Effects of field-contaminated sediments and related water soluble components on haemocyte function and Perkinsus marinus susceptibility and expression in oysters

This paper reviews and discusses our recent findings on the effects of contaminated sediments (CSs) and related water-soluble fractions (WSFs) on haemocyte function/activity and the onset and progression of an infectious disease caused by the protozoan parasite, Perkinsus marinus (Dermo) in the eastern oyster, Crassostrea virginica. Sediments used to generate WSFs and sediments used for the whole CS exposure experiments were collected in different areas of the southern branch of the Elizabeth River, a heavily polluted sub-estuary of the Chesapeake Bay, USA. The WSFs were dominated by low molecular weight polycyclic aromatic hydrocarbons (PAHs). The CSs used for whole CS exposure experiment had elevated concentrations of high molecular weight PAHs. Polychlorinated biphenyls (PCBs) and metals were also present in the CSs. No PCBs were detected in the WSFs. In vitro exposure of haemocytes to WSFs derived from CSs reduced to haemocytes' chemotaxic, phagocytic, and chemiluminescent responses to some extent. Exposure of oysters to suspended CSs stimulated neutral red uptake, mitochondrial dehydrogenase production and ³H-leucine incorporation in haemocytes. Exposure of oysters to 0, 15, 30% WSFs increased the oysters' susceptibility to laboratory-induced infection caused by P. marinus. Exposure of oysters to 15, and 30% dilutions of WSFs for 33 days or to 1.0, 1.5, and 2.0g CSs for 30 days significantly elevated the expression/progression of latent P. marinus infection in oysters in a dose-dependent manner.     

Effects of field-contaminated sediments and related water soluble components on haemocyte function and Perkinsus marinus susceptibility and expression in oysters

This paper reviews and discusses our recent findings on the effects of contaminated sediments (CSs) and related water-soluble fractions (WSFs) on haemocyte function/activity and the onset and progression of an infectious disease caused by the protozoan parasite, Perkinsus marinus (Dermo) in the eastern oyster, Crassostrea virginica. Sediments used to generate WSFs and sediments used for the whole CS exposure experiments were collected in different areas of the southern branch of the Elizabeth River, a heavily polluted sub-estuary of the Chesapeake Bay, USA. The WSFs were dominated by low molecular weight polycyclic aromatic hydrocarbons (PAHs). The CSs used for whole CS exposure experiment had elevated concentrations of high molecular weight PAHs. Polychlorinated biphenyls (PCBs) and metals were also present in the CSs. No PCBs were detected in the WSFs. In vitro exposure of haemocytes to WSFs derived from CSs reduced to haemocytes' chemotaxic, phagocytic, and chemiluminescent responses to some extent. Exposure of oysters to suspended CSs stimulated neutral red uptake, mitochondrial dehydrogenase production and ³H-leucine incorporation in haemocytes. Exposure of oysters to 0, 15, 30% WSFs increased the oysters' susceptibility to laboratory-induced infection caused by P. marinus. Exposure of oysters to 15, and 30% dilutions of WSFs for 33 days or to 1.0, 1.5, and 2.0g CSs for 30 days significantly elevated the expression/progression of latent P. marinus infection in oysters in a dose-dependent manner.     

Activity of conventional and reduced-risk insecticides for protection of grapevines against the rose chafer, Macrodactylus subspinosus (Coleoptera: Scarabaeidae)

Abstract:  Bioassays were conducted to compare the residual toxicity and leaf protection activity of conventional broad-spectrum and reduced-risk insecticides against the rose chafer, Macrodactylus subspinosus . Insecticides were applied to a Vitis labrusca (F.) vineyard and residues were aged for 1, 3 or 7 days before leaves were collected and exposed to beetles in no-choice tests. Azinphosmethyl caused rapid knockdown and mortality for up to 1 week after application, with 1-day-old residues providing 95.6% protection against feeding, dropping to 51.6% when residues were a week old. Fenpropathrin caused mortality and knockdown after beetles had been exposed to fresh residues for 72 h. Although these effects diminished as residues aged, this compound provided the best protection of leaves against beetle feeding, with 77.9% reduction in feeding compared with the control after 7 days of aging in the vineyard. Of the reduced-risk insecticides, imidacloprid caused the greatest initial mortality and knockdown of beetles, providing protection against feeding that was equivalent to azinphosmethyl. Exposure to azadirachtin caused a low level of knockdown and mortality when residues were 1- and 3-days old. Protection against feeding was also low, lasting for only 3 days. Beetles were minimally affected by capsaicin and kaolin, with mortality and knockdown seen only when beetles were exposed to 1-day-old residues for 72 h. Foliage protection from these compounds was minimal, with between 10 and 15% reduction in feeding injury. Results are discussed in relation to development of semi-field bioassay methods for evaluating reduced-risk insecticides, and the management of M. subspinosus within grape pest management programs.     

A Perspective on the Toxicity of Low Concentrations of Petroleum-Derived Polycyclic Aromatic Hydrocarbons to Early Life Stages of Herring and Salmon

This article presents a critical review of two groups of studies that reported adverse effects to salmon and herring eggs and fry from exposure to 1 μg/L or less of aqueous total polycyclic aromatic hydrocarbons (TPAH), as weathered oil, and a more toxic aqueous extract of “very weathered oil.” Exposure media were prepared by continuously flowing water up through vertical columns containing gravel oiled at different concentrations of Prudhoe Bay crude oil. Uncontrolled variables associated with the use of the oiled gravel columns included time- and treatment-dependent variations in the PAH concentration and composition in the exposure water, unexplored toxicity from other oil constituents/degradation products, potential toxicity from bacterial and fungal activity, oil droplets as a potential contaminant source, inherent differences between control and exposed embryo populations, and water flow rate differences. Based on a review of the evidence from published project reports, peer-reviewed publications, chemistry data in a public database, and unpublished reports and laboratory records, the reviewed studies did not establish consistent dose (concentration) response or causality and thus do not demonstrate that dissolved PAH alone from the weathered oil resulted in the claimed effects on fish embryos at low μg/L TPAH concentrations. Accordingly, these studies should not be relied on for management decision-making, when assessing the risk of very low–level PAH exposures to early life stages of fish.     

Swimming performance and blood chemistry in Atlantic salmon spawners exposed to acid river water with elevated aluminium concentrations

Exposure of sexually mature pre-spawning Atlantic salmon Salmo salar to Fossbekk water (pH 5·2) for 7 days led to a significant reduction in critical swimming speed (U-crit) in females but not in males. Exposure to Fossbekk water +A1 (as AlCl₃) for 24 h led to a significant reduction in U-crit in both males and females. In contrast to fish exposed for 7 days to Fossbekk water, fish exposed to Fossbekk+A1 had accumulated much more aluminium and mucus on their gills. Losses of plasma ions were similar in both groups exposed to acid water. Blood glucose was twice as high in fish exposed to Fossbekk water for 7 days compared with fish exposed to Fossbekk+A1 for 24 h. Plasma cortisol was still elevated compared with controls after exposure to Fossbekk water for 7 days. Possible mechanisms for the observed decrease in U-crit at the different exposures are discussed.     

Phenanthrene Biodegradation in Soils Using an Antarctic Bacterial Consortium

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in Antarctic soils is limited by low temperatures, lack of adequate levels of nutrients, low number of PAH-tolerant members in the autochthonous microbiota and low bioavailability of contaminants. In the present work, microcosms systems (performed in 1-L glass flasks containing Antarctic soil supplemented with 1744 ppm of phenanthrene) were used to study (i) the effect of biostimulation with a complex organic source of nutrients (fish meal) combined with a surfactant (Brij 700); (ii) the effect of bioaugmentation with a psychrotolerant PAH-degrading bacterial consortium (M10); (iii) the effect of the combination of both strategies. The authors found that combination of biostimulation and bioaugmentation caused a significant removal (46.6%) of phenanthrene after 56 days under Antarctic environmental conditions. When bioaugmentation or biostimulation were applied separately, nonsignificant reduction in phenanthrene concentration was observed. Microtox test showed a low increase in toxicity only in the most efficient system. Results proved that “in situ” bioremediation process of phenanthrene-contaminated soils is possible in Antarctic stations. In addition, inoculation with a psychrotolerant PAH-degrading bacterial consortium in association with a mix of fish meal and a high-molecular-weight surfactant improved phenanthrene removal and should be the selected strategy when the number of hydrocarbons degrading bacteria in the target soil is low.     

Effects of Melatonin on Memory and Learning Deficits Induced by Exposure to Thinner

The neurotoxic effects of thinner, a mixture including aromatic compounds (in particular, toluene) and widely used as an industrial solvent, were examined. Exposure of rats to high inhalation concentrations (3000 p.p.m.) of thinner for 45 days (1 h per day) significantly influenced the cognitive functions and levels of neural cell adhesion molecules (NCAM) in the hippocampus, cortex, and cerebellum of experimental animals. These exposures also caused dramatic increases in levels of LPO (malondialdehyde and 4-hydroxyalkenals) in these cerebral structures, while melatonin administration significantly reduced the LPO amounts in these brain regions. The level of NCAM (180 kDa) decreased significantly in the hippocampus and cortex of thinner-exposed rats. Furthermore, thinner-exposed rats showed cognitive deficits in the passive avoidance and Morris water maze tasks; these negative effects were considerably compensated in rats additionally chronically treated with melatonin. It is concluded that treatment with melatonin prevents the development of learning and memory deficits caused by thinner exposure, possibly by reducing oxidative stress and normalizing the neural plasticity.     

Residues of Aliphatic and Polycyclic Aromatic Hydrocarbons in Some Fish Species of Lake Temsah,Ismailia, Egypt: An Analytical Search for Hydrocarbon Sources and Exposure Bioindicators

Residues of aliphatic and polycyclic aromatic hydrocarbons (PAHs) were monitored in some fish species collected from Temsah Lake, near Ismailia, Egypt. Fish were selected to represent different feeding habits and ecological niches in the lake's ecosystem. Fish samples were extracted using organic solvents, and residues of aliphatic and PAHs were separated using column chromatography and detected using gas liquid chromatography. Fish species were Clupea sirm, Mugil sehli, Mugil capito, Morone labrax, and Sciasna sp. Clupea sirm, a surface feeder fish had the highest concentration of aliphatic hydrocarbons, 320 ± 54 ng g^?1, while Morone labrax, a predatory fish that live in the water column, had the highest concentration of PAHs, 315.87 ± 46 ng g^?1. Even-number aliphatic hydrocarbons were more frequently detected in all fish species in comparison to odd-number aliphatic hydrocarbons, suggesting a petrogenic origin of these compounds. Meanwhile, the pattern of PAHs detected in the present study suggested that they originate from atmospheric deposition rather than land-based runoff. Morone labrax fish and Clupea sirm fish were the most suitable candidate bioindicators of exposure to aliphatic hydrocarbons and PAHs through fish consumption of the five fish species examined in this study.     

The effect of pH and/or calcium-enriched freshwater on gill Ca2+-ATPase activity and osmotic water inflow in rainbow trout (Salmo gairdneri)

Rainbow trout (Salmo gairdneri) were exposed to pH 5.0-5.1, 6.6 and/or calcium-enriched freshwater for 14 days. Hematocrit, gill Ca2+-ATPase enzyme activities, gill osmotic water inflow, plasma calcium and osmolarity were measured. No significant changes in plasma calcium ion levels were found. The typical increase in hematocrit usually associated with exposure of fish to acidified water was not found in the present study and is discussed. Plasma osmolarity decreased in fish exposed to calcium-enriched freshwater (60 mg Ca2+ X 1(-1) ) in comparison to fish exposed to control freshwater conditions (2 mg Ca2+ X 1(1) ), irrespective of the pH level. Gill Ca2+-ATPase enzyme activities were measured for both low affinity (3 mM Ca2+) and high affinity (100 microM) activity. Exposure of rainbow trout to low pH (pH 5.0-5.1) did not affect the specific activity of Ca2+-ATPase enzyme. However, low affinity Ca2+-ATPase activity in fish exposed to calcium-enriched freshwater did show a significant reduction. The increase in gill osmotic water permeability in fish exposed to calcium-enriched freshwater is interpreted as a result of the increase in osmolarity of the ambient media.     

Effect of the organophosphorous insecticide, chlorpyrifos (Dursban), on growth, fecundity and mortality of Biomphalaria alexandrina and on the production of Schistosoma mansoni cercariae in the snail.

Exposure of Biomphalaria alexandrina to sublethal concentrations (0.125, 0.25 and 0.05 ppm) of the organophosphorous insecticide, chlorpyrifos (Dursban), induced a reduction in egg production and egg hatchability. Exposure of Schistosoma mansoni miracidia to the insecticide (60 min, 0.50 ppm) prior to infection of B. alexandrina did not affect the subsequent production of cercariae. However, exposure of S. mansoni-infected snails to the insecticide until day 55, from day 20 to day 62 and from day 35 to 62 following infection resulted in blockage of cercarial shedding. Cercarial shedding commenced in some snails when the treatment stopped. Exposure to the insecticide in concentrations of 0.125 and 0.25 ppm during the first 20 days following infection did not affect the subsequent production of cercariae, but exposure to 0.5 ppm during the first 20 days affected markedly the production of cercariae due to a high snail mortality. The findings indicate that the cercaria is the target stage for the activity of chlorpyrifos on the intramolluscan larval development. It is suggested that S. mansoni cercarial production in B. alexandrina may be a useful system for monitoring the effect of low concentrations of pesticides on the aquatic environment, and that the ability by chemical means to interrupt the cercarial production might be a useful tool in further analyses of important aspects of the snail/parasite relationship.     

The effect of thermal acclimation on the activity of arylhydrocarbon hydroxylase in rainbow trout (Oncorhynchus mykiss)

1. The possibility that temperature acclimation (to 10 or 18 degrees C for 28 days) would alter the cytochromes P-450 of rainbow trout was addressed. 2. The specific content of LM4b (P-450 IA1), the trout isozyme responsible for activation of polynuclear aromatic hydrocarbons, was lower in 18 degrees C fish than it was in 10 degrees C fish. 3. Kinetic analysis of aryl hydrocarbon hydroxylase indicated that, while thermal acclimation caused no change in Vmax, it lowered the apparent Km of this enzyme for benzo[a]pyrene when assayed at acutely shifted temperatures. 4. Thermal acclimation of fish may have significance when feral populations are subjected to acute temperature shifts.     

Bioelectronic monitoring of parasite-induced stress in brown trout and roach

A non-contact bioelectronic monitoring system was used to record changes in heart rate, ventilation rate and swimming activity in brown trout Salmo trutta and roach Rutilus rutilus , following exposure to two species of cercariae of digenean trematodes: Diplostomum pseudospathaceum which is a common parasite in the lens of fishes and xiphidiocercariae of Plagiorchis elegans , a parasite of anatid birds, both of which have the snail Lymnaea stagnalis as their first intermediate host. Swimming activity increased significantly in roach exposed to Diplostomum cercariae at densities as low as 3.8l⁻¹ and remained elevated for 24–36 h post exposure. Brown trout showed no response when exposed to low densities of Diplostomum (2.9 and 5.7 cercariae 1⁻¹) but there was a significant response at higher cercarial exposure densities (>501⁻¹). Total activity of brown trout increased immediately, peaking after 2 h and returning to pre-exposure levels within 5–6 h. There was a simultaneous increase in heart and ventilation rates which both declined following the reduction in activity of the fish. Heart rate then increased for a second time to a maximum level at 14–16 h and thereafter declined to reach a steady state at 3 days post-exposure. During this period there was no significant change in total activity. The qualitative nature of the heart response was identical in five infected brown trout although the magnitude of the response varied widely among fish. Exposure to cercariae of P. elegans elicited no response in either fish species. (c) 1996 The Fisheries Society of the British Isles     

Cost of exposure to trematode cercariae and learned recognition and avoidance of parasitism risk by fathead minnows Pimephales promelas

Fathead minnows Pimephales promelas exposed to cercariae of the trematode Ornithodiplostomum sp. incurred a significant mass loss 17 days after exposure to 20 or 120 cercariae. Parasite‐naïve P. promelas showed no evidence of innate recognition or avoidance of cercariae. After a single exposure to cercariae, however, fish responded to chemical and visual cues of dead (thawed) cercariae with a reduction in activity. Encounter rate with cercariae, and hence infection rate, increased with fish activity. The data indicated that experienced P. promelas associated parasitism risk with novel chemical and visual cues that later triggered avoidance behaviour. Parallels and interactions between antiparasite behaviour and antipredator behaviour open new avenues for behavioural ecological research in risk‐sensitive decision‐making.     

Xenobiotic metabolizing enzymes in spawning English sole (Parophrys vetulus) exposed to organic-solvent extracts of marine sediments from contaminated and reference areas

Female English sole (Parophrys vetulus) within 1-2 days of spawning were exposed by i.m. injection to organic-solvent extracts of marine sediments at the following doses: Eagle Harbor (EHSE, contaminated site)--6.8 mg aromatic hydrocarbons (AHs)/kg body wt; Duwamish Waterway (DSE, contaminated site)--0.52 mg AHs and 0.040 mg chlorinated hydrocarbons (CHs)/kg body wt; Hood Canal (HCSE, reference site)--0.00090 mg AHs/kg body wt. Hepatic aryl hydrocarbon hydroxylase (AHH) activity, measured at spawning, was induced 10-, 23-and 2-fold by EHSE, DSE and HCSE, respectively, compared to sham and vehicle controls. Hepatic glutathione-S-transferase and epoxide hydrolase activities were not affected by any treatment. EHSE, but not DSE or HCSE, inhibited spawning (P less than 0.01) in 36% of the exposed fish and hepatic AHH activity in the non-spawning fish was significantly (P less than 0.05) higher than in the fish that did spawn. These results suggest a potential for reproductive toxicity in benthic fish after exposure to sediment-associated contaminants.     

Influence of some environmental variables on the ascorbic acid status of striped mullet, Mugil cephalus Linn., tissues. III. Effects of exposure to oil

Exposure to water-soluble fractions (WSF) of a crude and two fuel oils altered the ascorbic acid (AsA) content of several striped mullet, Mugil cephalus , tissues. Exposure to sublethal concentrations of all three WSFs caused a depletion of AsA reserves in brain, gill, kidney and liver tissues, but not in muscle. There was a marked decline in AsA stores in kidney and gill tissues after only one day of exposure to WSFs of both crude and fuel oils. Liver AsA concentrations were significantly depleted after one week of oil exposure. Brain AsA content was only significantly depleted during chronic exposure to the highest oil concentration (20% WSF). A dose-dependent depletion of AsA reserves in the liver but not in the other tissues was observed one week after a single exposure to 2–20% WSFs of a No. 2 fuel oil. Exposure to 20% WSF of the No. 2 fuel oil caused a 47% decrease in liver AsA content one week later. Hepatic concentrations were still significantly depleted after 15 days, but had returned to control levels 20 days after the initial exposure. The data suggest that the depletion of tissue AsA reserves in fish inhabiting oil-contaminated environments could be sufficient on occasions to lead to AsA deficiency.     

Measurement of metabolites of polycyclic aromatic hydrocarbons in fish bile for comparative assessment of coastal water pollution

In the laboratory experiment and in fish caught in conditionally clean and polluted areas, the presence of metabolites of polycyclic aromatic hydrocarbons in bile by methods of conventional and synchronous spectrofluorimetry was determined. The study showed that this approach is well suited for the detection of metabolites of polycyclic aromatic hydrocarbons with 2–3, 4, and 5–6 aromatic rings. If the content of metabolites of polycyclic aromatic hydrocarbons with 4 and 5–6 rings in bile is low, the method of synchronous spectrofluorimetry is less sensitive than conventional spectrofluorimetry. Both methods of monitoring are simple and fast and reflect the real picture of water pollution.     

Detection of water toxicity using cytochrome P450 transgenic zebrafish as live biosensor: for polychlorinated biphenyls toxicity

Cytochrome P450 (CYPs) is significant in degradation of endogenous substrates and detoxification of carcinogens, therefore it is a biomarker for assessment of polycyclic aromatic hydrocarbons (PAHs) level in aquatic environment. In the present study, a transgenic line of zebrafish had been generated using a CYP-green fluorescence protein (CYP-GFP) construct, driven by CYP1A1 promoter. Polychlorinated biphenyls (PCBs) were used as toxicant, in concentrations of 0.02 μg/ml, 0.04 μg/ml, 0.08 μg/ml, 0.4 μg/ml, and 0.8 μg/ml. The transgenic control fish showed low intensity of fluorescence in the liver. After exposed to PCBs, zebrafish had morphological changes such as expansion of yolk, contortion of tails and inflation of pericardial area. Green fluorescence signals were found to express according to concentrations and time. The green fluorescence signal was most intense after treatment with 0.08 μg/ml PCBs. However, the maximum area of green fluorescent signal was found at 0.04 μg/ml PCBs. GFP started to express at 3h exposure to PCBs, increasing its intensity until 6 h exposure, and then level off. Since the GFP expression is fast responding and is sensitive to low PAHs concentrations, transgenic fish is a good tool for live imaging and monitoring of aquatic contamination.     

p-Nitrophenol and glutathione response in medaka (Oryzias latipes) exposed to MX,a drinking water carcinogen

When chlorine is introduced into public drinking water for disinfection, it can react with organic compounds in surface waters to form toxic by-products such as 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX). We investigated the effect of exposure to MX on cytochrome P450 2E1 (CYP2E1)-like activity and total glutathione (GSH) in the liver of the small fish model, medaka (Oryzias latipes). The multi-site carcinogen methylazoxymethanol acetate (MAMAc) was the positive control compound. Both medaka liver microsome preparations and S-9 fractions catalyzed the hydroxylation of p-nitrophenol (PNP), suggesting CYP2E1-like activity in the medaka. Male medaka exposed for 96 h to the CYP2E1 inducers ethanol and acetone under fasted conditions showed significant increases in PNP-hydroxylation activity. Furthermore, total reduced hepatic GSH was reduced in fish fasted for 96 h, indicating that normal feeding is a factor in maintaining xenobiotic defenses. Exposure to MX and MAMAc induced significant increases in hepatic CYP2E1-like activity, however MX exposure did not alter hepatic GSH levels. These data strengthen the role of the medaka as a suitable species for examining cytochrome P450 and GSH detoxification processes and the role these systems play in chemical carcinogenesis.     

Determination of aromatic hydrocarbons and their metabolites in human blood and urine

Methods for the biological monitoring of aromatic hydrocarbons and their metabolites in the human blood and urine are reviewed. For the determination of the unchanged aromatic hydrocarbon in blood, gas chromatographic head-space analysis is recommended. The metabolites can be monitored by photometric, thin-layer chromatographic, high-performance liquid chromatographic and gas chromatographic methods. For the assessment of health risks caused by aromatic hydrocarbons, reference values and occupational limit values, expressed as biological tolerance values and biological exposure indices, have to be considered.     

Seasonal variation in trypsin activity in juvenile Atlantic salmon upper and lower modal groups

Juvenile salmon in their first year of growth showed a bimodal distribution of body lengths by December. For experimental purposes samples of fish from the upper 2% of body lengths were taken as representing the upper modal group (UMG), whilst fish from the bottom 5% of body lengths were taken to represent the lower modal group (LMG). The population of fish from which the samples were taken were fed ad libitum from December to July. During the winter months to March, neither group increased in weight. Growth resumed between March and July. The LMG fish had a very low food intake, as indicated by the relative weight of digesta in the stomach, in the winter months. However, following resumption of feeding, the relative weight of stomach digesta of the LMG fish exceeded that of the UMG fish between May and July. The activity of trypsin in the intestinal digesta followed a similar pattern, the LMG fish showing a higher trypsin activity in the spring months. Starvation of UMG fish for 5 days in winter resulted in accumulation of trypsin in the pancreatic tissues, whilst injection of the trypsin releasing hormone cholecystokinin (CCK) into starving UMG fish resulted in reduction of trypsin in the secretory tissues. CCK also caused reduction of trypsin in the pancreatic tissues of LMG fish, suggesting that the pancreas of this group is potentially fully functional during the winter period. Ultrastructure studies of the pancreatic acinar cells showed evidence for lower secretory activity in the LMG fish, as indicated by smaller numbers of zymogen granules, less well developed Golgi systems and a smaller number of active secreting cells. It appears that trypsin secretion by the pancreas in LMG fish is at a low level during the winter, in response to the reduced amounts of food passing through the gut, which is ultimately controlled by changes in food intake, lowered metabolic level and lowered appetite levels.