Comparison of biomarker responses after acute and chronic bioassays in Lumbriculus variegatus.

Differential biomarker responses may occur after acute or chronic bioassays when animals are unfed. In many aquatic species, food deprivation may lead to a pro-oxidant condition. However, information about its effects on the oligochaete Lumbriculus variegatus, a bioindicator organism for water and sediment toxicity tests, is almost completely lacking. Acute (48 h) and chronic (21 days) bioassays were performed using unfed L. variegatus to assess the impact of food deprivation on several biomarkers related to the redox cellular status. Results showed that food deprivation inhibited the antioxidant enzymes superoxide dismutase and catalase, whilst levels of total glutathione (t-GSH) and lipid peroxidation processes increased with respect to controls. The same biomarkers were evaluated in unfed organisms exposed to tributyltin (TBT), nano-goethite or a binary mixture of both, for 48 h and 21 days. After 48 h, the organisms were able to cope with chemical stressors by enhancing antioxidant defences and lipid peroxidation processes were not observed. After 21 days, both TBT and the binary mixture induced the most noxious effects. At this time, the antioxidant enzymatic defences were still higher than controls, but levels of t-GSH were significantly decreased and lipid peroxidation was found. Therefore, differential biomarker responses were observed between starved organisms for 21 days and those simultaneously exposed to other chemical stressors. The activity of the enzyme acetylcholinesterase was also determined, but it remained unchanged in all cases.

Tributyltin bioaccumulation and toxic effects in freshwater gastropods Pomacea canaliculata after a chronic exposure: field and laboratory studies.

Freshwater samples and gastropod mollusks (Pomacea canaliculata) were collected at 5 sampling stations located along the lower Río de la Plata basin, Argentina, to assess the extent of tributyltin (TBT) contamination. Determined data revealed the presence of TBT and some of its breakdown products (dibutyltin: DBT, and monobutyltin: MBT) in all freshwater samples and also in soft tissues of P. canaliculata gastropods. Chronic bioassays (6 months) were performed using female gastropods that had been reared under laboratory conditions and exposed to a similar TBT concentration than the value determined in freshwater samples (1 µg L-1). The aims of this study were to evaluate the extent of TBT accumulation, the tissue distribution, and the effects on selected biomarkers (activity of superoxide dismutasa: SOD, activity of catalase: CAT, levels of total glutathione: t-GSH, lipid peroxidation, and activity of acetylcholinesterase: AChE). Gonads presented the highest accumulation, followed by the cephalopedal region, albumin gland, and finally hepatopancreas. Both metabolites, DBT and MBT, were also found. All exposed female animals presented development of a penis reflecting the potential of TBT as an endocrine disrupting chemical for this gastropod species. Results on the selected biomarkers confirmed additional adverse effects induced by TBT. An increase in CAT activity and changes in t-GSH levels are indicative of alterations on the cellular redox status. The inhibition of AChE could reflect signs of neurotoxicity. Altogether, these results reveal a negative impact on the health of this gastropod population.

Azinphos-methyl and chlorpyrifos, alone or in a binary mixture, produce oxidative stress and lipid peroxidation in the freshwater gastropod Planorbarius corneus.

Azinphos-methyl (AZM) and chlorpyrifos (CPF) are broad-spectrum organophosphate insecticides used for pest control on a number of food crops in many parts of the world that have been shown to inhibit cholinesterase activity in the non-target freshwater gastropod Planorbarius corneus. The present study was undertaken to determine: (a) whether AZM and CPF induce oxidative stress in P. corneus, and (b) whether a mixture of both organophosphates that causes a higher neurotoxicity than single pesticides also causes an enhanced oxidative stress. To this end, non-enzymatic and enzymatic parameters were measured in the soft tissues of snails acutely exposed to the insecticides in single-chemical (2.5 mg AZM L(-1) and 7.5 µg CPF L(-1)) and a binary-mixture (1.25 mg AZM L(-1) plus 3.75 µg CPF L(-1)) studies. At 24 h, all pesticide-exposed groups showed significantly decreased glutathione (GSH) and glutathione disulfide (GSSG) levels when compared to control animals. At 48 h, all exposed groups showed an alteration of the redox status (GSH/GSSG ratio) and a significant increase in malondialdehyde levels. The exposure for 48 h to AZM and CPF, alone or in the binary mixture, also resulted in a significant decrease of the antioxidant superoxide dismutase activity. The greatest decrease was observed with CPF exposure (59% of decrease relative to the control group). A significant increase in catalase and glutathione S-transferase activities was observed in CPF group and in CPF and AZM+CPF groups, respectively. The activities of glutathione reductase and glucose 6-phosphate dehydrogenase did not show significant changes with respect to controls in any treatment group. In conclusion, the data shown in the present study provide evidence that AZM, CPF and a mixture of both organophosphates are able to induce oxidative stress and oxidative damage in P. corneus tissues. However, no similarities between the degree of neurotoxicity and the degree of alterations of the measured oxidative stress parameters were found.

Binary mixtures of azinphos-methyl oxon and chlorpyrifos oxon produce in vitro synergistic cholinesterase inhibition in Planorbarius corneus.

In this study, the cholinesterase (ChE) and carboxylesterase (CES) activities present in whole organism homogenates from Planorbarius corneus and their in vitro sensitivity to organophosphorous (OP) pesticides were studied. Firstly, a characterization of ChE and CES activities using different substrates and selective inhibitors was performed. Secondly, the effects of azinphos-methyl oxon (AZM-oxon) and chlorpyrifos oxon (CPF-oxon), the active oxygen analogs of the OP insecticides AZM and CPF, on ChE and CES activities were evaluated. Finally, it was analyzed whether binary mixtures of the pesticide oxons cause additive, antagonistic or synergistic ChE inhibition in P. corneus homogenates. The results showed that the extracts of P. corneus preferentially hydrolyzed acetylthiocholine (AcSCh) over propionylthiocholine (PrSCh) and butyrylthiocholine (BuSCh). Besides, AcSCh hydrolyzing activity was inhibited by low concentrations of BW284c51, a selective inhibitor of AChE activity, and also by high concentrations of substrate. These facts suggest the presence of a typical AChE activity in this species. However, the different dose-response curves observed with BW284c51 when using PrSCh or BuSCh instead of AcSCh suggest the presence of at least another ChE activity. This would probably correspond to an atypical BuChE. Regarding CES activity, the highest specific activity was obtained when using 2-naphthyl acetate (2-NA), followed by 1-naphthyl acetate (1-NA); p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). The comparison of the IC(50) values revealed that, regardless of the substrate used, CES activity was approximately one order of magnitude more sensitive to AZM-oxon than ChE activity. Although ChE activity was very sensitive to CPF-oxon, CES activity measured with 1-NA, 2-NA, and p-NPA was poorly inhibited by this pesticide. In contrast, CES activity measured with p-NPB was equally sensitive to CPF-oxon than ChE activity. Several specific binary combinations of AZM-oxon and CPF-oxon caused a synergistic effect on the ChE inhibition in P. corneus homogenates. The degree of synergism tended to increase as the ratio of AZM-oxon to CPF-oxon decreased. These results suggest that synergism is likely to occur in P. corneus snails exposed in vivo to binary mixtures of the OPs AZM and CPF.

Evidencias de la presencia de plomo en efluentes cloacales del Río de la Plata / Evidence of the presence of lead in sewage in the Río de la Plata

El análisis de actividad de la enzima ALA-D reactivada constituye una herramienta muy eficaz para evaluar el grado de contaminación por plomo en muestras de sangre de P. lineatus.

ALA-D and ALA-D reactivated as biomarkers of lead contamination in the fish Prochilodus lineatus.

ALA-D activity and lead concentrations were measured in blood and liver tissues of the fish Prochilodus lineatus, collected from three locations along the coast of the La Plata River, Argentina. Two of them, Berazategui and Berisso, were located nearby the main ducts that discharge the urban and domestic waste disposal from Buenos Aires and La Plata cities, respectively, while the third station (Atalaya) was free of sewage discharges. For both tissues, the levels of lead in fish from Berazategui and Berisso were higher than those found in the samples from Atalaya. For blood, but not for liver, a significant negative correlation was found between ALA-activity and tissue levels of lead considering all the data. However, no good correlations were observed at each location. Therefore, an enzyme reactivation technique was optimized. The blood enzyme, but not the liver one, could be effectively reactivated with zinc (Zn(II)). The values of the reactivated ALA-D in samples from Berazategui and Berisso, but not from Atalaya, were significantly higher than the original values, indicating that the enzyme was actually inhibited. In addition, the reactivation index showed significant correlations with the blood lead levels. It is proposed that the reactivation index, rather than the ALA-D activity, may reflect better the extent of lead contamination, especially for field monitoring programs where many confounding factors may affect the biomarker response.

Trace metal levels in Prochilodus lineatus collected from the La Plata River, Argentina.

Most of the industrial, urban and sewage discharges are released into the La Plata River, Argentina without any previous treatment. However, few works have investigated the extent of metal contamination. The aim of this study was to assess the levels of cadmium, copper, lead and zinc in liver and gills of adults Prochilodus lineatus collected from three sampling stations along the coast of the La Plata River: Berazategui, Berisso and Atalaya (from north to south). Samplings were performed during 2002 and 2004. Berazategui and Berisso were located nearby the main ducts that discharge the urban and domestic waste disposal from the cities of Buenos Aires and La Plata, respectively. The third station, Atalaya, was free of sewage discharges. Levels of cadmium and copper in liver were always higher than those found in gills. Instead, for lead and zinc, high levels were observed either in liver or gills, depending on the sampling station and the sampling period. In both tissues, the concentrations of metals did not differ significantly between male and female fish. In liver samples, the concentrations of cadmium, copper and zinc tended to increase from north to south. Instead, the levels of lead followed an opposite pattern. No clear tendencies were observed in gill samples. The data may be useful as reference levels of metal contaminants in P. lineatus, the most important fish species in the La Plata River system.

Effects of azinphos-methyl exposure on enzymatic and non-enzymatic antioxidant defenses in Biomphalaria glabrata and Lumbriculus variegatus.

Azinphos-methyl is an organophosphate insecticide used for pest control on a number of food crops in many parts of the world. The oligochaete Lumbriculus variegatus and pigmented and non-pigmented specimens of the gastropod Biomphalaria glabrata are freshwater invertebrates that have been recommended for contamination studies. Recently, it has been shown that L. variegatus worms exhibit a higher cholinesterase (ChE) activity and a greater sensitivity to in vivo ChE inhibition by azinphos-methyl than pigmented B. glabrata snails. The aims of the present study were (1) to investigate if, in addition to its anticholinesterase action, azinphos-methyl has also pro-oxidant activity in L. variegatus and B. glabrata, and (2) to examine if species that are highly susceptible to the neurotoxic effects of organophosphates also suffer a greater degree of oxidative stress. Therefore, total glutathione (t-GSH) levels and activities of cholinesterase (ChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glucose 6-phosphate dehydrogenase (G6PDH) were measured in the whole body soft tissue of organisms exposed for 48 and 96 h to a level of azinphos-methyl that produces 50% of inhibition on ChE. Results showed different patterns of antioxidant responses between the gastropods and the oligochaetes, and even between the two phenotypes of gastropods: (1) in exposed L. variegatus t-GSH levels increased and CAT and SOD activities decreased with respect to control organisms, (2) in pigmented gastropods, SOD decreased while CAT transiently diminished, and (3) in non-pigmented gastropods, SOD activity showed a biphasic response. GST and G6PDH were not altered by azinphos-methyl exposure. Of note, t-GSH levels were 4-fold times higher in L. variegatus than in both phenotypes of B. glabrata. This may suggest that GSH could play a more important role in antioxidant defense in L. variegatus than in B. glabrata.

The impact of particle-bound cadmium on bioavailability and bioaccumulation: A pragmatic approach.

Studying the bioavailability of sediment-bound contaminants is complicated by many reasons, such as the variable composition of the particles, their temporal variations, the low levels of contaminant concentrations, their partitioning between diverse aqueous and particulate phases, and the variety of uptake routes that may involved with the biota. Therefore, simple and innovative methodologies should be tested as analogues for natural sediments. Among them, a diverse selection of artificial particles with well-defined surface properties, in the presence and absence of commercially available humic acids, has been proposed and used to investigate the bioavailability of several organic pollutants. For this work, this model was applied to investigate the uptake and accumulation of cadmium by the freshwater oligochaete Lumbriculus variegatus. The results showed that the uptake of the metal depended on the free dissolved Cd(II) species, while the contribution from the particles was negligible. Thus, the extent of cadmium bioaccumulated from each test system could be predicted as a function of the rate of absorption of the free dissolved Cd(II) species. These species were calculated either from the particle-water partition coefficients, or by using the MINEQL+ computer program. In general, the estimated accumulation levels were in good agreement with the experimental results.

Modeling lead bioavailability and bioaccumulation by Lumbriculus variegatus using artificial particles. Potential use in chemical remediation processes.

Artificial particles, specifically a diverse selection of chromatographical resins, have been recommended and used as a useful experimental model to predict the bioavailability and bioaccumulation of sediment-bound organic chemicals. In this work the same experimental model was adopted to investigate the bioavailability and bioaccumulation of lead by the freshwater oligochaete Lumbriculus variegatus. Particle-water partition coefficients were also determined. Sand particles and the anionic exchange resin promoted a similar uptake and bioaccumulation of lead. Instead, in the presence of the cationic exchanger the metal was not detected in the animals. For neutral particles, the uptake and accumulation depended on the chemistry of the functional groups at the active sites. In addition, a significant negative correlation was found between bioaccumulation and the particle-water partition coefficients. These studies may help to develop alternative methods for chemical remediation of lead-contaminated aquatic systems.

Comparative study on two freshwater invertebrates for monitoring environmental lead exposure.

Two freshwater invertebrate organisms, Biomphalaria glabrata and Lumbriculus variegatus, were tested as potential experimental animal models to assess Pb exposure using acute laboratory bioassays. Since long, the enzyme delta-aminolevulinic acid dehydratase (ALA-D) has been recognised as a useful biomarker of Pb exposure and effect. Therefore, determinations of ALA-D activity were performed in the whole body soft tissues of pigmented and non-pigmented gastropods B. glabrata and in the oligochaete L. variegatus. The organisms were exposed to varying concentrations of Pb for 48 h. Levels of Pb in the invertebrates were also analysed. Highly significant negative correlations were found between the enzymatic activity and the levels of Pb exposure, and also between the enzymatic activity and the metal incorporated by the invertebrates. No significant differences were found in the enzyme activity nor in the resulting metal accumulation based on gastropod pigmentation. The values of Pb concentration that produce 50% of inhibition on the enzyme activity (EIC50) were 0.023 and 0.029 mg Pb/L for pigmented and non-pigmented B. glabrata, respectively. A much higher value was found for L. variegatus (0.703 mg Pb/L). The non-observed effect concentration (NOEC) on enzyme activity for the oligochaetes was 0.05 mg Pb/L, about twice the EIC50 calculated for the gastropods. These data showed that both pigmented and non-pigmented B. glabata were much more sensitive organisms than the oligochaetes. The differences in enzyme inhibition could be attributed, at least partly, to differences in the metal body burden found between the organisms, since levels of Pb accumulated by B. glabrata were approximately three times higher compared to those observed in L. variegatus. Therefore B. glabrata showed to be a more suitable and reliable bioindicator organism for assessing Pb contamination in aquatic ecosystems, especially at low levels of metal exposure.