Cardiovascular changes and physiological response during nitrite exposure in rainbow trout

Nitrite-exposed (1 mM) rainbow trout Oncorhynchus mykiss fell into two distinct groups with regard to susceptibility and physiological response. Group 1 accumulated nitrite in plasma to a concentration of 2·9 mM within 24 h and died before 48 h. Group 2 survived for 96–144 h, and the accumulation of nitrite was slower, levelling off at a concentration c. 2·3 mM at 72 h. Methaemoglobin (metHb) formation was faster in group 1 than in group 2, but both groups had a metHb fraction c. 70% before dying. The extracellular electrolyte balance was perturbed significantly only in group 1, where plasma [Cl^-] decreased and plasma [K⁺] increased. Heart rate increased rapidly, more in group 1 than in group 2. The tachycardia occurred before any significant changes in metHb or [K⁺] had developed, suggesting that it was due to nitrite-induced vasodilation, possibly via nitric oxide generated from nitrite, that was countered by an increased cardiac pumping to re-establish blood pressure. Arterial blood pressure and pulse pressure were accordingly kept reasonably constant. Heart rate variability was significantly depressed in group 1. The ventilation rate was significantly increased after 9 h of nitrite exposure in group 1, while the ventilation in group 2 did not increase significantly before 21 h. The data reveal that nitrite has substantial influence on the cardio-respiratory function in fishes.     

Effects on acid-base balance, methaemoglobinemia and nitrogen excretion of European eel after exposure to elevated ambient nitrite

Haemoglobin, methaemoglobin, blood nitrite concentration and acid-base balance were measured in European eel Anguilla anguilla following exposure to 0 (control), 0·142, 0·356, 0·751 and l·549 mM nitrite in fresh water for 24 h. Blood GOT (glutamate oxaloacetate transaminase) and GPT (glutamate pyruvate transaminase) activities and whole animal ammonia-N and urea-N excretions were also measured. Blood nitrite, methaemoglobin, PO ₂ (oxygen partial pressure), GOT, and whole animal ammonia-N excretion and urea-N excretion increased directly with increasing ambient nitrite concentrations, whereas blood pH, PCO ₂, and HCO⁻₃ were inversely related to ambient nitrite concentration. An accumulation of nitrite in the blood of A. anguilla following 24 h exposure to elevated ambient nitrite as low as 0·751 mM increased its blood methaemoglobin, PO ₂, GOT and nitrogen excretion, but decreased its PCO ₂ (carbon dioxide partial pressure), HCO⁻₃ and functional haemoglobin.     

Effects of cultural conditions on denitrification by Pseudomonas stutzeri measured by Membrane Inlet Mass Spectrometry

Denitrification is a globally important process leading to loss of fertiliser efficiency and the production of the greenhouse gas nitrous oxide and nitric oxide, an ozone depleter. Membrane inlet mass spectrometry (MIMS) was employed to study the effect of different variables on the process of denitrification by Pseudomonas stutzeri in a defined salts medium. MIMS was used for concomitant measurements of nitrous oxide, nitrogen and oxygen and showed that denitrification occurred in the presence of dissolved oxygen. A nitrate concentration of 15 mmol l⁻¹ and a nitrite concentration of 5 mmol l⁻¹ were found to be optimum for complete denitrification of nitrate or nitrite to nitrogen and varying these concentrations had a marked effect on the ratio of gaseous products released. Denitrification products were also dependant on pH with neutral or alkaline conditions being best for production of gaseous end products. Our results suggest that under nutrient rich conditions the most important factor in the regulation of denitrification by Ps. stutzeri is the amount of nitrite generated at the first enzymatic stage of the process. This appears to cause inhibition of the denitrification pathway above 5 mmol l⁻¹ and at high enough concentrations (15 mmol l⁻¹) restricts growth.     

Metabolic adjustments in the common carp during prolonged hypoxia

Biochemical and respiratory changes in the common carp Cyprinus carpio , were studied 6, 24, 96 and 168 h upon exposure to hypoxia (0·5 mgO₂ l⁻¹). Modification of kinetic properties of phosphofructokinase (PFK-1), coupled with a decreased in PFK-1 activities, were evident in muscle. No changes in kinetics and activities could be observed in muscle pyruvate kinase (PK) and lactate dehydrogenase (LDH). A decrease in muscle citrate synthase (CS) and an increase in muscle cytochrome c oxidase (CCO) were found. The common carp was able to maintain a constant level of muscle glycogen, muscle ATP, and liver CS throughout the 168-h experimental period. Changes in activities of liver LDH and muscle CCO were observed only at 168 h, which indicates that common carp may switch to alternative metabolic pathway to deal with prolonged hypoxia. A severe decrease in liver glycogen was accompanied by increases in lactate levels in both the muscle and liver. Oxygen consumption rate was reduced under hypoxia, but resumed to normoxic levels within 2 h upon return to normoxic condition. Overall, these results indicate that carp adopt different strategies in an attempt to deal with short term and long term hypoxia in the natural environment.     

The behavioural and physiological response of Atlantic cod Gadus morhua L. to short-term acute hypoxia

The average rate of swimming speed and the physiological status or stress of individual Atlantic cod Gadus morhua was monitored in response to short-term acute (STA) hypoxia ( i.e. partial pressure of oxygen,     , reduced from 20·9 to 4·3 kPa within 1 h at 10° C). The STA hypoxic response of Atlantic cod was associated with a large primary increase (+29%) and a large secondary decrease (−54%) in swimming speed as well as major physiological stress ( e.g. plasma cortisol = 214·7 ng ml⁻¹ and blood lactate = 2·41 mmol l⁻¹).     

Oxygen requirements of larvae of the smallmouth bass, Micropterus dolomieui Lacépède

Smallmouth bass larvae became highly sensitive to oxygen deficiency on the second day after hatching and continued so to the 10th day. During this period they could not survive exposure to 1 mg O₂ l^–1 for 3 h at 20° C, and many were killed within 1 h. At 2 mg O₂ l^–1 half the larvae survived 3 h at 20° C; at 2.5 mg l^–1 most survived, and at 3.5 mg l^–1 all survived. Resistance to oxygen deficiency was regained by the 11th day, the majority of the larvae withstanding a 3-h exposure at 1 mg O₂ l^–1. At 25° C the effects of low oxygen concentration were intensified. At 3 and 4 mg O₂ l^–1 and 20° C the normally quiescent larvae became very active, even swimming to the surface 5 or 6 cm above the substrate. Increasing the temperature increased this response. Smallmouth larvae were more sensitive than large-mouth bass larvae to oxygen deficiency.     

Effects of pollutants in fresh waters on European non-salmonid fish I: Non-metals

As part of a programme of acquiring data for preparing standards to safeguard European, nonsalmonid, freshwater fish from pollution, toxicity tests were carried out in hard, well-aerated water. Asymptotic median lethal concentrations (LC50s) of undissociated ammonia, cyanide, nitrite and phenol to one or more of three species were determined. The LC50s were as follows: to common carp 16mg1^-1 as NO₂–N; to perch 0.1 mg1^-1 as HCN; to roach 0.35 mg1^-1 as NH₃-N, 0-11mg1^-1 as HCN, 10.1 mg1^-1 asNO₂-Nand 10mg^-1 as phenol. In order to define these LC50s, exposure periods within the range 0.3 days (phenol) to 14 days (nitrite) were required. Comparisons are made with other data and the tentative water quality 'criteria' proposed by the European Inland Fisheries Advisory Commission.     

Haematology of juvenile Acipenser oxyrinchus and Acipenser brevirostrum at rest and following forced activity

In vivo experiments were conducted to examine the haematology of juveniles from two relic bony fishes, Atlantic sturgeon Acipenser oxyrhinchus and shortnose sturgeon Acipenser brevirostrum . Oxygen transport characteristics (haematocrit, haemoglobin and mean erythrocytic haemoglobin concentration), ionic composition (Na⁺, Cl⁻, K⁺ and osmolality), metabolite concentration (lactate, cortisol and glucose) and protein content in blood were measured or calculated at rest and during recovery from forced activity. Under resting conditions, plasma osmolality and concentrations of Na⁺, Cl⁻, lactate, cortisol and total protein were significantly different between Atlantic and shortnose sturgeon. All other resting variables were not different between species. Following forced activity, plasma lactate levels were significantly higher in both species than at rest. Plasma cortisol levels in both species were only significantly higher 1 h following forced activity compared to resting values. Plasma lactate levels were significantly higher in Atlantic sturgeon than in shortnose sturgeon, but these levels returned to resting levels by 1 h in both species. Cortisol increases were greater in shortnose sturgeon than in Atlantic sturgeon. In general, oxygen transport characteristics, blood glucose, plasma protein and plasma osmolality were not altered by forced activity in either sturgeon species. Overall, both species had reduced responses ( i.e . the magnitude of changes in measured variables) to forced activity compared with teleosts.     

The respiration of young coho Oncorhynchus kisutch at gradually declining oxygen levels and during recovery from oxygen deprivation

The respiration of coho salmon, Oncorhynchus kisutch , weighing between 15 and 50 g was measured at gradually declining oxygen levels and at temperatures ranging between 14 and 17°C. The maximum and minimum oxygen concentrations tested were 250 and 40 μmol L⁻¹, respectively. Respiration rates were measured for 1 h periods before oxygen concentration was lowered by 12.5 or 25.0 μmol oxygen L⁻¹. At the end of these endurance tests the oxygen level was returned to normoxic conditions and respiration rates were determined for the recovery period. Under normoxic conditions (> 200 μmol L⁻¹) the respiration of coho levelled around 5.1 μmol g⁻¹ wet weight h⁻¹. At intermediate levels between 150 and 200 μmol oxygen L⁻¹, the average rate increased to 5.8 μmol g⁻¹ h⁻¹, which could be attributed to higher spontaneous activity of the test animals. At low oxygen levels (< 150 μmol⁻¹) average respiration rates dropped to values between 5.5 and 5.7 μmol g⁻¹ h⁻¹, reaching a minimum of 3.8 μmol g⁻¹ h⁻¹ at oxygen levels below 50 μmol L^μ. First mortality was observed in this range. After exposure to reduced oxygen levels the fish maintained a higher respiration rate when again exposed to normoxic oxygen levels above 200 μmol L⁻¹. Increased respiration rates were observed for a recovery period of 6 h.     

Genetic polymorphism and isoenzyme patterns of lactate dehydrogenase in tench (Tinea tinea), crucian carp (Carassius carassius) and carp (Cyprinus carpio)

Isoenzyme patterns and the polymorphism of lactate dehydrogenase (LDH) were investigated in 3 fish species of family Cyprinidae, i.e. tench ( Tinea tinea ), crucian carp ( Carassius carassius ) and carp ( Cyprinus carpio). The isoenzyme patterns were tissue and species specific. In crucian carp subunits with different electro-phoretic mobility are present, which are genetically controlled from the B¹, B², A¹, A²and C loci, while the set of loci in carp is B¹, B², A, C¹and C²and in tench B, A, C. The locus B of LDH in tench, the locus B²in crucian carp, and the loci B¹, C¹and C²in carp are polymorphic and have two different alleles in each case. The polymorphism did not affect the total LDH activity in the tissues. All the populations investigated were in Hardy-Weinberg equilibrium. The genetic control of the polymorphism in B¹and C¹loci in carp was proved by test matings. The polymorphism in B loci tested in erythrocytes may be utilized as genetic markers in the fish breeding.     

The effects of acid and acid/aluminum exposure on circulating plasma cortisol levels and other blood parameters in the rainbow trout, Salmo gairdneri

Softwater (Ca²⁺=50, Na⁺= 50(μequiv. l⁻¹) acclimated rainbow trout were fitted with chronic arterial catheters to allow for repetitive blood sampling. After 48 h recovery they were then exposed to either control (pH 6.5, Al = 0μg l⁻¹), acid (pH 4.8, Al = 0μg l⁻¹) or acid plus aluminum (pH 4.8, A1 = 112 μg l⁻¹) conditions for 72 h. Parameters measured included blood glucose, lactate, haemoglobin, haematocrit and plasma Na⁺, Cl⁻, protein and cortisol.
Exposure to pH 4'8 alone caused no mortality, a moderate ionoregulatory disturbance and a transient elevation in plasma cortisol. All other parameters were not significantly different from controls. Addition of aluminum to this exposure caused 100% mortality with a mean survival time of only 27.0 h. There was a marked decrease in plasma ions, hyperglycemia, lactate accumulation, haemoconcentration, red cell swelling, and a sharp rise in plasma cortisol becoming greatly increased as the fish neared death. The mechanism of toxicity of acute acid/aluminum exposure, the role for cortisol under such conditions, and the validity of cortisol and glucose as indicators of stress in fish are discussed.     

Influence of pH and lactic acid concentration on Clostridium tyrobutyricum during continuous growth in a pH-auxostat

The aim of this project was to establish the minimal inhibitory concentration (MIC) of lactic acid for growth of Clostridium tyrobutyricum. A pH-auxostat was used to maintain a constant pH and to allow continuous growth at the highest possible rates at fixed, but adjustable concentrations of lactate. By raising the concentration of lactic acid and keeping the pH constant, the growth rate was shown to decrease linearly with increasing lactic acid concentration. The p K _a of lactic acid, measured in the actual growth medium at 37°C, was 3.40 (±0.03). Based on this value, the MIC_undiss values for each pH were estimated. The MIC of total lactic acid (MIC_tot) ranged from 150 mmol l⁻¹ to 1510 mmol l⁻¹ at pH 4.6–6.25, respectively. The corresponding MIC values of undissociated lactic acid (MIC_undiss) ranged from 8.9 to 2.1 mmol l⁻¹ at the same pH values. These results emphasize the importance of a rapid pH decrease and an equally rapid initial lactic acid fermentation of the ensilage, in order to sufficiently suppress clostridial growth.     

The effect of dissolved oxygen and salinity on the toxicity of ammonia to smolts of salmon, Salmo salar L.

The survival of Atlantic salmon smolts on exposure to constant concentrations of ammonia has been measured under laboratory conditions. At concentrations of dissolved oxygen close to the air-saturation value, the 24-h LC50 of un-ionised ammonia is 0.15 mg NH₃1⁻¹ in fresh water (hardness 264 mg 1⁻¹ as CaCO₃) and 0.3 mg NH₃1⁻¹ in 30% sea water; at concentrations of dissolved oxygen of 3.5 mg 1⁻¹ in fresh water and 3.1 mg 1⁻¹ in 30% sea water, the 24-h LC50 is 0.09 mg NH₃ 1⁻¹ and 0.12 mg NH₃ 1⁻¹ respectively; for fish acclimated for 1 day to a concentration of ammonia close to the 24-h median for un-acclimated fish, the median is increased between 38 and 79%, depending on test conditions.     

Photosynthesis in ozone-exposed duckweed (Lemna gibba)

The photosynthetic light saturation curve in duckweed was lowered by 20–25% after ozone exposure (300 nmol mol⁻¹, 1 h). The light flux and oxygen concentration during ozone-exposure had no effect on reduction of net photosynthesis. Net photosynthesis and photorespiration were both depressed by about 40% after exposure for 1 h to 360 nmol mol⁻¹ ozone. We could not find any change in dark respiration after ozone exposure below 300 nmol mol⁻¹. When the concentration of ozone was doubled from 150 nmol mol⁻¹ to 300 nmol mol⁻¹, the uptake of ozone in duckweed changed from 100 nmol m⁻² s⁻¹ to 170 nmol m⁻² s⁻¹. We found no differences in fluorescence (pattern) between ozone treated plants and the control plants during a period of 150 min after ozone treatment, but there was an increase in synthesis of the Dl-protein and a significant reduction in degradation after ozone treatment (300 nmol mol⁻¹, 1 h). These results, together with fluorescence measurements, indicate that photochemical electron transport was not responsible for the ozone-induced reduction in net photosynthesis.     

Intracellular ion levels in erythrocytes and hepatocytes isolated from three teleost species

Intracellular concentrations of Na⁺ and K⁺ were similar (∼75 mmol l⁻¹) in rainbow trout Oncorhynchus mykiss hepatocytes directly following isolation by collagenase digestion, but partial recovery occurred over 6 h with K⁺ levels increasing to 110 mmol l⁻¹ and Na⁺ levels decreasing to 42 mmol l⁻¹. Black bullhead Ameiurus melas hepatocytes exhibited higher intracellular concentrations of K⁺ (90 mmol l⁻¹) than Na⁺ (55 mmol l⁻¹) with no recovery occurring over 6 h following cell isolation. Concentrations of Na⁺, K⁺ and Cl⁻ in eel Anguilla rostrata hepatocytes were similar (∼ 55 mmol l⁻¹) following isolation, with no recovery occurring over time. Erythrocytes from all species apparently did not experience an intracellular ion imbalance following isolation as indicated by high K⁺ levels (<140 mmol l⁻¹) and low Na⁺ levels (<40 mmol l⁻¹) during the entire 24-h monitoring period. Although hepatocytes from all species exhibited an ion imbalance post-isolation, comparison of their in vitro intracellular Na⁺ and K⁺ concentrations with those in plasma demonstrated that directionally correct ion gradients still exist across the cell membrane, albeit differing from those that would be found in the tissue in vivo .     

Effects of aluminium in hard, acid water on metabolic rate, blood gas tensions and ionic status in the rainbow trout

Rainbow trout, Salmo gairdneri , cannulated in the dorsal aorta, were exposed to 2 mgl⁻¹ aluminium (added as Al₂(SO₄)₃, 18H₂O) at pH = 5.0 in hard water of very high calcium concentrations (3.3 mmol 1⁻¹). No changes were observed in the concentration of the major plasma ions. The fish, however, became hypoxic, as seen from a fall in the dorsal aortic oxygen tension from around 100 mmHg to 30–40 mmHg, and a simultaneous increase in carbon dioxide tension. Moreover, exposure resulted in a 15% increase in standard oxygen uptake. The ventilation frequency doubled and swelling of the erythrocytes was observed. The results are discussed in the light of some earlier data on the effects of acid/aluminium exposure in water of low calcium concentration, and reveal an important role for calcium in the physiological responses to low pH and aluminium.     

Effect of acute and chronic ammonia and nitrite exposure on oxygen consumption and growth of juvenile big bellied seahorse

Juvenile big bellied seahorse Hippocampus abdominalis were exposed acutely and chronically to elevated ammonia and nitrite {24 h exposure: 0·01, 5·0, 10·1, 14·8 and 19·9 mg l⁻¹ total ammonia-nitrogen [TA-N] and <0·001, 74·4, 99·2 and 123·6 mg l⁻¹ [NO₂-N] nitrite-nitrogen and 35 days exposure: 0·11, 0·55, 1·67 and 3·07 mg l⁻¹ TAN and <0·001, 0·92, 4·67 and 9·10 mg NO₂-N l⁻¹}. Significant ( P <0·001) increases in oxygen consumption rate and ventilation frequency occurred at 14·8, 19·9 mg l⁻¹ TA-N and 99·2, 123·6 mg l⁻¹ NO₂-N for acutely exposed fish. Oxygen consumption rate was significantly ( P <0·05) elevated at 1·67 and 3·07 mg l⁻¹ TA-N in chronically treated fish and ventilation frequency increased significantly ( P <0·05) at 0·55, 1·67, 3·07 mg l⁻¹ TA-N and 4·59, 9·10 mg l⁻¹ NO₂-N. There were no significant differences in growth between controls and ammonia exposed fish. Mortalities occurred at 14·8, 19·9 mg l⁻¹ TA-N.     

The acute lethal toxicity of mixtures of cyanide and ammonia to smolts of salmon, Salmo salar L. at low concentrations of dissolved oxygen

The survival of Atlantic salmon smolts on exposure to constant concentrations of cyanide and ammonia, singly and together, has been measured under laboratory conditions at a concentration of 5 mgl^-1 of carbon dioxide. The 24-h LC50 values of cyanide and of un-ionised ammonia, in fresh water, were 0·073 mg HCN l^-1 and 0·20 mg NH₃l^-1 respectively at a concentration of dissolved oxygen of 10 mg l^-1, and 0·024 mg HCN l^-1 and 0·08 mgNH₃l^-1 respectively at a concentration of dissolved oxygen of 3·5 mg l^-1. In 30% sea water the corresponding values were similar for cyanide but markedly higher for ammonioa. In 80% sea water the values were intermediate between those of fresh water and 30% sea water. Prior acclimation of the fish to the respective toxicant increased the resistance of the fish only slightly to cyanide, but with ammonia the 24-h LC50 was increased between 1·4 and 2-fold after acclimation for 1–3 days to between 0·2 and 0·5 of the 24-h LC50 value. Mixtures of cyanide and ammonia were between 0·6 and 1·25 times as toxic as expected, assuming simple additivity of toxicity.     

Methylxanthines as inducers of pectin lyase in Penicillium griseoroseum cultured on sucrose

Pectin lyase (PL) from Penicillium griseoroseum can be induced by xanthine, theobromine, theophylline and especially by caffeine and hypoxanthine (5 mmol l⁻¹ with 0·01% yeast extract (YE)). For caffeine and hypoxanthine, PL activity was, respectively, 5·2 and 3·7 times higher than with YE alone. The simultaneous addition of caffeine or hypoxanthine (5 mmol l⁻¹) and YE (0·1%) had a synergistic effect on PL activity as compared to the addition of these substances alone (0·2% YE; 10 mmol l⁻¹ caffeine; 10 mmol l⁻¹ hypoxanthine). Increasing caffeine concentrations (0–10 mmol l⁻¹) for a constant YE content of 0·01%, resulted in an increase in PL activity and a decrease in mycelial mass. For a constant caffeine concentration (5 mmol l⁻¹) and increasing YE contents (0–0·2%), a higher PL activity and mycelial mass were detected. The addition of caffeine (10 mmol l⁻¹) at the beginning of incubation increased PL activity and decreased mycelial mass, while caffeine added after 12 and 24 h resulted in decreases in PL activity and increases in mycelial mass. The results presented here indicate that methylxanthines, especially caffeine, can induce PL in P. griseoroseum .     

Changes in circulating blood cell levels of rainbow trout, Salmo gairdneri Richardson, following acute and chronic exposure to copper

Significant leucopenia, predominantly the result of a large reduction in lymphocyte concentration, was observed in rainbow trout exposed to 301 μg1⁻¹ copper for 24h. A strong trend ( P = 0–085) toward reduced erythrocyte concentration was also apparent. The pattern of haematological change after chronic exposure (118 μg 1⁻¹ copper for 16 weeks) was substantially different. With the exception of a significant increase in neutrophil concentration (neutrophilia), all blood cell concentrations had returned to control levels.