Activities of biomarkers in multiple life stages of the model crustacean, Palaemonetes pugio

Increased urbanization of coastal areas has led to increased contaminant levels in adjacent sediments and waters. Consequently, many studies have been conducted to determine the potential impacts on estuarine organisms, including the grass shrimp, Palaemonetes pugio. This study investigated baseline levels of four cellular biomarkers (glutathione (GSH), lipid peroxidation (LPx), acetylcholinesterase (AChE), and cholesterol (CHL)) in multiple life stages (stage IV, V, VI, and VII embryos, newly-hatched larvae, 18-day-old larvae, juveniles, and adults) of P. pugio to determine which biomarkers may potentially be useful as indicators of contaminant exposure. There was a similar pattern in both LPx and AChE levels, with a clear increase occurring from the embryonic to the adult stages. Detectable levels of AChE did not occur until embryo stage V. Glutathione shared a similar pattern to LPx and AChE from the newly-hatched larvae through the adult stage, however, it did not exhibit any distinguishable pattern overall with highly variable levels among all life stages. Likewise, CHL did not exhibit any distinguishable pattern, but in contrast, CHL levels were similar throughout all life stages. This research provides valuable background information that may be used in future assessments of grass shrimp population health.     

Effect of temperature and salinity on acetylcholinesterase activity, a common pollution biomarker, in Mytilus sp. from the south-western Baltic Sea

Acetylcholinesterase (AChE) activity is a well established biomarker to monitor environmental pollution caused by neurotoxic compounds, such as organophosphorus and carbamate pesticides. The presence of these compounds results in a measurable inhibition of the enzyme. This has been shown for different marine species including blue mussels. Besides pollution, environmental variables may also have a direct or indirect effect on AChE activity, particularly in estuarine and brackish water environments. To assess the impact of abiotic factors on the AChE activity the seasonal course of gill AChE activity was followed in relation to temperature and salinity in Mytilus sp. collected from the south-western Baltic Sea. In addition, the effect of salinity on AChE activity was investigated in an experimental study. The AChE activity showed significant seasonal differences with maximum activities during the summer period and minimum activities in winter. These changes correlate significantly (p<0.001) and positively with water temperature. The experimental exposure of Mytilus sp. to different salinities (5, 7, 16 or 20 psu) resulted in changes in the gill AChE activity. Empirical orthogonal function (EOF) analysis revealed that AChE activity was significantly and negatively correlated with salinity (p<0.01). These results clearly demonstrate the need to consider the potential influence of temperature and salinity on AChE activity when applying this biomarker to monitor exposure to and effect of neurotoxic substances in estuarine and brackish water blue mussels.     

Effects of salinity and temperature on the expression of enzymatic biomarkers in Eurytemora affinis (Calanoida, Copepoda)

In order to establish effective enzymatic biomarkers that could provide in situ early warning of contaminant exposure in estuarine ecosystems, the potential effects of the principal abiotic factors (temperature and salinity) were investigated on common biomarkers, the acetylcholinesterase (AChE) and the glutathione S-transferase (GST) in Eurytemora affinis. Short term salinity stress effects simulated during an experimental tide indicated that enzymatic activities of this species are characterized by maximum expression related to an optimal salinity range (between 5 and 15 psu). Moreover, longer time exposure to various salinity tanks confirmed the effects of this factor on both AChE and GST activities. Therefore, optimal AChE activity was measured at 10 psu, while optimal GST activity was measured at 5 psu. Furthermore, significant effects of temperature were also recorded, particularly for AChE expression (slight effects were measured on GST expression) with an optimal condition at 11 degrees C. These experiments indicated a more pronounced effect of salinity over temperature especially on the AChE expression and confirmed the need to standardize sampling procedures in relation with environmental parameters for biomonitoring studies based on enzymatic analyses.     

Biomarker-enhanced assessment of reproductive disorders in Monoporeia affinis exposed to contaminated sediment in the Baltic Sea

Introducing biomarkers into monitoring programs requires understanding of their responses in relation to higher-level biological effects as well as modulating effects of confounding environmental factors. We evaluated relationships between the general toxicity biomarkers (acetylcholinesterase [AChE], lysosomal membrane stability [LMS], oxygen radical absorbance capacity [ORAC]) and reproductive performance (fecundity and embryo aberrations) in the amphipod Monoporeia affinis in the Baltic Sea. To further link biomarker response to contaminant (PCBs, PAHs and metals) levels in the surrounding sediments as well as environmental factors (salinity, bottom depth and total organic carbon in sediments [TOC]), correlation and partial least square regression (PLSR) analyses were applied. The observed contaminants levels were frequently elevated for heavy metals and PAHs, but not PCBs. In the amphipod populations, female ORAC values were positively related to the occurrence of females carrying malformed or membrane-damaged embryos and to the percentage of such embryos in their broods, but also to the fecundity. Female AChE activity was negatively related to the frequency of the membrane-damaged embryos, and positively to the frequency of embryos with arrested development in the broods. Moreover, higher AChE activity and ORAC values in the females occurred at elevated concentrations of metals and PAHs, while there was a negative correlation between embryo ORAC and some PCB congeners. The PLSR models explained over 80% of the variation in the female ORAC and AChE values by variation in contaminant concentrations in combination with environmental variables. Specifically, CB180 and PAM4,9 were identified as negative predictors for ORAC, whereas many PAHs and some metals were positive predictors. The AChE activity was positively related to some metals and negatively to PCBs. In the PLSR models, environmental factors had significant modulating effects, with positive effect of salinity on female ORAC and AChE, and negative effect of TOC on the AChE. The LMS data were less informative, with no apparent relation to any of the contaminants. Linking subcellular responses to the reproduction effects facilitates environmental stress assessment and understanding of the response mechanisms, but also calls for more experimental and field data providing a mechanistic understanding to these linkages.     

绿原酸对凡纳滨对虾抗氧化系统及抗低盐度胁迫的影响

对绿原酸调节凡纳滨对虾(Litopenaeus vannamei)血淋巴抗氧化系统功能及抗低盐度胁迫的效果进行了评价。360尾凡纳滨对虾随机分为4组,分别投喂含有0、100、200和400 mg/kg绿原酸的饲料28 d,随后将对虾从盐度为32的天然海水直接转入至盐度为10的水中胁迫72 h。结果表明,在正常养殖条件下,绿原酸对凡纳滨对虾的成活率、血淋巴总抗氧化能力(Total antioxidative capacity, T-AOC)、超氧化物歧化酶(Superoxide dismutase, SOD)及过氧化氢酶(Catalase, CAT)活力均无明显影响,然而投喂含有绿原酸的饲料14 d,对虾血淋巴谷胱甘肽过氧化物酶(Glutathione peroxidase, GPx)活性和血淋巴细胞GPx和CAT基因表达水平均显著高于对照组(P<0.05);低盐度胁迫24 h,绿原酸组凡纳滨对虾的存活率较对照组提高10%,但各组之间无显著性差异(P>0.05);低盐度胁迫24 h,各组凡纳滨对虾血淋巴T-AOC、SOD和GPx活性与胁迫前相比均显著增加,说明低盐度胁迫条件下机体产生了抗氧化胁迫反应,同时绿原酸组对虾血淋巴GPx、CAT活性均显著高于对照组(P<0.05);低盐度胁迫72 h,绿原酸组对虾血淋巴T-AOC、GPx和CAT活性和血淋巴细胞GPx和CAT基因表达水平均明显高于对照组。上述结果表明绿原酸可有效调节凡纳滨对虾的抗氧化系统功能,增强对虾对于低盐度胁迫下的适应能力。     

Biomarker responses and accumulation of hazardous substances in mussels (Mytilus trossulus) transplanted along a pollution gradient close to an oil terminal in the Gulf of Finland (Baltic Sea)

Baltic Sea blue mussels (Mytilus trossulus) were used as sentinel organisms to detect the biological effects of chemical contamination in the low salinity environment. Mussels naturally adapted to a salinity of ca. 6.0 PSU were caged for 30 days at four sites along an assumed pollution gradient (salinity ca. 4.5 PSU) in the vicinity of Finland's largest oil refinery and harbor Kilpilahti in the Gulf of Finland. Tissue concentrations and accumulation rates of especially organic contaminants (PAHs, PCBs and organotins) were clearly elevated at the innermost coastal stations near the harbor area. Biological effects of contaminant exposure on caged mussels were evaluated by measuring a suite of biomarkers including catalase, glutathione S-transferase, superoxide dismutase, glutathione reductase, lipid peroxidation, acetylcholinesterase activity and lysosomal membrane stability. Mussels transplanted near the harbor area were able to elevate their antioxidant defense in response to environmental contamination. Reduced morphometric condition index and soft tissue growth rate together with increased lipid peroxidation and low lysosomal membrane stability were also observed at the most contaminated site. The results suggest that caging of M. trossulus for four weeks at lower salinity is a feasible method for the detection of environmental pollution also in low salinity areas of the Baltic Sea.     

Ecological niche differentiation between native and non-native shrimps in the northern Baltic Sea

Invasions of non-native species are modifying global biodiversity but the ecological mechanisms underlying invasion processes are still not well understood. A degree of niche separation of non-native and sympatric native species can possibly explain the success of novel species in their new environment. In this study, we quantified experimentally and in situ the environmental niche space of caridean shrimps (native Crangon crangon and Palaemon adspersus, non-native Palaemon elegans) inhabiting the northern Baltic Sea. Field studies showed that the non-native P. elegans had wider geographical range compared to native species although the level of habitat specialization was similar in both Palaemon species. There were clear differences in shrimp habitat occupancy with P. elegans inhabiting lower salinity areas and more eutrophicated habitats compared to the native species. Consequently, the non-native shrimp has occupied large areas of the northern Baltic Sea that were previously devoid of the native shrimps. Experiments demonstrated that the non-native shrimp had higher affinity to vegetated substrates compared to native species. The study suggests that the abilities of the non-native shrimp to thrive in more stressful habitats (lower salinity, higher eutrophication), that are sub-optimal for native shrimps, plausibly explain the invasion success of P. elegans.     

Variation in penaeid shrimp growth rates along an estuarine salinity gradient: Implications for managing river diversions

Freshwater inflows from river diversions may affect nekton populations by altering the salinity and temperature of estuarine waters. To investigate the influence of these environmental variables on the growth and survival rates of brown shrimp Farfantepenaeus aztecus and white shrimp Litopenaeus setiferus, we conducted field experiments in May and September 2007 to expose experimental animals to the range of different combinations of salinity and water temperature that commonly occur in an estuarine environment. Growth rates for shrimp held in mesocosms for approximately 7 days were compared among four locations and three treatments; locations were identified by the dominant marsh vegetation and distance from the Gulf of Mexico (low to high salinity: Intermediate, Brackish, Saline UE = Saline Up Estuary, Saline DE = Saline Down Estuary). At each location, the treatments were replicated four times and included shallow water with additional food, shallow water without food added, and deeper water (an attempt to expose animals to lower temperatures). Our experiments were designed to test the null hypothesis that shrimp growth and survival rates did not differ by location or treatment. Both brown shrimp and white shrimp grew more slowly at the Intermediate than higher salinity locations. Potential prey (benthic infauna) biomass was relatively low at both the Intermediate and Brackish locations in May, and both shrimp species consistently grew faster in mesocosms where food was added. We conclude that reduced growth in low salinity environments is likely due to the combined effects of increased metabolic costs and less food in these areas. River diversions that reduce estuarine salinities over a large portion of available habitat during peak recruitment periods may reduce overall growth rates and shrimp productivity in the affected areas.     

Effects of hypoxia on osmotic and ionic regulation in the brown shrimp Crangon crangon (L.) from brackish water

When exposed to hypoxia (P_wO₂, <P_cr) and low salinity conditions, the shrimp, Crangon crangon (L.) shows a progressive loss of blood chloride until a new, stable value, still higher than that of the medium, is reached. Simultaneously, circulating levels of calcium progressively increase. Under identical experimental conditions, circulating levels of sodium and magnesium and the blood total osmolarity (Δ °C) are unchanged. The possible explanations for such changes are considered along with their adaptive significance.     

An energetic and conceptual model of the physiological role of dietary carbohydrates and salinity on Litopenaeus vannamei juveniles

We are reporting results directed to explain the relation between carbohydrates (CHO), protein metabolism, and the energetic balance of Litopenaeus vannamei juveniles. The interaction of dietary CHO and salinity was measured to try to understand the relation between osmotic control and metabolism, both from a biochemical and energetic point of view. Two experiments were done. In the first experiment, shrimp were fed with 0%, 5%, 33%, and 61% CHO and maintained at 15‰ and 40‰ salinity. Glucose, lactate protein, hemocyanin, ammonia concentration, and osmotic pressure were measured in blood. Digestive gland glycogen (DGG) was measured also. In the second experiment, shrimp were fed with 0% and 38% dietary CHO and maintained at 15‰ and 40‰ salinity. From that shrimp, absorbed energy (Abs) was calculated as: Abs=respiration (R)+ammonia excretion (U) and production (P); assimilated energy (As) was calculated as the product of R×P. Osmotic pressure, hemocyanin, protein, lactate, and blood ammonia increased with the reduction in dietary CHO. In contrast, an increase in blood glucose was observed with an increase in dietary CHO. Digestive gland glycogen (DGG) increased following a saturation curve with a DGG maximum at 33% dietary CHO. Blood metabolites of fasting and feeding shrimp showed the same behavior. Energy balance results showed that shrimp maintained in low salinity and fed without CHO waste more energy in U production than for shrimp maintained in high salinity and fed with high CHO levels. Notwithstanding, the production efficiency was higher in shrimp fed without CHO than that observed in shrimp fed with high CHO independent of salinity. A scheme trying to integrate the relation between CHO and protein metabolism and the way in which both are modulated by salinity is presented. From published and present results, there are two factors that apparently control the use of high dietary CHO levels; α-amylase enzyme-dietary CHO level capacity and glycogen saturation in DG. Production of glucose is limited in shrimp because of saturation of α-amylase when shrimp are fed with diets above 33% CHO. This is the first control point of starch metabolism. The digestive gland is saturated with glycogen in shrimp fed with dietary CHO levels >33%. This is apparently the second control point of CHO metabolism that limits growth rate in such conditions. The high metabolic cost related to high CHO diets could explain why shrimp are well adapted to use protein as a source of energy.     

The use of Fulvia fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: A mutimarkers approach

Five biomarkers, catalase (CAT) activity, glutathione-S-transferase (GST) activity, the neural transmitter enzyme acetylcholinesterase (AChE), reduced glutathione (GSH) and malondialdehyde (MDA) levels were measured in specimens of Fulvia fragilis collected from Bizerta lagoon (Tunisia). Results demonstrated that F. fragilis showed differential biomarker response according to the importance of the anthropogenic pressure and the nature of pollutants that affect the lagoon. A clear organotropism was also observed with a higher biomarker response in digestive gland than in gills of this bivalve. These results indicate that F. fragilis constitutes a useful tool as sentinel organism for biomonitoring of aquatic pollution.     

Modular estuarine mesocosm validation: ecotoxicological assessment of direct effects with the model compound endosulfan

This study represents the first in a series of validation experiments for the modular estuarine mesocosm testing direct pesticide effects. Endosulfan, an agricultural insecticide, was selected as a model contaminant for studying direct toxic effects as well as uptake of this model contaminant by estuarine biota. The grass shrimp, Palaemonetes pugio, and the mummichog, Fundulus heteroclitus, showed significant mortality (96 h LC₅₀: 0.12 and 2.2 μg/l, respectively) to endosulfan as predicted by laboratory bioassays. There was no effect on fiddler crabs and eastern oysters. The mesocosm was also useful in demonstrating the bioconcentration of endosulfan by eastern oysters (bioconcentration factor [BCF]=375) similar to results reported in field studies. This study illustrates the modular estuarine mesocosms' ability to detect direct effects of pesticide exposure and the uptake of a pesticide by estuarine fauna.     

Use of Biomarkers in Oil Spill Risk Assessment in the Marine Environment

Numerous molecular, cellular, and physiological biomarkers have been used to assess the responses of marine animals to petroleum compounds. To be used in ecological risk assessment after an oil spill, a biomarker response needs to be linked to petroleum exposure and not strongly influenced by internal and external confounding factors. Biomarker responses to petroleum PAH, dominated by alklated two-and three-ringed aromatics, can be quite different than responses to pyrogenic PAH, dominated by four-and five-ringed aromatics. In many field sites there is a mixture of petrogenic and pyrogenic PAH, along with other contaminants, making it difficult to relate biomarker responses to a particular contaminant class. Biomarkers used to assess marine animal responses in the field include the cytochrome P450 system, heat stress protein, histopathology, and bile fluorescent compounds (FAC). Other biomarkers, including DNA/chromosomal damage and phase 2 enzymes, have been shown to respond after laboratory exposure, but more work needs to be done to demonstrate their usefulness in the field. One of the most useful biomarkers of petroleum exposure are the FAC responses in fish, which can be used to distinguish between petrogenic and pyrogenic PAH exposure. Few of the presently used biomarkers are linked to higher order biological effects, e.g. toxicity, reproductive failure.     

Brown shrimp (Crangon crangon, L.) functional response to density of different sized juvenile bivalves Macoma balthica (L.)

Variability in infaunal bivalve abundance in the Wadden Sea is largely determined by recruitment variability. Post-settlement, but pre-recruitment bivalve mortality is high and related to the occurrence of their most abundant predator, the brown shrimp Crangon crangon. To investigate if the mortality patterns of newly settled bivalves can be explained by the foraging behavior of brown shrimp, we carried out experiments on shrimp functional response to three size classes of juveniles of the Baltic Tellin Macoma balthica. The functional response curves for all three prey sizes (0.62 mm, 0.73 mm, and 0.85 mm) were the hyperbolic Holling's type II. The attack rate was highest for the smallest prey size (a = 0.31, medium and large prey a = 0.22); the handling time was longest for the largest prey size (T_h = 29 s, small and medium prey T_h = 15 s). Thus, a large body size is advantageous for the bivalves over the whole density range. Knowledge of individual foraging behavior is needed to model predation mortality of bivalves. The consumption rates in the experiment were theoretically high enough to account for M. balthica mortality in the field.     

Biomarkers in Ruditapes decussatus: a potential bioindicator species.

The clam Ruditapes decussatus is distributed worldwide and due to its ecological and economical interest has been proposed as a bioindicator in areas where mussels are not available. The accumulation of several anthropogenic compounds in their tissues suggests that they possess mechanisms that allow them to cope with the toxic effects of these contaminants. Besides pollutant uptake, the use of biomarkers is pointed out in this paper since it is a promising approach to monitor the effect of these contaminants in the marine environment. Biomarkers complement the information of the direct chemical characterization of different types of contaminants. Therefore, the aim of this paper is to review the role of several biomarkers: (metallothioneins (MT), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and selenium-dependent), lipid peroxidation (measured as MDA, one of the final products of lipid peroxidation), glutathione S-transferase (GST) and acetylcholinesterase (AChE), measured in different tissues of the clam R. decussatus, in laboratory conditions and under various environmental stresses, in two ecosystems (Ria Formosa lagoon- Portugal) and Bizerta lagoon (Tunisia) in a perspective of a multibiomarker approach to assess environmental changes. Experiment and field studies are in good agreement since MT levels, especially in the gills, the first target tissue of these contaminants, can be used as biomarker of exposure to Cd. GPx and MDA may also be determined in this respect. AChE activity is inhibited by pesticide and, to a less extent, by metal exposure in the gills and whole soft body of clams. However, the induction of GST isoforms experimentally demonstrated is not observed in the field because only global GST activity was determined. The whole set of results opens new research perspectives for the use of this species to assess the effect of mixtures of pollutants in the aquatic environment.     

Establishing Causality between Pollution and Effects at Different Levels of Biological Organization: The VALIMAR Project

The study summarizes the objectives of the VALIMAR project and gives selected examples of biomarker responses that allow causal relationships to be established between exposure and biological effects at different levels of biological organization. In this project, active and passive biomonitoring experiments with brown trout (Salmo trutta f. fario) and stone loach (Barbatula barbatula) were performed in two small streams in southern Germany between 1995 and 1999 in parallel with investigations on contaminant mixtures in the laboratory in order to evaluate the suitability of biomarkers representing different levels of biological organization for the assessment of pollution in small streams. In addition to biomarker studies, the morphology of the test streams was characterized and limnological and chemical parameters were monitored. Early life stage tests and ecological studies of brown trout and stone loach population demography, of the fish assemblages, and the macro- and meiozoobenthos communities in the two test streams were included in the project. Several causality criteria were addressed by means of combined (1) laboratory and field studies, (2) chemical, biological, and statistical investigations, and (3) in vivo and in vitro studies that allowed establishment of cause-effect relationships at different biological levels. The comparison of results obtained at these levels allowed identification of mechanisms responsible for the respective effects (coherence of association, biological plausibility). Finally, individual responses (biomarkers, bioindicators) could be extrapolated to higher biological levels (population, community) thus addressing the criteria of ‘time order’ and ‘coherence of association’.     

Effects of dibutyl phthalate and di‐ethylhexyl phthalate on acetylcholinesterase activity in bagrid catfish,Pseudobagrus fulvidraco (Richardson)

Acetylcholinesterase (AChE) activity is a well‐known biomarker for exposure to organophosphate or carbamate compounds in aquatic organisms. However, the effect of dibutyl phthalate (DBP) and di‐ethylhexyl phthalate (DEHP), widely used as a plasticizer, on the change of AChE activity is not yet known. Bagrid catfish Pseudobagrus fulvidraco were administrated with 100, 500 and 1000 mg kg^?1 diet of DBP or DEHP and the effects on AChE activity were assessed in the liver, gill, kidney, heart, brain, muscle and eye of the exposed fish. All tissues contained different background AChE activity in non‐treated bagrid catfish: the highest was observed in the brain, followed by muscle, heart, and kidney. The enzyme activities in various tissues were significantly inhibited after exposure to DBP or DEHP in a concentration‐dependent manner, especially in brain and muscle. A similar, but less pronounced, inhibition was also observed in liver and kidney when exposed to DBP and DEHP. Although AChE activity in gill and heart was also affected by DBP and DEHP, the decrease in these organs was least marked in these organs. Exposure to 1000 mg kg^?1 led to mortalities of 8.0% with DBH and 14% with DEHP; both seemed to be ascribable to phthalate toxicity. This study is the first report that the measurement of AChE activity in bagrid catfish is a valuable biomarker of DBP and DEHP exposure. This biomarker could be incorporated into a battery of biomarkers to strengthen the confidence with which ecotoxicologists can assess the impact of phthalate ester pollution in the aquatic environment.     

Weak meiofaunal trophic linkages in Crangon crangon and Carcinus maenus

To document the relative importance of meiofauna as prey for juvenile Crangon crangon and Carcinus maenus, short interval (1.5-2 h) collections were made in the muddy Lynher Estuary (Plymouth, Great Britain) and in the sandy-bottom Ythan Estuary (Aberdeenshire, Scotland) in 1990. Gut passage times of Crangon fed flaked fish food and fluorescent tracer in the laboratory at 13 °C ranged from 4 to 20 h. Wild shrimp exhibited feeding periodicity, with guts fullest during high tide in both locations. Visual and immunological gut contents analyses revealed that meiofaunal nematodes and harpacticoid copepods were present only in recently settled shrimp from 8 to 12 mm total length on muddy bottoms. Larger shrimp collectively consumed up to 33 different macrobenthic prey types. Shrimp were fullest at night (mean gut contents weight = 8% wet body weight, Lynher) or at dawn (6%, Ythan). The Lynher Carcinus gut contents—from animals 8 to 30 mm carapace width, examined visually only—contained mostly fluids, green benthic algae, sediment particles, and masses of unidentifiable prey remains plus digestion-resistant hard parts visually identifiable as macrobenthic in origin. None of the 203 crabs examined from the 24-h collection contained meiofaunal prey. Crangon shrimp probably eat meiofaunal prey for only a brief period of time after their initial settlement to the bottom. Evidence for significant top-down impacts on meiofauna from these two abundant shallow-water predators was weak. More trophic studies are needed on newly settled epibenthic predators to test the hypothesis that biological control of shallow-water meiofauna is important.     

Metallothionein and cellular energy allocation in the estuarine mysid shrimp Neomysis integer exposed to cadmium at different salinities

In the present study the induction of metallothioneins (MTs) and effects on the cellular energy allocation (CEA) in euryhaline crustacean Neomysis integer exposed to Cd at different salinities were studied. N. integer was exposed to the same sub-lethal concentration of free cadmium ion (1/5 of the cadmium activity of the reported 96 h LC₅₀ value) in hypo-osmotic (7.2 μg Cd/L at 5 psu), isosmotic (23.0 μg Cd/L at 16 psu) and hyper-osmotic media (38.1 μg Cd/L at 25 psu) for 7 days. By using the free Cd concentration as the basis for conducting the exposures, the effect of salinity on cadmium speciation was eliminated and therefore the true effect of salinity as an abiotic factor on the MT induction and CEA could be studied.MT content was quantified by differential pulse voltammetry (DPV); this is the first time that this method is applied to assays with N. integer. No significant differences in MT levels between the control and Cd-exposed groups were observed. The measured MT levels (ranging from 32.3 to 75.7 μg/mg_{(cytosolic protein)}) probably represent the constitutive MT levels responsible for binding essential metals. The differences in MT levels observed at the different salinities indicate a possible relationship between some physiological process (possibly osmoregulation) other than detoxification and MT induction. A decrease in salinity caused an increase in MT level in N. integer, and this was significantly correlated to the CEA values (Spearman correlation coefficient r = − 0.56, p = 0.016). Our results indicate that salinity can change the energy status and MT content of N. integer. These findings should be taken into account when using these biomarkers - and especially MTs - in field studies and environmental monitoring.     

Effects of temperature and salinity on the standard metabolic rate (SMR) of the caridean shrimp Palaemon peringueyi

The standard metabolic rate (SMR) of the caridean shrimp Palaemon peringueyi to changes in temperature (15-30 °C), salinity (0-45‰) and a combination thereof was investigated. The rate of oxygen consumption of the shrimp was determined using a YSI oxygen meter. At a constant salinity of 35‰ the respiration rate of P. peringueyi increased with an increase in temperature and ranged between 0.260 and 0.982 μl O₂ mg wwt^− 1 h^− 1. The Q₁₀ value over the temperature range 15-25 °C was estimated at 3.13. At a constant temperature of 15 °C the respiration rate of P. peringueyi also increased with an increase in salinity and ranged between 0.231 and 0.860 μl O₂ mg wwt^− 1 h^− 1. For combination experiments the absence of any significant difference in the respiration rate of P. peringueyi at the four temperatures over the salinity range 15-35‰ suggests that the shrimp is well adapted to inhabiting environments characterised by variations in salinity and temperature such as those encountered within the middle and lower reaches of permanently open estuaries with substantial freshwater inflow. On the other hand, the total mortality of the shrimp recorded at salinities < 5‰ at all four temperatures suggests that the upper distribution of the shrimp may reflect physiological constraints. Similarly, the increase in the respiration rate of the shrimp at the four temperatures at salinities > 35‰ suggests that the shrimp may experience osmotic stress in freshwater deprived permanently open and intermittently open estuaries where hypersaline conditions may develop.