Morpho-histochemical study of the biological effects of sodium dodecyl sulphate on the digestive gland of the Portuguese oyster

In the present work we have studied the effects on survival as well as histopathological and histochemical alterations on the digestive gland of the Portuguese oyster (Crassostrea angulata, Lmk.) induced by acute action of the anionic tensioactive Sodium Dodecyl Sulphate (SDS) at different concentrations (50, 100, 200 and 400 mg/L). Firstly, the LC 50 at 96 h was found to be 136 mg/L SDS. Secondly we calculated the exposure time required for 50% mortality of the specimens exposed to different concentrations, and thirdly we examined histopathological alterations (degenerative processes ranging from inflammatory responses to extreme vacuolation) and histochemical changes in the distribution of carbohydrates and proteins that appeared at each employed concentration. The degree of these alterations and the effects on survival were dependent upon the SDS concentration. The above established modifications indicate that exposure to SDS has a detrimental effect on oyster digestive gland, perturbing metabolic and nutritional functions, what may have some influence on oyster survival.     

The effect of sub-lethal exposure to copper and the time course of recovery in clean water on biochemical changes in juvenile fish (Acipenser persicus)

This study reports the 96-h LC₅₀ value and tissue copper (Cu) levels and biochemical changes in juvenile fish (Acipenser persicus) exposed to 0.026?mg/l ambient Cu for 1, 7 and 14?days. It then examined the recovery of the same parameters after placing the juvenile fish in clean water for a further period of 28?days. The intestine, kidney and gill Cu levels, plasma glucose, total protein, triglyceride, cortisol, triiodothyronine and thyroxine concentrations, liver protein contents, liver catalase, superoxide dismutase (SOD) and glutathione S-transferase activities were studied. The 96-h LC₅₀ value of Cu was 0.502?mg/l for juvenile A. persicus. The results indicate that Cu exposure produced significant accumulations of Cu in gills and kidney over the treatment time. Sublethal dose of Cu resulted in a short-term increase in plasma glucose, total protein and cortisol levels that decreased with time. After the 28-day recovery phase, there were significant differences in kidney Cu levels and triglyceride concentrations as well as SOD activities between recovery fish treatments and their control groups on day 42. The 28-day recovery phase caused significant decreases in total protein levels and SOD activities of Cu-exposed fish on day 42 compared to day 14. The results suggest that 28?days are insufficient for complete recovery to Cu exposure by juveniles and a longer period would be required for full recovery. Moreover, the study showed that the recovery phase following Cu exposure could change biochemical parameters to levels that are not close to those seen during exposure or control levels.     

Immunolocalization of metallothioneins in different tissues of turbot (Scophthalmus maximus) exposed to Cd

Metallothioneins (MT) were localized by immunochemistry in different organs and cell compartments of turbot exposed to sublethal concentrations (100 ppb) of Cd for 7 days. The polyclonal rabbit anti-cod MT antibody (NIVA, Norway) applied herein exhibited positive cross-reactivity with turbot MTs. Immunoreactive MTs were localized in the branchial epithelium, in the liver and in the kidney of turbot. In Cd exposed fishes MTs were demonstrated mainly in branchial chloride cells (CC) and to a lesser extend in the area where progenitor cells are located and in the cells of the respiratory epithelium (secondary lamellae). A higher staining intensity for MTs was observed in CC of the interlamellar space of the main branchial epithelium in comparison with control CC. MT-staining was also observed in the chondroblasts of the cartilage and in the erythrocytes within blood vessels both in control and Cd-exposed specimens. MT immunoreaction was high in the liver hepatocytes and weak in the epithelium of the proximal portion of the kidney in exposed turbot. The tegument, spleen and muscle were devoid of any immunolabelling in both treatments. Ultrastructural studies at the transmission electron microscope revealed that Cd-induced MTs were mainly located in the cytoplasm of gill CC, the lysosomes and the cytoplasm of hepatocytes and in the basal labyrinth of kidney proximal nephrocytes. The differential localization/induction of MTs in different cell types described hereby suggests that the quantification of the specific expression of MT may be used in biomonitoring programs as a biomarker of Cd exposure in aquatic environments.     

Effects of some pesticides on the vital organs of juvenile rainbow trout (Oncorhynchus mykiss)

Gill, trunk kidney, spleen, and liver of rainbow trout (Oncorhynchus mykiss) were examined after exposure to different sublethal concentrations of carbosulfan (25, 50 and 200 μg L⁻¹), propineb (3, 6 and 24 mg L⁻¹), and benomyl (2, 5 and 20 mg L⁻¹) for 14 days. Lesions were observed in gill, trunk kidney, spleen, and liver of rainbow trout exposed to either concentration of pesticides. The most important lesions were determined in the highest concentrations of pesticides. Lamellar fusion, lamellar hyperplasia, epithelial lifting, vacuolization of epithelial tissue, epithelial necrosis, hypertrophy and sloughing of epithelium were observed on fish exposed to carbosulfan, propineb and benomyl. Fish had cell necrosis, degeneration and oedemas in liver, trunk kidney and spleen. None of these lesions were seen in control fish.     

Response of Ig-positive cells to Enteromyxum scophthalmi (Myxozoa) experimental infection in turbot, Scophthalmus maximus (L.): A histopathological and immunohistochemical study

In recent years, a new parasite that causes severe losses has been detected in farmed turbot, Scophthalmus maximus (L.), in Northwestern Spain. Dead fish showed emaciation and cachexia caused by severe necrotizing enteritis that affected all portions of the digestive tract. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. This study was set up to gain insights into the immune response of fish against this parasitic infection. The kinetics of immunoglobulin positive (Ig+) cells in spleen, kidney and intestine from turbot experimentally infected with E. scophthalmi was studied. For evaluating both the progress of infection and the lesions induced by the parasite, we performed histopathological studies and for detecting Ig+ cells in situ we used an indirect immunohistochemical method. In fish exposed to the parasite, the number of Ig+ cells significantly increased in the intestine, the target organ of the parasite, whereas in spleen and kidney, haematopoietic organs where the parasite was not detected, the number of Ig+ cells decreased. Furthermore, the pattern of distribution of Ig+ cells changed in all three organs examined in recipient/infected fish compared with control fish (not exposed to the parasite). The results obtained in this study indicate that the infection by E. scophthalmi in turbot induced an immune response that involved changes in the number and distribution of Ig+ cells.     

The effect of sublethal concentrations of the molluscicide niclosamide on the infectivity of Schistosoma mansoni cercariae.

Experiments were conducted to assess the effect of sublethal concentrations of niclosamide on the infectivity of Schistosoma mansoni cercariae. Exposure of cercariae to 0.02 mg/l and 0.05 mg/l of niclosamide, respectively, for only two hours increased their mortality during penetration of mammalian host skin. The observed increase in mortality in the skin resulted in a consequent reduction of adult worm recovery from the liver and mesenteric veins of animals infected with the treated cercariae.     

Toxic effects of detergents on the alga Plagioselmis prolonga (Cryptophyta)

The effect of sodium dodecyl sulfate (SDS) and the household synthetic detergents (HSDs) Kristall and Tix (0.1, 1, and 10 mg/l) on cell motility, cell number dynamics, and the growth rate of the alga Plagioselmis prolonga (Cryptophyta) is studied. Algal cell motility proved to be the most sensitive indicator of detergent toxicity. SDS was the least toxic: 1 mg SDS/l caused a short-term loss of motility in 10% of the algal cells. The HSD Tix was the most toxic: only 70% of the cells recovered motility after a 24 h exposure to 1 mg/l. The substances tested in a concentration of 10 mg/l caused mortality of the P. prolonga population. According to their toxic effect on P. prolonga, the investigated toxicants can be arranged as follows: SDS < Kristall < Tix.     

Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass

When juvenile turbot Scophthalmus maximus and sea bass Dicentrarchus labrax were fed to satiation, growth and food intake were depressed under hypoxia (3·2±0·3 and 4·5±0·2 mg O₂ l^-1). However, no significant difference in growth was observed between fishes maintained in hypoxia and fed to satiation and fishes reared in normoxia (7·4±0·3 mg O₂ l^-1) and fed restricted rations (same food intake of fishes at 3·2 mg O₂ l^-1). Routine oxygen consumption of fishes fed to satiation was higher in normoxia than in hypoxia due to the decrease in food intake in the latter. Of the physiological parameters measured, no significant changes were observed in the two species maintained in hypoxia. This study confirms the significant interaction between environmental oxygen concentrations, feeding and growth in fishes. Decrease in food intake could be an indirect mechanism by which prolonged hypoxia reduces growth in turbot and sea bass, and may be a way to reduce energy and thus oxygen demand.     

Disposition of 14C-flumequine in eel Anguilla anguilla, turbot Scophthalmus maximus and halibut Hippoglossus hippoglossus after oral and intravenous administration.

The absorption, distribution and elimination of 14C-labelled flumequine were studied using whole body autoradiography and liquid scintillation counting. Flumequine was administered to eel Anguilla anguilla, turbot Scophthalmus maximus and halibut Hippoglossus hippoglossus intravenously and orally as a single dose of 5 mg kg(-1), corresponding to 0.1 mCi kg(-1). The turbot and halibut studies were performed in salt water (salinity of 32%) at temperatures of 16 +/- 1 degrees C (turbot) and 9.5 +/- 0.5 degrees C (halibut). The eel study was conducted in fresh water at 23 +/- 1 degrees C. In the intravenously administered groups flumequine was rapidly distributed to all major tissues and organs. After oral administration flumequine also appeared to have rapid and extensive absorption and distribution in all 3 species. After the distribution phase, the level of flumequine was higher in most organs and tissues than in the blood, except in muscle and brain. The most noticeable difference between the species was the slow elimination of flumequine from eel compared to turbot and halibut. In orally administered eels, substantial amounts of flumequine remained in all major organs/tissues for 7 d. At 28 d significant levels of flumequine were present in liver, kidney and skin (with traces in muscle), and at the last sampling point (56 d) in eye, bone, bile and posterior intestine. In orally administered turbot significant levels of flumequine were observed over 96 h in bile, urine, bone, skin, intestine and eye, and traces were detected over 28 d in bone and eye in addition to a significant level in bile. In orally administered halibut, significant levels of flumequine were observed in bile, skin, intestine and eye over 96 h. Traces were present in skin and eye over 7 d. The maximal flumequine concentrations in blood were calculated to be 2.5 mg equivalents l(-1) (eel at 12 h), 0.8 mg l(-1) (turbot at 6 h) and 0.6 mg l(-1) (halibut at 6 h) after oral administration.     

Morpho-histochemical changes in the liver and intestine of young giltheads (fish-nursery), Sparus aurata, L., induced by acute action of the anionic tensioactive alkylbenzene sulphonate.

In the present study we have assessed the effect on the survival and the morpho-histochemical changes in the liver and intestine of young giltheads (fish-nursery), Sparus aurata, L., induced by acute action of the anionic tensioactive, alkyl benzene sulphonate (ABS). Firstly, the LC50 of ABS at 96 hours was found to be 0.6 mg/L. Secondly, lots with 50 young giltheads (fish-nursery) were exposed to ABS concentrations of 0.5, 1, 3 and 5 mg/L, to obtain the surface tension value and exposure time required for 50% mortality of the specimens at each tested concentration. Exposure to ABS caused several forms of histopathological damage in the liver (the radial arrangement of hepatocytes was lost) and intestine (destruction of the structure of villi and increase in thickness of the other three layers). In addition, changes in bio-macromolecule components (proteins in general, siderophile proteins, neutral mucopolysaccharides, glycogen and acid mucopolysaccharides) were observed. The degree of these alterations was dependent upon the ABS concentration. These changes could have detrimental effects on the growth and survival of the species.     

Effects of Juvenile Hormone and Precocene on the Reproduction of Brachionus calyciflorus

We studied the effects of juvenile hormone and precocene on reproduction of the rotifer Brachionus calyciflorus. Amictic females of B. calyciflorus that were 2‐4 hours old were exposed to different concentrations of juvenile hormone (0.004, 0.02, 0.1, 0.5, 2.5, 12.5 mg/L) and/or precocene (0.05, 0.25, 0.75, 3.75, 7.5 mg/L) for 24 h. They were then transferred to a new medium without hormone and checked every 2 h during the next 48 h, and thereafter monitored daily until the individual died. Precocene had no effects on the length of the rotifer juvenile period, hatching time of the first neonate, lifetime reproduction, or the mixis ratio. In contrast, juvenile hormone at 0.5, 2.5, and 12.5 mg/L significantly prolonged the juvenile period by 6.1, 9.2, and 8.6%, respectively. When 26‐28‐h‐old amictic females were exposed to the same concentration series of juvenile hormone or precocene, precocene at 3.75 mg/L resulted in an increase in lifetime reproduction of 30.39%. However, at 0.75 and 3.75 mg/L precocene, a significantly lower percentage of mictic females was found, whereas juvenile hormone had no effect on the lifetime reproduction or mixis ratio. The population growth test showed that juvenile hormone had significant effects on the population growth rate and mixis ratio, but no effect on resting egg production. In comparison, precocene had no effect on any of these parameters. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Short-term physiological changes in turbot and seabream juveniles exposed to exogenous ammonia

Seabream and turbot juveniles (40-520 g) were exposed to constant exogenous NH5-N concentrations (1.27-4.27 mmol/l; pH, 8.15). In 96 hr acclimated fish, plasma TA-N (total ammonia nitrogen) contents were positively correlated to ambient ammonia concentrations. The LD50 were 2.2-2.5 mmol/l TA-N in both species. There were no marked osmoregulatory disturbances and plasma urea-N, thyroid hormones levels and gill (Na-K)-ATPase activities were only affected at the highest concentrations. Liver GOT, GPT and GIDH activity dose-response were low and species dependent. In cannulated and non-cannulated turbot exposed to half 96 hr LC50 (lethal ambient concentration for 50% of the population), there was a rapid, pronounced and prolonged increase in plasma TA-N, followed by an immediate decline when exogenous ammonia supply was stopped. Maximum loading and unloading were observed within 1-3 hr. Plasma cortisol levels indicated a stressful situation in exposed fish (150 ng/ml) and a quick recovery capacity. In dose and time response experiments, the most relevant physiological indicator of ammonia stress was blood TA-N content. Other parameters tested led either to transient or low amplitude responses except when fish approached death.     

Composition of the seminal Fluid and ultrastructure of the spermatozoon of turbot (Scophthalmus maximus)

Mean values of the composition of the seminal fluid of 30 turbots were: osmotic pressure =306 mosml⁻¹, pH =7–31, total protein =8.8mg ml¹, Na⁺=133.0 mmoll¹, K⁺=3.80mmoll⁻¹, Cl⁻=129mmol l⁻¹. The rounded nucleus, the reduced middle piece and the typical '9 + 2' structure of the flagellum mean that the spermatozoon of turbot can be considered to be of a primitive type.     

Histopathological changes induced by maneb and carbaryl on some tissues of rainbow trout, Oncorhynchus mykiss

Acute toxicity of the pesticides, maneb and carbaryl, to juvenile rainbow trout were evaluated under static-renewal test conditions. Actual concentrations of maneb ranged from 0.10 mg/L to 2.00 mg/L and carbaryl ranged from 0.20 mg/L to 3.90 mg/L. The concentrations of maneb that killed 50% of the rainbow trout (3.27 ± 0.9 g) within 24-h (24-h; LC₅₀), 48-h, 72-h and 96-h were 1.19 ± 0.12, 1.04 ± 0.11, 0.92 ± 0.12 and 0.81 ± 0.14 mg/L (95% confidence limits), respectively. LC₅₀ values of carbaryl for 24-h, 48-h, 72-h and 96-h were 2.52 ± 0.71, 2.16 ± 0.63, 1.71 ± 0.46 and 1.39 ± 0.15 mg/L, respectively. None of the unexposed control fish died and the first fish died 6 h after exposure to maneb (≥1.30 mg/L), and carbaryl (≥2.60 mg/L). Lamellar edema, separation of epithelium from lamellae, lamellar fusion, swelling of the epithelial cells and epithelial cell necrosis were observed on maneb and carbaryl exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to pesticides had inflammation and focal necrosis in liver, trunk kidney and spleen. Maneb and carbaryl had similar histopathological lesions. In order, the most affected organs were gill, trunk kidney and liver.     

Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus

Growth of Vibrio alginolyticus was not affected by TSB medium containing ammonia-N concentration in the range of 0-20 mg l(-1). White shrimp Litopenaeus vannamei (7-12 g in the intermolt stage) were challenged with V. alginolyticus, which had been incubated for 24 h in the TSB medium containing different concentrations of ammonia-N (0, 1, 5. 10 and 20 mg l(-1)). There was no significant difference in cumulative mortality for shrimp incubated in the TSB medium containing 0, 1, 5, 10 and 20 mg l(-1)ammonia-N after 120 h of challenge. The shrimps were challenged with V. alginolyticus previously incubated in the TSB medium for 24 h, then placed in water containing concentrations of ammonia-N at 0.01 mg l(-1)(control), 1.10, 5.24, 11.10 and 21.60 mg l(-1). Mortality of shrimp in 5.24, 11.10 and 21.60 mg l(-1)was significantly higher than those in the control solution (0.01 mg l(-1)) after 48-168 h. Shrimps which had been exposed to control, 1.10, 5.24, 11.10 and 21.60 mg l(-1)ammonia-N for 7 days were examined for THC (total haemocyte count), granular cells, hyaline cells, phenoloxidase activity, release of superoxide anion, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V. alginolyticus. No significant difference in THC, hyaline cells and granular cells were observed among shrimps at different ammonia-N concentrations. Phenoloxidase activity however, decreased when the shrimps were exposed to 5.24 mg l(-1)ammonia-N and greater after 7 days. The release of superoxide anion increased significantly, whereas SOD activity decreased significantly at 21.60 mg l(-1)ammonia-N. With shrimps exposed to 11.21 and 21.22 mg l(-1)ammonia-N for 7 days, phagocytic activity and clearance efficiency to V. alginolyticus significantly decreased. It is therefore suggested that ammonia in water caused a depression in the immune response and an increase in mortality of L. vannamei from the V. alginolyticus infection.     

Effects of fluoxetine exposure on serotonin-related activity in the sheepshead minnow (Cyprinodon variegatus) using LC/MS/MS detection and quantitation

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor and is among the top 100 drugs prescribed yearly in the United States and the United Kingdom. Tissue and water extraction methods were developed to detect and quantify FLX, norfluoxetine and the associated biological compounds serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), tryptophan (TRP) and melatonin (MEL) using LC/MS/MS. Acute mortality and sublethal physiological effects of FLX were assessed using standard static renewal toxicity tests in which juvenile sheepshead minnows (Cyprinodon variegatus) were exposed to FLX. Fluoxetine did not cause significant mortality at levels near currently reported environmental concentrations. Significant changes in neurotransmitter levels were observed within the serotonergic system in juvenile sheepshead minnows exposed at concentrations approximately one order of magnitude above those currently reported in the environment. Transformation activity ratios of a product to a precursor compound (5-HT/TRP, 5-HIAA/5-HT and MEL/5-HT) also exhibited significant changes with FLX treatment. Fluoxetine exposure did not only affect 5-HT but had additional effects both upstream and downstream of 5-HT within its synthesis and metabolic pathways. These sublethal changes within the serotonergic pathway may result in behavioral changes which could, in turn, have implications for the ecological response of populations to additional environmental stressors.     

Effects of salinity on the ionic balance and growth of juvenile turbot

The effects of salinity changes (27, 19 and 10‰) on seawater-adapted juvenile turbot were studied on their plasma osmolarity and ion concentrations, on oxygen consumption, on gill Na⁺,K⁺-ATPase activity after 3 months and on growth parameters. All plasma concentrations (except chloride) were unchanged, suggesting that fish were well adapted to their environment. Oxygen consumption was significantly decreased in the 19 and 10‰ groups, where fish weighed significantly more 105 days after transfer than fish maintained in sea water. These results, and the fact that apparent food conversion rates were lower in a diluted environment, suggest that on a long term schedule growth conditions could be improved by adaptation to brackish waters (salinities between 10 and 19‰). The effects of transfer from sea water to 27, 19, 10 and 5‰ were also followed during the first 3 weeks. With salinity 10‰ a steady state was reached on day 21 with all plasma values within the same range. The significant differences observed in osmolarity, plasma ion concentrations and Na⁺,K⁺-ATPase activity 3 weeks after transfer of juveniles to 5‰ salinity, compared with transfers in higher salinities, suggest that there is a threshold of acclimation of turbot to a hypotonic environment.     

Physiological responses to handling in the turbot

Turbot Scophthalmus maximus were cannulated via the afferent branchial artery and were either net-confined in sea water or in air for 9 min to monitor the stress response of a hatchery-reared marine flatfish. No mortality was observed. Aerial exposure appeared to mobilize plasma free fatty acids and stimulate the interrenal tissue but had no effect on circulating glucose or lactate levels. This pattern was qualitatively similar to that induced by net-confinement in sea water, although the magnitude and duration of the changes were more marked in the turbot handled in sea water. Aerial exposure had no effect on plasma osmolality, protein, or Cl⁻ concentrations and only a minor effect on plasma Na⁺ concentrations, which was in sharp contrast to the ionoregulatory disturbance noted in turbot which were net-confined in sea water. However, plasma K⁺ and cortisol concentrations were similarly elevated by both handling procedures. The results from these experiments suggest that whilst similarities with the salmonid physiological stress response are apparent, the lack of a plasma glucose response may represent a fundamental difference in turbot stress physiology.     

Effect of ammonia on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalone Haliotis diversicolor supertexta held in 30 parts/per thousand seawater and 26 degrees C were injected with TSB-grown Vibrio parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then placed in water containing different concentrations of ammonia-N (un-ionized plus ionized ammonia) at 0.01 mg l(-1) (control), 1.12, 3.22, 5.24 and 10.18 mg l(-1). Mortality of abalone increased directly with ambient ammonia-N concentration. After 12 h, the mortality of V. parahaemolyticus-injected abalone held in 3.22 mg l(-1) ammonia-N was significantly higher than those placed in 1.12 mg l(-1) ammonia-N and the control solution. In another experiment, the abalone which had been exposed to control, 1.08, 3.16, 5.37 and 10.34 mg l(-1) ammonia-N for 24, 72 and 120 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), and phagocytic activity and clearance efficiency to V. parahaemolyticus. The abalone when exposed to 3.16 mg l(-1) ammonia-N had decreased THC after 72 h, and decreased phenoloxidase activity, phagocytic activity and clearance efficiency after 24 h. However, the abalone when exposed to 3.16 mg l(-1) ammonia-N had increased respiratory burst after 24 h. The immune parameters except superoxide anion seemed to be suppressed in a dose-dependent fashion after 24 h. It is concluded that ammonia caused a depression in immune parameters and an increase in mortality of H. diversicolor supertexta from V. parahaemolyticus infection.