Effect of Gold Nanoparticles and Ions Exposure on the Aquatic Organisms.

An increase in the production and usage of gold nanoparticles (AuNPs) triggers the necessity to focus on their impact on ecosystems. Therefore, the purpose of this study was to investigate the acute toxicity of AuNPs and ionic gold (Au (III)) to organisms representing all trophic levels of the aquatic ecosystem, namely producers (duckweed Lemna minor), consumers (crustacean Daphnia magna, embryos of Danio rerio) and decomposers (bacteria Vibrio fischeri). The organisms were exposed according to a standardized protocol for each species and endpoints. The AuNPs (1.16 and 11.6 d.nm) were synthesized using citrate (CIT) and polyvinylpyrrolidone (PVP) as capping agents, respectively. It was found, that Au (III) was significantly more toxic than AuNPs PVP and AuNPs CIT. AuNPs showed significant toxicity only at high concentrations (mg/L), which are not environmentally relevant in the present time, but a cautious approach is advised, due to the possibility of interactions with other contaminants.

Effects of Silver Nanoparticles and Ions Exposure on the Soil Invertebrates Folsomia candida and Enchytraeus crypticus.

The aim of the study was to assess the effects of silver nanoparticles (AgNPs1 = 2.7 d·nm, AgNPs2 = 6.5 d·nm) and silver nitrate (AgNO3) on Enchytraeus crypticus and Folsomia candida using toxicity tests (OECD Guideline 220, 232). A 28-day chronic toxicity study was performed to evaluate the reproduction and mortality rate. E. crypticus reproduction was more sensitive to AgNO3 with a 28dEC50 of 86.40 (62.52-119.4) mg·kg-1 dry weight (d.w.) compared to AgNPs1 (28dEC50 = 119.3 (60.4-235.6) mg·kg-1 d.w). Similarly, the reproduction of F. candida was inhibited the most by AgNO3 with a 28dEC50 of 126.2 (104.2-152.9) mg·kg-1 d.w. followed by AgNPs1 (28dEC50 = 158.7 (64.05-393.2) mg·kg-1 d.w.) and AgNPs2 (28dEC50 = 206.4 (181.9-234.1) mg·kg-1 d.w.). No mortalities were observed for tested soil invertebrates exposed to AgNPs at concentrations up to 166 mg·kg-1 d.w. of AgNPs1 and 300 mg·kg-1 d.w. of AgNPs2, respectively. It was found that silver ions are more toxic in comparison with AgNPs.

Evaluation of platinum nanoparticles ecotoxicity using representatives of distinct trophic levels of aquatic biocenosis.

OBJECTIVES: The aim of this study was to clarify the influence of three different sizes of platinum nanoparticles on aquatic ecosystem and assess the toxic effect in term of particle size. Tests were conducted on organisms representing all trophic levels of the aquatic ecosystem, namely producers (duckweed Lemna minor), consumers (water fleas Daphnia magna) and decomposers (bacteria Vibrio fischeri). DESIGN: Experiments were carried out methodologically in accordance with the following standards: OECD 221 guideline (Lemna sp. Growth Inhibition test), OECD 202 guideline (Inhibition of the mobility of Daphnia magna) and ISO 11348-2 (Inhibitory effect of platinum nanoparticles on the light emission of Vibrio fischeri). RESULTS: The most toxic have been the smallest sized platinum nanoparticles for all tested organisms. The highest toxicity of all tested samples (Pt1, Pt2, Pt3) was observed in bacteria (30´EC50 = 135.47; 167.94; 254.64 µg.L-1), respectively. The lowest toxicity was recorded for Daphnia (48hEC50 = 405.74; 413.24; 514.07 µg.L-1), respectively. CONCLUSION: The ecotoxicity of platinum nanoparticles varies considerably according to the test organisms and particle size.

Development of New Silver Nanoparticles Suitable for Materials with Antimicrobial Properties.

Silver nanoparticles are the most important nanoparticles in connection with the antimicrobial effect. Nowadays, the green synthesis of various types of nanoparticles is rapid, effective and produce less toxic nanoparticles often with specific properties. In our experiment we have developed and described in details various types of silver nanoparticles synthesized chemically or by the green synthesis. Nine different silver nanoparticles were synthesized, three by citrate method at different pHs (8; 9; 10), four using gallic acid at alkaline pHs (10; 11), and two by green synthesis using green tea and coffee extracts, both at pH 9. Characterisation of silver nanoparticles was performed using dynamic light scattering, scanning electron microscopy, and ultraviolet-visible absorption spectroscopy. Silver nanoparticles prepared by green synthesis showed the highest antioxidant activity and also ability for quenching of free radicals. Antibacterial activity of silver nanoparticles was determined on bacterial cultures such as Staphylococcus aureus and Escherichia coli. Silver nanoparticles synthesized using green tea and coffee extracts showed the highest antibacterial activity for both bacterial strains. Minimal inhibition concentration for both strains was found to be 65 µM at each silver nanoparticle synthesized using green synthesis.

Ecotoxicity of selected antibiotics for organisms of aquatic and terrestrial ecosystems.

OBJECTIVES: The aim of this study was to assess the ecotoxicity of selected antibiotics (i.e. penicillin G, vancomycin and tetracycline) using ecotoxicological tests. Tests were conducted on organisms representing all trophic levels of the aquatic ecosystem, namely producers (green freshwater algae Pseudokirchneriella subcapitata), consumers (water fleas Daphnia magna) and decomposers (bacteria Vibrio fischeri). The effect of antibiotics on the representative of edaphon was measured by testing the inhibition of the reproduction of springtails Folsomia candida and earthworms Eisenia fetida. DESIGN: Methodologically, the procedure was carried out in accordance with the following standards: OECD 201 (Fresh water algal growth inhibition test), OECD 202 (Inhibition of the mobility of Daphnia magna), ISO 11348-2 (Inhibitory effect of antibiotics on the light emission of Vibrio fischeri), OECD 232 (Inhibition of reproduction of Collembola Folsomia candida) and OECD 222 (Inhibition of reproduction of Eisenia fetida). RESULTS: In aquatic organisms the highest level of toxicity was shown by tetracycline to algae (72hEC50 = 1.82 mg.l-1) and daphnia (48hEC50 = 8.16 mg.l-1). The least toxic for all test organisms was penicillin G. The results of the tests performed on the representative of edaphon, Folsomia candida, showed that its reproduction was most inhibited by penicillin G (28dEC50 = 328 mg.kg-1) and least by tetracycline (28dEC50 = 2560 mg.kg-1). Similar results were observed in Eisenia fetida (56dEC50 = 348 mg.kg-1 for penicillin G and 56dEC50 = 2735 mg.kg-1 for tetracycline. CONCLUSION: The ecotoxicity of antibiotics differed significantly depending on the test organism and testing conditions.

Exposure to 17ß-Oestradiol Induces Oxidative Stress in the Non-Oestrogen Receptor Invertebrate Species Eisenia fetida.

BACKGROUND: The environmental impacts of various substances on all levels of organisms are under investigation. Among these substances, endocrine-disrupting compounds (EDCs) present a threat, although the environmental significance of these compounds remains largely unknown. To shed some light on this field, we assessed the effects of 17ß-oestradiol on the growth, reproduction and formation of free radicals in Eisenia fetida. METHODOLOGY/PRINCIPAL FINDINGS: Although the observed effects on growth and survival were relatively weak, a strong impact on reproduction was observed (50.70% inhibition in 100 µg/kg of E2). We further demonstrated that the exposure of the earthworm Eisenia fetida to a contaminant of emerging concern, 17ß-oestradiol (E2), significantly affected the molecules involved in antioxidant defence. Exposure to E2 results in the production of reactive oxygen species (ROS) and the stimulation of antioxidant systems (metallothionein and reduced oxidized glutathione ratio) but not phytochelatins at both the mRNA and translated protein levels. Matrix-assisted laser desorption/ionization (MALDI)-imaging revealed the subcuticular bioaccumulation of oestradiol-3,4-quinone, altering the levels of local antioxidants in a time-dependent manner. CONCLUSIONS/SIGNIFICANCE: The present study illustrates that although most invertebrates do not possess oestrogen receptors, these organisms can be affected by oestrogen hormones, likely reflecting free diffusion into the cellular microenvironment with subsequent degradation to molecules that undergo redox cycling, producing ROS, thereby increasing environmental contamination that also perilously affects keystone animals, forming lower trophic levels.

Doxorubicin interactions with bovine serum albumin revealed by microdialysis with on-line laser-induced fluorescence detection at subpicogram level.

Doxorubicin (DOX) is an effective antitumor drug employed for treatment of a wide range of cancers types such as neuroblastoma, osteosarcoma, breast and esophageal carcinomas. On the other hand, the cumulative dose is restricted (300-550 mg/m(2)) and its amount administered to a patient has to be closely controlled due to its cardiotoxicity. To understand the mechanisms of the DOX side effects as well as to reveal the ways how to reduce its adverse impact on cardiomyocytes, the interactions with particular components of the blood and tissues have to be studied in greater detail. In this work, microdialysis technique was optimized to extract DOX from samples and subsequently monitor its interaction with BSA. Finally, the microdialysis probe was connected on-line to the LIF detector to ensure the real-time detection. The best flow rate was 1 µL/min and after 120 min of microdialysis 28% of the DOX was dialyzed out from the sample. The results from investigation of the DOX-BSA interaction indicate that the interaction occurs in less than 30 min, causing marked decrease in the amount of DOX extracted by microdialysis.

17ß-estradiol-containing liposomes as a novel delivery system for the antisense therapy of ER-positive breast cancer: An in vitro study on the MCF-7 cell line.

The present study suggests and describes the application of a delivery system for antisense oligonucleotides against mRNA encoding estrogen receptor proteins α and ß. The delivery system is composed of a cationic liposome envelope containing 17ß-estradiol (E2) in its structure. Cationic liposomes protect cargo against the extracellular matrix, and E2 can increase its shuttling efficiency into cells. Using MCF-7 cells derived from estrogen receptor-positive ductal carcinoma, treatment with liposomes against ERα was found to decrease MCF-7 proliferation, and importantly the application of both the antisense against ERα and ß exhibited an antiproliferative effect expressed as cell viability. Using qRT-PCR, it was shown that MT1A, NF-κB1 and K-ras genes, but not TFF1, were downregulated using E2-based liposomes (evaluated at P=0.05). Further indicators of oxidative stress were employed to assess the effect on treatment efficiency. Glutathione (GSH/GSSG redox ratio), metallothionein (MT) and malondialdehyde (MDA) confirmed a positive effect of antisense therapy resulting in their decreased levels in the MCF-7 cells. Based on these data, we suggest that E2-based liposomes offer sufficient transfer efficiency and moreover, due to the effect on NF-κB1, MT and GSH, tumor cells can be chemosensitized to increase treatment effectiveness.

In vitro interactions between 17ß-estradiol and DNA result in formation of the hormone-DNA complexes.

Beyond the role of 17ß-estradiol (E2) in reproduction and during the menstrual cycle, it has been shown to modulate numerous physiological processes such as cell proliferation, apoptosis, inflammation and ion transport in many tissues. The pathways in which estrogens affect an organism have been partially described, although many questions still exist regarding estrogens' interaction with biomacromolecules. Hence, the present study showed the interaction of four oligonucleotides (17, 20, 24 and/or 38-mer) with E2. The strength of these interactions was evaluated using optical methods, showing that the interaction is influenced by three major factors, namely: oligonucleotide length, E2 concentration and interaction time. In addition, the denaturation phenomenon of DNA revealed that the binding of E2 leads to destabilization of hydrogen bonds between the nitrogenous bases of DNA strands resulting in a decrease of their melting temperatures (Tm). To obtain a more detailed insight into these interactions, MALDI-TOF mass spectrometry was employed. This study revealed that E2 with DNA forms non-covalent physical complexes, observed as the mass shifts for app. 270 Da (Mr of E2) to higher molecular masses. Taken together, our results indicate that E2 can affect biomacromolecules, as circulating oligonucleotides, which can trigger mutations, leading to various unwanted effects.

Identification of estrogen receptor proteins in breast cancer cells using matrix-assisted laser desorption/ionization time of flight mass spectrometry (Review).

Estrogen receptors [ERs (subtypes α and ß)], classified as a nuclear receptor super family, are intracellular proteins with an important biological role as the transcription factors for estrogen target genes. For ER-induced transcription, an interaction must exist between ligand and coregulators. Coregulators may stimulate (coactivators) or inhibit (corepressors) transcription, following binding with a specific region of the gene, called the estrogen response element. Misbalanced activity of coregulators or higher ligand concentrations may cause increased cell proliferation, resulting in specific types of cancer. These are exhibited as overexpression of ER proteins. Breast cancer currently ranks first in the incidence and second in the mortality of cancer in females worldwide. In addition, 70% of breast tumors are ERα positive and the importance of these proteins for diagnostic use is indisputable. Early diagnosis of the tumor and its classification has a large influence on the selection of appropriate therapy, as ER-positive tumors demonstrate a positive response to hormonal therapy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) has been hypothesized to have great potential, as it offers reliable, robust and efficient analysis methods for biomarker monitoring and identification. The present review discusses ER protein analysis by MALDI TOF MS, including the crucial step of protein separation.

Flow injection analysis with electrochemical detection for rapid identification of platinum-based cytostatics and platinum chlorides in water.

Platinum-based cytostatics, such as cisplatin, carboplatin or oxaliplatin are widely used agents in the treatment of various types of tumors. Large amounts of these drugs are excreted through the urine of patients into wastewaters in unmetabolised forms. This phenomenon leads to increased amounts of platinum ions in the water environment. The impacts of these pollutants on the water ecosystem are not sufficiently investigated as well as their content in water sources. In order to facilitate the detection of various types of platinum, we have developed a new, rapid, screening flow injection analysis method with electrochemical detection (FIA-ED). Our method, based on monitoring of the changes in electrochemical behavior of analytes, maintained by various pH buffers (Britton-Robinson and phosphate buffer) and potential changes (1,000, 1,100 and 1,200 mV) offers rapid and cheap selective determination of platinum-based cytostatics and platinum chlorides, which can also be present as contaminants in water environments.

Fullerene as a transporter for doxorubicin investigated by analytical methods and in vivo imaging.

Carbon nanomaterials, including fullerenes, exhibit not only unique structure and electronic properties but also a significant potential to serve as radical scavengers and/or anti-oxidants. Their conjugation with anticancer drugs such as doxorubicin (DOX) may help to balance severe negative side effects of these cytostatics and also improve the delivery of the drug taking advantage of the enhanced cellular uptake, selectivity to cancer cells, and pH regulated release. In this study, the fullerene (C60) surface was oxidized by concentrated nitric acid, which enabled simple DOX-fullerene conjugation based on π-π stacking and hydrophilic interactions with carboxylic groups. The strength of this noncovalent binding is pH dependent. At a low pH, the amino group of DOX is protonated, however at a higher pH, the amino group is deprotonated, resulting in stronger hydrophobic interactions with the fullerene walls. CE and HPLC were employed for characterization of resulting complexes. The cell toxicity of the conjugates was evaluated using Staphylococcus aureus and finally they were administered into the chicken embryo to assess the applicability for in vivo imaging.

Study of the influence of platinum, palladium and rhodium on duckweed (Lemna minor).

OBJECTIVES: Road traffic pollutants and the residues of cytostatics that are widely used in anti-cancer therapy are a significant sources of platinum group elements (PGE; Pt, Pd and Rh) in environment. These metals can migrate into sewage and thus pollute surface waters. The purpose of our study was to evaluate the effect of PtCl4 on the antioxidant and enzymatic activity of duckweed (Lemna minor), a bioindicator of the aquatic environment. METHODS: The study was performed using a 7-day conventional test based on the OECD 221 (CSN EN ISO 20079)--Lemna sp. Growth Inhibition Test. We also conducted a microbiotest to analyse the effects of PtC4, PdCl2 and RhCl3 on the morphology and vegetative growth of colonies of this plant and compared their inhibitory effects during the microbiotest. RESULTS: We observed inhibition of colony growth and clear morphological changes. Antioxidant and enzymatic activities increased with platinum doses increased. The 168hEC50 of PtCl4 was 12.16 µM (95% confidence interval = 9.88-14.44) and the 168hEC50 of PdCl2 was 50.39 (95% confidence interval = 23.83-76.96). The greatest inhibition of growth by RhCl3 was observed at 25 µM. CONCLUSIONS: The obtained results suggest that L. minor phytotoxicity tests should be widely used in the biomonitoring.

Impact of platinum group elements on the soil invertebrate Enchytraeus crypticus.

OBJECTIVES: The platinum group elements (PGE) platinum (Pt), palladium (Pd), and rhodium (Rh) are used in automobile catalytic converters, from which they have been emitted into the environment to an increasing degree over the last 20 years. Despite the bioavailability of these metals to plants and animals, studies determining the effects of PGE on organisms are extremely rare. Enchytraeids are ecologically relevant soil organisms, due to their activity in decomposition and bioturbation in many soil types worldwide. DESIGN: The experiments were carried out as described in the OECD Guideline 220 [CSN EN ISO 16387--Soil quality--Effects of pollutants on Enchytraeidae (Enchytraeus sp.)--Determination of effects on reproduction]. The reproductive effects of platinum (PtCl4), palladium (PdCl2) and rhodium (RhCl3) were examined. The concentrations of PGE tested were as follows: 5, 10, 25, 50 and 100 µmol x L(-1) PdCl2; 50, 100, 150, 200 and 250 µmol x L-1 PtCl4/RhCl3. The EC50 (medium effective concentration) was determined after 28 days of exposure. The inhibition of the reproduction of PGE-exposed enchytraeids was compared against controls. RESULTS: Values of 28dEC50 of PtCl4, PdCl2 and RhCl3 amounted to 161.9 µmol x L(-1), 70.0 µmol x L(-1) and 246.6 µmol x L(-1), respectively. We can confirm that the relative order of toxicities is Pd (II) >Pt(IV) >>Rh(III). CONCLUSION: To the best of our knowledge, this is the first study to use Enchytraeus crypticus as an indicator species to assess the risk of soil contamination by platinum, palladium and rhodium. Results of this study contribute important data on the ecotoxicity of a rarely studied elements.

Impact of platinum group elements on the soil invertebrate Folsomia candida.

OBJECTIVES: Trace concentrations of the platinum group elements (PGE; Pt, Pd and Rh) are nowadays an irreplaceable part of environmental analysis and assessment. These rare elements are used as effective substances in automotive catalysts to reduce pollution by emissions originating from fuel combustion. Due to their harmful potential, it is necessary to monitor their content and behaviour in different samples. Effect assessment using ecotoxicological bioassays with organisms at different trophic levels can provide valuable pieces of information on the risk of chemical substances in the ecosystem. DESIGN: The experiments were carried out as described in the OECD Guideline 232 [CSN ISO 11267 - Soil quality - Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants]. The reproductive effects of palladium (PdCl2) and rhodium (RhCl3) were examined. Concentrations of PGE tested were as follows: 5, 10, 25, 50 and 100 µmol.L-1. The EC50 (medium effective concentration) was determined after 28 days of exposure. Inhibition of reproduction of PGE-exposed collembolans was compared against controls. RESULTS: Values of 28dEC50 of PdCl2 and RhCl3 amounted to 21.0 µmol.L-1 and 266.22 µmol.L-1, respectively. We can confirm that the relative order of toxicities is Pd (II) > Pt(IV) >> Rh(III). CONCLUSION: To the best of our knowledge, this is the first study to use Folsomia candida as an indicator species to assess the risk of soil contamination by palladium and rhodium. However, more toxicity data for various species are needed to evaluate the environmental risks of PGEs in soils.

Molecular biology of beta-estradiol-estrogen receptor complex binding to estrogen response element and the effect on cell proliferation.

Group of estrogen pollutants, where the highest estrogen activity is reported at estradiol, is characterized by the fact that even at very low concentrations have potential to cause xenoestrogenic effects. During exposure of excessive amounts of estradiols may be produced undesirable effects resulting in the feminization of males of water organisms. The presence of estradiols in drinking water implies also a risk for the human population in the form of cancers of endocrine systems, abnormalities in reproduction or dysfunctions of neuronal and immune system. Currently, the research is focused mainly to uncover the relationship between the estrogen receptors binding affinity with an estrogen response element and estradiol. In this review we summarized facts about molecular biological principles of ß estradiol-estrogen receptor complex binding with estrogen response element and its successive effect on cancer genes expression.

Yew poisoning of olive baboons (Papio anubis) in captivity: laboratory diagnosis.

OBJECTIVES: Toxic effects of the yew have been known since ancient times. Yew toxicity is due to the content of cyanogenic glycosides and a mixture of alkaloids known as taxines. Taxine B is probably responsible for the most part of adverse effects in poisoned organisms. This particular taxoid is common in body fluids of the yew-poisoned. The present study is engaged with laboratory examination to confirm substances that lead to fatality of a pair of olive baboons (Papio anubis) following ingestion of yew seeds. When both cage mates (male and female) died suddenly, poisoning was suspected because many berries had fallen into the cage from a nearby fruiting yew tree (Taxus baccata) during the windy night before. METHODS: The analysis was performed using electrospray ionization quadrupole time-of-flight mass spectrometry. A flow injection analysis/mass spectrometry setting was prepared for this purpose. RESULTS: The above mentioned mass spectrometry analysis of taxoids confirmed poisoning by taxanes. The presence of taxin B/isotaxin B was confirmed in all investigated samples. Apparently in urine and bile there were concentrations ranging 150-220 ng.mL-1 and in blood serum concentrations 25-30 ng.mL-1. CONCLUSION: It follows from the results obtained that we confirmed that baboons were deadly intoxicated by yew fruits.

Lead ions encapsulated in liposomes and their effect on Staphylococcus aureus.

The aim of the study was the preparation of a liposome complex with encapsulated lead ions, which were electrochemically detected. In particular, experiments were focused on the potential of using an electrochemical method for the determination of free and liposome-encapsulated lead and determination of the encapsulation efficiency preventing the lead toxicity. Primarily, encapsulation of lead ions in liposomes and confirmation of successful encapsulation by electrochemical methods was done. Further, the reduction effect of the liposome matrix on the detected electrochemical signal was monitored. Besides encapsulation itself, comparison of toxicity of free lead ions and lead ions encapsulated in liposome was tested. The calculated IC50 values for evaluating the lead cytotoxicity showed significant differences between the lead enclosed in liposomes (28 µM) and free lead ions (237 µM). From the cytotoxicity studies on the bacterial strain of S. aureus it was observed that the free lead ions are less toxic in comparison with lead encapsulated in liposomes. Liposomes appear to be a suitable carrier of various substances through the inner cavity. Due to the liposome structure the lead enclosed in the liposome is more easily accepted into the cell structure and the toxicity of the enclosed lead is higher in comparison to free lead ions.

Ion exchange chromatography and mass spectrometric methods for analysis of cadmium-phytochelatin (II) complexes.

In this study, in vitro formed Cd-phytochelatin (PC2) complexes were characterized using ion exchange chromatography (IEC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The ratio of both studied compounds as well as experimental conditions were optimized. The highest yield of the complex was observed under an applied concentration of 100 µg·mL(-1) PC2 and 100 µg·mL(-1) of CdCl2. The data obtained show that IEC in combination with MALDI-TOF is a reliable and fast method for the determination of these complexes.

Lead toxicosis of captive vultures: case description and responses to chelation therapy.

BACKGROUND: Lead, a serious threat for raptors, can hamper the success of their conservation. This study reports on experience with accidental lead intoxication and responses to chelation therapy in captive Cinereous (Aegypius monachus) and Egyptian (Neophron percnopterus) Vultures. RESULTS: Soil contamination by lead-based paint sanded off the steel aviary resulted in poisoning of eight Cinereous and two Egyptian Vultures. A male Egyptian Vulture developed signs of apathy, polydipsia, polyuria, regurgitation, and stupor, and died on the next day. Liver, kidney and blood lead concentrations were 12.2, 8.16 and 2.66 µg/g, respectively. Laboratory analyses confirmed severe liver and kidney damage and anaemia. Blood Pb levels of Pb-exposed Cinereous Vultures were 1.571 ± 0.510 µg/g shortly after intoxication, decreased to 0.530 ± 0.165 µg/g without any therapy in a month and to 0.254 ± 0.097 µg/g one month after CaNa(2)EDTA administration. Eight months later, blood lead levels decreased to close to the background of the control group. Blood parameters of healthy Pb-non-exposed Cinereous Vultures were compared with those of the exposed group prior to and after chelation therapy. Iron levels in the lead-exposed pre-treatment birds significantly decreased after chelation. Haematocrit levels in Pb-exposed birds were significantly lower than those of the controls and improved one month after chelation. Creatine kinase was higher in pre-treatment birds than in the controls but normalised after therapy. Alkaline phosphatase increased after chelation. A marked increase in the level of lipid peroxidation measured as thiobarbituric acid reactive species was demonstrated in birds both prior to and after chelation. The ferric reducing antioxidant power was significantly lower in pre-treatment vultures and returned to normal following chelation therapy. Blood metallothionein levels in lead-exposed birds were higher than in controls. Reduced glutathione dropped after CaNa(2)EDTA therapy, while oxidised glutathione was significantly lower in both pre- and post-treatment birds. A chick in an egg produced by a Cinereous Vulture female two months after lead toxicosis died on day 40 of artificial incubation. Lead concentrations in foetal tissues were consistent with levels causing avian mortality. CONCLUSIONS: The reported blood parameters and reproduction impairment in captive birds may have implications for professionals dealing with lead exposure in wild birds.