Neutral Red Retention Time Research Articles

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.

Study on the acute toxicity and metabolic changes caused by cadmium exposure on the fish Hyphessobrycon Callistus used as an indicator of environmental health

In Brazil, Hyphessobrycon callistus is an important commercially exploited species and it is an ideal animal for studyingdamages caused by the effects of heavy metal that are often detected in the aquatic environment. The main purpose of thepresent study was to detect the acute toxicity of Cd to H. callistus and investigate its effects on oxygen consumption, ammoniumexcretion, and the neutral red retention time assay to estimate effects at the cellular level. Such investigations havenot been carried out before with this species. Acute toxicity of Cd to H. callistus in 24h, 48h, 72h, and 96h medium lethalconcentration (LC50) was showed to be 45.95, 32.49, 28.28, and 15.16 mg/l, respectively. Furthermore, it was found thatexposure of fish to 1.5 mg/l Cd caused reduction in oxygen consumption and ammonium excretion of 59% and 71.5%,respectively as compared to controls.

Effects of Cry1Ab Transgenic Maize on Lifecycle and Biomarker Responses of the Earthworm, Eisenia Andrei

A 28-day study was conducted to determine the effects of the Bacillus thuringiensis Cry1Ab toxin on the earthworm Eisenia andrei. Previously, investigations have been limited to life-cycle level effects of this protein on earthworms, and mostly on E. fetida. In this study several endpoints were compared which included biomass changes, cocoon production, hatching success, a cellular metal-stress biomarker (Neutral Red Retention Time; NRRT) and potential genotoxic effects in terms of Randomly Amplified Polymorphic DNA sequences (RAPDs). NRRT results indicated no differences between treatments (p > 0.36), and NRRT remained the same for both treatments at different times during the experiment (p = 0.18). Likewise, no significant differences were found for cocoon production (p = 0.32) or hatching success (p = 0.29). Conversely, biomass data indicated a significant difference between the control treatment and the Bt treatment from the second week onwards (p < 0.001), with the Bt treatment losing significantly more weight than the isoline treatment. Possible confounding factors were identified that might have affected the differences in weight loss between groups. From the RAPD profiles no conclusive data were obtained that could link observed genetic variation to exposure of E. andrei to Cry1Ab proteins produced by Bt maize.

Utilizing Eisenia andrei to assess the ecotoxicity of platinum mine tailings disposal facilities

South Africa is an important platinum mining country which results in environmental impacts due to the construction of tailing disposal facilities (TDFs). It is unclear what the effects of ageing are on the ecotoxicity of TDFs and whether it increases or decreases over time. The aim of this study was to determine the ecotoxicity of differently aged TDFs by investigating earthworm (Eisenia andrei) responses viz. growth, reproduction, neutral red retention times (NRRT) and tissue metal concentrations. Further, to evaluate the status of these in terms of a geoaccumulation index (I(geo)), pollution index and integrated pollution index. Results indicated that earthworms showed reduced reproductive success (hatchlings per cocoon) and decreased NRRT in all the sites. Juveniles per cocoon between all of the different treatment groups were; control (2.83 ± 0.54) > site 2 (20 years old; 1.83 ± 0.27) > sites 1 and 3 (40 years old; 1.06 ± 0.15 and 6 years old; 0.88 ± 0.39). This might be ascribed to the elevated levels of Cr (±200 to 1,166 μg g(-1)) and Ni (±100 to 316 μg g(-1)) in all of the sites. Earthworms did not bioaccumulate metals with bioconcentration factors for all the different treatments <0.01. Studies like these could be useful when establishing a ranking of TDFs in the future to provide legislative institutions with an indication of the environmental liabilities of platinum mines.

The effect of extended wet-storage on the condition, physiology and stress response of cultured blue mussels (Mytilus edulis L. 1758) during summer and fall in northeastern Newfoundland

In order to determine the effects of extended wet-storage under ambient conditions we investigated the physiological response of blue mussels held for up to 3months in a commercial holding facility. During the summer and the fall there was a significant decline in dry weight and condition of mussels held over time when compared to field controls. Neutral red retention time decreased significantly in holding for both seasons but not in the field controls, indicating an increase in stress response for held mussels. Little variation was noted in the expression of the oxidative stress genes superoxide dismutase and GSH-peroxidase during the summer or fall regardless of treatment. Expression of the antimicrobial peptide MGD2 was evident in the summer holding samples as early as 1week but did not occur in the fall holding samples until 3months. This suggests that holding conditions have stimulated the immune response in a manner potentially related to time in holding and environmental temperature. Based on our results we recommend that one month should be the maximum time allowable in ambient extended holding during warm water seasons. During the warmest part of the summer this could be reduced to two weeks.

Stability of lysosomal membrane in Carcinus maenas acts as a biomarker of exposure to pharmaceuticals

The presence of pharmaceuticals in the environment is now a major concern given their potential adverse effects on organisms, particularly human beings. Because the feeding style and habitat of the crab Carcinus maenas make this species vulnerable to organic contaminants, it has been used previously in ecotoxicological studies. Lysosomal membrane stability (LMS) in crabs is a general indicator of cellular well-being and can be visualized by the neutral red retention (NRR) assay. LMS in crab hemolymph has been evaluated as a cellular biomarker of adverse effects produced by exposure to pharmaceutical compounds. Crabs were exposed in the laboratory to four different pharmaceuticals for 28 days in a semistatic 24-h renewal assay. Filtered seawater was spiked every 2 days with various concentrations (from 0.1 to 50 μg · L(-1)) of caffeine, ibuprofen, carbamazepine, and novobiocin. Results showed that NRR time, measured at day 28, was significantly reduced (p < 0.05) after exposure to environmental concentrations of each pharmaceutical (caffeine = 15 μg · L(-1); carbamazepine = 1 μg · L(-1); ibuprofen = 5 μg · L(-1); and novobiocin = 0.1 μg · L(-1)) when compared with control organisms. The predicted "no environmental effect" concentration/measured environmental concentration results showed that the selected pharmaceuticals are toxic at environmental concentrations and need further assessment. LMS monitoring in crabs is a sensitive tool for evaluating exposure to concentrations of selected drugs under laboratory conditions and provides a robust tier 1 testing approach (screening biomarker) for rapid assessment of marine pollution and environmental impact assessments for analyzing pharmaceutical contamination in aquatic environments.

Enantioselective Effects of Methamidophos on the Coelomocytes Lysosomal Membrane Stability of Eisenia fetida

Many of organophosphorous insecticides are chiral compounds. In this study, the enantioselective effects of organophosphate insecticide methamidophos on the coelomocytes lysosomal membrane stability of earthworm Eisenia fetida were studied: (1) The enantiomers of methamidophos were absolutely separated by high-performance liquid chromatography with a commercial chiral column; (2) The neutral red retention assay was used to judge the lysosomal membrane stability. The results showed that with the concentration increasing, lysosomal membranes have been significantly destroyed by individual stereoisomers and racemate of methamidophos. The neutral red retention times were significantly descended from 76.88 to 29.78 min. Both (+)- and (-)-methamidophos showed more prone to destroy the integrity of the lysosomal membrane than the racemate. However, the different effect between stereoisomers is slight.

Total thiol redox status as a potent biomarker of PAH-mediated effects on mussels

This laboratory study describes phenanthrene (Ph) and/or anthracene (An) ability to alter the total thiol redox status (TRS), via depletion of protein free thiols (PSH) and glutathione (GSH) levels, in gills of mussel Mytilus galloprovincialis, after a short-term (7 days) exposure period to each contaminant (at a final concentration of 0.1 mg L−1) or in a mixture (ration 1:1, at a final concentration of 0.2 mg L−1). A number of observable changes, like lysosomal membrane impairment (as detected via the neutral red retention time assay, primarily performed in haemocytes), enhancement of lipid peroxidation byproducts, increased nuclear abnormalities, inhibition of AChE and ALP activity, as well as a significant depletion of PSH and GSH were detected in gills of exposed mussels, in any case. Significant relationships occurred among TRS parameters with each change/stress indices measured in tissues of mussels, could reinforce the use of PSH as a potent biomarker.

Investigation of landfill leachate toxic potency: An integrated approach with the use of stress indices in tissues of mussels

The present study investigates the harmful impacts of landfill leachate release and/or disposal into the marine environment, as well as its ability to induce lethal and pre-pathological alterations in marine organisms, such as the mussel Mytilus galloprovincialis. In specific, mortality test (96h), performed first in order to estimate leachate lethal endpoints, showed increased levels of mussel mortality after exposure to leachate higher than 0.5%, v/v (96h LC50=0.526%, v/v), while the exposure to 0.01 and 0.1% (v/v) of leachate showed negligible levels of mortality (96h LC10=0.167%, v/v). Furthermore, the estimation of lysosomal membrane integrity in hemocytes of exposed mussels (Neutral Red Retention Time assay) showed increased levels of lysosomal destabilization in cells of mussels exposed to sub-lethal concentrations of leachate (0.01, 0.1 and 0.5%, v/v) for 4 days. In order to exclude parameters, such as mussel mortality and cell death, which could interfere with the obtained results, leachate at final concentrations of 0.01 and 0.1% (v/v) were finally used for the estimation of a battery of stress indices in target tissues of mussels, such as hemolymph, gills and digestive gland. According to the results, leachate-exposed mussels showed a significant inhibition of acetylcholinesterase activity, increased levels of nuclear abnormalities, as well as increased levels of metallothionein, superoxide anion and lipid peroxides (in terms of malondialdehyde equivalents) in each tissue tested. The results of the present study clearly indicate leachate-induced lethal effects as well as the ability of leachate to induce disturbances on different levels of organism function before mortality occurs.

Biological effects of environmentally relevant concentrations of the pharmaceutical Triclosan in the marine mussel Perna perna (Linnaeus, 1758)

Triclosan (5-Chloro-2-(2,4-dichlorophenoxy) phenol) is an antibacterial compound widely employed in pharmaceuticals and personal care products. Although this emerging compound has been detected in aquatic environments, scarce information is found on the effects of Triclosan to marine organisms. The aim of this study was to evaluate the toxicity of a concentration range of Triclosan through fertilization assay (reproductive success), embryo-larval development assay (early life stage) and physiological stress (Neutral Red Retention Time assay - NRRT) (adult stage) in the marine sentinel organism Perna perna. The mean inhibition concentrations for fertilization (IC50 = 0.490 mg L−1) and embryo-larval development (IC50 = 0.135 mg L−1) tests were above environmental relevant concentrations (ng L−1) given by previous studies. Differently, significant reduction on NRRT results was found at 12 ng L−1, demonstrating the current risk of the continuous introduction of Triclosan into aquatic environments, and the need of ecotoxicological studies oriented by the mechanism of action of the compound.

Biomarker responses of earthworms (Eisenia fetida) exposured to phenanthrene and pyrene both singly and combined in microcosms

Microcosm studies were undertaken to relate biomarker responses to the toxicities in soil ecosystems contaminated by phenanthrene (Phe) and pyrene (Pyr), both singly and combined. Growth inhibition, enzyme activity, MDA content, sperm count, neutral-red retention time (NRRT) and annetocin and TCTP gene transcriptions were determined in earthworm Eisenia fetida exposed to Phe and Pyr, both singly and combined pollution in microcosm. Exposure to 0.5 and 2.5mgkg−1 Phe or 50 and 100mgkg−1 Pyr alone significantly decreased E. fetida growth, NRRT and sperm count. Two-way ANOVA analysis shows that the combination of these two compounds decreased growth, SOD activities, NRRT and sperm count synergistically, but increased the CAT activities and MDA content. The highest suppression rate of growth was 48.12%, the lowest levels of SOD activities and NRRT were 51.66% and 45.57% of the control, respectively. The highest increase in CAT activities and MDA content were 120.05% and 121.03% greater than that of the control when exposed to 0.5 (Phe)+100 (Pyr) mgkg−1 soils. A clear dose-related response with exposure concentration was established for the NRRT. Real-time PCR shows that Phe and Pyr increased the expression levels of annetocin and TCTP gene synergistically. These results demonstrate that earthworms were under physiological stress at field dose of 0.5 (Phe)+100 (Pyr) mgkg−1 soils. Phe and Pyr synergistically decreased sperm count and NRRT, but antagonistically caused changes in antioxidant enzyme activities to disrupt the detoxification functions and inhibit earthworm growth.

Acute and sublethal effects of sequential exposure to the pesticide azinphos-methyl on juvenile earthworms (Eisenia andrei)

The use of organophosphate pesticides is an integral part of commercial farming activities and these substances have been implicated as a major source of environmental contamination and may impact on a range of non-target fauna. The extent to which soil dwelling non-target organisms are affected by exposure to the organophosphate azinphos-methyl was investigated through monitoring selected biomarker responses and life cycle effects under laboratory conditions in the earthworm Eisenia andrei. Standard acute toxicity tests were conducted followed by a sequential exposure regime experiment, in order to assess the effects of multiple pesticide applications on biomarker (cholinesterase activity and neutral red retention time), life-cycle (growth and reproduction) and behaviour (avoidance and burrowing activity) responses. The present study indicates that the time between exposure events was a more important variable than concentration and that a longer interval between exposures may mitigate the effects of pesticide exposure provided that the exposure concentration is low. Additionally, it was shown that E. andrei was unable to avoid the presence of azinphos-methyl in soil, even at concentrations as high as 50% of the LC(50) value, indicating that the presence of azinphos-methyl in the soil pose a realistic threat to earthworms and other soil dwelling organisms. The ChE inhibition test showed a high percentage inhibition of the enzyme in all exposure groups that survived and NRR times of exposed organisms were lower than that of the controls. The present study yielded important results that contribute to the understanding of biological impacts of pesticide pollution on the environment. Extrapolating these results can aid in optimising pesticide application regimes to mitigate the environmental effects thereof and thus ensuring sustained soil biodiversity in agricultural areas.

Application of neutral red retention assay to caged clams (Ruditapes decussatus) and crabs (Carcinus maenas) in the assessment of dredged material

Dredged material management is a key issue for the protection of aquatic environments. The in situ approach using caged bioindicator species has been chosen lately as a new methodology for the assessment of dredged material. In a tier testing approach, neutral red retention (NRR) assay has been applied as a screening tool to detect adverse changes in health status associated with contamination. Nevertheless, to authors' knowledge, little is known about the application and validation of this technique in sediment bioindicator species and under field conditions. Caged Ruditapes decussatus and Carcinus maenas were exposed during 28days to potentially contaminated sediments at three sites in Algeciras Bay (SW Spain) and one site in Cádiz Bay (SW Spain). Lysosomal membrane stability was measured over time in haemolymph samples of exposed clams and crabs using the NRR assay. Sediment characterization of the study sites was performed in parallel. NRR time did not vary significantly (p>0.05) over time in organisms from Cádiz Bay. Conversely, significant differences (p<0.05) in NRR time were found in clams and crabs exposed to sediments from Algeciras Bay, which exhibited a 30-70% decrease in haemocyte lysosome membrane stability compared to day 0. Statistical analysis showed a strong correlation between the drop of haemocyte lysosome membrane stability, in both crabs and clams, and the presence of metals (p<0.05) and PAHs (p<0.01) in the studied sediments. The results obtained confirmed the use of NRR assay as a suitable and sensitive method to be used in the assessment of sediment quality using as bioindicator species the clam R. philippinarum and the crab C. maenas.

Effects of spawning on immune functions in the surf clam Mactra veneriformis (Bivalvia: Mactridae)

The production of surf clam, Mactra veneriformis, an important fishery resource in Korea, has recently been decreasing. This study was carried out to examine effects of spawning on immune functions of this species. Total hemocyte count (THC), phenoloxidase (PO) activity, phagocytic activity, neutral red retention (NRR) time and antibacterial activity were assessed. Spawned clams showed reduction in THC, PO, phagocytic activity and NRR times compared with unspawned ones. While spawning event did not elicit any change of antibacterial activity in both spawned and unspawned ones. This study indicates that spawning process decreases immune functions in the surf clams which could cause mortality increment and yield reduction.

Effect of movement stress on immune function in farmed Australian abalone (hybrid Haliotis laevigata and Haliotis rubra)

Current abalone mariculture practices include movement of stock to reduce stocking density in tanks and to grade abalone into similar sizes. Australian farmers report that this procedure leads to increased mortality rates in the days following movement. A standard movement procedure as currently done in Australia was investigated on farm as to its effects on haemocyte counts, phagocytic rates, antibacterial activity and lysosomal membrane stability (neutral red retention assay). The experimental design divided the movement process into the main causes of stress, comparing movement without anaesthesia (manual detachment or chipping), anaesthesia without movement and anaesthesia with subsequent movement. Each of the 3 treatment groups had elevated haemocyte counts and depressed phagocytic rates, neutral red retention times and antibacterial activity. The most severely affected abalone were those anaesthetised and moved, followed by those that were anaesthetised but not moved. Abalone manually detached without anaesthesia showed non-significant change in most of the parameters. Recovery back to baseline levels occurred within 1 day for most parameters, but not for the neutral red retention times. The experiment was then repeated with very similar results. These experiments indicate a practical means to compare on farm the effects that husbandry methods have on the immune system, which in turn will allow for the development of better husbandry procedures to reduce stresses incurred during intensive mariculture.

Suitability of lysosomal membrane stability in Eisenia fetida as biomarker of soil copper contamination

Accumulated metals in soils negatively affect dwelling organisms. Earthworms, which are widespread and perform various essential functions, are able to accumulate metals that can damage the coelomic cells. The aim of this work was to evaluate the effect on Eisenia fetida lysosomal membrane stability both during and after copper exposure, and finally to link this to internal concentrations. E. fetida specimens were exposed to a reference soil and two Cu-spiked soils (35 and 350 mg kg −1 d.w.) for 14 days (uptake period) and then transferred into the reference soil for other 18 days (elimination period). After 3 days of uptake, internal Cu concentrations increased and were higher in the specimens exposed to soils spiked with 350 mg Cu kg −1 d.w. After 2 days of elimination, a strong decrease of internal Cu concentrations was always observed. The lysosomal membrane stability, measured as neutral red retention-times, was approximately 50 min for the earthworms exposed to the reference soils, whereas it decreased, at the end of the uptake period, to 21 and 13 min, respectively, for the organisms exposed to soils spiked with 35 and 350 mg Cu kg −1 d.w. A full recovery of the lysosomal membrane stability was reached after 14 and 18 days of the elimination period, respectively, for the organisms exposed to soils spiked with 35 and 350 mg Cu kg −1 d.w. The neutral-red assay would seem a good biomarker since the lysosomal membrane stability of E. fetida appeared to respond rapidly and strongly to soil copper contamination.

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.

An ecotoxicological approach for the Boka Kotorska Bay (south-eastern Adriatic Sea): First evaluation of lysosomal responses and metallothionein induction in mussels

Mytilus galloprovincialis was used as a biomonitoring organism in Boka Kotorska Bay, a coastal transitional ecosystem in south-western Montenegro. Native mussels were collected in June 2008 at four sites thought to be differently impacted. Biological effects were investigated analysing both generic and specific biomarkers at cellular level (metallothionein content, lysosomal membrane stability, lipofuscin and neutral lipid accumulation, lysosomal structural changes). Trace element levels in mussels were quite low, only Cu and Zn exhibiting slight increases in the two sampling sites of Tivat Bay, the first one in front of a small working dockyard and the other in the water area of a former Naval dockyard. Mussels collected from these sites exhibited the highest values in neutral lipid and lysosomal volume density and the lowest neutral red retention times. Metallothionein content was always low, reflecting the minor body burden of the trace elements and suggesting a non-relevant environmental induction.

Coelomocyte biomarkers in the earthworm Eisenia fetida exposed to 2,4,6-trinitrotoluene (TNT)

Contamination by 2,4,6-trinitrotoluene (TNT) is a global environmental problem at sites of former explosive production, handling, or storage, and could have deleterious consequences for human and ecological health. We investigated its sublethal effects to Eisenia fetida, using two nonspecific biomarkers. In coelomocytes of earthworms exposed 24, 48, or 72 h, we evaluated DNA damage (comet assay) and neutral red retention time (NRRT), using the filter paper contact test. Both percentage of damage (D%) and calculated damage index showed significant DNA damage at almost all concentrations, at all time points assayed. Along exposure time, two different patterns were observed. At the lower TNT concentrations (0.25-0.5 μg/cm2) an increased DNA migration at 48 h, with a decrease close to initial levels after 72 h exposure, was observed. This decrease could be attributed to activation of the DNA repair system. At higher concentrations (1.0-2.0 μg/cm2), the high DNA damage observed remained constant during the 72 h exposure, suggesting that the rate of DNA repair was not enough to compensate such damage. Analysis of NRRT results showed a significant interaction between time and treatment. After 48 h, a significant decrease was observed at 4.0 μg/cm2. After 72 h, NRRT presented a concentration-dependent decrease, significantly different with respect to control at 0.5, 1.0, 2.0, and 4.0 μg/cm2. The two assayed methods, performed on the same sample, showed clear responses to sublethal TNT exposure in E. fetida, providing sensitive unspecific biomarkers of cell injury and DNA damage.

Bioaccumulation and biological effects in the earthworm Eisenia fetida exposed to natural and depleted uranium

The accumulations of both natural (U) and depleted (DU) uranium in the earthworms ( Eisenia fetida) were studied to evaluate corresponding biological effects. Concentrations of metals in the experimental soil ranged from 1.86 to 600 mg kg −1. Five biological endpoints: mortality, animals’ weight increasing, lysosomal membrane stability by measuring the neutral red retention time (the NRRT), histological changes and genetic effects (Comet assay) were used to evaluate biological effects in the earthworms after 7 and 28 days of exposure. No effects have been observed in terms of mortality or weight reduction. Cytotoxic and genetic effects were identified at quite low U concentrations. For some of these endpoints, in particular for genetic effects, the dose (U concentration)-effect relationships have been found to be non-linear. The results have also shown a statistically significant higher level of impact on the earthworms exposed to natural U compared to depleted U.