Neutral Red Retention Research Articles

Sublethal effects induced by morphine to the freshwater biological model Dreissena polymorpha.

Opioids are considered as emerging contaminants in aquatic ecosystems, mainly due to their large illicit consume worldwide. Morphine (MOR) is the main opiate and it was commonly found at measurable concentrations in freshwaters. Even though its occurrence is well documented, just limited information is available regarding its hazard to nontarget organisms. The aim of this study was of the evaluation of sublethal effects induced by MOR to the freshwater bivalve Dreissena polymorpha. We exposed mussels to two MOR concentrations (0.05 µg/L and 0.5 µg/L) for 14 days and we investigated the sublethal effects by a suite of biomarkers. The Neutral Red Retention Assay (NRRA) was used as a test of cytotoxicity, while the oxidative stress was evaluated by the activity of antioxidant and detoxifying enzymes, namely catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and by measuring the levels of lipid peroxidation (LPO) and protein carbonylation (PCC). The genetic damage was assessed by the Single Cell Gel Electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test). Finally, the filtration rate of D. polymorpha was evaluated in order to investigate possible physiological effects. Both tested concentrations reduced the lysosome membrane stability of bivalves, but only the highest MOR concentration induced significant changes in the activity of antioxidant enzymes (SOD, CAT, and GPx) and increase in lipid peroxidation levels. Slight increase in primary DNA fragmentation was noticed, while no fixed genetic damage and alterations of the filtering rate were found.

Environmental concentrations of 3,4-methylenedioxymethamphetamine (MDMA)-induced cellular stress and modulated antioxidant enzyme activity in the zebra mussel.

Recent monitoring studies showed measurable levels of the 3,4-methylenedioxymethamphetamine (MDMA) in aquatic environments. However, no information is currently available on its potential hazard to aquatic non-target organisms. The aim of this study was to investigate the potential sub-lethal effects induced by 14-day exposures to low MDMA concentrations (0.05 and 0.5 μg/L) to zebra mussel (Dreissena polymorpha) specimens through the application of a biomarker suite. The trypan blue exclusion method and the neutral red retention assay (NRRA) were used to assess MDMA cytotoxicity. The activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), as well as the lipid peroxidation (LPO) and protein carbonyl content (PCC), were measured as oxidative stress indexes. The single cell gel electrophoresis (SCGE) assay, the DNA diffusion assay, and the micronucleus test (MN test) were applied to investigate DNA damage, while filtration rate was measured as physiological parameter. Despite significant decrease in lysosome membrane stability, hemocyte viability and imbalances in CAT and GST activities pointed out at the end of the exposure to 0.5 μg/L, no significant variations for the other end points were noticed at both the treatments, suggesting that environmentally relevant MDMA concentrations did not induce deleterious effects to the zebra mussel.

Utilizing an earthworm bioassay (Eisenia andrei) to assess a South African soil screening value with regards to effects from a copper manufacturing industry.

Metal contamination of soil due to industrialization has become an increasingly important problem in South Africa. This study aimed to investigate the potential impact of a copper (CuSO4·5H2O) production company on the soil environment. Bioassays using Eisenia andrei were performed to assess changes in biomass, reproduction and a biomarker, neutral red retention time, over a 28 day period. Earthworms exposed to soils from the Cu production site differed significantly (p < 0.05) from those exposed to soils 500 m and 5 km away in terms of the measured endpoints. These findings are consistent with the results from the chemical analysis which showed an elevated soil Cu content for both sites closest to the chemical production company compared to the reference site. The results confirm the importance and predictive value of using bioassays in conjunction with chemical analysis during soil quality assessments.

Responses of the earthworm Eisenia andrei exposed to sublethal aluminium levels in an artificial soil substrate

Industrial discharges of Al are increasingly common. In this study, the activities of three antioxidases, malondialdehyde (MDA) content and changes in coelomocytes were investigated in earthworms (Eisenia andrei) exposed to different concentrations of aluminium chloride (range 0–200 mg Al kg−1 dry soil) in artificial soils for 32 days. Within the first 16 days of Al exposure, catalase activity was elevated in most treatments, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were inhibited in all treatments. After 16 days exposure, SOD and GSH-Px activities returned to normal at lower Al concentrations, but were still inhibited at higher concentrations, hence the inhibition of SOD and GSH-Px showed some exposure-level dependence during the late exposure phase. MDA content was significantly elevated from 16 days after initial exposure. Neutral Red retention time of coelomocytes decreased at the highest exposure concentrations after 32 days, and an increased proportion of cells showing membrane damage was also observed at this time using flow cytometry. We conclude that excessive Al in soils can cause oxidative stress and cell damage in earthworms. Hence, Al accumulation in the environment may present an ecological hazard through suppression of the important functions of earthworms in soil ecosystems.

Effects of severe heat stress on immune function, biochemistry and histopathology in farmed Australian abalone (hybrid Haliotis laevigata×Haliotis rubra)

High summer temperatures are one of the most stressful environmental problems confronted by the abalone mariculture industry and are commonly associated with outbreaks of infectious disease. We tested the effect of extreme but non-lethal elevated temperatures on abalone immunology, biochemistry and quantitative histology. We subsequently compared the haemolymph results to the histology to gain increased understanding of how heat stress impacts abalone health. Abalone were kept in water that was heated from the ambient 16°C temperature to 26°C within 5h and then held at 26°C for one week to determine the effects of this acute heat stress on the day of temperature elevation and whether there was acclimatization or deterioration 2 and 7days later. Antibacterial activity, phenoloxidase activity and neutral red retention times declined significantly with heat and did not recover. The total haemocyte count was elevated significantly during heat stress and was highest on day 1. The phagocytic rate was elevated on day 1 but had recovered by the following day. Acid phosphatase activity, leucine aminopeptidase, haemolymph protein and haemolymph electrolytes (calcium, phosphate, magnesium, sodium, and chloride) were not significantly affected by heat stress. This indicates that severe heat stress causes changes in some, but not all haemolymph parameters. The sublethal immunologic effects seen in haemolymph samples occurred concurrently with histological changes. The digestive gland had significantly increased haemocyte infiltrates in heat stressed abalone. Heat stressed abalone had significantly greater loss of epithelium lining from the gills, with no recovery. The gill goblet cell numbers declined significantly on day 2 and had recovered by day 7. There was no significant change in the volume of fluid or protein concentration of the haemolymph in the gill sinuses between treatment groups. These results indicate that immunosuppression and organ damage are likely to be involved in the increased incidence of bacterial disease reported by abalone farmers during summer.

Short- and long-term responses and recovery of mussels Mytilus edulis exposed to heavy fuel oil no. 6 and styrene

Biomarkers have the potential to be used to assess the impact of anthropogenic discharges in marine waters. We have used a suite of biomarkers spanning from enzymatic to histopathological alterations and general stress responses to assess the short- and long-term impact on mussels Mytilus edulis of heavy fuel oil no. 6 and styrene. Mussels were exposed for 5 months, with a refilling of the exposure system, to a water soluble fraction of heavy fuel and, then, kept for a month in clean water for recovery. In a second experiment, mussels were exposed to styrene for 19 days and maintained in clean water for up to 4 months. Chemical body tissue levels reflected the weathering processes of these compounds. Acyl-CoA oxidase activity was induced in oil-exposed mussels after refilling, whereas styrene inhibited it after 19 days of exposure and after 2 weeks in clean water. Gamete development and alkali-labile phosphate levels suggest that neither oil nor styrene behaved as endocrine disruptors. Neutral red retention time was lower in treated groups than in controls. Lysosomal membrane stability was significantly reduced in exposed groups and recovered after withdrawal of oil but not after removal of styrene. Neither oil nor styrene exposure affected the condition index except for the reduction seen in mussels exposed to oil for 1 month. Biomarker response index discriminated exposed mussels, which showed higher values, and returned to control levels after recovery. Results obtained from these pilot experiments can help to identify relevant monitoring tools to assess the impact of oil and chemicals in marine spill scenarios.

In vitro effects of the nonsteroidal anti-inflammatory drug, ibuprofen, on the immune parameters of the colonial ascidian Botryllus schlosseri

In this study, in vitro effects of ibuprofen (IBU) on the immune parameters of the colonial ascidian Botryllus schlosseri were evaluated. Haemocytes were exposed for 1h to 0 (control), 100 and 1000μg IBU/L and the effects on haemocyte viability and morphology (shape factor), lysosomal membrane stability (Neutral Red Retention Assay), phagocytic activity, apoptosis (TUNEL reaction), hydrolytic (acid phosphatase) and oxidative (phenoloxidase and peroxidase) enzyme activities were evaluated. The exposure of haemocytes to IBU did not affect significantly their viability, but increased the percentage of cells with round shape. IBU caused a significant reduction in both phagocytic activity and lysosomal membrane stability. The percentage of haemocytes positive to TUNEL reaction (indicative of DNA fragmentation) increased significantly after IBU exposure. Significant decreases in the percentage of haemocytes positive to acid phosphatase were recorded at 1000μg/L of IBU. Conversely, no significant variations were recorded in the percentage of haemocytes positive to phenoloxidase and peroxidase. Results obtained indicate that exposure of ascidian haemocytes to IBU induces marked alterations in cell functionality. Immunomarkers measured in this study are sensitive, rapid and reproducible. However, their responsiveness and biological relevance will need to be verified for in vivo exposure.

Toxicity of copper oxide nanoparticles in the blue mussel, Mytilus edulis: A redox proteomic investigation

Relatively little is known about the fate and effects of nanomaterials even in relatively simple organisms such as Mytilus edulis. Here, copper oxide nanoparticles (CuO NP) are shown to induce dose-dependent toxic effects at the biochemical, physiological and tissue levels in the blue mussel. Stable CuO NP suspensions were sized by differential light scattering and nanoparticle tracking analysis to yield average particle diameters of approximately 100nm. These were administered to M. edulis, at doses of 400, 700 and 1000ppb. Ingested copper was predominantly located in the gill tissue with small amounts in digestive gland. Fifteen coomassie-stained spots were excised from two dimensional gel electrophoresis separations of gill tissue extacts and identified by peptide mass fingerprinting. These contained six unique proteins (alpha- and beta-tubulin, actin, tropomyosin, triosephosphate isomerase and Cu–Zn superoxide dismutase). Of these, two spots (actin and triosephosphate isomerase) showed decreased protein thiols while three (alpha-tubulin, tropomyosin and Cu–Zn superoxide dismutase) showed increased carbonylation which is indicative of protein oxidation of cytoskeleton and enzymes in response to CuO NP. The neutral red retention time (NRRT) assay revealed toxicity due to the CuO NPs which was comparable with toxic metal oxide nanoparticles such as chromium and cobalt. In contrast, non-toxic titanium and gold metal oxide nanoparticles gave no NRRT effects at similar NP concentrations. Histology revealed deposition of pigmented brown cells in response to CuO NP, located predominantly along the mantle and gill margin but also lining digestive tubules and some of the sinuses and distributed throughout the connective tissue and in the adductor muscle.

Interaction of citrate‐coated silver nanoparticles with earthworm coelomic fluid and related cytotoxicity in Eisenia andrei

Understanding the interaction of nanoparticles with biological fluid is important for predicting the behavior and toxicity of nanoparticles in living systems. The earthworm Eisenia andrei was exposed to citrate-coated silver nanoparticles (cAgNPs), and the interaction of cAgNPs with earthworm coelomic fluid (ECF), the cytotoxicity of cAgNPs in earthworm coelomocytes was assessed. The neutral red retention assay showed a reduction in lysosomal stability after exposure. The toxicity of silver ions dissolved from cAgNPs in the soil medium was not significant. The aggregation and dissolution of cAgNPs increased in ECF, which contains various electrolytes that alter the properties of nanoparticles, and their subsequent toxicity. Microscopic and dissolution studies demonstrated that the aggregation of cAgNPs rapidly increased, and readily dissolved in ECF. The bioavailability of cAgNPs to earthworms induced lysosomal cytotoxicity. This is the first report to test the interaction and lysosomal cytotoxicity of nanoparticles in earthworm biofluids.

Condition Index and Neutral Red Assay Response of Cultured Mytilus edulis L. Stored in a Wet Holding Facility during Winter and Spring in Northeastern Newfoundland

In order to determine the effects of extended wet-holding on cultured blue mussels under ambient conditions during the winter and spring in Newfoundland, we investigated physiological stress response using the Neutral Red Assay, change in dry and wet weight and in overall condition based on the dry tissue weight/dry shell weight ratio and the dry tissue weight/wet tissue weight ratio of animals held for up to three months in a commercial holding facility and compared with field controls. During the winter season, dry weight and condition of mussels in the holding facility increased significantly over the three month period when compared with samples from the field. Spring-held mussels were observed to lose dry weight and condition in holding as compared with field controls. The Neutral Red Assay indicated an observed but not statistically significant change in physiological stress response for mussels held during the winter season. No observed change was noted for field controls. During the spring no overall change was noted in Neutral Red response for held or field control samples Dry weight and condition analysis indicated that the winter season was the most stable for long term holding of harvested mussels. Overall analysis of lysosomal neutral red retention time did not reveal a statistically significant response for mussels held during winter or spring suggesting that low temperatures may affect the haemocyte lysosomal response to Neutral Red retention. Based on the observed change in condition and dry weight across season, we recommend that mussels can be held for atleast two months during the winter season and at a limit of one month for spring (especially late spring i.e. May and June) before a significant loss in condition and potential quality is observed.

Earthworm biomarker responses on exposure to commercial cypermethrin

Cypermethrin is a synthetic pyrethroid insecticide used worldwide in agriculture, home pest control, disease vector control, and food safety. It accumulates in soil. Therefore, traces of cypermethrin may frequently appear in vegetables grown in contaminated soil. There is a push now to develop biomarkers as early warning indicators of environmental pollution. In this study, DNA damage (tail DNA%, tail length, and olive tail moment), the micronucleus, neutral red retention (NRR) time, and pinocytic adherence ability of coelomocytes were investigated in Pheretima peguana earthworms exposed to cypermethrin in filter paper tests. The NRR time of earthworm coelomocytes decreased significantly at a concentration of 3.5 × 10(-3) µg · cm(-2) (1/100 LC50 ) after 48 h exposure, with a highly negative correlation with cypermethrin concentration. Pinocytic adherence ability of coelomocytes also declined significantly at a cypermethrin concentration of 3.5 × 10(-2) µg · cm(-2) (1/10 LC50 ). The DNA damage to earthworm coelomocytes (tail DNA%, tail length, and olive tail moment) increased considerably at the highest concentration (3.5 × 10(-1) µg · cm(-2) ) although the correlation between tail DNA% and cypermethrin concentration was low. Thus, physiological biomarkers were more sensitive than the genotoxic effects in earthworms exposed to commercial cypermethrin. Although a suite of earthworm biomarkers could be used to evaluate cypermethrin terrestrial pollution, the NRR test is easier to conduct and a more sensitive indicator.

Effects of dietary organic selenium on immune responses, total selenium accumulation and digestive system health of marron,Cherax cainii(Austin, 2002)

A 90-day feeding trial was conducted to determine the effects of dietary organic selenium (OS) supplementation on immune responses, total selenium accumulation in various tissues and the digestive system health of marron Cherax cainii. A group of marron (initial weight 3.65 � 0.05 g) were fed 0.2 g kg � 1 of Sel-Plex as a source of OS and were compared to a control group of marron that were not fed OS. After 90 days of feeding OS, total haemocyte counts (THC) and the percentages of granular and semigranular cells in marron were significantly higher than in marron fed a control diet. However, dietary OS did not affect the proportion of hyaline cells. The marron fed OS had significantly lower bacteraemia and longer neutral red retention times than the control group. Total selenium levels in the haemolymph, hepatopancreas and muscle tissues of the OS-fed marron group were significantly higher (P < 0.05) than in control diet group. The highest total soluble selenium levels were found in the muscle tissues of OS-fed marron on the final day of the feeding trial. Amylase and protease activity, soluble protein, and numbers of microvilli were significantly higher (P < 0.05) in marron fed OS. These findings demonstrate the benefits of including 0.2 g kg � 1 OS in marron diet to enhance immune parameters, increase total selenium accumulation in muscle and improve marron digestive system health.

Oxidative and genetic responses induced by Δ-9-tetrahydrocannabinol (Δ-9-THC) to Dreissena polymorpha

Cannabis is the most used illicit substance worldwide and its main psychoactive compound, the Δ-9-tetrahydrocannabinol (Δ-9-THC), is detected in aquatic environments at measurable concentrations. Even though its occurrence is well documented, no information is available on its hazard to aquatic organisms. The aim of this study was to assess the adverse effects induced to zebra mussel (Dreissena polymorpha) specimens by 14day exposures to environmentally relevant Δ-9-THC concentrations (0.05μg/L and 0.5μg/L) by means of the application of a biomarker suite. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities, as well as the lipid peroxidation (LPO) and protein carbonyl content (PCC), were measured as oxidative stress indices. The single cell gel electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test) were applied to investigate DNA injuries, while the neutral red retention assay (NRRA) was used to assess Δ-9-THC cytotoxicity. The lowest treatment induced negligible adverse effects to bivalves, while 0.5μg/L Δ-9-THC exposure caused remarkable alterations in D. polymorpha oxidative status, which lead to significant increase of lipid peroxidation, protein carbonylation and DNA damage.

Comparing the effects of dietary selenium and mannan oligosaccharide supplementation on the growth, immune function, and antioxidant enzyme activity in the cultured marron Cherax cainii (Austin, 2002)

This study was conducted to compare the effects of dietary supplementation of selenium (Se, both inorganic and organic) and mannan oligosaccharide (MOS) on the growth, immune function, and antioxidant enzyme activities in the haemolymph of the marron Cherax cainii (Austin, 2002); 0.4 mg kg−1 of sodium selenate, 0.2 g kg−1 Sel-Plex®, or 0.4 % Bio-MOS® was added to the basal diet as sources of inorganic selenium (IS), organic selenium (OS), and MOS, respectively. After 90 days, marron fed all 3 supplemented test diets had significantly higher final weight, specific growth rate (SGR), survival, total haemocyte count (THC), percentage of hyaline cells, glutathione-S-transferase (GST), and glutathione peroxidase (GPx) activities than marron fed without any supplements. The marron fed OS showed significantly higher THC and percentage of hyaline cells than those fed IS and MOS. Marron fed MOS showed the highest final weight, SGR, and percentage of granular cells; however, there was no significant difference in the neutral red time retention between marron fed IS and OS, whereas marron fed OS resulted in the highest GPx activity and total Se levels in the haemolymph but the lowest lipid peroxidase activity; therefore, it is suggested that dietary supplementation with any source of Se and MOS is beneficial to improving growth, survival, and antioxidant activity.

Using lysosomal membrane stability of haemocytes in Ruditapes philippinarum as a biomarker of cellular stress to assess contamination by caffeine, ibuprofen, carbamazepine and novobiocin

Although pharmaceuticals have been detected in the environment only in the range from ng/L to μg/L, it has been demonstrated that they can adversely affect the health status of aquatic organisms. Lysosomal membrane stability (LMS) has previously been applied as an indicator of cellular well-being to determine health status in bivalve mussels. The objective of this study is to evaluate LMS in Ruditapes philippinarum haemolymph using the neutral red retention assay (NRRA). Clams were exposed in laboratory conditions to caffeine (0.1, 5, 15, 50 μg/L), ibuprofen (0.1, 5, 10, 50 μg/L), carbamazepine and novobiocin (both at 0.1, 1, 10, 50 μg/L) for 35 days. Results show a dose-dependent effect of the pharmaceuticals. The neutral red retention time measured at the end of the bioassay was significantly reduced by 50% after exposure to environmental concentrations (p < 0.05) (caffeine = 15 μg/L; ibuprofen = 10 μg/L; carbamazepine = 1 μg/L and novobiocin = 1 μg/L), compared to controls. Clams exposed to these pharmaceuticals were considered to present a diminished health status (retention time < 45 min), significantly worse than controls (96 min) (p < 0.05). The predicted no environmental effect concentration (PNEC) results showed that these pharmaceuticals are very toxic at the environmental concentrations tested. Measurement of the alteration of LMS has been found to be a sensitive technique that enables evaluation of the health status of clams after exposure to pharmaceuticals under laboratory conditions, thus representing a robust Tier-1 screening biomarker.

Comparison of intermittent and continuous exposures to cadmium in the blue mussel, Mytilus edulis: Accumulation and sub-lethal physiological effects

Little is known about the bioaccumulation responses of shellfish to metals during intermittent compared to continuous exposure. There is also the concern that the toxicity of intermittent events may not be the same as that from the steady-state continuous exposures. The aim of the present study was to determine whether there was any difference between cadmium (Cd) accumulation, or Cd-dependent biological responses, in tissues of blue mussels (Mytilus edulis) during intermittent compared to continuous Cd exposure. Tissues and hemolymph were collected from M. edulis exposed for 14 days to either control (no added Cd, only seawater), or 50µg/l Cd as CdCl2 in continuous or intermittent profile (2 day exposure, 2 days in clean seawater alternately); and sub-lethal responses examined using a suite of assays including total glutathione, thiobarbituric acid reactive substances (TBARS), neutral red retention, total hemocyte counts, hemolymph Na+ and K+, plasma glucose and histopathology. A time-dependent accumulation of the Cd was observed in tissues of mussels after continuous exposure, while the intermittent exposure showed step-wise changes in the hemolymph and gonad. Tissue Cd concentration in the continuous exposure was significantly increased (≥2 fold) for most tissues compared to the intermittent exposure. No clear differences were seen between the continuous and intermittent exposure for most end points measured apart from a 2 fold significant increase in hemocyte infiltration in the digestive gland of the continuous exposure compared to the intermittent exposure. Overall, the data showed that the Cd accumulation was generally greater in the continuous exposure regime, but despite this, most of the biological responses being similar in both regimes.

Effect of starvation and hibernation on the values of five biomarkers of general and specific stress using the land snail Eobania vermiculata

Values of five biomarkers related to cell stress or pollution were evaluated in tissues of the land snail Eobania vermiculata under starvation or hibernation conditions. The biomarkers applied were lysosomal membrane stability in digestive gland cells (LMS) or in haemocytes (neutral red retention assay (NRR)), acetylcholinesterase activity (AchE; EC 3.1.1.7), metallothionein content (MTs), and cyclic AMP content (cAMP). Three groups of snails were studied that were kept under starvation, hibernation and normal conditions, respectively. The results indicated statistically lower values of LMS and NRR in snails kept under starvation or hibernation compared to control ones. Higher values of AChE activity were measured in snails under hibernation compared to controls. MT contents were statistically higher in snails under starvation compared to controls. Measurement of cAMP contents showed no significant differences among the tested groups. The values of the first four biomarkers may be affected by factors other than pollution, such as starvation or hibernation. Therefore, these factors should be taken into consideration when biomonitoring studies are performed in time intervals of hibernation or starvation.

Dietary organic selenium improves growth, survival and resistance to Vibrio mimicus in cultured marron, Cherax cainii (Austin, 2002)

To determine the effects of dietary organic selenium (OS) supplementation on the growth performance and immune competence of marron, Cherax cainii (Austin, 2002), a group of marron were fed 0.2 g kg−1 of Sel-Plex® supplemented basal diet and then compared with another group (control) of marron fed basal diet without any supplementation. After 90 days of feeding, final weight, average weekly gains (AWG), relative gain rate (RGR), specific growth rate (SGR), survival, total and differential haemocyte counts (THC and DHC), were compared between the two groups. Surviving marron from each group were then divided into three sub-groups (three tanks per sub-group with seven marron per tank); (1) first sub-group was injected with 20 μL of 3.24 × 106 cfu Vibrio mimicus; (2) the second sub-group was injected with 20 μL normal saline and (3) the third sub-group was not subjected to injection and became the control group. THC, DHC, neutral red retention time (NRRT) and Vibrio ranks of post-injected marron were evaluated for 96 h, at every 24-h interval. The results showed that after 90 days of feeding, final weight, AWG, RGR, SGR, survival, THC, proportion of hyaline cells of OS-fed marron were significantly higher (P < 0.05) than the control group, whereas proportion of granular and semigranular cells were not affected by dietary OS. After challenging with V. mimicus, survival rate of marron without dietary OS significantly decreased (P < 0.05) as compared to the control group of marron. THC of marron in all sub-groups were significantly reduced (P < 0.05) after the challenge. However, THC and granular cells of sub-groups fed OS were higher than other sub-groups. Vibrio ranks and NRRT of marron fed OS were significantly lower and slower, respectively, than marron fed without OS. These findings demonstrated the benefits of OS inclusion in the marron diet in terms of growth, health and disease resistance.

Identification of biomarkers responsive to chronic exposure to pharmaceuticals in target tissues of Carcinus maenas

A 28-day bioassay was performed with Carcinus maenas to evaluate chronic effects caused by exposure to caffeine and ibuprofen (0.1–50 μg L−1) in sea water. Lysosomal membrane stability (LMS) was evaluated in hemolymph applying the neutral red retention assay (NRRA); several biomarkers including ethoxyresorufin O-deethylase (EROD), dibenzylfluorescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPX), lipid peroxidation (LPO) and DNA damage were studied in gill, hepatopancreas, muscle and gonad tissues. In crabs exposed to environmental concentrations of the drugs, retention time was reduced by 50%. EROD and DBFOD activities were induced by caffeine in muscle and hepatopancreas tissues (p < 0.05); GST activity was activated by ibuprofen in gill, hepatopancreas and muscle at the highest concentrations tested (p < 0.05). All tissues showed GPX activity and LPO induction (p < 0.05). Crabs exposed to caffeine and ibuprofen showed evidence of DNA damage mainly in hepatopancreas tissues (p < 0.05). Environmental concentrations of pharmaceuticals induce LMS and the biochemical responses studied in this crab. This methodology is a suitable technique for assessing pharmaceutical toxicity in the marine environment.

Flow cytometry detection of lysosomal presence and lysosomal membrane integrity in the three-spined stickleback (Gasterosteus aculeatus L.) immune cells: applications in environmental aquatic immunotoxicology.

The neutral red retention assay has been proposed to determine the lysosomal membrane stability in immune cells. Nevertheless, this assay implies many examinations under a microscope at short time intervals and therefore the analysis of few samples. The present study proposes two more rapid, efficient, and sensitive sample analyses using flow cytometry method. Lysosomal presence and lysosomal membrane integrity (LMI) were evaluated on the three-spined stickleback, Gasterosteus aculeatus (L.), a well-described model fish species for aquatic ecotoxicology studies. After development of the two biomarkers, they were validated by ex vivo contamination with endosulfan and copper and by in situ sampling. These immunomarkers were clearly modulated by pollutants and their variations seemed to be correlated with leucocyte mortality. Thus, from a practical point of view, lysosomal presence and LMI may provide novel and efficient means of evaluating immune capacities and indicating the toxic effects of environmental pollution.