Characterisation of esterases as potential biomarkers of pesticide exposure in the lugworm Arenicola marina (Annelida: Polychaeta)

Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC50 of 5 microg l(-1) for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 microg l(-1) azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F=2.75, P=0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F=15.33, P<0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species.     

B-type esterases in the snail Xeropicta derbentina: an enzymological analysis to evaluate their use as biomarkers of pesticide exposure

The study was prompted to characterize the B-type esterase activities in the terrestrial snail Xeropicta derbentina and to evaluate its sensitivity to organophosphorus and carbamate pesticides. Specific cholinesterase and carboxylesterase activities were mainly obtained with acetylthiocholine (K_m = 77.2 mM; V_max = 38.2 mU/mg protein) and 1-naphthyl acetate (K_m = 222 mM, V_max = 1095 mU/mg protein) substrates, respectively. Acetylcholinesterase activity was concentration-dependently inhibited by chlorpyrifos-oxon, dichlorvos, carbaryl and carbofuran (IC50 = 1.35 × 10⁻⁵-3.80 × 10⁻⁸ M). The organophosphate-inhibited acetylcholinesterase activity was reactivated in the presence of pyridine-2-aldoxime methochloride. Carboxylesterase activity was inhibited by organophosphorus insecticides (IC50 = 1.20 × 10⁻⁵-2.98 × 10⁻⁸ M) but not by carbamates. B-esterase-specific differences in the inhibition by organophosphates and carbamates are discussed with respect to the buffering capacity of the carboxylesterase to reduce pesticide toxicity. These results suggest that B-type esterases in X. derbentina are suitable biomarkers of pesticide exposure and that this snail could be used as sentinel species in field monitoring of Mediterranean climate regions.     

Imidacloprid induced alterations in oxidative stress,biochemical, genotoxic,and immunotoxic biomarkers in non-mammalian model organism Galleria mellonella L. (Lepidoptera: Pyralidae)

Imidacloprid (IMI), a neonicotinoid insecticide, is widely used to control pests in agriculture. We investigated the changes in antioxidant enzyme activities, lipid peroxidation levels, biochemical effects, genotoxic effect, and immunotoxic effect of sublethal doses (0.25, 0.50, 0.75, and 1.00?µg) of IMI at different time periods (24, 48, 72, and 96?h) on a model organism, Galleria mellonella L. The results indicated that there were dose-dependent increases in both antioxidant enzyme activities (SOD and CAT) and MDA levels. Protein content was not affected by IMI at 24th and 48th, whereas it was decreased by the highest dose of IMI (1.00?µg) at 72nd and 96th h. Lipid and carbohydrate contents were reduced with increasing doses of IMI. Micronucleus frequency significantly increased in all IMI doses. All IMI doses caused a significant decrease in THC at 24th, 48th, and 72nd h. Our results can help to illustrate the effects of IMI in target organisms and indirectly may aid to discover potential risk of it on nontarget organisms. Future studies, at molecular levels, will be helpful in understanding the mechanism of action of IMI on these biomarkers.     

Analysis of lipid peroxidation in animal and plant tissues as field-based biomarker in Mediterranean irrigated agroecosystems (Extremadura,Spain)

ABSTRACT

The development of field-based biomarkers can allow for a more reliable assessment of the exposure of organisms to pollutants. Different sampling sites, along two streams running through an irrigable agricultural area, were selected to evaluate the effect of agrochemical load on the measured endpoints. The levels of lipid peroxidation were evaluated in several organs of Procambarus clarkii. The same method was applied to leaves of two woody species. Determining levels of MDA (malonaldehyde) by thiobarbituric acid reactive substances assay allows measuring the levels of lipid peroxidation. Differences in lipid peroxidation levels were observed in P. clarkii individuals collected at different sites; however, the patterns varied depending on the organ (when accounting for variations due to sex). The use of a MDA-gills/MDA-hepatopancreas index allowed for discrimination between reference and polluted sites. Significant differences in oxidative damage between sites were found in the leaves of Quercus rotundifolia but not in Salix sp. The lipid peroxidation of crayfish organs and holm oak leaves as a suitable biomarker of environmental pollution deserves further investigation.     

Plasma esterases in the tegu lizard Tupinambis merianae (Reptilia, Teiidae): impact of developmental stage, sex, and organophosphorus in vitro exposure

Purpose  

In this study, we determined normal serum butyrylcholinesterase (BChE) and carboxylesterase (CbE) activities in Tupinambis merianae in order to obtain reference values for organophosphorus pesticide monitoring.     

Vitellogenin and zona radiata proteins as biomarkers of endocrine disruption in peregrine falcon (Falco peregrinus)

The aim of this study was to test a specific method for the detection of Vitellogenin (Vtg) and Zona Radiata Proteins (Zrp) in plasma from peregrine falcon (Falco peregrinus) as specific biomarkers for the evaluation of the effects of endocrine disruptors. The method was assayed with different peregrine falcon individuals (including mature and immature birds of both sexes) from a Spanish population being studied in terms of their contamination with organochlorine compounds with endocrine disrupting properties. This study shows that mouse anti bird Vtg monoclonal antibody ND3C3 (Biosense) seems to be the most specific antibody in binding plasmatic lipoproteins in peregrine falcon when compared to other anti Vtg antibodies. Rabbit anti salmon Zrp polyclonal antibodies O146 (Biosense) show cross-reactivity with Zrp in the samples studied. These preliminary results confirm the applicability of both of these diagnostic tools assayed (induction of Vtg and Zrp) in detecting exposure to Endocrine Disrupting Chemicals (EDCs) in this species. The increase of Vtg and Zrp detected in male specimens suggest a potential hazard to EDCs in the peregrine falcon which represents a species still affected by organochlorine compounds, and in particular those with estrogenic activity.     

Stress biomarkers and alkali-labile phosphate level in mussels (Mytilus galloprovincialis) collected in the urban area of Venice (Venice Lagoon, Italy)

In this study, a spatial and temporal survey at three sites located in the "canals" of the Venice historic centre (Italy) and at a reference site was undertaken to evaluate stress effects on mussels sampled in the Venice urban area, where raw sewage is discharged without treatment directly into the water. A battery of biomarkers (metallothionein, micronuclei, condition index and survival in air) was used to evaluate the stress condition of the animals. At the same time the alkali-labile phosphate assay (ALP) was performed in mussel' hemolymph with the aim to find an estrogenic effect biomarker in this mussel species. Biomarker results showed an impairment of the general health condition in the mussels coming from the urban area, in agreement with the chemical analysis. Significantly higher level of the ALP was found in male mussels sampled in April in the urban area, in comparison with the ones from the reference site (P<0.001). Finally, the PCA proved an easy and useful tool to summarize the obtained results, also able to classify the data to indicate a pollution gradient in the Venice urban area.     

Lysosomal and lipid alterations in the digestive gland of mussels, Mytilus galloprovincialis (L.) as biomarkers of environmental stress

Lysosomal and lipid alterations in the digestive gland cells of the common mussel Mytilus galloprovincialis collected from six stations along Thermaikos Gulf (Northern Greece) were monitored through a 1-year period. The ultrastructural observations of the digestive cells of sampled mussels indicated a higher incidence of "granular" or "dark" residual bodies in the relatively more polluted stations compared to less polluted ones. Alterations in the lysosomal system of digestive cells were assessed: (1) using the lysosomal membrane stability test (2) quantifying lysosomal structural alterations of the residual bodies and the heterolysosomes of the digestive cells at the ultrastructural level, and (3) determining the elemental composition of lysosomes by means of X-ray microanalysis on epoxy resin sections. Evaluation of neutral lipids was realized using image analysis on histochemical resin sections. Mussels from the most heavily polluted stations exhibited reduced lysosomal membrane stability, higher volume density and diameter of heterolysosomes and residual bodies, and, in some cases, relatively higher lysosomal heavy metal content compared to mussels from less polluted stations. The results showed that the lysosomal membrane stability and the volume density of the residual bodies are more sensitive to general stress compared to the other biomarkers used, and could be used successfully as early warning tools in biomonitoring programs.     

Mixing ratios of volatile organic compounds (VOCs) in the atmosphere of Karachi,Pakistan

Mixing ratios of carbon monoxide (CO), methane (CH₄), non-methane hydrocarbons, halocarbons and alkyl nitrates (a total of 72 species) were determined for 78 whole air samples collected during the winter of 1998–1999 in Karachi, Pakistan. This is the first time that volatile organic compound (VOC) levels in Karachi have been extensively characterized. The overall air quality of the urban environment was determined using air samples collected at six locations throughout Karachi. Methane (6.3 ppmv) and ethane (93 ppbv) levels in Karachi were found to be much higher than in other cities that have been studied. The very high CH₄ levels highlight the importance of natural gas leakage in Karachi. The leakage of liquefied petroleum gas contributes to elevated propane and butane levels in Karachi, although the propane and butane burdens were lower than in other cities (e.g., Mexico City, Santiago). High levels of benzene (0.3–19 ppbv) also appear to be of concern in the Karachi urban area. Vehicular emissions were characterized using air samples collected along the busiest thoroughfare of the city (M.A. Jinnah Road). Emissions from vehicular exhaust were found to be the main source of many of the hydrocarbons reported here. Significant levels of isoprene (1.2 ppbv) were detected at the roadside, and vehicular exhaust is estimated to account for about 20% of the isoprene observed in Karachi. 1,2-Dichloroethane, a lead scavenger added to leaded fuel, was also emitted by cars. The photochemical production of ozone (O₃) was calculated for CO and the various VOCs using the Maximum Incremental Reactivity (MIR) scale. Based on the MIR scale, the leading contributors to O₃ production in Karachi are ethene, CO, propene, m-xylene and toluene.     

Haematology, genotoxicity, enzymatic activity and histopathology as biomarkers of metal pollution in the shrew Crocidura russula

Haematological (WBC, RBC, Hgb and Hct) and genotoxicity (MNT) parameters, hepatic enzymatic activities (GST, GPx and GR), and a histopathological evaluation of liver, kidneys and gonads were assessed as general biomarkers of metal pollution in the shrew Crocidura russula inhabiting a pyrite mining area. Specimens exposed to metals presented a few significant alterations when compared with reference animals: GST activity decreased; micronuclei increased; and evident liver alterations related to metal exposure were observed. On the basis of all the parameters studied, age was an important factor that partly explained the observed variation, whereas sex was the least important factor. Significant correlations were also found between heavy metal concentrations and biomarkers evaluated, demonstrating the great influence of these metals in the metabolic alterations. To the best of our knowledge, these data constitute the first measurements of a battery of biomarkers in shrews from a mine site and are among the few available for insectivorous mammals.     

Dioxin exposure in contaminated sawmill area: the use of molar teeth and bone of bank vole (Clethrionomys glareolus) and field vole (Microtus agrestis) as biomarkers

Developmental disorders of teeth are among the most sensitive targets of polychlorinated dibenzo-p-dioxin and -furan (PCDD/F) exposure. In rats, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces dose-dependently the size of molars, most severely the third lower molars. Dioxins also have effects on developing bone, including altered bone mineral density as well as reduced bending breaking force and stiffness. The aim of this study was to evaluate the use of the third lower molar and long bones as biomarkers of PCDD/F exposure in two wild vole species, the bank vole (Clethrionomys glareolus) and the field vole (Microtus agrestis) collected from a PCDD/F contaminated former sawmill area. Survey of soil and biota of the sawmill area indicated a PCDD/F contamination with a congener profile characteristic for the chlorophenol wood preservative Ky-5. The PCDD/F concentration in the bank vole was notably higher than in the field vole. The third molar of the bank vole was significantly smaller in dioxin-exposed animals compared to control group, while there was no difference between these two groups in the field vole. No significant alterations were observed in bone density and strength in either species except for reduced bending strength of the femur neck in bank vole males exposed to dioxins. Even though the bone changes are among the sensitive endpoints of dioxin-exposure, high variability due to age, size and gender limits their use as biomarkers of wildlife exposure. In conclusion, the size of molar teeth seems to be a sensitive and robust biomarker for PCDD/F exposure in wild bank vole populations and thus worth of further studies.     

Polychlorinated organic compounds (PCOCs) in waters, suspended solids and sediments of the Yangtse River

The contamination levels of polychlorinated organic compounds (PCOCs) in waters, suspended solids and sediments of the Yangtse River (Nanjing part) were analyzed in this paper. Their concentrations determined by GC/MS were very low in comparison with those in European River. The average concentration of total HCH (alpha-HCH, beta-HCH, gamma-HCH) was much higher than that of other PCOCs in all waters, which made up 65% of total amount of PCOCs. Due to the complete dilution and mixture of pollutants in the mighty Yangtse River, the content of PCOCs at each sampling station demonstrated very similar spatial pattern for waters and suspended solids. Since the small suspended solid (<0.7 microm) passed through the filter was also considered as dissolved part, the dominant parts of HCHs, PCA and PCBs were found in dissolved phase with percentage proportion of 85-94%, 72-85% and 61-78%, respectively. For DDTs, HCB and PeCB, their contents in dissolved phase were slightly higher than in particulate phase. The contents of PCOCs in sediments were also very low and varied with high fluctuation at different sampling points, indicating the heterogeneous deposition. HCB and its metabolite (PeCB) presented the highest contamination levels among PCOCs in sediments.     

Antioxidant response versus selenium accumulation in the liver and kidney of the Siberian sturgeon (Acipenser baeri)

The aim of this study was to examine the effects of selenium on concentrations of metabolites and enzyme activities acting as antioxidant markers in liver and kidney of Siberian sturgeon Acipenser baeri. Sturgeons were fed selenium cysteine for 30 and 60 d at 1.25, 5, 20 mg Se kg⁻¹. Selenium level in the control feed was 0.32 mg kg⁻¹. Se concentration was measured in liver, kidney and muscle of every specimen. Sturgeon accumulated Se in tissues with a clear dose-response relationship and the highest Se concentration was recorded in liver. This outcome is lined up with the findings obtained on the antioxidant markers evaluated in both tissues, and in which a dose-response for several biomarkers was recorded in liver. The superoxide dismutase activity in Se-treated fish was generally induced, while catalase activity was lower in liver or unaltered in kidney. The concentrations of glutathione S-transferase, glutathione reductase and total glutathione responded differently for both tissues and were induced in a different way at both endpoints. No changes of glyoxalase I activity were noted for both Se-treated tissues, while for glyoxalase II enzyme in liver a dose-related pattern was found showing a reversible effect (decreased and increased counteractive response) only in the 5 mg kg⁻¹ group. Moreover, the highest Se concentrations did not cause marked changes in malondialdehyde levels of liver and kidney. The enhancement of glutathione peroxidase activity in Se-treated sturgeon might have prevented the lipid peroxidation in both tissues, providing to the Siberian sturgeon a great defense ability versus the prooxidant effect of selenium.     

Trace analysis of dissolved polychlorinated organic compounds in the water of the Yangtse River (Nanjing, China)

Eighteen organochlorine contaminants from the water samples of the Yangtse River have been qualitatively and quantitatively analyzed. The analysis of polychlorinated organic compounds (PCOCs) extracted by C18 Solid Phase Extract (SPE) was performed using HP6890 gas chromatograph coupled by ECD detector. The analytical results demonstrate that the analytical methods are reliable based on the fact that most of the recoveries for the targeted compounds are 60-90%. Most of the concerned contaminants, i.e. chlorinated pesticides and PCBs were found in the water samples of the Yangtse River, but their concentration was very low, <2.97 ng l(-1), which is far less than 1 microg l(-1)--the standard of drinking water of China. It is apparent that the PCOCs were in high concentration during the dry season, and in low concentration during the rainy season. The situation of PCOCs contamination in the water of the Yangtse River in 1999 was greatly improved in comparison with 10 years ago.     

In vivo evaluation of three biomarkers in the mosquitofish (Gambusia yucatana) exposed to pesticides

In this study, the acute toxicity and the in vivo effects of commercial chlorpyrifos, carbofuran and glyphosate formulations on cholinesterase (ChE), glutathione S-transferase (GST) and lactate dehydrogenase (LDH) activities of the mosquitofish (Gambusia yucatana) were investigated. In a first phase of the study, head and muscle ChE were characterized with different substrates (acetylthiocholine iodide, s-butyrylthiocholine iodide and propionylthiocholine iodide) and the selective inhibitors eserine hemisulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284C51), and N,N'-diisopropylphosphorodiamic acid (iso-OMPA). The results obtained suggest that the enzyme present in both head and muscle of G. yucatana is mainly acetylcholinesterase (AChE). Acute toxicity was evaluated by exposing fish to several concentrations of single pesticides and of a mixture of chlorpyrifos/glyphosate. LC50 values were determined after 96 h of exposure, except in the case of carbofuran for which LC50 was calculated after 24 h since almost all the fish died within this period. LC50 values were 0.085 mg/l for chlorpyrifos, 17.79 mg/l for glyphosate, 0.636 mg/l for carbofuran and 0.011 mg/l for the chlorpyrifos/glyphosate mixture. A Toxic Unit approach was used to compare the toxicity of chlorpyrifos and glyphosate when occurring in a mixture with their toxicities as single compounds. Synergistic effects of chlorpyrifos and glyphosate when present in a mixture were found. At the end of each bioassay (24 h for carbofuran, 96 for the other substances/mixture), effects on biomarkers were analyzed. Muscle LDH activity was not altered by any of the three pesticides tested. Gill GST activity was significantly inhibited (40%) by carbofuran after 24 h of exposure to concentrations equal or higher than 0.06 mg/l. ChE muscle and head activity were significantly inhibited (50% and 30%, respectively) by carbofuran at concentrations equal or higher than 0.25 mg/l. Chlorpyrifos induced a significant inhibition of both muscle and head ChE (80% and 50%, respectively) after 96 h of exposure to concentrations equal or higher than 0.05 mg/l. Carbofuran did not induce significant alterations of fish ChE. The ChE EC50 determined for chlorpyrifos/glyphosate mixture (0.070 mg/l) was higher than the correspondent value calculated for chlorpyrifos alone (0.011 mg/l) suggesting an antagonistic effect of glyphosate on ChE inhibition by chlorpyrifos. ChE activity of G. yucatana seems to be a good biomarker to diagnose the exposure of wild populations of this species exposed to anticholinesterase pesticides.     

Analysis of perfluorooctanoate (PFOA) and other perfluorinated compounds (PFCs) in the River Po watershed in N-Italy

C7-C11 perfluorinated carboxylates (PFACs) and perfluorooctansulfonate (PFOS) were analysed in selected stretches of the River Po and its major tributaries. Analyses were performed by solid-phase extraction (SPE) with Oasis HLB cartridges and methanol elution followed by LC-MS-MS detection using 13C-labelled internal standards. High concentration levels ( approximately 1.3 microg l(-1)) of perfluorooctanoate (PFOA) were detected in the Tánaro River close to the city Alessandria. After this tributary, levels between 60 and 337 ng l(-1) were measured in the Po River on several occasions. The PFOA concentration close to the river mouth in Ferrara was between 60 and 174 ng l(-1). Using the river discharge flow data in m3 s(-1) at this point (average approximately 920 m3 s(-1) for the year 2006), a mass load of approximately 0.3 kg PFOA per hour or approximately 2.6 tons per year discharged in the Adriatic Sea has been calculated. PFOS concentration levels in the Po River at Ferrara were approximately 10 ng l(-1).     

Comparing the response of biochemical indicators (biomarkers) and biological indices to diagnose the ecological impact of an oil spillage in a Mediterranean river (NE Catalunya, Spain)

Three biomarkers of hydrocarbon exposure, liver 7-ethoxyresourfin-O-deethylase activity (EROD), fluorescent hydrocarbon compounds (FACs) in bilis, and the liver antioxidant enzyme catalase (CAT) were examined in the autochthonous fish species Barbus meridionalis collected in the river Fluvià (NE Catalunya, Spain) after an oil spillage. Four different locations were sampled, including the impacted site, upstream and downstream sites and a reference site. Biomarker responses were compared with diatom and macroinvertebrate community assemblage metrics (Specific Pollution Sensitivity index - IPS, and Iberian Biological Monitoring Working Party - IBMWP, respectively). Chemical analyses denoted that polycyclic aromatic hydrocarbon levels in sediment were much higher at the impacted site than in downstream reaches. Four fold increase of EROD activity together with increased levels of biliary FACs in barbs collected at the spilled site indicated exposure of inhabiting fish to the oil. Additionally, CAT activity was significantly depressed (four fold) when compared to other stations, thus suggesting that fish collected from the impacted sites could be more susceptible to suffer oxidative stress. Biological indices (particularly that of the diatom community IPS) showed slight significant effects between control and impacted sites, indicating that more tolerant taxa were favoured because of the oil spillage. These results support the need to include biochemical responses measured in local species in monitoring programmes aimed to diagnose specific pollution effects in stressed river ecosystems.     

Natural attenuation of xenobiotic organic compounds in a landfill leachate plume (Vejen,Denmark)

Demonstration of natural attenuation of xenobiotic organic compounds (XOCs) in landfill leachate plumes is a difficult task and still an emerging discipline within groundwater remediation. One of the early studies was made at the Vejen Landfill in Denmark in the late 1980s, which suggested that natural attenuation of XOCs took place under strongly anaerobic conditions within the first 150 m of the leachate plume. This paper reports on a revisit to the same plume 10 years later. Within the strongly anaerobic part of the plume, 49 groundwater samples were characterized with respect to redox-sensitive species and XOCs. The analytical procedures have been developed further and more compounds and lower detection limits were observed this time. In addition, the samples were screened for degradation intermediates and for toxicity. The plume showed fairly stationary features over the 10-year period except that the XOC level as well as the level of chloride and nonvolatile organic carbon (NVOC) in the plume had decreased somewhat. Most of the compounds studied were subject to degradation in addition to dilution. Exceptions were benzene, the herbicide Mecoprop (MCPP), and NVOC. In the early study, NVOC seemed to degrade in the first part of the plume, but this was no longer the case. Benzyl succinic acid (BSA) was for the first time identified in a leachate plume as a direct indicator, and as the only intermediate of toluene degradation. Toxicity measurements on solid phase-extracted (SPE) samples revealed that toxic compounds not analytically identified were still present in the plume, suggesting that toxicity measurements could be helpful in assessing natural attenuation in leachate plumes.     

Temporal trends of polyfluoroalkyl compounds (PFCs) in liver tissue of grey seals (Halichoerus grypus) from the Baltic Sea, 1974-2008

Temporal trends of polyfluoroalkyl compounds (PFCs) were examined in grey seal (Halichoerus grypus) liver from the Baltic Sea over a period of 35 years (1974-2008). In total, 17 of 43 PFCs were found, including the perfluoroalkyl sulfonates (C₄-C₁₀ PFSAs), perfluorooctanesulfinate (PFOSi), long chain perfluoroalkyl carboxylates (C₇-C₁₄ PFCAs), and perfluoroalkyl sulfonamides (i.e., perfluorooctane sulfonamide (FOSA) and N-ethyl perfluorooctane sulfonamide (EtFOSA)), whereas saturated and unsaturated fluorotelomer carboxylates, shorter chain PFCAs and perfluoroalkyl phosphonic acids were not detected. Perfluorooctane sulfonate (PFOS) was the predominant compound (9.57-1444 ng g⁻¹ wet weight (ww)), followed by perfluorononanoate (PFNA, 0.47-109 ng g⁻¹ ww). C₆-C₈ PFSAs, PFOSi and C₇-C₁₃ PFCAs showed statistically significant increasing concentrations between 1974 and 1997, with a peak in 1997 and then decreased or levelled off (except for C₁₂ and C₁₃ PFCAs). FOSA had a different temporal trend with a maximum in 1989 followed by significant decreasing concentrations until 2008. Toxicological implications for grey seals are limited, but the maximal PFOS concentration found in this study was about 40 times lower than the predicted lowest observed effect concentrations (LOEC). The statistically significant decreasing concentrations or levelling off for several PFCs in the relative closed marine ecosystem of the Baltic Sea indicate a rapidly responding to reduced emissions to the marine environment. However, the high concentrations of PFOS and continuing increasing concentrations of the longer chain PFCAs (C₁₂-C₁₄) shows that further work on the reduction of environmental emissions of PFCs are necessary.