Harold L. Bergman

Relationship between copper exposure duration, tissue copper concentration, and rainbow trout growth

Abstract Rainbow trout fry were exposed in soft water to sublethal concentrations of copper for 60 days under controlled laboratory conditions. At 20-day intervals, fish were sampled for weight, length, and whole-body copper and metallothionein concentrations. Exposures to waterborne copper concentrations as low as 4.6 μg 1−1 resulted in significantly reduced growth and significantly elevated whole-body copper concentrations after 20 days. Whole-body metallothionein concentrations did not differ significantly from controls. Fish did not recover or return to control growth rates throughout the entire exposure period; a 45% reduction in mean weight relative to controls observed on day 40 in the 9.0 μg 1−1 Cu exposure was sustained through day 60. Whole-body accumulation rates of copper in fish exposed to 4.6 μg 1−1 and higher levels of Cu increased significantly between 0 and 40 days and appeared to reach steady-state after 40 days. Copper accumulation was found to depend on dose and time. Trout exposed to higher copper concentrations accumulated more whole-body copper, with longer times to reach steady-state. Our data suggest that both accumulation capacity and copper depuration rates from a slowly exchangeable pool are concentraion-dependent. A linear model was developed for the relationship between exposure duration, copper accumulation, and fish weight: In (wet wt., μg) = 4.8 + 0.03 (exposure duration, days)—0.04 (whole-body copper, mg g−1 dry wt.) (P

Structural changes in gills of Lost River suckers exposed to elevated pH and ammonia concentrations

The Lost River sucker (Deltistes luxatus) is a federally listed, endangered fish that occurs primarily in Upper Klamath Lake-a hypereutrophic lake in southern Oregon, USA. A decline of the sucker population in the lake over the past few decades has been partly attributed to adverse water quality conditions, including elevated pH and ammonia concentrations that occur during summer cyanobacterial blooms. We quantitatively analyzed structural changes in gills of larval Lost River suckers after they were exposed to elevated pH and ammonia concentrations for 30 d. Exposure to pH as high as 10 caused no observed structural changes. However, lamellar thickness and O(2) diffusion distance increased significantly (P<0.05) at ammonia concentrations that did not significantly decrease survival, growth, whole-body ion concentrations, or swimming performance. Additionally, we qualitatively observed increases in the frequency of hyperplasic and hypertrophic mucous cells, tissue damage, epithelial lifting, and infiltration of white blood cells into paracellular lymphatic spaces at the highest sublethal ammonia concentration. These observed gill changes typically indicate compromised respiratory and ionoregulatory capacity, although such effects were not manifested in the assays we performed. Regardless, these structural gill changes appear to be a more sensitive indicator of exposure to elevated ammonia concentrations than are more traditional sublethal indices. Therefore, gill histopathology might be a relevant early-warning monitoring tool of the health of Lost River suckers in Upper Klamath Lake, and other species in similar eutrophic systems.

Toxicity of cobalt and copper to rainbow trout : application of a mechanistic model for predicting survival

Abstract We conducted 14-day laboratory toxicity tests with rainbow trout to evaluate, (1) the acute toxicity of Co and Cu, and (2) the effects of selected Co concentrations on the toxicity of Cu in Co/Cu mixtures. A one-compartment, uptake-depuration model was used to estimate incipient lethal levels (ILLs) and quantify differences in lethality responses among the Co-only, Cu-only and Co/Cu exposures. Additionally, we compared mortality percentages observed in Co/Cu mixtures to those predicted from two models of joint toxicity. Co was a slower-acting and less potent toxicant than was Cu. For Co, the ILL for 50% mortality was 346 μg l −1 ; for Cu, the ILL for 50% mortality was 14 μg l −1 . Moreover, in Co/Cu mixtures, Co acted as an antagonist during the first 48–96 h, but later acted as an additive or slightly synergistic toxicant—making it difficult to predict short-term mortality of fish in Co/Cu mixtures. Lethality thresholds for Cu were reduced by 11–26% and 37–45%, respectively, in the presence of 50 and 250 μg l −1 Co, although those differences were not statistically significant. The ordering of ILLs for the metal exposures tested was: 250 μg l −1 Co+Cu>50 μg l −1 Co+Cu>Cu-only>Co-only.

Effects of Water pH and Calcium Concentration on Ion Balance in Fish of the Rio Negro, Amazon

We examined the effects of acute low‐pH exposure on ion balance (Na+, Cl−, K+) in several species of fish captured from the Rio Negro, a dilute, acidic tributary of the Amazon. At pH 5.5 (untreated Rio Negro water), the four Rio Negro species tested (piranha preta, Serrasalmus rhombeus; piranha branca, Serrasalmus cf. holandi; aracu, Leporinus fasciatus; and pacu, Myleus sp.) were at or near ion balance; upon exposure to pH 3.5, while Na+ and Cl− loss rates became significant, they were relatively mild. In comparison, tambaqui (Colossoma macropo‐mum), which were obtained from aquaculture and held and tested under the same conditions as the other fish, had loss rates seven times higher than all the Rio Negro species. At pH 3.0, rates of Na+ and Cl− loss for the Rio Negro fish increased three‐ to fivefold but were again much less than those observed in tambaqui. Raising water Ca2+ concentration from 10 mmol L−1 to 100 mmol L−1 during exposure to the same low pHs had no effect on rates of ion loss in the three species tested (piranha preta, piranha branca, aracu), which suggests that either they have such a high branchial affinity for Ca2+ that all sites are saturated at 10 mmol L−1 and additional Ca2+ had no effect, or that Ca2+ may not be involved in regulation of branchial ion permeability. For a final Rio Negro species, the cardinal tetra (Paracheirodon axelrodi), we monitored body Na+ concentration during 5 d of exposure to pH 6.0, 4.0, or 3.5. These pHs had no effect on body Na+ concentration. These data together suggest that exceptional acid tolerance is a general characteristic of fish that inhabit the dilute acidic Rio Negro and raise questions about the role of Ca2+ in regulation of branchial ion permeability in these fish.

Responses of an Amazonian Teleost, the Tambaqui (Colossoma macropomum), to Low pH in Extremely Soft Water

Our goal was to compare the internal physiological responses to acid challenge in an acidophilic tropical teleost endemic to dilute low-pH waters with those in nonacidophilic temperate species such as salmonids, which have been the subject of most previous investigations. The Amazonian tambaqui (Colossoma macropomum), which migrates between circumneutral water and dilute acidic “blackwater” of the Rio Negro, was exposed to a graded low-pH and recovery regime in representative soft water (Na+ = 15, Cl− = 16, Ca2+ = 20 μmol L−1). Fish were fitted with arterial catheters for repetitive blood sampling. Water pH was altered from 6.5 (control) to 5.0, 4.0, 3.0, and back to 6.5 (recovery) on successive days. Some deaths occurred at pH 3.0. Throughout the regime, there were no disturbances of blood gases (O2 and CO2 tensions and contents) or lactate levels, and only very minor changes in acid-base status of plasma and red cells. However, erythrocytic guanylate and ade-nylate levels increased at pHs less than or equal to 5.0. Down to pH 4.0, plasma glucose, cortisol, and total ammonia levels remained constant, but all increased at pH 3.0, denoting a stress response. Plasma Na+ and Cl− levels declined and plasma protein concentration increased at pH 3.0, indicative of ionore-gulatory and fluid volume disturbance, and neither recovered upon return to pH 6.5. Cortisol and ammonia elevations also persisted. Transepithelial potential changed progressively from highly negative values (inside) at pH 6.5 to highly positive values at pH 3.0; these alterations were fully reversible. Experimental elevations in water calcium levels drove the transepithe-lial potential positive at circumneutral pH, attenuated or prevented changes in transepithelial potential at low pH, and reduced Na+ and Cl− loss rates to the water during acute low-pH challenges. In general, tambaqui exhibited responses to low pH that were qualitatively similar but quantitatively more resistant than those previously documented in salmonids.

Ion and Acid‐Base Balance in Three Species of Amazonian Fish during Gradual Acidification of Extremely Soft Water

Sensitivity to acid water was assessed in three species of Amazonian fish that encounter naturally acidic blackwaters to differing degrees in the wild: tambaqui (Colossoma macropomum), matrincha (Brycon erythropterum), and tamoatá (Hoplosternum littorale), in decreasing order of occurrence in blackwater. Fish were exposed to a graded reduction in water pH, from pH 6 to 5 to 4 to 3.5, followed by return to pH 6. Fish were exposed to each new pH for 24 h. During these exposures, net transfers of ions (Na+, K+, Cl−, and Ca2+) and acid‐base equivalents to and from the external water were used as physiological indicators of acid tolerance. Exposure to pH 5 had a minimal effect on net ion fluxes. Significant net losses of all ions (except Ca2+) were recorded in all three species during the first few hours of exposure to pH 4. However, ion balance was usually restored within 18 h at pH 4. Exposure to pH 3.5 caused even greater ion losses in all three species and proved to be acutely lethal to tamoatá. Matrincha sustained irreversible physiological damage at pH 3.5, as ion fluxes did not recover following return to pH 6 and there was some mortality. Tambaqui suffered the least ionoregulatory disturbances at pH 3.5 and was the only species to make a full recovery on return to pH 6. In all species, there was a tendency for ammonia excretion to increase at low water pH, but even at pH 3.5, there was no significant net uptake of acid from the water. Overall, there was a strong relationship between the magnitude of ionic disturbances and the lethality of exposure to low pH. The relative insensitivity of the ionoregulatory system of tambaqui to low pH indicates that this is a feature of fish native to blackwater systems rather than one that is common to all Amazon fish.

Respiratory Physiology of the Lake Magadi Tilapia (Oreochromis alcalicus grahami), a Fish Adapted to a Hot, Alkaline, and Frequently Hypoxic Environment

The tilapia Oreochromis alcalicus grahami is a unique ureotelic teleost, the only fish that lives in the alkaline hotsprings of Lake Magadi, Kenya. Physical conditions and fish behavior were monitored in the Fish Springs Lagoon area, a site where the tilapia were particularly abundant. Water Po2 and temperature fluctuated more or less in parallel in a diurnal cycle from less than 20 Torr and less than 25° C at night to greater than 40° Torr and 38° C during the day, whereas pH remained constant at approximately 9.8. Field laboratory tests demonstrated that routine Ṁo2 (under normoxia) increased greatly from 27° C to 36° C (Q10 = 62) but then stabilized at a very high level (~34.5 μmol g−1 h−1) up to the lethal temperature (~ 42.5° C), a pattern that was adaptive to the natural diurnal regime. The Po2 threshold for survival during acute exposure (≤ 1 h) was approximately 16 Torr. Ṁo2 from water was well maintained down to a Po₂ of 60 Torr, below which it declined. Under such hypoxic conditions, the fish performed supplementary surface breathing when allowed access to air. Both the better oxygenated surface layer and air bubbles were inspired, resulting in significant uptake of O2. The Po2 threshold for surface breathing was 1.8-fold higher at 37.5° C than at 31° C. Surface breathing and voluntary entry of fish into air were observed in the field. The blood O2 dissociation curve at 30°-32° C was h3perbolic, with a high afinity (P50 = 6 Torr), low cooperativity (Hill coeficient = 1.18), and no Bohr effect over the extracellular pH range 8.2-8.6.

Obligatory urea production and the cost of living in the Magadi tilapia revealed by acclimation to reduced salinity and alkalinity.

Alcolapia grahami is a unique ureotelic tilapia that lives in the highly alkaline, saline Lake Magadi, Kenya (pH, ∼10.0; alkalinity, ∼380 mmol L−1; Na+, ∼350 mmol L−1; Cl−, ∼110 mmol L−1; osmolality, ∼580 mosm kg−1). The fish survived well upon gradual exposure to dilute lake water (down to 1%, essentially freshwater). Urea excretion continued, and there was no ammonia excretion despite favorable conditions, indicating that ureotelism is obligatory. Levels of most ornithine‐urea cycle enzymes in the liver were unchanged relative to controls kept for the same period in 100% lake water. The fish exhibited good abilities for hypo‐ and hyperregulation, maintaining plasma Na+, Cl−, and osmolality at levels typical of marine and freshwater teleosts in 100% and 1% lake water, respectively. Plasma total CO2 did not change with environmental dilution. Routine oxygen consumption (Mo2) was extremely high in 100% lake water but decreased by 40%–68% after acclimation to dilute lake water. At every fixed swimming speed, Mo2 was significantly reduced (by 50% at high speeds), and critical swimming speed was elevated in fish in 10% lake water relative to 100% lake water. Osmotic and Cl− concentration gradients from water to plasma were actually increased, and osmotic and Na+ gradients were reversed, in 10% and 1% dilutions relative to 100% lake water, whereas acid‐base gradients were greatly reduced. We suggest that approximately 50% of the animal’s high metabolic demand originates from the cost of acid‐base regulation in the highly alkaline Lake Magadi. When this load is reduced by environmental dilution, the energy saved can be diverted to enhanced swimming performance.

Discordance Between Genetic Structure and Morphological, Ecological, and Physiological Adaptation in Lake Magadi Tilapia

The Magadi tilapia (Alcolapia grahami, formerly Oreochromis alcalicus grahami) is a remarkable example of teleost life in an extreme environment. Typical conditions include water pH = 10, titration alkalinity > 300 mM, osmolality = 525 mOsm, temperatures ranging from 23° to 42°C, and O2 levels fluctuating diurnally between extreme hyperoxia and anoxia. A number of relatively small tilapia populations are present in various thermal spring lagoons around the margin of the lake separated by kilometers of solid trona crust (floating Na2CO3) underlain by anoxic water. Despite the apparent isolation of different populations, annual floods may provide opportunities for exchange of fish across the surface of the trona and subsequent gene flow. To assess the question of isolation among Lake Magadi populations, we analyzed the variable control region of the mitochondrial DNA (mtDNA) from six lagoons. A total of seven mtDNA haplotypes, including three common haplotypes, were observed in all six populations. Several of the Lake Magadi populations showed haplotype frequencies indicative of differentiation, while others showed very little. However, differentiation among lagoon populations was discordant with their geographical distribution along the shoreline. All populations exhibited the unusual trait of 100% ureotelism but specialized morphological and physiological characteristics were observed among several of the lagoon systems. In addition, distinct differences were observed in the osmolality among the lagoons with levels as high as 1,400–1,700 mOsm kg−1, with corresponding differences in the natural levels of whole‐body urea. These levels of osmotic pressure proved fatal to fish from less alkaline systems but remarkably were also fatal to the fish that inhabited lagoons with this water chemistry. Upon more detailed inspection, specific adaptations to differential conditions in the lagoon habitat were identified that allowed survival of these cichlids. Additional evidence against potential for gene flow among lagoons despite the sharing of common mtDNA haplotypes was that the osmolality of floodwaters following a heavy rain showed lethal levels exceeding 1,700 mOsm kg−1. In isolation, different mtDNA haplotypes would be predicted to go to fixation in different populations due to rapid generation times and the small effective population sizes in a number of lagoons. We propose a model of balancing selection to maintain common mtDNA sequences through a common selection pressure among lagoons that is based on microhabitats utilized by the tilapia.

Rh Proteins and NH4+ –activated Na+-ATPase in the Magadi Tilapia (Alcolapia grahami), a 100% Ureotelic Teleost Fish.

SUMMARY The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l−1). The Magadi tilapia is the only 100% ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins (Rhag, Rhbg and Rhcg2) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg, Rhcg2 and Na+,K+-ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH4+ (versus K+), suggesting it can function as an NH4+-transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH4+-activated Na+-ATPase function.