The effect of pH and/or calcium-enriched freshwater on gill Ca2+-ATPase activity and osmotic water inflow in rainbow trout (Salmo gairdneri)

Rainbow trout (Salmo gairdneri) were exposed to pH 5.0-5.1, 6.6 and/or calcium-enriched freshwater for 14 days. Hematocrit, gill Ca2+-ATPase enzyme activities, gill osmotic water inflow, plasma calcium and osmolarity were measured. No significant changes in plasma calcium ion levels were found. The typical increase in hematocrit usually associated with exposure of fish to acidified water was not found in the present study and is discussed. Plasma osmolarity decreased in fish exposed to calcium-enriched freshwater (60 mg Ca2+ X 1(-1) ) in comparison to fish exposed to control freshwater conditions (2 mg Ca2+ X 1(1) ), irrespective of the pH level. Gill Ca2+-ATPase enzyme activities were measured for both low affinity (3 mM Ca2+) and high affinity (100 microM) activity. Exposure of rainbow trout to low pH (pH 5.0-5.1) did not affect the specific activity of Ca2+-ATPase enzyme. However, low affinity Ca2+-ATPase activity in fish exposed to calcium-enriched freshwater did show a significant reduction. The increase in gill osmotic water permeability in fish exposed to calcium-enriched freshwater is interpreted as a result of the increase in osmolarity of the ambient media.     

Estrogenic octylphenol affects seminal fluid production and its biochemical composition of eelpout (Zoarces viviparus)

Estrogenic chemicals such as alkylphenols (APs) have been shown to disrupt the reproductive system of male fish. In the present study, the effects of the estrogenic chemical octylphenol (100 microg g(-1)) and 17 beta-estradiol on the semen production and the biochemical composition of the seminal fluid of the viviparous eelpout (Zoarces viviparus) were investigated at the time of spawning. After 10 days of octylphenol or estrogen treatment, vitellogenin (Vtg) synthesis was induced as indicated by increased plasma vitellogenin concentration. In accordance with the increased vitellogenin concentration, hepatosomatic index (HSI), total protein concentration, and total calcium concentration were also increased, and free amino acids concentration was decreased in blood plasma. Octylphenol treatment caused a decrease in the gonadosomatic index (GSI) and the milt volume and an increase in the spermatocrit. The histological examination revealed that octylphenol affected the normal lobular structure, including the Sertoli cells. In the majority of the octylphenol-treated fish, trapped sperm cells were observed in parts of the seminiferous lobules and the sperm ducts. The biochemical composition of the seminal fluid was also affected by the octylphenol or estrogen. The seminal plasma concentrations of magnesium, calcium, and total protein were elevated, and the concentration of free amino acids was reduced in the treated fish. This study indicates that octylphenol inhibits the seminal fluid production and changes its biochemical composition in eelpouts.     

Catecholamine release and interrenal response of brown trout, Salmo trutta, exposed to aluminium in acidic water

Cannulated brown trout, Salmo trutta , were exposed for 36 h to synthetic water with a low calcium content of pH 5 and similar synthetic water dosed with aluminium to raise the filterable A1 from 5 to 290 μg 1⁻¹ over the 36-h period. There were no significant disturbances of plasma concentrations of glucose, cortisol or catecholamine (adrenaline and noradrenaline) in fish held in water of pH 5. The addition of aluminium to this acidic synthetic water resulted in a generalized endocrine stress response with a four-fold increase in plasma glucose concentration after 18 h and a significant increase in plasma cortisol concentration from 24 h onwards when filterable A1 exceeded 200 μg 1⁻¹. Plasma catecholamine concentration indicated an adrenergic stress response in aluminium-exposed brown trout. A transient doubling in noradrenaline after 6 h in A1 was followed by a larger increase in both plasma adrenaline and noradrenaline concentrations in fish surviving the 36-h exposure to A1.     

Vitellogenic effects of 17beta-estradiol in male Chinese loach (Misgurnus anguillicaudatus)

Male Chinese loaches (Misgurnus anguillicaudatus) were exposed to 17beta-estradiol (E2) in the ambient water at nominal concentrations of 0.5, 1, 5 and 10 mug/L continuously for 8 weeks using a semi-static system. The gonadosomatic index (GSI), total plasma protein, total plasma calcium, magnesium and zinc, and plasma vitellogenin (Vtg) were determined as test endpoints. Our results indicate no significant changes in the GSI between exposed and control groups (P<0.05). All exposure concentrations of E2 dramatically induced the production of Vtg within 7 days. The vitellogenic response of male loach was time- and dose-dependent. At higher concentrations of E2 (5 and 10 microg/L), total plasma protein, total plasma calcium, and total plasma magnesium presented a significant time- and dose-dependent increase, and the three parameters were significantly correlated with plasma Vtg. We conclude that Chinese loaches are sensitive to estrogenic compounds and may be chosen as potential sentinel species in field and laboratory studies. In addition, total plasma protein, total plasma calcium, and total plasma magnesium can be used as indirect indicators to predict Vtg levels in estrogenized Chinese loach.     

Assessing environmental conditions of the Río Champotón (México) using diverse indices and biomarkers in the fish Astyanax aeneus (Günther, 1860)

The upper reaches of the Río Champotón in southwestern Mexico are largely unknown to science. This river is sustaining anthropogenic and natural disturbances by deforestation, substitution of natural vegetation, and agricultural non-point source pollution. Furthermore, hurricanes provoke flooding of these areas, exposing the aquatic biota to pollutants. Several approaches were used to assess the effects of environmental conditions along the freshwater portion of the Río Champotón in the native fish Astyanax aeneus and to determine the feasibility of extrapolating responses between levels of organization, and to link spatial and seasonal fish responses with water quality features. A water quality index (WQI) was employed as an indicator of environmental conditions, a set of sub-organismal biomarkers in A. aeneus (lipid peroxidation, glutathione S-transferase, ethoxyresorufin-O-deethylase and lactate dehydrogenase) was monitored to determine the integrated biomarker response (IBR), and somatic indices (individual and population-level responses; gonadosomatic, hepatosomatic, and condition factor) were characterized. Canonical correspondence analysis revealed spatial and seasonal variations in all the approaches. The dry and the wet season (April and July) exhibited low WQI scores, but decreased hardness and conductivity values as well as increased WQI values occurred during the post-hurricane season when crop fields adjacent to the riverbed were inundated. Although the WQI suggested low levels of contamination, the biomarkers did not remain static; wide fluctuations reflected fish exposure to seasonal stressors that provoked impairments in biological responses. All biomarker values were high, mainly in the post-hurricane season (November). The IBR exhibited two peaks that may be related to stress periods (April and November), with decreasing post-stress values suggesting the existence of homeostatic processes in the fish. The somatic indices reflected some seasonal and spatial differences at the population level – the gonadosomatic and hepatosomatic indices peaked in the middle of July and November, respectively – but the condition factor remained constant among sites and study periods. Future studies are needed to explore the link between the fish biomarker responses and possible causal stressors (natural or anthropogenic).     

Interaction between dietary calcium supplementation and chronic waterborne zinc exposure in juvenile rainbow trout, Oncorhynchus mykiss

This study investigated the effects of dietary Ca2+ on branchial Ca2+ and Zn2+ uptake, new and total zinc accumulation in target tissues (gill, liver and kidney), calcium and zinc homeostasis, and acute tolerance to waterborne zinc in fish chronically exposed to waterborne zinc. Juvenile rainbow trout (Oncorhynchus mykiss) were maintained on a calcium-enriched diet [41.2 mg vs. 21.2 mg (control) calcium/g dry wt. of food] and chronic waterborne zinc exposure (2.3 micromol/L), both separately and in combination, for 28 days. Calcium-supplemented diet in the absence of waterborne zinc significantly reduced branchial Ca2+ and Zn2+ influx rates, and new and total zinc accumulations in target tissues relative to control. However it did not protect against the acute zinc challenge. In contrast, waterborne zinc exposure significantly increased branchial Ca2+ and Zn2+ influx rates, new and total zinc concentrations in target tissues, and acute zinc tolerance relative to control. Interestingly, no such changes in any of these parameters were recorded in fish treated simultaneously with elevated dietary Ca2+ and waterborne zinc, except acute zinc tolerance which was highest among all the treatments. Thus, we conclude that the interactions between elevated dietary Ca2+ and waterborne zinc can protect freshwater fish against waterborne zinc toxicity.     

Some effects of low pH and calcium on the growth and tissue mineral content of yearling brown trout, Salmo trutta

Yearling brown trout, Salmo trutta , were exposed to low mineral content water (nominal concentrations of 20μmol 1⁻¹ magnesium, 7.7 μmol 1⁻¹ potassium, 44 μmol 1⁻¹ sodium) over a pH range of 4.0–5.2 with ambient calcium concentrations of 2.5–60 μmol 1⁻¹. All fish died at pH 4.0 and 4.2 irrespective of ambient calcium concentration and also at pH 4.4 with only 2–3 μmol 1 ⁻¹ calcium (that is calcium-free water except for that leached from the diet or excreted by the fish). Good growth rates were obtained over the remaining treatments which extended down to pH 4.4 with as little as 7 μmol 1⁻¹ calcium. When starved, weight loss was inversely correlated with pH. Effects on plasma chloride, percentage dry weight and calcium, potassium sodium, and phosphorus contents of skin, muscle and bone tissue were also investigated. These demonstrated pH effects on mineral metabolism in starved fish, but no effects were detected in fed fish.     

Effect of rearing density on thyroid and interrenal gland activity and plasma and hepatic metabolite levels in rainbow trout, Salmo gairdneri Richardson

There were significant inverse correlations between rearing density of rainbow trout, Salmo gairdneri Richardson, and final body weight, plasma L-thyroxine (T₄), trüodo-L-tryronine (T₃), cortisol and protein concentrations, plasma T₄/T₃ ratios and thyroid epithelial cell height. In addition, hepatosomatic indices and plasma free fatty acid concentrations were higher in fish reared at low (134 g 1⁻¹) density compared with groups reared at medium (210g1⁻¹) or high density (299g 1⁻¹), and the post-feeding (3.5-4h) elevation in plasma glucose and triglyceride levels evident in trout maintained at low rearing density was not found in those fish reared at higher densities. There were no significant effects of rearing density on hematocrit, carcass composition, hepatic glycogen and lipid levels and interregnal nucleus size.     

Response of PAS-positive cells of the pituitary pars intermedia in the teleost Carassius auratus to acid water

Summary The PAS-positive or PIPAS cells in the pars intermedia of goldfish are activated after reduction of the pH of the ambient freshwater from 7.5 to 3.5. The cells increase in number and exhibit a five-fold increase in cell volume. Granular endoplasmic reticulum occupies most of the cytoplasm. Goldfish PIPAS cells (also termed calciumsensitive cells) are thought to have a hypercalcemic function. Therefore, their activation in acid water may be caused by the severe drop in plasma calcium concentration following exposure of the fish to low water pH. However, activation of the PIPAS cells in response to acidification of the water is not prevented when the calcium concentration of the water is increased to levels that result in hypercalcemia instead of hypocalcemia. Activation of the PIPAS cells occurs also in fish exposed to acidified freshwater enriched with NaCl to an osmolarity similar to that of the blood. This prevents the reduction in plasma osmolarity and Na⁺ and Cl^- concentrations that follow exposure of goldfish to acidified normal freshwater. Our observations do not support the hypothesis that the PIPAS cells in goldfish produce a hypercalcemic hormone, or indeed any hormone involved in calcium metabolism or osmoregulation. The cells may be implicated in acid-base regulation (a characteristic of many types of fish when exposed to acidified water) but the evidence is indirect.     

Estrogenicity and Genotoxicity Detection in Different Contaminated Waters

Owing that Oreochromis niloticus is resistant to different aquatic environments, four contaminated sites were selected: decentralized engineered ecosystem (DEE) and conventional wastewater treatment plants (WWTP); urban lagoon; and gasoline-contaminated marshland. Endocrine disruption was assessed by alkali-labile phosphate (ALP) quantification, genotoxicity by micronuclei frequency, and morphological changes by hepatosomatic and gonadosomatic indexes. The ALP baseline of non-exposed O. niloticus males was low, compared with other fish species in the literature. Endocrine disruption was observed in fish exposed to raw wastewater of WWTP and DEE, discharge point of channeled river in the urban lagoon, and water from gasoline-contaminated marshland. After treatment in the DEE, the endocrine disruption effect was removed. The micronuclei frequency corroborated with the ALP results in most cases and proved to be a useful tool to assess genotoxicity in fish. The exposure time of 15 days was not enough to provoke morphological effects in most samples. However, in all gasoline-contaminated samples, the fishes showed an increase in the hepatosomatic index followed by a decrease in the gonadosomatic index. The tested biomarkers showed feasibility, sensibility, reproducibility, and were complementary in the assessment of chronic ecotoxicity; therefore, we recommend them to compose a suitable protocol for aquatic monitoring programs.     

The effects of elevated summer temperature and sublethal pollutants (ammonia, low pH) on protein turnover in the gill and liver of rainbow trout (Oncorhynchus mykiss) on a limited food ration.

Protein synthesis, degradation and growth of the liver and gills were determined in juvenile rainbow trout (Oncorhynchus mykiss) fed a limited ration and exposed for 90 days to normal or elevated summer temperatures (+2 degrees above ambient) and either low pH (5.2) in softwater or 70 microM total ammonia in hardwater. The limited ration resulted in low rates of growth (< 0.80% per day) and protein synthesis in all fish. In softwater, whole-body growth was significantly inhibited by elevated temperature but stimulated by low pH, although tissue protein metabolism was generally unaffected by these treatments. There was no significant difference in final size between the groups of fish in hardwater, but liver protein synthesis and degradation were significantly lower at +2 degrees C, the reduction in synthesis being due to an inhibition of both the capacity for protein synthesis, Cs and the RNA translational efficiency, kRNA. Gill protein metabolism was unaffected by the experimental treatments in trout in hardwater. The authors conclude that a global warming scenario would be detrimental to protein synthesis and growth in freshwater fish under conditions of food limitation in summer, and when late summer temperatures approached the upper thermal limit of the species, regardless of food availability.     

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.     

Effects of epinephrine on branchial and renal calcium handling in the rainbow trout

Acute exposure of rainbow trout (Salmo gairdneri) to low external calcium (25 microM) caused an immediate but transient increase in plasma epinephrine concentration that may have been related to a concomitant depression of blood pH. Intra-arterial infusion of epinephrine at normal ambient calcium levels (0.35 mM) for 4 h caused circulating levels of epinephrine to rise from 2.9 X 10(-9) to 8.0 X 10(-8) M but did not affect norepinephrine levels, or branchial unidirectional calcium fluxes. Active (ATP-dependent) calcium transport across basolateral plasma membranes prepared from gill epithelial cells was not affected by pretreatment of fish with epinephrine or by direct application of epinephrine or cAMP, in vitro. Epinephrine infusion elevated urine flow rate, decreased urine pH, and increased urine phosphate levels significantly. Net renal calcium efflux increased significantly as a result of the increased urine flow rate. It is concluded that epinephrine does not stimulate branchial calcium uptake or renal conservation of calcium in rainbow trout at normal external calcium levels and therefore we cautiously suggest that epinephrine is unlikely to be involved in calcium balance during periods of exposure to low external calcium. Instead, epinephrine may play a role in compensating the acid-base disturbances and the increased branchial water influx that are associated with exposure to low ambient calcium.     

Time-course effects of repetitive oestradiol-17β and thyroxine injections on the natural spring smolting of Atlantic salmon, Salmo salar L.

The impact of thyroxine and oestradiol injections on the smolting process of Salmo salar was investigated over 39 days. The increase in the degree of silvering was advanced by thyroxine but inhibited by oestradiol, as compared to controls. Oestradiol treatment increased the level of total calcium in the plasma, the hepatosomatic index and the concentration of RNA in the liver, indicating the induction of vitellogenin synthesis. The level of plasma chloride and protein in the liver remained unchanged in all experimental groups. Gill Na⁺/K⁺-ATPase activity was low initially in the control fish, but increased steadily during the 39 days. The enzyme activity was advanced in the thyroxine-treated fish, but inhibited by oestradiol. Gill chloride cell number increased in control fish and in the thyroxine-treated fish until 25 days after the hormone treatment was initiated. Oestradiol-treatment induced a lower chloride cell number on days 25 and 39. Altogether the present study demonstrates a reciprocal action of exogenous thyroxine and oestradiol during smolting in S. salar.     

Salmon lice, Lepeophtheirus salmonis, infestation as a causal agent of premature return to rivers and estuaries by sea trout, Salmo trutta, juveniles

A field experiment conducted in the River Lønningdalselven in spring 1992 supports the hypothesis that salmon lice, Lepeophtheirus salmonis, infestations may cause premature return of sea trout, Salmo trutta, juveniles, either to estuaries or to rivers. When lice infested (exposed) and uninfested (control) sea trout juveniles (post smolts) were released simultaneously into the sea, exposed fish returned to the estuarine area earlier compared with controls. Within the following two days, exposed sea trout migrated further into freshwater. At that time they were infested with a median of 62.5 lice, dominated by chalimus larvae and late juveniles. Exposed sea trout suffered from an osmoregulatory failure in sea water and this is considered one reason for infested fish returning to brackish water. While only a few control fish returned to the estuary on the day of release, some more returned to freshwater the following four days. During this time they had become heavily infested with copepodids, and carried a median of 150.0 lice. It is suggested that physiological stress and high infection pressure in the sea results in sea trout juveniles returning to estuaries and freshwater.     

The cost of living for freshwater fish in a warmer, more polluted world

Little of the vast literature on the temperature physiology of freshwater fish is useful in predicting the effects of global warming. In the present review a series of laboratory experiments is reviewed in which rainbow trout (Oncorhynchus mykiss) were exposed to simulated global warming, a 2 °C increment superimposed upon the natural thermal regime, in the presence and absence of two common freshwater pollutants, ammonia and acidity (low pH). Simulated global warming had little effect on the growth and physiology of trout fed to satiation over much of the summer. However, in late summer, when ambient water temperature was at its highest, the addition of 2 °C caused a marked inhibition of appetite and growth, although this impact was not exacerbated by a reduction in food availability. In winter, + 2 °C stimulated metabolism, appetite and growth by approximately 30–60%. Exposure of satiation‐fed trout to low levels of pollutants produced unexpected results. Ammonia (NH₃ + NH₄⁺ = 70 μm) stimulated summer growth and energy conversion efficiency, whilst acidification (pH 5.2) increased appetite and growth but caused no disturbance of electrolyte balance. These pollutant effects were additive upon, but not synergistic with, the effects of + 2 °C. The ability of the fish to acclimate to the experimental conditions was tested with acute lethal temperature and/or toxicant challenges. Fish exposed to + 2 °C had a slightly (0.2–1.0 °C) but significantly higher lethal temperature than those exposed to ambient temperature when fed to satiation. However, there was no evidence of acclimation to either ammonia or low pH. It is concluded that the impact of global warming on freshwater fish will vary seasonally. The additional temperature may provide growth benefits in winter, but may threaten fish populations living towards the upper end of their thermal tolerance zone in (late) summer.     

Effects of external osmolality,calcium and prolactin on growth and differentiation of the epidermal cells of the cichlid teleost Sarotherodon mossambicus

Summary Osmolality and concentrations of divalent cations calcium, and to a lesser extent magnesium of the water are the main environmental factors that determine development and degree of mucification of the skin epithelium of Sarotherodon mossambicus. Epithelial thickness and number of mucocytes in fish exposed to low (freshwater level) concentrations of calcium and magnesium are directly related to the height of the osmotic gradient between water and blood plasma. No such relationship is found in fish exposed to a high (seawater level) concentration of calcium in the water, irrespective of the height of the osmotic gradient.The results strongly indicate that the effects of osmolality and divalent cations are indirect, and mediated by prolactin, since administration of ovine or fish prolactin stimulates growth and multiplication of the cells of the basal layer of the epidermis, and promotes the differentiation of the mucocytes.     

Plasma Ca2+ concentration limits melatonin night production in two fish species

In freshwater (FW) rainbow trout Oncorhynchus mykiss of spontaneously low plasma calcium concentrations ([Ca²⁺]_pl), plasma melatonin at night was significantly lower than that measured in FW fish with the highest [Ca²⁺]_pl. In brackish water adapted rainbow trout with originally high [Ca²⁺]_pl, plasma melatonin concentration at night was elevated. In cannulated flounder Platichthys flesus , night plasma melatonin increases (ΔMel) corresponded to [Ca²⁺]_pl. It is postulated that in physiological steady-state conditions, melatonin synthesis capacity is coupled to free calcium concentration in plasma of O. mykiss and P. flesus .     

Cortisol and thyroid hormone responses to acid stress in the brown trout, Salmo trutta L.

Individual cannulated brown trout monitored during exposure to acidic water showed increased plasma cortisol after 3 h at pH 4.0 with low (0.05 mm) or high (2.8 mm) calcium (Ca) content, and after 2 days in acidic water with a high Ca content. Most fish did not survive for 2 days in acidic water with a low Ca content. Non-cannulated fish showed a similar increase in mean plasma cortisol after 2 days in high-Ca acidic water (pH 4.0–4.6), but not in acidic water of a low Ca content. After 7 days of exposure to acidic water, plasma cortisol appeared to recover when there was a high Ca content but increased 20-fold when Ca content was low. In cannulated fish severe acid stress resulted in a marked and rapid thyroid response. Plasma thyroxine (T₄) was elevated after 3 h exposure to acidic water of both low and high Ca content and remained elevated for 2 days of acid exposure with high Ca. In non-cannulated fish an increase in mean T₄ was apparent only after 7 days in low-Ca acidic water. Plasma triiodothyronine (T₃) levels were not significantly altered by any of the acid regimes. Plasma glucose of cannulated fish was elevated within 3 h of acid-exposure and remained elevated after 2 days in high-Ca acidic water.     

Vitellogenin levels in male and female rainbow trout (Salmo gairdneri Richardson) at various stages of the reproductive cycle

The immunoassayable vitellogenin (VTG) in plasma from male rainbow trout had the same molecular weight as authentic VTG from female fish. The VTG level in male trout was low (usually nanograms, occasionally up to a few micrograms, per ml) and did not correlate with the stage of sexual maturity. The plasma VTG level of female trout that were two years from first spawning was 200-fold higher than males of the same strain and age. The plasma VTG level of female rainbow trout rose approximately a million-fold during the two or three years required to attain sexual maturity.