Carl Haux

Fish physiology and metal pollution: results and experiences from laboratory and field studies.

Physiological methods, previously used as health indicators in laboratory investigations on fish exposed to toxicants, have been applied to feral fish living in metal-polluted waters. A background to the use of a physiological approach for detecting early-arising effects of toxic chemicals on fish is given, together with a description of the clinical parameters used and their physiological relevance. The results and experiences from the field application show that certain clinical methods may be very useful in detecting and diagnosing sublethal disturbances in natural fish populations in polluted areas. Furthermore, the results indicate a good agreement between metal-induced physiological effects detected in fish exposed under natural field conditions and those found in laboratory experiments.

Abrupt changes in photoperiod affect age at maturity, timing of ovulation and plasma testosterone and oestradiol-17β profiles in Atlantic salmon, Salmo salar

Abstract Atlantic salmon ( Salmo salar L.), reared in sea cages for 18 months (age 36 months from hatching), were exposed to natural light (NL, 61°N), or continuous additional light from January (ALJ) or March (ALM) until July. On July 13, the fish were moved to indoor raceways with brackish water (2–19‰) and ambient temperature (declining from 13.0 to 5.6°C). Fish from each treatment were subjected to either simulated natural photoperiod (SNP), continuous light (24L), or short photoperiod (8L=8 L:16D), creating a total of nine experimental groups with approx. 50 fish in each. The proportion of sexually maturing females was reduced from 91% in the NL groups, to 67% and 9% in the ALM and ALJ groups, respectively ( p ≤0.005). A similar reduction was observed among the males, from 74% in the NL groups, to 57% and 16% in the ALM and ALJ groups, respectively ( p ≤0.001). Ovulation commenced in late October in the control group (NL-SNP). Compared with control, median ovulation time was advanced by 5, 4 and 3 weeks in the ALM-8L, NL-8L and ALM-SNP groups, respectively, whereas ovulation was delayed by 1 and 6 weeks in the ALM-24L and NL-24L groups, respectively. The altered timing of ovulation among the groups was paralleled by similar shifts in the seasonal plasma oestradiol-17 β and testosterone profiles. Survival of eggs to the eyed stage was lower in the ALM-8L group (mean=64.2%) compared with the NL-SNP group (mean=92.5%), indicating a negative effect on egg quality in the most advanced group. Although abrupt changes in photoperiod can be used to control timing of ovulation in Atlantic salmon to obtain off-season eggs, the decrease in egg survival and proportion of maturing fish may set constraints on how much maturation can be advanced by use of continuous light during winter and spring. However, the effect on age at maturity may also be exploited to reduce the problem with unwanted early maturation in salmon farming.

Increased hepatic metallothionein content correlates to cadmium accumulation in environmentally exposed perch (Perca fluviatilis)

Abstract The subcellular distribution of cadmium, copper and zinc in the liver, and the binding of these heavy metals to metallothionein (MT) were studied in a wild population of perch (Perca fluviatilis). The fish were caught in two areas of the cadmium-contaminated river Eman in the southeast of Sweden. The livers were analyzed for zinc, copper and cadmium and the subcellular distribution of the heavy metals in mitochondrial, microsomal and cytosolic fractions was determined. The cytosols were chromatographed on Sephadex G-75 columns to determine the partition of heavy metals between high molecular weight and MT fractions. Metallothionein was determined, in tissue extracts, by differential pulse polarography. The elevated hepatic cadmium levels found in perch from the contaminated region of the river did not significantly alter the distribution of zinc and copper in the liver. With increasing amounts of cadmium present in the liver cytosol, the distribution of cadmium was altered. The increased cadmium content correlated with an increased MT level in the liver (r=0.84). The relationship between cadmium and MT is described by the formula [MT] = 7.2 + 0.53 [Cd]. Chromatography of the liver revealed that all of the applied cadmium was bound to MT. The variations in zinc and copper showed low correlation to the MT content of the liver (r=0.51 and r=−0.15, respectively).

Disturbed ion balance in flounder, Platichthys flesus L. exposed to sublethal levels of cadmium

Abstract The ion balance in the flounder ( Ptatichthys flesus L.) was studied after 4 and 9 wk of exposure to sublethal cadmium levels (5–500 μg Cd/l) in brackish water. The cadmium exposure had no effect on the major blood plasma electrolytes, sodium and chloride, indicating an intact osmoregulation. In contrast, cadmium seriously affected the regulation of other ions. Potassium and calcium concentrations showed a strong and dose-dependent depression in blood plasma, whereas the plasma concentrations of inorganic phosphate and magnesium were significantly elevated. It is suggested that these ion disturbances might be associated with cadmium-induced pathological changes in ion-regulating tissues. The reduced concentrations of plasma calcium and potassium might be responsible for neuromuscular disturbances, such as hyperexcitability, spasms and tetanic body contractions, observed in some flounders exposed to the highest cadmium concentration (500 μg Cd/l). In spite of the pronounced disturbance of the calcium metabolism, the spinal columns of the cadmiumexposed flounders did not show any signs of demineralization, fractures or other deformations. This might be due to the fact that the flounder probably has an acellular bone tissue, which only to a small degree seems to be affected by the disturbed calcium balance. It is suggested that fish species with acellular bone tissue run a minor risk of suffering from skeletal damage after cadmium exposure than fish species with an active cellular bone tissue.

Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids

An improved straight-phase HPLC method for the separation and quantification of lipid classes is described. Two binary gradient solvent systems were used, one for polar and one for neutral lipids, and detection was performed with a light-scattering detector. The developed HPLC methods were highly reproducible and allowed base-line separation of all investigated polar lipid classes (phosphatidic acid, diphosphatidylglycerol. phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine) and neutral lipid classes (triacylglycerol, free fatty acid, diacylglycerol, cholesterol and monoacylglycerol) except of cholesterol ester and wax ester. Application of the chromatographic systems demonstrated that the methods are suitable for quantitative analysis of the major lipid classes present in lipid extracts from livers and eggs of Atlantic salmon (Salmo salar).

Variations in hepatic metallothionen, zinc and copper levels during an annual reproductive cycle in rainbow trout,Salmo gairdneri

The normal variations in hepatic levels of metallothionein, zinc and copper were studied during an annual reproductive cycle in rainbow trout of both sexes. In female fish, the total hepatic zinc levels closely followed the estradiol-17β and the LSI levels. Hence, the zinc levels rose in September, peaked in December and dropped in January. No distinct peaks were, however, observed in the whole-liver copper content. The hepatic metallothionein levels in female fish began to increase at the onset of exogenous vitellogenesis. Maximum levels were reached after estradiol-17β and LSI levels had dropped in January. In male fish no distinct peaks in either zinc or copper levels were observed. The metallothionein levels increased somewhat during the time of spermatogenesis. It is suggested that metallothionein may regulate the hepatic zinc distribution during the annual reproductive cycle in female rainbow trout, thereby ensuring the organism of a control mechanism to keep the pool of available zinc at an appropriate level.

Changes in plasma vitellogenin, sex steroids, calcitonin, and thyroid hormones related to sexual maturation in female brown trout (Salmo trutta).

Female brown trout (Salmo trutta) from a wild strain (Baltic sea trout) and a cultured strain were sampled individually for blood plasma at regular intervals during the period around final sexual maturation. The plasma samples were analyzed for vitellogenin (VTG), estradiol-17 beta, testosterone, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), calcitonin, tri-iodothyronine (T3), thyroxine (T4), and total and free plasma calcium. In the wild fish, VTG, estradiol-17 beta, and testosterone peaked 30 days before ovulation, while 17,20 beta-P had a sharp peak at ovulation. Both T3 and T4 declined at the beginning of the sampling period, reached minimal levels 30 days before ovulation, and rose sharply at the time of ovulation. Calcitonin levels were elevated during final maturation. Total plasma calcium correlated with plasma VTG levels. In the cultured strain, sampling was started 2 weeks before ovulation. The levels of VTG, estradiol-17 beta, and testosterone decreased throughout the sampling period. 17,20 beta-P and calcitonin concentrations were high during the period close to ovulation. Plasma thyroxine remained at basal levels in cultured trout. The discrepancies observed between wild and cultured females may be due to differences in stress susceptibility, environmental conditions, life cycles, or to genetic divergence between the strains.

Long-term sublethal physiological effects on rainbow trout, Salmo gairdneri, during exposure to cadmium and after subsequent recovery

Abstract The effects on hematology, blood plasma ion balance and carbohydrate metabolism were studied in rainbow trout, Salmo gairdneri, kept in brackish water, and after 18 and 30 wk of exposure to sublethal levels of cadmium (10 and 100 μg Cd/liter), after 25 and 57 wk of subsequent recovery in clean water. Cadmium exposure caused anemia, hypocalcemia and hypermagnesemia. These effects have previously been observed in fish during shorter periods of exposure, but disappeared in the present study within 25 wk of recovery. This suggests that it is the presence of free cadmium ions during the exposure that is responsible for most of the hematological and biochemical effects. Contrary to these findings, the hyperglycemia, that was present during the exposure, persisted throughout the recovery period. Further evidence for a disturbed carbohydrate metabolism after cessation of the exposure was indicated by a dose-dependent decrease in muscle glycogen content after 25 wk of recovery, and by a dose-dependent increase in liver glycogen content after 57 wk of recovery. These effects on the carbohydrate metabolism could be mediated via the selective uptake of cadmium ions in the pancreas and an inhibitory effect on the insulin secretion, thus resulting in an abnormal endocrine control of the metabolism. Analyses of cadmium revealed a dose-dependent accumulation of cadmium in the muscle, that had reached equilibrium after 18 wk of exposure, while a continuing and dose-dependent accumulation was found in the liver throughout the exposure. The levels attained in the liver were about 100 times higher than in the muscle. The slow elimination from liver and kidney suggest a biological half-life for cadmium of more than 1 yr in these tissues. A close positive correlation was found between cadmium levels in liver and in kidney (r = 0.90) in fish after 57 wk of recovery.

Biotransformation enzyme activities and histopathology in rainbow trout Salmo gairdneri, treated with cadmium

Abstract The effects of intraperitoneal administration of cadmium and exposure to cadmium in the water on rainbow trout, Salmo gairdneri , were studied. The results of intraperitoneal injection of cadmium indicate that this metal has the potential to inhibit phase I and phase II xenobiotic biotransformation activities in the liver and kidney. The exposure to cadmium through water resulted in different responses in the liver and kidney. The marked inhibition of the glucuronidation reaction is of toxicological importance, since this may potentiate the toxic action of organic xenobiotics biotransformed through this pathway. This response in the kidney was accompanied by histopathological changes in the proximal renal tubules, which are probably associated with the plasma hypocalcaemic response observed in cadmium-poisoned fish.

Metallothionein as an indicator of heavy-metal exposure in two subtropical fish species☆

Induction of metallothionein (MT) and levels of cadmium, zinc and copper in liver of the subtropical fish blue-striped grunt Haemulon sciurus were studied after cadmium injection. Hepatic levels of MT, zinc and copper were also analysed in blue-striped grunt and tomtate H. aurolineatum caught at heavy-metal-polluted areas of Hamilton Harbour and Castle Harbour, Bermuda, respectively. In addition, a comparison of the hepatic content of MT, cadmium, zinc and copper was made between blue-striped grunt, tomtate and squirrelfish Holocentrus rufus, caught at unpolluted locations. Hepatic MT was assayed by a radioimmunoassay (RIA), using an antiserum raised against MT from perch Perca fluviatilis and partially purified MT from blue-striped grunt as standard. Hepatic MT was significantly (P < 0.001) induced in a dose-dependent manner in response to a single i.p. injection of CdCl2. The doses were 2200, 740, 250, 82, 27 and 0 μg Cd · kg−1 body wt, respectively. Significantly elevated hepatic MT levels (P < 0.01) were found 2 days after injection of 2200 μg Cd · kg−1 body wt and maximum content of MT in liver was reached after 6 days. MT values in fish 6 days after treatment were increased five-fold compared to the control group. Significantly increased MT levels (P < 0.05) were also found in livers of blue-striped grunt and tomtate caught at contaminated sites compared to control fish. The increased MT levels were in good accordance with the hepatic zinc and copper levels in these fish. The levels of MT, cadmium, zinc and copper in liver from blue-striped grunt and tomtate, caught at clean sites, were within the range found in other fish species, such as rainbow trout Oncorhynchus mykiss and P. fluviatilis. Squirrelfish, caught at an uncontaminated site, had exceptionally high levels of hepatic MT, zinc and copper.