Development of enzyme-linked immunosorbent assay (ELISA) for glutathione S-transferase (GST-S) protein in the intertidal copepod Tigriopus japonicus and its application for environmental monitoring

To utilize the GST-S protein as a useful biomarker for environmental contamination, we developed a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) in the intertidal copepod Tigriopus japonicus. Two polyclonal antibodies, TJ-GST-S1 and TJ-GST-S2, were raised against two TJ-GST-S synthetic peptides. Also a recombinant TJ-GST-S protein was purified as a standard for ELISA development. Each polyclonal antibody was tested by Western blot analysis and indirect ELISA. Of two polyclonal antibodies, TJ-GST-S2 ELISA was further employed due to its wide range of detection and the limit of specificity compared to those of TJ-GST-S1 ELISA system. After exposure to 4 metals (Ag, As, Cd, and Cu) to T. japonicus, the amount of TJ-GST-S protein was significantly elevated in a concentration-dependent manner. Also, TJ-GST-S protein was upregulated at relative high concentrations of B[α]P, PCB, and TBT. In this paper, we suggest that T. japonicas ELISA for TJ-GST-S2 is useful as a potential indicator system for marine contaminants.     

Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers

In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24 h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).     

AHH activity, tissue dose and DNA damage in different tissues of the scallop Chlamys farreri exposed to benzo[a]pyrene

A collaborative study was performed on scallops (Chlamys farreri) exposed to 0.5, 3 and 10 microg/L benzo[a]pyrene (B[a]P) for 20 days. The levels of aryl hydrocarbon hydroxylase (AHH) activity, B[a]P accumulation and DNA strand break were assayed in the gill and digestive gland. Results showed that AHH activity and B[a]P accumulation were significantly related to B[a]P dose. AHH activity was induced and then became stable gradually. B[a]P accumulation increased first and showed an incoming plateau. DNA strand break levels in the 0.5 and 3 microg/L B[a]P groups remained high and significantly different from control values until day 6, followed by a reduction in the gill and no recovery in the digestive gland. The 10 microg/L B[a]P groups remained significantly lower than control until the end. These results suggested that the application of comprehensive indices may give information on the actual exposure of organisms to pollutants, and also information on toxic effects.     

Detecting points as developmental delay based on the life-history development and urosome deformity of the harpacticoid copepod, Tigriopus japonicus sensu lato, following exposure to benzo(a)pyrene

To identify ecotoxicological responses to an endocrine disrupter, benzo(a)pyrene, we examined the life-history of the harpacticoid copepod, Tigriopus japonicus sensu lato. Based on the life-history of copepods, survival rate of nauplii (NSR) and copepodites (CSR), copepodite emergence day (CED) and adult male emergence day (AMED), sex ratio (MER), brooding success rate (BSR), and first brooding day of adult females (FBD) were measured. Significant differences were observed in the survival and development of nauplii (NSR and CED) and sex ratio (MER) of exposed and non-exposed copepods. Moreover, high concentration of BaP can be lethal to copepodite and exhibited a delay of growth. In this study, the CED and AMED among ecotoxicological response based on life-history developments were delayed and the body characteristics decreased in response to exposure to benzo(a)pyrene. The dwarfism and urosome deformity of the T. japonicus s.l. was exhibited in response to chemical exposure. Specifically, the body characteristics and biomass of dwarf copepods that had been exposed to benzo(a)pyrene were 30% and 50% lower than the control group, respectively. The incidence of abnormal urosomes was divided into two types. The first deformity type was signs of shrinkage in the middle of the urosome or the entire urosome was narrower than those of the control organisms. In the second type, the anal somite and the distal side of the urosome had abnormally swelled. Taken together, the nauplii and copepodid development of T. japonicus s.l. can be used as a useful biomaker for detecting developmental delay based on their entire life-history. In addition, the urosome deformity was used a good potential monitoring tool invading various chemicals and environmental contamination into water system.     

Influence of environmental factors on the response of a natural population of Daphnia magna (Crustacea: Cladocera) to spinosad and Bacillus thuringiensis israelensis in Mediterranean coastal wetlands

The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17 and 33 μg L⁻¹) on a field population of Daphnia magna under natural variations of water temperature and salinity, using Bti (0.16 and 0.50 μL L⁻¹) as the reference larvicide. Microcosms (125 L) were placed in a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during the 21-day observation period may have been partly responsible for the decrease of daphnid population density in all the microcosms. It is also probably responsible for the absence of recovery in the microcosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two days following treatment whereas Bti had no effect. These results suggest that it may be difficult for a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and anthropogenic (larvicides) stressors.     

Bioaccumulation of persistent organic pollutants in female common dolphins (Delphinus delphis) and harbour porpoises (Phocoena phocoena) from western European seas: geographical trends, causal factors and effects on reproduction and mortality

Concentrations of polychlorinated biphenyls (PCBs) in blubber of female common dolphins and harbour porpoises from the Atlantic coast of Europe were frequently above the threshold at which effects on reproduction could be expected, in 40% and 47% of cases respectively. This rose to 74% for porpoises from the southern North Sea. PCB concentrations were also high in southern North Sea fish. The average pregnancy rate recorded in porpoises (42%) in the study area was lower than in the western Atlantic but that in common dolphins (25%) was similar to that of the western Atlantic population. Porpoises that died from disease or parasitic infection had higher concentrations of persistent organic pollutants (POPs) than animals dying from other causes. Few of the common dolphins sampled had died from disease or parasitic infection. POP profiles in common dolphin blubber were related to individual feeding history while those in porpoises were more strongly related to condition.     

NDL-PCBs in muscle of the European catfish (Silurus glanis): An alert from Italian rivers

The non-dioxin-like polychlorinated biphenyls (NDL-PCBs) highly contribute to the PCB dietary intake of total PCBs. Most of the NDL-PCBs are assumed through ingestion of contaminated fish and fishery products. Therefore, it is important to quantify their presence in aquatic organisms to evaluate human risks associated with fish consumption. The European catfish is a top food-chain predator and is considered a reliable bio-monitoring tool reflecting the state of the environmental organic pollution. From 2006 to 2009, 54 European catfish were captured in four sites covering the area of the Po River (North Italy), and their muscles were analysed to determine the levels of 18 PCBs congeners. All samples presented detectable levels of 18 congeners and, on average, results showed an important presence of NDL-PCBs. The sum of the six congeners (28, 52, 101, 138, 153, 180 IUPAC) was used as indicator of the total PCBs concentration. The 33% of the samples analysed exceeded the maximum levels of 125 ng g⁻¹ set by European regulations in fish. The values measured ranged from 19.7 to 1015.4 ng g⁻¹ (mean 135.6 ± 149.8 ng g⁻¹).     

(210)Po and (210)Pb activity concentrations in Mytilus galloprovincialis from Croatian Adriatic coast with the related dose assessment to the coastal population

Activity concentrations of ²¹⁰Po and ²¹⁰Pb in the species Mytilus galloprovincialis from Croatian Adriatic coast were determined. The samples were collected in thirteen sampling places which are also a part of Croatian Mediterranean mussel watch project. The results of ²¹⁰Po and ²¹⁰Pb activity concentrations were found to vary between (22.1 ± 2.5)-(207 ± 21) Bq kg⁻¹ fresh weight and (2.8 ± 1.4)-(9.3 ± 0.7) Bq kg⁻¹ fresh weight, respectively. ²¹⁰Po/²¹⁰Pb ratio ranged between 6.2 and 30.7. The highest measured activities were found in the enclosed basins of the Neretva channel, where the majority of Croatian mussel cultivation is taking place. The estimated consequent average total annual effective ingestion dose due to ²¹⁰Po and ²¹⁰Pb from mussel consumption in Croatian coastal region is 202 ± 99 μSv with 96% contribution of ²¹⁰Po to the total effective dose.     

Persistence of organochlorine chemical residues in fish from the Tombigbee River (Alabama, USA): Continuing risk to wildlife from a former DDT manufacturing facility

Organochlorine pesticide and total polychlorinated biphenyl (PCB) concentrations were measured in largemouth bass from the Tombigbee River near a former DDT manufacturing facility at McIntosh, Alabama. Evaluation of mean p,p′- and o,p′-DDT isomer concentrations and o,p′- versus p,p′-isomer proportions in McIntosh bass indicated that DDT is moving off site from the facility and into the Tombigbee River. Concentrations of p,p′-DDT isomers in McIntosh bass remained unchanged from 1974 to 2004 and were four times greater than contemporary concentrations from a national program. Total DDT in McIntosh bass exceeded dietary effect concentrations developed for bald eagle and osprey. Hexachlorobenzene, PCBs, and toxaphene concentrations in bass from McIntosh also exceeded thresholds to protect fish and piscivorous wildlife. Whereas concentrations of DDT and most other organochlorine chemicals in fish have generally declined in the U.S. since their ban, concentrations of DDT in fish from McIntosh remain elevated and represent a threat to wildlife.     

Changes in Lumbriculus variegatus metabolites under hypoxic exposure to benzo(a)pyrene,chlorpyrifos and pentachlorophenol: Consequences on biotransformation

The regulation of endogenous metabolites is still not fully understood in aquatic invertebrates exposed concurrently to toxicants and hypoxia. Despite the prevalence of hypoxia in the aquatic environment, toxicity estimations seldom account for multiple stressors thereby differing from natural conditions. In this study, we examined the influence of hypoxia (<30% O₂) on contaminant uptake and the composition of intracellular metabolites in Lumbriculus variegatus exposed to benzo(a)pyrene (B(a)P, 3 μg L⁻¹), chlorpyrifos (CPF, 100 μg L⁻¹) or pentachlorophenol (PCP, 100 μg L⁻¹). Tissue extracts of worms were analyzed for 123 metabolites by gas chromatography–mass spectrometry and metabolite levels were then related to treatments and exposure time. Hypoxia markedly increased the accumulation of B(a)P and CPF, which underlines the significance of oxygen in chemical uptake. The oxygen effect on PCP uptake was less pronounced. Succinate and glycerol-3-phosphate increased significantly (p < 0.0001) following hypoxic treatment, whereas sugars, cysteine, and cholesterol were effectively repressed. The buildup of succinate coupled with the corresponding decline in intracellular 2-oxo- and 2-hydroxy glutaric acid is indicative of an active hypoxia inducible factor mechanism. Glutamate, and TCA cycle intermediates (fumarate, and malate) were disturbed and evident in their marked suppression in worms exposed concurrently to hypoxia and PCP. Clearly, hypoxia was the dominant stressor for individuals exposed to B(a)P or CPF, but to a lesser extent upon PCP treatment. And since oxygen deprivation promotes the accumulation of different toxicants, there may be consequences on species composition of metabolites in natural conditions.     

New cytochrome P450 1B1, 1C1, 2Aa, 2Y3, and 2K genes from Chinese rare minnow (Gobiocypris rarus): Molecular characterization,basal expression and response of rare minnow CYP1s and CYP2s mRNA exposed to the AHR agonist benzo[a]pyrene

Cytochrome P450 (CYP450) genes play an important role in catalyzing oxidative metabolism of toxicants. Recently, CYP1 subfamily were discovered and reported in fish, however, little is known regarding the CYP2 isoforms in fish. In the present study, the cDNA fragments of CYP 1B1 and 1C1 and CYP2Aa, 2Y3, and 2K of rare minnow were cloned and exhibited a high amino acid sequence identity compared with their zebrafish orthologs. Basal expression showed CYP1C1 and CYP 2Aa expression were observed in all eight tissues analyzed (liver, gill, intestine, kidney, spleen, brain, skin, and muscle). CYP 1A, and 1B1 expression was found in all tissues except for muscle and skin. However, CYP 2Y3 was expressed in liver, spleen, intestine and muscle whereas CYP 2 K in liver, kidney and intestine. 4 and 100 μg L⁻¹ Benzo[a]pyrene (BaP) induced patterns showed that CYP 1A, 1B1 and 1C1 expression in liver, gill, and intestine was strongly up-regulated (p < 0.05). Furthermore, CYP 2Y3 was strongly induced in liver from BaP treatments (p < 0.05). The high induction on mRNA level of CYP1s and CYP 2Y3 by BaP could be associated with catalyzing detoxification and indicated that CYP2s may also be potential biomarker to screen AHR agonist. The high responsiveness of CYP1 and 2 genes suggested Chinese rare minnow is feasible to screen and assess pollution with AHR agonist.     

Copper desorption in flooded agricultural soils and toxicity to the Florida apple snail (Pomacea paludosa): implications in Everglades restoration

Copper (Cu) desorption and toxicity to the Florida apple snail were investigated from soils obtained from agricultural sites acquired under the Comprehensive Everglades Restoration Plan. Copper concentrations in 11 flooded soils ranged from 5 to 234 mg/kg on day 0 and from 6.2 to 204 mg/kg on day 28 (steady-state). The steady-state Cu concentration in overlying water ranged from 9.1 to 308.2 microg/L. In a 28-d growth study, high mortality in snails occurred within 9 to 16 d in two of three soil treatments tested. Growth of apple snails over 28 d was affected by Cu in these two treatments. Tissue Cu concentrations by day 14 were 12-23-fold higher in snails exposed to the three soil treatments compared to controls. The endangered Florida snail kite and its main food source, the Florida apple snail, may be at risk from Cu exposure in these managed agricultural soil-water ecosystems.     

Protection of palak (Beta vulgaris L. var Allgreen) plants from ozone injury by ethylenediurea (EDU): Roles of biochemical and physiological variations in alleviating the adverse impacts

Ameliorative effects of ethylenediurea (N-[2-(2-oxo-1-imidazolinidyl) ethyl]-N′ phenylurea, abbreviated as EDU) against ozone stress were studied on selected growth, biochemical, physiological and yield characteristics of palak (Beta vulgaris L. var Allgreen) plants grown in field at a suburban site of Varanasi, India. Mean eight hourly ozone concentration varied from 52 to 73 ppb which was found to produce adverse impacts on plant functioning and growth characteristics. The palak plants were treated with 300 ppm EDU at 10 days after germination at 10 days interval up to the plant maturity. Lipid peroxidation in EDU treated plants declined significantly as compared to non-EDU treated ones. Significant increment in F_v/F_m ratio in EDU treated plants as compared to non-EDU treated ones was recorded. EDU treated plants showed significant increment in ascorbic acid contents and reduction in peroxidase activity as compared to non-EDU treated ones. As a result of the protection provided by EDU against ozone induced stress on biochemical and physiological characteristics of palak, the morphological parameters also responded positively. Significant increments were recorded in shoot length, number of leaves plant⁻¹, leaf area and root and shoot biomass of EDU treated plants as compared to non-EDU treated ones. Contents of Na, K, Ca, Mg and Fe were higher in EDU treated plants as compared to non-EDU treated ones. The present investigation proves the usefulness of EDU in partially ameliorating ozone injury in ambient conditions.