Toxic effects of mercury on the hard clam,Meretrix lusoria,in various salinities

1. The 96-hr lc₅₀ values for juvenile hard clams, Meretrix lusoria, were 328, 392 and 194 μg/l Hg in 10, 20 and 30 ppt salinities at 25 ± 1°C, respectively; for adult hard clams 341 and 140 μg/l Hg in 20 and 30 ppt salinities, respectively.2. Acclimatizing the adult clams to low salinity of 10 ppt lessened the toxicity of mercury. However, juvenile animals appeared to be more sensitive to mercury poisoning after 96 hr exposure in 10 ppt salinity.3. All embryos exposed to 40 μg/l Hg and above died within 30 hr. In the control, 44% of hatched embryos had developed into D-stage larvae, while those exposed to 20 μg/l Hg were still in the trochophore stage. Most of the retarded larvae developed into abnormal forms within 30 hr at 28°C in 15 ppt salinity.4. In order to maintain water quality and protect natural resources, the recommended safe level of mercury is 0.046 (0.039–0.053) μg/l Hg, based on the estimated 30-hr EC₅₀ for the clam embryos, with an application factor of 0.01.     

Acute toxicity of ammonia on Litopenaeus vannamei Boone juveniles at different salinity levels

Litopenaeus vannamei juveniles (total length 22+/-2.4 mm) were exposed to different concentrations of ammonia-N (un-ionized plus ionized ammonia as nitrogen), using the static renewal method at different salinity levels of 15 per thousand, 25 per thousand and 35 per thousand at pH 8.05 and 23 degrees C. The 24, 48, 72, 96 h LC50 values of ammonia-N on L. vannamei juveniles were 59.72, 40.58, 32.15, 24.39 mg l(-1) at 15 per thousand; 66.38, 48.83, 43.17, 35.4 mg l(-1) at 25 per thousand; 68.75, 53.84, 44.93, 39.54 mg l(-1) at 35 per thousand, respectively. The 24, 48, 72, 96 h LC50 values of NH(3)-N (un-ionized ammonia as nitrogen) were 2.95, 2.00, 1.59, 1.20 mg l(-1) at 15 per thousand; 2.93, 2.16, 1.91, 1.57 mg l(-1) at 25 per thousand; 2.78, 2.18, 1.82, 1.60 mg l(-1) at 35 per thousand, respectively. As the salinity decreased from 35 per thousand to 15 per thousand, susceptibility of ammonia-N increased by 115%, 132%, 140% and 162% after 24, 48, 72 and 96 h exposure, respectively. The "safety level" for rearing L. vannamei juveniles was estimated to be 2.44, 3.55, 3.95 mg l(-1) for ammonia-N and 0.12, 0.16, 0.16 mg l(-1) for NH(3)-N in 15 per thousand, 25 per thousand and 35 per thousand, respectively.     

Effects of endosulfan on survival,growth and reproduction of Daphnia magna

1. Acute and chronic toxicity tests with endosulfan were conducted on Daphnia magna. The 24-hr static lc₅₀ was 0.62 mg/l with a coefficient of variation of 14.2%.2. The sublethal effects of 0.12, 0.15, 0.20, 0.25 and 0.31 mg/l endosulfan on the survival, reproduction and growth of D. magna were monitored for 21 days.3. The parameters used to determined the effect of the pesticide on reproduction were: mean total young per female, maximum number of broods, mean brood size, mean number of broods, mean time to first reproduction and intrinsic rate of natural increase (r).4. Growth, as measured by body length, was depressed significantly at all endosulfan concentrations tested. The highest concentrations used (0.20, 0.25 and 0.31 mg/l) caused a decrease in survival and mean total young per female, and an increase in the time to first reproduction. The intrinsic rate of natural increase (r) and the mean number of broods were decreased significantly at all the sublethal concentrations of endosulfan tested.     

Toxicity of colchicine towards a marine fish,Dicentrarchus labrax

1. Hematology and blood biochemistry parameters were examined in order to determine the metabolic or endocrine disturbances caused by colchicine intoxication in sea bass (Dicentrarchus labrax) aged 1 + . The detection of non-lethal effects of colchicine requires the use of a broad concentration range (6.25–1020 mg 1⁻¹).2. The 48-hr lc₅₀, was 1020 mg 1⁻¹, a value which is close to that previously reported in the reference fresh water fish Brachydanio rerio. Exposure to 50 and 125 mg ⁻¹ colchicine for 48 hr resulted in 100% mortality one week after treatment.3. Colchicine involves the concomitant increase of packed cell volume and hemoglobin concentration in whole blood, in the concentration range of 12.5–1020 mg 1⁻¹.4. The activity of erythrocyte antioxidant enzymes undergoes low amplitude variations after 48-hr exposure to colchicine, regardless of concentration. Two days after recovery, decreased SOD, and increased catalase and peroxidase activities were shown.     

Dinitrogen oxide production by a mixed culture of nitrifying bacteria during ammonia shock loading and aeration failure

A number of experiments was conducted in order to establish if N₂O in the exhaust gas from an aerobic consortium of nitrifiers could be used as an indicator for monitoring the nitrification process. Laboratory-scale experiments with an activated sludge system showed a strong correlation between ammonia shock loads and both the concentration of N₂O and the rate of increase of N₂O in the exhaust gas for shock loads less than 1.60 mg ammonical nitrogen (NH₃-N) per g total suspended solids (TSS). For greater ammonia shock loads, correlation was found between build-up of nitrite in the aeration tank and the concentration of N₂O in the exhaust gas from the tank. When subjecting the system to aeration failure, a similar pattern was seen, with a correlation between nitrite build-up in the aeration tank and increases in the concentration of N₂O in the exhaust gas. The results from this work suggest that the changes in N₂O concentration in the exhaust gas from a nitrifying process may be a useful parameter for monitoring such processes. Received 15 October 2001/ Accepted in revised form 05 June 2002     

Simultaneous oxidation of ammonium and p-cresol linked to nitrite reduction by denitrifying sludge

The metabolic capability of denitrifying sludge to oxidize ammonium and p-cresol was evaluated in batch cultures. Ammonium oxidation was studied in presence of nitrite and/or p-cresol by 55 h. At 50 mg/L NH₄⁺-N and 76 mg/L NO₂⁻-N, the substrates were consumed at 100% and 95%, respectively, being N₂ the product. At 50 mg/L NH₄⁺-N and 133 mg/L NO₂⁻-N, the consumption efficiencies decreased to 96% and 70%, respectively. The increase in nitrite concentration affected the ammonium oxidation rate. Nonetheless, the N₂ production rate did not change. In organotrophic denitrification, the p-cresol oxidation rate was slower than ammonium oxidation. In litho-organotrophic cultures, the p-cresol and ammonium oxidation rates were affected at 133 mg/L NO₂⁻-N. Nonetheless, at 76 mg/L NO₂⁻-N the denitrifying sludge oxidized ammonium and p-cresol, but at different rate. Finally, this is the first work reporting the simultaneous oxidation of ammonium and p-cresol with the production of N₂ from denitrifying sludge.     

Accumulation of urea in the haemolymph and ammonia excretion of Penaeus japonicus exposed to ambient nitrite

Penaeus japonicus (15.7 ± 1.4 g) were exposed individually in 30 ppt seawater to 0.01 (control), 5, 10, 20 and 50 mg/l nitrite-N for 24 hr. Haemolymph ammonia, urea, nitrite and whole shrimp ammonia-N excretion and nitrite-N uptake were then determined. Ammonia excretion of P. japonicus increased with increased ambient nitrite, and with a concomitant decrease of haemolymph ammonia, as occurring increased concentrations of nitrite. Concentrations of nitrite-N and urea-N in the haemolymph of shrimp increased with increased ambient nitrite-N. However, no urea-N excretion was observed for shrimp exposed to any nitrite treatments.     

Isolation of an aboriginal bacterial community capable of utilizing cyanide,thiocyanate, and ammonia from metallurgical plant wastewater

An aboriginal bacterial community capable of degrading cyanide (10 mg/l) and thiocyanate (2 g/l) and eliminating ammonia (120 mg/l) had been isolated from recycled water samples after blast-furnace gas purification of a metallurgical plant wastewater. It was shown that the optimal conditions for this bacterial community were as follows: temperature, 34°C; pH, 8.8–9.0; available organic matter concentration (glucose equivalent), 5 g/l; and dissolved O₂ concentration, 8–10 mg/l. This aboriginal community was formed by the bacteria belonging to the genus Pseudomonas.     

Chronic toxicity of ammonia to juvenile gilthead seabream Sparus aurata and related histopathological effects

Effect of 20 days exposure of juvenile gilthead seabream ( Sparus aurata ) to elevated levels of ammonia on growth and survival was examined in a continuous flow system. Suppressed growth and reduced survival were observed at concentrations of 8.2 and 13 mg l⁻¹ total ammonia-N (0.5 and 0.7 mg l⁻¹ un-ionized ammonia-N, respectively) and higher. The maximum acceptable toxic concentration (MATC) for growth was between 4.8 to 8.2 mg l⁻¹ total ammonia-N (0.3 and 0.5 mg l⁻¹ un-ionized ammonia-N, respectively). Fish exposed to high ammonia levels (13 mg l⁻¹ total ammonia-N, 0.7 mg l⁻¹ un-ionized ammonia-N) displayed clear signs of liver pathology. Existing evidence suggests that S. aurata is less sensitive to ammonia than other reported marine and freshwater fish.
Under certain conditions ammonia concentration in the intensive fish ponds in Eilat may exceed the no observed effect concentration for S. aurata .     

Nutritional Status as the Key Modulator of Antioxidant Responses Induced by High Environmental Ammonia and Salinity Stress in European Sea Bass (Dicentrarchus labrax)

Salinity fluctuation is one of the main factors affecting the overall fitness of marine fish. In addition, water borne ammonia may occur simultaneously with salinity stress. Additionally, under such stressful circumstances, fish may encounter food deprivation. The physiological and ion-osmo regulatory adaptive capacities to cope with all these stressors alone or in combination are extensively addressed in fish. To date, studies revealing the modulation of antioxidant potential as compensatory response to multiple stressors are rather lacking. Therefore, the present work evaluated the individual and combined effects of salinity challenge, ammonia toxicity and nutritional status on oxidative stress and antioxidant status in a marine teleost, European sea bass (Dicentrarchus labrax). Fish were acclimated to normal seawater (32 ppt), to brackish water (20 ppt and 10 ppt) and to hypo-saline water (2.5 ppt). Following acclimation to different salinities for two weeks, fish were exposed to high environmental ammonia (HEA, 20 mg/L representing 50% of 96h LC₅₀ value for ammonia) for 12 h, 48 h, 84 h and 180 h, and were either fed (2% body weight) or fasted (unfed for 7 days prior to HEA exposure). Results show that in response to decreasing salinities, oxidative stress indices such as xanthine oxidase activity, levels of hydrogen peroxide (H₂O₂) and lipid peroxidation (malondialdehyde, MDA) increased in the hepatic tissue of fasted fish but remained unaffected in fed fish. HEA exposure at normal salinity (32 ppt) and at reduced salinities (20 ppt and 10 ppt) increased ammonia accumulation significantly (84 h–180 h) in both feeding regimes which was associated with an increment of H₂O₂ and MDA contents. Unlike in fasted fish, H₂O₂ and MDA levels in fed fish were restored to control levels (84 h–180 h); with a concomitant increase in superoxide dismutase (SOD), catalase (CAT), components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase), ascorbate peroxidase (APX) activity and reduced ascorbate (ASC) content. On the contrary, fasted fish could not activate many of these protective systems and rely mainly on CAT and ASC dependent pathways as antioxidative sentinels. The present findings exemplify that in fed fish single factors and a combination of HEA exposure and reduced seawater salinities (upto 10 ppt) were insufficient to cause oxidative damage due to the highly competent antioxidant system compared to fasted fish. However, the impact of HEA exposure at a hypo-saline environment (2.5 ppt) also defied antioxidant defence system in fed fish, suggesting this combined factor is beyond the tolerance range for both feeding groups. Overall, our results indicate that the oxidative stress mediated by the experimental conditions were exacerbated during starvation, and also suggest that feed deprivation particularly at reduced seawater salinities can instigate fish more susceptible to ammonia toxicity.     

Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha

Nitrosomonas eutropha, an obligately lithoautotrophic bacterium, was able to nitrify and denitrify simultaneously under anoxic conditions when gaseous nitrogen dioxide (NO₂) was supplemented to the atmosphere. In the presence of gaseous NO₂, ammonia was oxidized, nitrite and nitric oxide (NO) were formed, and hydroxylamine occurred as an intermediate. Between 40 and 60% of the produced nitrite was denitrified to dinitrogen (N₂). Nitrous oxide (N₂O) was shown to be an intermediate of denitrification. Under an N₂ atmosphere supplemented with 25 ppm NO₂ and 300 ppm CO₂, the amount of cell protein increased by 0.87 mg protein per mmol ammonia oxidized, and the cell number of N. eutropha increased by 5.8 × 10⁹ cells per mmol ammonia oxidized. In addition, the ATP and NADH content increased by 4.3 μmol ATP (g protein)^–1 and 6.3 μmol NADH (g protein)^–1 and was about the same in both anaerobically and aerobically grown cells. Without NO₂, the ATP content decreased by 0.7 μmol (g protein)^–1, and the NADH content decreased by 1.2 μmol (g protein)^–1. NO was shown to inhibit anaerobic ammonia oxidation. Received: 9 October 1996 / Accepted: 5 December 1996     

Revision of N2O-Producing Pathways in the Ammonia-Oxidizing Bacterium Nitrosomonas europaea ATCC 19718

Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N₂O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N₂O production by ammonia oxidizers. The growth phenotypes of the Nitrosomonas europaea ATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O₂ tensions. Anoxic resting-cell assays and instantaneous nitrite (NO₂⁻) reduction experiments were done to assess the ability of the wild-type and mutant N. europaea strains to produce N₂O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation of N. europaea and showed that NorB is involved in N₂O production during growth at both atmospheric and reduced O₂ tensions. Anoxic resting-cell assays and measurements of instantaneous NO₂⁻ reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N₂O production by N. europaea and will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.     

Anaerobic metabolism of Nitrosomonas europaea

Nitrosomonas europaea is capable of maintaining an anaerobic metabolism, using pyruvate as an electron donor and nitrite as an electron acceptor; utilization of nitrite depends upon supply of both pyruvate and ammonia. The role of ammonia in this reaction was not determined. N europaea also assimilates CO₂ anaerobically into cell material in the presence of nitrite (0.5–1.0 mM), pyruvate and ammonia. This reaction was partially inhibited by nitrite which apparently competed with CO₂ for reducing power. Anaerobic nitrite respiration is sensitive to ionophores, FCCP being the most effective.Non-standard-abbreviations TCA trichloroacetic acid - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazon     

Nitrous oxide production by cyanobacteria

Evidence is presented here that axenic cultures of Nostoc spp., Aphanocapsa (PCC 6308), and Aphanocapsa (PCC 6714) but not Anacystis nidulans R-2 (PCC 7942) produce N₂O and ammonia when grown on nitrite. The data suggest that the cyanobacteria produce N₂O by nitrite reduction to ammonia.Nonstandard abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - NIR nitrite reductase     

Organophosphorus diazinon induced toxicity in the fish anguilla angvilla L.

1. Acute toxicity effects of diazinon on European eel (Anguilla anguilla) were examined using short-term exposures in static conditions.2. The lc₅₀ values found were: 0.16, 0.11, 0.09 and 0.08 mg/1 at 24, 48, 72 and 96 hr exposure, respectively.3. Eels were exposed to 0.056 mg/l of diazinon and the bioaccumulation and elimination of this insecticide in liver, muscle, gill and blood tissues were studied.4. BCF were 800 in liver, 1600 in muscle tissue and 2300 and 2730 in gill and blood tissue, respectively.5. The BCF₁ were 0.30 for liver, 0.60 for muscle and 0.84 for gill. Higher accumulation capacity of the gill was observed for the first hour of exposure.6. Diazinon elimination from the selected tissues was rapid, diazinon levels were not detected in any tissue after 24 hr in clean water.7. The excretion rate constants (K₂) of this insecticide were 0.023 hr⁻¹ for liver, 0.005 hr⁻¹ for gill and 0.019 ⁻¹ for muscle.8. Diazinon half-lives were calculated as 30.6, 32.2 and 38.3 hr for liver, muscle and gill, respectively.     

Effects of low-salinity on the growth and development of spotted halibut Verasper variegatus in the larva-juvenile transformation period with reference to pituitary prolactin and gill chloride cells responses

Low-salinity adaptability was investigated in a flatfish spotted halibut Verasper variegatus during the period from late metamorphic larvae to early juveniles by a 20-day rearing experiment under different salinity regimes (1, 4, 8, 16 and 32 ppt). Effects of low-salinity on growth and development were examined and the changes in the prolactin (PRL) production level in the pituitary and the gill chloride cell morphology were examined as physiological backgrounds for low salinity adaptation. PRL cells and chloride cells were identified by immunocytochemistry with a specific antiserum for PRL₁₈₈ and Na⁺,K⁺-ATPase. Most of the fish exposed to over 4 ppt survived for 20 days, but all the fish exposed to 1 ppt died within 5 days. Fish kept in intermediate salinities (8, 16 ppt) grew significantly better than those in the control group (32 ppt). Fish exposed to 4 ppt attained almost the same body length as the control group at 20 days after transfer, although these fish showed an abnormally dark body color as well as delayed development. These results suggested that spotted halibut has a high-adaptability to low-salinity environments and prefers an intermediate salinity near iso-osmolality (about 12 ppt) from the late metamorphic larval stage, but does not completely adapt to a hypoosmotic of 4 ppt salinity or less than half of the osmolality. The percentage of PRL-cell volume to pituitary volume was significantly higher at 4 ppt than in the control group. The chloride cells in gill filaments were significantly larger at 4 ppt than in the control group. These results suggest that juveniles could adapt to a low-salinity environment due to the activation of PRL production and enlargement of chloride cells. These laboratory findings suggest that late metamorphic larvae and early juveniles of spotted halibut may utilize a low salinity environment such as estuarine tidal flats or very shallow coastal areas as their nursery grounds in the sea.     

Exchange of N2O and CH4 between the atmosphere and soils in spruce-fir forests in the northeastern United States

We measured the exchange of N₂O and CH₄ between the atmosphere and soils in 5 spruce-fir stands located along a transect from New York to Maine. Nitrous oxide emissions averaged over the 1990 growing season (May–September) ranged from 2.1 ug N₂O-N/m²-hr in New York to 0.4 ug N₂O-N/m²-hr in Maine. The westernmost sites, Whiteface Mtn., New York and Mt. Mansfield, Vermont, had the highest nitrogen-deposition, net nitrification and N₂O emissions. Soils at all sites were net sinks for atmospheric CH₄ Methane uptake averaged over the 1990 growing season ranged from 0.02 mg CH₄-C/M²-hr in Maine to 0.05 mg CH₄-C/m²-hr in Vermont. Regional differences in CH₄ uptake could not be explained by differences in nitrogen-deposition, soil nitrogen dynamics, soil moisture or soil temperature. We estimate that soils in spruce-fir forests at our study sites released ca. 0.02 to 0.08 kg N₂O-N/ha and consumed ca. 0.74 to 1.85 kg CH₄ C/ha in the 1990 growing season.     

Hybrid wastewater treatment system using anaerobic microorganisms and reed (Phragmites communis)

A small hybrid wastewater treatment system consisting of a settling tank in series with an anaerobic filter-reed (Phragmites communis) treatment cell was evaluated and compared with a similar plant-free system. Data demonstrated that by combining anaerobic filters, also referred to as attached film filters, and vascular aquatic plants a synergistic effect is produced which increases the treatment efficiency of each individual system. The plant-free system reduced the BOD₅ from 114 to 31 mg/l in 6 h as compared to a reduction of 110 to 9 mg/l in the anaerobic filter-reed system in the same length of time. The BOD₅ and TSS after 24 h in each component of plant-free system were reduced from 114 mg/l to 14 mg/l and 51 to 15 mg/l, respectively. Under the same conditions, the hybrid system reduced the BOD₅ from 110 to 3 mg/l and the TSS from 68 to 6 mg/l. The hybrid system also reduced the total Kjeldahl nitrogen (TKN) from 16.1 to 2.9 mg/l, total phosphorus (TP)from 4.4 to 2.0 mg/l, and the ammonia (NH₃-N)from 12.4 to 0.6 mg/l after 24 h of exposure while the plant-free system demonstrated insignificant reduction of these components.     

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N₂O and N₂ from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with ¹⁵N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N₂O, and finally N₂O was converted to N₂. At pH 8.5, nitrate was converted directly to N₂, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.     

Stoichiometry and kinetics of microbial toluene degradation under denitrifying conditions

Batch experiments were carried out to investigate the stoichiometry and kinetics of microbial degradation of toluene under denitrifying conditions. The inoculum originated from a mixture of sludges from sewage treatment plants with alternating nitrification and denitrification. The culture was able to degrade toluene under anaerobic conditions in the presence of nitrate, nitrite, nitric oxide, or nitrous oxide. No degradation occurred in the absence of Noxides. The culture was also able to use oxygen, but ferric iron could not be used as an electron acceptor. In experiments with¹⁴C-labeled toluene, 34%±8% of the carbon was incorporated into the biomass, while 53%±10% was recovered as¹⁴CO₂, and 6%±2% remained in the medium as nonvolatile water soluble products. The average consumption of nitrate in experiments, where all the reduced nitrate was recovered as nitrite, was 1.3±0.2 mg of nitrate-N per mg of toluene. This nitrate reduction accounted for 70% of the electrons donated during the oxidation of toluene. When nitrate was reduced to nitrogen gas, the consumption was 0.7±0.2 mg per mg of toluene, accounting for 97% of the donated electrons. Since the ammonia concentration decreased during degradation, dissimilatory reduction of nitrate to ammonia was not the reductive process. The degradation of toluene was modelled by classical Monod kinetics. The maximum specific rate of degradation, k, was estimated to be 0.71 mg toluene per mg of protein per hour, and the Monod saturation constant, K _s , to be 0.2 mg toluene/l. The maximum specific growth rate, _max , was estimated to be 0.1 per hour, and the yield coefficient, Y, was 0.14 mg protein per mg toluene.Abbreviations NVWP Non Volatile Water-soluble Products