In vitro effects of acephate on carbonic anhydrase activity in the blood and gills of rainbow trout, Salmo gairdneri

Acephate, a water-soluble organophosphate pesticide used to control terrestrial insect pests, may enter aquatic ecosystems in the course of its use and adversely affect fish populations. The in vitro effects of this insecticide on gill and red blood cell (RBC) carbonic anyhdrase (CA) activity in rainbow trout were investigated over a range of 100 mg/1 (0.55 mM) to 50,000 mg/l (273 mM) to assess the manner in which acephate might affect respiratory capacity in exposed fish. Concentrations required to produce 50% inhibition of CA activity in the gill and RBC preparations were 38,000 mg/l (207 mM) and 8,900 mg/l (48 mM) respectively. The toxic action of acephate may be related to inhibition of CA activity in the blood and gills with resultant disturbances of respiratory capacity and salt balance.     

Formation of N-nitrosodimethylamine (NDMA) by ozonation of dyes and related compounds

Formation of N-nitrosodimethylamine (NDMA) by ozonation of commercially available dyes and related compounds was investigated. Ozonation was conducted using a semi-batch type reactor, and ozone concentration in gas phase and the ozone gas flow were 10 mg L(-1) and 1.0 L min(-1), respectively. NDMA was formed by 15 min of ozonation of seven out of eight selected target compounds (0.05 mM) at pH 7. All the target compounds with N,N-dimethylamino functions were NDMA precursors in ozonation. The lowest and highest NDMA concentrations after ozonation of the target compounds were 13 ng L(-1) for N,N-dimethylformamide (DMF) and 1600 ng L(-1) for N,N-dimethyl-p-phenylenediamine (DMPD), respectively. NDMA concentrations after 15 min of ozonation of 0.05 mM methylene blue (MB) and DMPD increased with an increase in pH in its range of 6-8. The effects of coexisting compounds on NDMA concentrations after 15 min of ozonation of 0.05 mM MB and DMPD were examined at pH 7. NDMA concentrations after ozonation of MB and DMPD increased by the presence of 0.05 mM (0.7 mg L(-1) as N) nitrite (NO(2)(-)); 5000 ng L(-1) for MB and 4000 ng L(-1) for DMPD. NDMA concentration after MB ozonation decreased by the presence of 5mM tertiary butyl alcohol (TBA), a hydroxyl radical (HO.) scavenger, but that after DMPD ozonation was increased by the presence of TBA. NDMA concentrations after ozonation of MB and DMPD were not affected by the presence of 0.16 mM (5.3 mg L(-1)) hydrogen peroxide (H(2)O(2)). When 0.05 mM MB and DMPD were added to the Yodo and Tone river water samples, NDMA concentrations after 15 min of their ozonation at pH 7 increased compared with those in the case of addition to ultrapure water samples.     

Spatial distribution of organotins in sediments of lowland river catchments

A method is described for the measurement of nanogram quantities of a range of methyl and butyltin compounds. The method utilises a modified purge and trap/boiling point separation technique for the determination of organotin compounds in sediment samples. A spatial survey of sediment samples within 5 river systems in the East Anglia region of the United Kingdom was undertaken and identified a range of organotin compounds with concentrations of tributyltin (TBT) ranging from <3.9 ng g(-1) up to 1291 ng g(-1). The highest values were found in areas connected with boating activities. Wherever TBT was present, dibutyltin and monobutyltin were most commonly found along with small concentrations of certain methyltins.     

Sublethal toxicity of two wastewater treatment polymers to lake trout fry (Salvelinus namaycush)

Lake trout fry (Salvelinus namaycush) were exposed in laboratory experiments to two wastewater treatment polymers, one anionic (MagnaFloc 156) and one cationic (MagnaFloc 368; Ciba Speciality Chemical), to determine if these chemicals which are used and discharged by mining operations in Canada's North pose a significant hazard to juvenile fishes. The cationic polymer was substantially more toxic to lake trout fry than the anionic polymer, with 96-h LC50 estimates of 2.08 and >600 mg/l, respectively. Separate 30-d exposure experiments yielded no observed and lowest observed effect concentrations, respectively, of 0.25 and 0.5mg/l for MagnaFloc 368, and 75 and 150 mg/l for MagnaFloc 156. In both cases, behavioural responses, especially startle response, were the most sensitive test endpoints. Histopathological assessment revealed that gill pathology appeared within a few days of exposure to both polymers, apparently as a result of localized hypoxia. Acute (4 d) effects included cloudy swelling of epithelial cells, increased gill vascularization, and thickening and shortening of the gill lamella. Chronic (30 d) polymer exposure produced only slightly greater pathological effects than acute exposure, with comparable responses observed only at >1.0mg/l MagnaFloc 368 and 150 mg/l MagnaFloc 156, suggesting that the fish displayed some level of both behavioural and physiological adaptation to the respiratory stress imposed by the two polymers.     

Mortality, growth, swimming activity and gill morphology of brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar) exposed to low pH with and without aluminum

Measurements were made of mortality, growth, swimming activity, and gill morphology of young-of-the-year brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar), exposed for 30 days to pH 5.6 +/- 0.5 (mean +/- 1 standard deviation) with and without addition of 107 +/- 57 microg liter(-1) exchangeable (labile monomeric) aluminum. The experiment was conducted in artificial stream channels adjacent to a natural stream and subject to daily and seasonal changes in temperature, light, and chemical conditions. There were no differences in survival or growth for brook trout in any treatment; Atlantic salmon survival and growth were significantly decreased in the acid + Al treatment. Scanning electron microscopy showed no damage to gills of either species in the acid treatments, but the acid + Al treatment caused slight swelling of brook trout gills near the filament tips and significant swelling and fusion of secondary lamellae of Atlantic salmon gills. The acid treatment increased swimming activity in brook trout, but both the acid and acid + Al treatments reduced activity in Atlantic salmon.     

Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds

Due to the numerous biotechnological applications of laccase enzyme, it is essential to know the influence of different agents usually present in the natural environment on its enzymatic action, especially for in situ treatment technologies. In the present work, a simple and rapid method to determine the inhibitory or inducer effect of different compounds on laccase activity was developed. The compounds tested were copper-chelating agents and heavy metals. It was found that using syringaldazine as a substrate, all copper-chelating agents (except EDTA) highly inhibited laccase activity (around 100%) at an inhibitor concentration lower than 20 mM. Moreover, 40% of inhibition, which was detected at a concentration of 20 mM for both Cd(2+) and Cu(2+) increased with concentration until nearly complete inhibition at 80 mM.     

Immunotoxicity in ascidians: Antifouling compounds alternative to organotins – II. The case of Diuron and TCMS pyridine

Using short-term hemocyte cultures of the colonial ascidian Botryllus schlosseri exposed to various sublethal concentrations of Diuron (3-(3,4-diclorophenyl)-1,1-dimethylurea) and TCMS pyridine (2,3,5,6-tetrachloro-4-(metylsulphonyl)pyridine), we evaluated their immunotoxic effects through a series of cytochemical assays previously used for organotin compounds. At concentrations higher than 250 μ M and 10 μ M for Diuron and TCMS pyridine, respectively, both biocides exerted immunosuppressant effects on Botryllus hemocytes, causing i) deep changes in the cytoskeleton that irreversibly affect cell morphology and phagocytosis, ii) induction of DNA damage, iii) leakage of oxidative and hydrolytic enzymes due to membrane alteration. Unlike organotin compounds, Diuron and TCMS pyridine do not inhibit cytochrome-c-oxidase, and only TCMS pyridine triggers oxidative stress. When co-present, they exert an antagonistic interaction on cytoskeletal components.     

Nitrate reduction by fluoride green rust modified with copper

Nitrate reduction by the fluoride form of green rust modified with copper (GR-F(Cu)) was investigated using a batch reactor system. The extent of nitrate reduction was measured by measuring the increase in concentration of ammonia, which is the final product of nitrate reduction by GR. This approach was required, because nitrate could be removed from solution by ion exchange without reduction. The rate of ammonium production was investigated over the range of pH 7.8-11. The fastest reaction was achieved at pH 9 when GR was present at a concentration of 0.083M as Fe(II) and 1mM of Cu(II) was added. The rate at pH 9 was enhanced by a factor of 2.5 compared to that at pH 7.8 by comparing the time elapsed to transform all nitrate to ammonium. Kinetics of nitrate reduction by GR-F at pH 7.8 were affected by the concentration of Cu(II) added. The rate constants for ammonium production increased from 0.012 to 1.52h(-1) as Cu(II) additions increased from 0 to 2.5mM, but the reaction rate at 5mM was slightly decreased to 1.25h(-1). The mechanism of enhanced rates of nitrate reduction by addition of Cu(II) could not be fully determined in this study. However, XRD results showed that magnetite was produced in the reaction of Cu(II) and GR-F and SEM shows the production of nano-size particles which were not fully identified in this study. In addition, the concentration of Fe(II) in GR was observed to linearly decrease with concentration of Cu(II) added.     

Water chemistry and fish community responses to episodic stream acidification in Pennsylvania, USA

Five streams were studied on the Northern Appalachian Plateau of Pennsylvania from October 1988 through June 1989 to determine chemical changes that occur during episodic storm run-off and the responses of fish to these events. These second-order streams flowed through undisturbed, wooded, sandstone bedrock catchments with surface areas ranging from 500 to 1000 hectares. Median pH of precipitations was about 4.2, and among streams it ranged from 5.0 to 6.2. During storm events, pH declined by as much as 1.2 units and peak concentrations of total monomeric Al ranged from < 0.01 to 0.75 mg litre(-1). Organically bound A1 was generally a minor component of total monomeric A1. Wild brook trout (Salvelinus fontinalis) were found in all streams, although only a remnant population existed in the most acidic stream. Sculpins (Cottus bairdi or C. cognatus) were collected only in the two streams with the least severe episodes. Mortality of brook trout and sculpins in in situ bioassays ranged from 0 to about 80% among streams during acidic episodes and was positively related to concentrations of total dissolved Al. Radio-tagged brook trout moved downstream during episodes when Al reached toxic concentrations. Some displaced trout were found near groundwater seeps, where pH was higher and dissolved Al was lower than in the main channel.     

Immunotoxicity in ascidians: antifouling compounds alternative to organotins - II. The case of Diuron and TCMS pyridine

Using short-term hemocyte cultures of the colonial ascidian Botryllus schlosseri exposed to various sublethal concentrations of Diuron (3-(3,4-diclorophenyl)-1,1-dimethylurea) and TCMS pyridine (2,3,5,6-tetrachloro-4-(metylsulphonyl)pyridine), we evaluated their immunotoxic effects through a series of cytochemical assays previously used for organotin compounds. At concentrations higher than 250 micro M and 10 micro M for Diuron and TCMS pyridine, respectively, both biocides exerted immunosuppressant effects on Botryllus hemocytes, causing i) deep changes in the cytoskeleton that irreversibly affect cell morphology and phagocytosis, ii) induction of DNA damage, iii) leakage of oxidative and hydrolytic enzymes due to membrane alteration. Unlike organotin compounds, Diuron and TCMS pyridine do not inhibit cytochrome-c-oxidase, and only TCMS pyridine triggers oxidative stress. When co-present, they exert an antagonistic interaction on cytoskeletal components.     

A study on organotin levels in Canadian drinking water distributed through PVC pipes

A study on organotin compounds in Canadian drinking water was carried out in winter-spring 1996 (28 sites) and autumn 1996 (21 sites). Approximately 29% and 40% of distribution waters supplied through PVC pipes installed recently (typically less than 6 months) contained organotin compounds in the winter-spring and autumn surveys respectively. Monomethyl-, dimethyl-, monobutyl- and dibutyltin levels ranged up to 291 ng Sn/L, 49.1 ng Sn/L, 28.5 ng Sn/L and 52.3 ng Sn/L, respectively. An additional study in summer 1996, of locations where the highest organotin levels were detected in the winter-spring survey, indicated that organotin levels had decreased in most distribution water samples. Samples of PVC pipe/tubing contained organotin compounds consistent with the organotin patterns found in the distribution water samples.     

Halofenate and clofibrate inhibition of pyruvate dehydrogenase from Fusarium culmorum

Pyruvate dehydrogenase (E1, E.C. 1.2.4.1) was obtained from Fusarium culmorum by ammonium sulfate precipitation. An eight-fold purification was obtained with a specific activity of 13 K units/mg protein. Both halofenate and clofibrate inhibited the enzyme complex non-competitively. The inhibitory effect of halofenate was greater than that of clofibrate being 42% higher at 20 mM concentration compared to the inhibition by clofibrate at 40 mM concentration. Both compounds disorganized the normal cytoplasmic lipids including the emptying of cells in the mycelium suggesting membrane disruption.     

Pilot study on the contamination of drinking water by organotin compounds from PVC materials

Raw and treated water samples and tap water samples from four to six houses located on distribution lines where PVC pipe/tubing had been recently installed were collected in five Canadian municipalities for the analysis of organotin compounds. After derivatisation with sodium tetraethylborate the ethylated organotin compounds were extracted with hexane and analysed by gas chromatography-microwave induced plasma atomic emission spectrometry using a wavelength (326.234 nm) specific for tin. Organotin compounds, mainly methyltin and dimethyltin at concentrations ranging respectively from 0.5 to 257 ng Sn/L and from 0.5 to 6.5 ng Sn/L, were detected in samples from ten of the twenty-two houses. No organotin compounds were detected in raw water or treated water leaving the treatment plant, indicating that the organotin compounds were leaching into the water from some component of the distribution system.     

Influence of pH and aluminum on developing brook trout in a low calcium water

Eyed embryos of brook trout (Salvelinus fontinalis) were exposed to nominal pHs of 4.5, 5.5 and 7.5 with and without aluminum (300 microg liter(-1)) in extremely soft water (hardness <9mg liter (-1)) at 12 degrees C. Embryo mortality exceeded 80% at pH 4.5, averaged 15 to 18% in the pH 5.5 treatments and was less than 2% in the pH 7.5 treatments. Aluminum significantly reduced embryo mortality (85.3% vs 99.5%) at pH 4.5 but did not affect mortality at pH 5.5 or pH 7.5. Percent hatch and poor hatch were pH dependent and were not significantly influenced by aluminum. Brook trout larvae cumulative mortalities were 100% within 30 days at pH 4.5, with or without aluminum; 69% after 60 days at pH 5.5; 100% in 15 days at pH 5.5 with aluminum and 20% after 60 days at pH 7.5 with or without aluminum. Fish that survived the pH 5.5 treatment showed decreased growth and behavioral impairments compared to the controls (pH 7.5 without aluminum).     

Assessment of tin and butyltin species in estuarine superficial sediments from Gipuzkoa,Spain

Ten superficial sediments from river estuaries of Gipuzkoa (North Spain) were collected and analyzed for nine metals (including tin, Sn) and butyltin compounds (monobutyltin, MBT; dibutyltin, DBT; tributyltin, TBT). Total metal concentration in the fine fraction (<63 micrometer) of the sediment was determined in aqua regia--hydrofluoric acid extracts by atomic absorption spectrometry. The butyltin species (BTs) were firstly extracted from the sediments with hydrochloric acid-methanol mixture. After derivatization with sodium tetraethyl borate, the organotin compounds were extracted by solid phase microextraction (SPME) in headspace mode. A fiber coated with 100 micrometer poly(dimethylsiloxane) was used for SPME. The organotin species were analyzed by gas chromatography-flame ionization detection. Analysis of the certified reference sediment (PACS-2) shown the suitability of the procedures for determination of metal contents and DBT-TBT species in contaminated sediments. Factorial analysis was used to examine the main sources of metals. Three factors represented more than 89% of the total variance of the metal system, and the Sn was related with Cu-Zn-Pb suggesting the same pollution source. The BTs concentrations in the area were high (TBT ranged from 0.05 to 5.48 mg Sn kg(-1)). The percentage of total butyltin species ( summation operator BTs) respect to the total Sn amount was higher than 4% in all the sediments, showing in the Bidasoa river estuary a remarkable value higher than 20%. Hence, the studied estuarine sediments reflect a pollution that is related with historical industrial and fishing activities of the area.     

Marine and farmed fish on the Polish market: comparison of the nutritive value and human exposure to PCDD/Fs and other contaminants

Chemical analyses were performed in nine fish species that are popular on the Polish market. These included Baltic fish (cod, herring, salmon), fish farmed in Poland (carp, trout), marine fish imported from China (Alaska pollock, sole), and farmed fish imported from Vietnam and China (sutchi catfish, tilapia). The nutritional composition (amino acid, micro- and macronutrients, fat-soluble vitamins - A₁, D₃, E) and certain contaminants (organochlorine pesticides, OCPs; indicator polychlorinated biphenyl, PCB₆; polychlorinated dibenzo-paradioxins and polychlorinated dibenzofurans, PCDD/Fs; dioxin-like polychlorinated biphenyls, dl-PCBs; organotin compounds, OCTs; dyes, malachite green and crystal violet; veterinary drug residues, nitrofurans and chloramphenicol; toxic metals, Cd, Pb, Hg) in the muscle tissues of fish were determined. It was confirmed that the fish species analyzed were excellent sources of amino acids, and were rich in phosphorous and selenium. Baltic Sea fish (salmon, herring), fish farmed in Poland (carp and trout), and tilapia were also rich in vitamin D₃.Traces of OCP, PCB₆, OCT, dyes, veterinary drug residues, and heavy metals were detected in concentrations which do not pose a threat to consumers at the current rate of fish consumption in Poland. However, the problem might arise from the content of PCDD/Fs and dl-PCBs in fatty Baltic fish. The fish species analyzed, differed in their nutritional values and degrees of contamination. We suggest that for optimum health and safety, it is advisable that consumers include a variety of different fish species in their diets.     

Toxicity evaluation of new antifouling compounds using suspension-cultured fish cells

A simple, rapid toxicity test was developed using the suspension-cultured fish cell line CHSE-sp derived from chinook salmon Oncorhynchus tshawytscha embryos in order to assess the toxicity of new marine antifouling compounds. The compounds tested were copper pyrithione, Diuron, Irgarol 1051, KH101, Sea-Nine 211, and zinc pyrithione, all of which have been nominated in Japan as possible replacements for organotin compounds. The in vitro acute toxicity (24-h EC50) of the six compounds to these fish cells was evaluated using the dye Alamar Blue to determine cell viability, and then correlated with the results of in vivo chronic toxicities (28-day LC50) to juvenile rainbow trout Oncorhynchus mykiss. The suspension-cultured fish cells were found to be suitable for the screening of such chemicals before performing an in vivo test. The toxicities of the test compounds obtained from both tests, shown in decreasing order, were as follows: copper pyrithione > zinc pyrithione > KH101 > or = Sea-Nine 211 > Diuron > Irgarol 1051. The herbicides Diuron and Irgarol 1051 showed the least toxicity, while the pyrithiones had the greatest toxicity.     

Monitoring contaminants from oil production at sea by measuring gill EROD activity in Atlantic cod (Gadus morhua)

An ex vivo gill EROD assay was applied in Atlantic cod (Gadus morhua) as a biomarker for waterborne CYP1A-inducing compounds derived from oil production at sea. Exposure to nominal concentrations of 1 ppm or 10 ppm North Sea crude oil in a static water system for 24 h caused a concentration-dependent gill EROD induction. Further, exposure of cod for 14 days to environmentally relevant concentrations of produced water (PW, diluted 1:200 or 1:1000) from a platform in the North Sea using a flow-through system resulted in a concentration-dependent induction of gill EROD. Crude oil (0.2 ppm) from the same oil field also proved to induce EROD. Finally, gill EROD activity in cod caged for 6 weeks at 500-10 000 m from two platforms outside Norway was measured. The activities in these fish were very low and did not differ from those in fish caged at reference sites.     

Organotin contamination in the Atlantic Ocean off the Iberian Peninsula in relation to shipping

Imposex in female snails, a bioindicator of TBT contamination, and the presence of organotins in snails' tissue and sediments were studied at nine sites off the western Iberian Peninsula. The study was part of a European project (acronym HIC-TBT) co-financed by the EU-LIFE programme, intending to investigate and communicate the impact of organotins from ships in marine ecosystems. Snails and sediments were sampled during two cruises in May/June 1999 and in January 2000 in areas of high, intermediate and low-shipping density. Imposex was found in female snails from several sampling sites, some of which had an imposex incidence of 100%. Differences in sensitivity were found between species; hence comparison of imposex levels between locations where different species were collected was not straightforward. Total organotin concentrations in sediments (sum of butyl and phenyltin compounds) ranged from 21 to 185 ng Sn g(-1) with higher values for most sites sampled in the vicinity of shipping lanes. Organotin concentration in snails' tissue ranged from <5 to 196 ng Sn g(-1), which are similar to those found in snails from other offshore areas contaminated by TBT. Overall, these results give further support to the recent ban on the use of organotin based antifouling paints to all ship size.     

Optimisation of pressurised liquid extraction for elimination of sulphur interferences during determination of organotin compounds in sulphur-rich sediments by gas chromatography with flame photometric detection

A simple method for species-selective analysis of organotin compounds (OTCs) (butyl and phenyl) in sediments was developed. The sample preparation procedure was specifically optimised for sulphur-rich sediments to eliminate interferences from elemental sulphur and organosulphur compounds. Tin species were extracted from sediment samples using pressurised liquid extraction technique (PLE), ethylated - with simultaneous extraction to isooctane - in aqueous phase with sodium tetraethylborate (NaBEt(4)) and separated/detected by gas chromatography with flame photometric detection (GC-FPD). PLE operational variables (extraction temperature and pressure, solvent composition and number of static extraction steps) and extract handling routine were fine-tuned to minimise the amount of extracted interferents while keeping OTCs recovery at an acceptable level. Best results were obtained after extraction of sediment samples with methanol/water (75% v/v methanol) solution of acetic acid/sodium acetate with tropolone addition (0.6 g l(-1)). Derivatisation of low temperature, high-pressure (50 degrees C, 13.8 MPa) extracts gives isooctane extracts which are clean enough to be directly analysed by GC-FPD without any further cleanup. Interferences from elemental sulphur were completely eliminated while concentrations of other interferents were reduced to the level not impairing quantitation of OTCs under the study. No negative effects in terms of chromatographic column deterioration were observed after repeated injections of such extracts. Two certified reference materials, BCR646 and PACS-2, were analysed to assess performance of the method. Recoveries of all OTCs under the study, except MBT, were in the range of 91-114%. MBT extraction efficiency was low (34-47%) therefore the method is unsuitable for precise determinations of this compound.