf of Australia by produced formation water

Normal operation of oil well platforms results in the discharge of produced formation water (PFW). The expression of CYP1A, CYP2M1- and 2K1-like proteins was examined for use as possible biomarkers of PFW exposure. A pilot study on the Northwest Shelf of Australia had indicated that PFW contamination possibly contributes to induction of CYP1A-like proteins in Gold-Spotted Trevally (Carangoides fulvoguttatus). The pilot study samples were re-examined for CYP1A, and, in addition, CYP2K1/2M1-like proteins. In a subsequent caged fish study in the same location a second species, Stripey seaperch (Lutjanus carponotatus), caught at a clean site, were distributed to three caging sites in a PFW gradient from the Harriet A production platform: A (near-field), B (far-field) and C (a non-impacted reference site). Fish were sampled at time (T) T = 0, T = 3 and T = 10 days. Significant increases of CYP1A, one CYP2K1- and two CYP2M1-like proteins were noted at Site A at T = 10d. For another CYP2K1-like protein, a significant increase was observed at Site A only at T = 3d. These results support a previous study indicating that CYP1A protein is sensitive to PFW exposure. Importantly, statistically significant environmental induction of both CYP2M1- and CYP2K1-like proteins in tropical fish due to PFW exposure had not previously been described and induction of enzymes in the CYP2 family suggest new biomarkers for PFW. In addition, the novel response of one CYP2K-like protein requires further verification, but offers promise for improved monitoring of sub-lethal responses in marine organisms.     

网箱养殖对海南新村澙湖海菖蒲生物学与生态学特征的影响

以海南新村澙湖海草床优势种之一的海菖蒲（Enhalus acoroides）为研究对象,于2005年4月至2006年1月间,分4个季节对海菖蒲的茎枝生物量、叶长、叶宽、叶单位面积附着藻类生物量、茎枝密度及生物量等进行了研究,初步探讨了网箱养殖对海菖蒲生物学和生态学特征的影响.其结果表明：（1）在春、夏、秋3个季节里,海菖蒲叶长、叶宽和茎枝生物量等均显示了与其所在样区海水和沉积物间隙水D IN含量呈负相关关系.（2）随着水体N负荷的增加,海菖蒲叶单位面积附着藻类生物量剧增.（3）网箱养殖区海菖蒲较小的地上生物量、茎枝密度及地上生物量与地下生物量比,均表明网箱养殖区海菖蒲受人为干扰的影响明显大于其他2个样区.     

Biomarkers in flounder (Platichthys flesus): an evaluation of their use in pollution monitoring

Flounder (Platichthys flesus) is among the most common fish-species in Norwegian and European estuaries. It lives in or on sediments from which it also finds most of its food. The aim of the present work was to evaluate biomarkers in flounder for possible future use in monitoring programmes. There were clear biomarker responses in flounder following injection of model contaminants benzo[a]pyrene (B[a]P), PCB #156 and Cd, singly or in sequence. Cytochrome P4501A responded following injection of the organic contaminants and metallothionein (MT) following Cd injection. All groups receiving B[a]P, either singly or in combination with other contaminants, accumulated high levels of B[a]P-metabolites in bile. There was little change in glutathione-S-transferase activity (measured using CDNB as substrate) following the treatments. Starvation appeared to affect the response of hepatic MT to Cd, but none of the other biomarkers. PAH in sediments elicited strong biomarker responses in caged flounder, whereas sediment-associated metals appeared to be largely unavailable to flounder in this study.     

Assessment of environmental quality and inland water pollution using biomarker responses in caged carp (Cyprinus carpio): Use of a bioactivation:detoxication ratio as a Biotransformation Index (BTI)

In the present study the bioaccumulation of poly chlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) as well as the responses of a suite of biochemical parameters were investigated in a standardized carp (Cyprinus carpio) fish line. Carps were caged for 2 to 8 weeks at two Amsterdam freshwater sites: the relatively unpolluted Outer IJ and the heavily polluted Volgermeerpolder. The primary objective of the experiments was to develop a reliable and reproducible method to monitor the inland water pollution and to classify the environmental quality of freshwater sites. The highest tissue levels of organic trace pollutants were generally found in carp that was caged for at least four weeks at the polluted site. Hepatic phase I enzymes showed the highest pollution-induced responses, while phase II enzymes and serum transaminases were less sensitive. The antioxidant enzymes showed virtually no response to pollutants. In order to assess effects on the balance between bioactivation and detoxication the ratio between phase I (EROD) and phase II (GST or UDPGT) activities was introduced as a biotransformation index (BTI). Highest BTI values were observed in carp that was caged for 4 to 6 weeks at the polluted site. The BTI values decreased after eight weeks of exposure due to elevated phase II activities.     

Evaluation of non-specific immune functions in dab,Limanda limanda L., following short-term exposure to sediments contaminated with polyaromatic hydrocarbons and/or polychlorinated biphenyls

Experimental exposure tanks were set up containing 'clean' marine sediment spiked with set doses of polyaromatic hydrocarbons (PAH), or polychlorinated biphenyls (PCB), or both. Reference tanks contained only 'clean' sediment. Female dab Limanda limanda L., exposed to these sediments for 7 days showed no differences in serum total protein concentration, serum lysozyme activity or body growth. Their kidney leucocytes were assayed for extracellular production of reactive oxygen species of the phagocytic respiratory burst. No significant differences in superoxide anion (O2-) production were found between fish exposed to the 'spiked' sediments and those on the 'clean' reference sediment. The production of hydrogen peroxide (H2O2) was, however, reduced in the fish held on the PAH and PAH/PCB spiked sediments. H2O2 levels for fish exposed to PCBs alone, did not differ significantly from those of control fish. Our results suggest that the decrease in H2O2 production was due to exposure to PAHs, rather than to PCBs.     

Unbleached pulp mill effluents affect cytochrome P450 monooxygenase enzyme activities

The polysubstrate monooxygenase system has been shown to be highly responsive to chemical pollution. The present study summarizes the enzyme based biomonitoring of the waste waters released by a pulp mill producing unbleached pulp and paperboard. Cytochrome P4501A enzyme activities of feral and caged fish, as well as cultures of fish hepatocytes, were tested. The 7-ethoxyresorufin-O-deethylase (EROD) activity was clearly induced in fish hepatocytes exposed to biotreated unbleached pulp mill effluent fractions in vitro. The effluent increased EROD activities also in feral perch, compared with controls. Caging experiments showed similar effects to those seen in feral fish: however, the maximal induction coefficients observed were higher. Unbleached effluents contain compounds that are able to affect the P4501A activities in fish.     

Trophic transfer of benzo[a]pyrene metabolites between benthic marine organisms

This study was undertaken to determine the potential for trophic transfer of polycyclic aromatic hydrocarbon (PAH) metabolites from infaunal organisms to bottom-feeding fish. Winter flounder, Pseudopleuronectes americanus, were given single oral doses of ground polychaetes (Nereis virens), either treated with pure [¹⁴C]henzo[a]pyrene (BaP) or containing a mixture of naturally produced radiolabeled BaP and BaP metabolites. Fish were sacrificed 24 h after feeding and total accumulated radioactivity and metabolite class profiles determined in major organs. Metabolites produced by worms were absorbed by flounder, although as a percentage of dose given they were less available than parent BaP. Comparison of metabolite profiles in the worm diet and in target organs in the fish indicated that metabolites accumulated through the diet can be further modified by the prey organism and can lead to the formation of bound residues. These results demonstrate that PAH metabolites in the diet are available for accumulation. Furthermore, metabolites absorbed appear to be susceptible to metabolic alteration by consumer organisms.     

Effects of wild fish and motile epibenthic invertebrates on the benthos below an open water fish farm

A manipulative caging experiment was carried out to evaluate the role of wild fish and motile epibenthic invertebrates on the benthic system influenced by an open water fish farm. Chemical and biological parameters of the sediment were measured as indicators of the ecological benthic status. The combination of wild fish and currents notably lowered aquaculture waste sedimentation below the fish farm. The limited waste sedimentation rate could have limited the scavenger and predation activity of wild fish on the benthos, whose role may be taken over by motile epibenthic invertebrates. The interaction of these motile epibenthic invertebrates with the sediment differed from that observed with fish. The motile epibenthic invertebrates led to more reduced conditions with lower redox values, significantly decreased the number of species of macrofaunal benthic assemblages and significantly modified macrofaunal benthic assemblages. Therefore, epibenthic invertebrates do not seem to have an ameliorative effect on the benthic status produced by fish farming. Since the effects of epibenthic species on the benthic system can greatly vary according to their identity, further experiments should be performed to better understand the drivers that influence the epibenthic species identity that modulate the benthic system affected by fish farming.     

Protein carbonyls and antioxidant defenses in corkwing wrasse (Symphodus melops) from a heavy metal polluted and a PAH polluted site

The use of fish in environmental monitoring has become increasingly important in recent years as anthropogenic substances, many of which function as prooxidants, are accumulating in aquatic environments. We have measured a battery of antioxidant defenses as a measure of oxidative status, as well as protein carbonylation as a measure of oxidative damage, in corkwing wrasse (Symphodus melops) captured near a disused copper mine, where water and sediment are contaminated with heavy metals, and an aluminum smelter, a site contaminated with PAHs. Results were compared to two different reference sites. Fish at the heavy metal site had lower glucose-6-phosphate dehydrogenase activity and elevated protein carbonyls (1.8 times) compared to fish from the reference site. At the PAH site, EROD was increased 2-fold, while total glutathione and methemoglobin reductase concentration, were decreased. No differences were seen in protein carbonyl levels at the PAH site. Measures of both antioxidant defenses and oxidative damage should be used when assessing effects of xenobiotics on oxidative stress in fish species.