Metal measurement in aquatic environments by passive sampling methods: Lessons learning from an in situ intercomparison exercise

Passive sampling devices (PS) are widely used for pollutant monitoring in water, but estimation of measurement uncertainties by PS has seldom been undertaken. The aim of this work was to identify key parameters governing PS measurements of metals and their dispersion. We report the results of an in situ intercomparison exercise on diffusive gradient in thin films (DGT) in surface waters. Interlaboratory uncertainties of time-weighted average (TWA) concentrations were satisfactory (from 28% to 112%) given the number of participating laboratories (10) and ultra-trace metal concentrations involved. Data dispersion of TWA concentrations was mainly explained by uncertainties generated during DGT handling and analytical procedure steps. We highlight that DGT handling is critical for metals such as Cd, Cr and Zn, implying that DGT assembly/dismantling should be performed in very clean conditions. Using a unique dataset, we demonstrated that DGT markedly lowered the LOQ in comparison to spot sampling and stressed the need for accurate data calculation.

In vitro studies of drug transformations: application to forensic toxicology

The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.

An aquatic biologist's view of water renovation

Caption title.

Recommended reporting standards for test accuracy studies of infectious diseases of finfish, amphibians, molluscs and crustaceans: the STRADAS-aquatic checklist

Complete and transparent reporting of key elements of diagnostic accuracy studies for infectious diseases in cultured and wild aquatic animals benefits end-users of these tests, enabling the rational design of surveillance programs, the assessment of test results from clinical cases and comparisons of diagnostic test performance. Based on deficiencies in the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines identified in a prior finfish study (Gardner et al. 2014), we adapted the Standards for Reporting of Animal Diagnostic Accuracy Studies—paratuberculosis (STRADAS-paraTB) checklist of 25 reporting items to increase their relevance to finfish, amphibians, molluscs, and crustaceans and provided examples and explanations for each item. The checklist, known as STRADAS-aquatic, was developed and refined by an expert group of 14 transdisciplinary scientists with experience in test evaluation studies using field and experimental samples, in operation of reference laboratories for aquatic animal pathogens, and in development of international aquatic animal health policy. The main changes to the STRADAS-paraTB checklist were to nomenclature related to the species, the addition of guidelines for experimental challenge studies, and the designation of some items as relevant only to experimental studies and ante-mortem tests. We believe that adoption of these guidelines will improve reporting of primary studies of test accuracy for aquatic animal diseases and facilitate assessment of their fitness-for-purpose. Given the importance of diagnostic tests to underpin the Sanitary and Phytosanitary agreement of the World Trade Organization, the principles outlined in this paper should be applied to other World Organisation for Animal Health (OIE)-relevant species.

Development of a new analytical approach based on cellulose membrane and chelator for differentiation of labile and inert metal species in aquatic systems

A new procedure was developed in this study, based on a system equipped with a cellulose membrane and a tetraethylenepentamine hexaacetate chelator (MD-TEPHA) for in situ characterization of the lability of metal species in aquatic systems. To this end, the DM-TEPHA system was prepared by adding TEPHA chelator to cellulose bags pre-purified with 1.0 mol L-1 of HCl and NaOH solutions. After the MD-TEPHA system was sealed, it was examined in the laboratory to evaluate the influence of complexation time (0-24 h), pH (3.0, 4.0, 5.0, 6.0 and 7.0), metal ions (Cu, Cd, Fe, Mn and Ni) and concentration of organic matter (15, 30 and 60 mg L-1) on the relative lability of metal species by TEPHA chelator. The results showed that Fe and Cu metals were complexed more slowly by TEPHA chelator in the MD-TEPHA system than were Cd, Ni and Mn in all pH used. It was also found that the pH strongly influences the process of metal complexation by the MD-TEPHA system. At all the pH levels, Cd, Mn and Ni showed greater complexation with TEPHA chelator (recovery of about 95-75%) than did Cu and Fe metals. Time also affects the lability of metal species complexed by aquatic humic substances (AHS); while Cd, Ni and Mn showed a faster kinetics, reaching equilibrium after about 100 min, and Cu and Fe approached equilibrium after 400 min. Increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS-metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems. (c) 2006 Elsevier B.V. All rights reserved.

Direct determination of Cu, Cd, Ni and Pb in aquatic humic substances by graphite furnace atomic absorption spectrometry

A method has been developed for the direct determination of Cu, Cd, Ni and Pb in aquatic humic substances (AHS) by graphite furnace atomic absorption spectrometry. AHS were isolated from water samples rich in organic matter, collected in the Brazilian Ecological Parks. All analytical curves presented good linear correlation coefficient. The limits of detection and quantification were in the ranges 2.5-16.7 mu g g(-1) and 8.5-50.0 mu g g(-1), respectively. The accuracy was determined using recovery tests, and for all analytes recovery percentages ranged from 93 - 98 %, with a relative standard deviation less than 4 %. The results indicated that the proposed method is a suitable alternative for the direct determination of metals in AHS.

Characterization of the Interactions between Endocrine Disruptors and Aquatic Humic Substances from Tropical Rivers

Interactions between two endocrine disruptors (ED) and aquatic humic substances (AHS) from tropical rivers were studied using an ultrafiltration system equipped with a 1 kDa cut-off cellulose membrane to separate free ED from the fraction bound in the AHS. Quantification of 17 alpha-ethynylestradiol and bisphenol A was performed using gas chromatography-mass spectrometry (GC-MS). The times required for establishment of equilibrium between the AHS and the ED were ca. 30 min, and complexation capacities for 17 alpha-ethynylestradiol and bisphenol A were 18.53 and 2.07 mg g(-1) TOC, respectively. The greater interaction of AHS with 17 alpha-ethynylestradiol, compared to bisphenol A, was due to the presence of hydrogen in the structure of 17 alpha-ethynylestradiol, which could interact with ionized oxygenated groups of the AHS. The results indicate that AHS can strongly influence the transport and reactivity of endocrine disruptors in aquatic systems.

BALD EAGLES (HALIAEETUS LEUCOCEPHALUS) AS INDICATORS OF GREAT LAKES ECOSYSTEM HEALTH

Environmental indicators have been proposed as a means to assess ecological integrity, monitoring both chemical and biological stressors. In this study, we used nestling bald eagles as indicators to quantify direct or indirect tertiary-level contaminant exposure. The spatial and temporal trends of polychlorinated biphenyl (PCB) congeners were evaluated in nestling plasma from 1999–2014. Two hexa-chlorinated congeners, PCB-138 and 153, were detected with the highest frequency and greatest concentrations throughout Michigan. Less-chlorinated congeners such as PCB-52 and 66 however, comprised a greater percentage of total PCB concentrations in nestlings proximate to urbanized areas, such as along the shorelines of Lake Erie. Toxic equivalents were greatest in the samples collected from nestlings located on Lake Erie, followed by the other Great Lakes spatial regions. Nestling plasma samples were also used to measure concentrations of the most heavily-used group of flame retardants, brominated diphenyl ethers (BDEs), and three groups of alternative flame retardants, non-BDE Brominated Flame Retardants (NBFRS), Dechloranes, and organophosphate esters (OPs). BDE-47, 99 and 100 contributed the greatest to total BDE concentrations. Concentrations of structurally similar NBFRs found in this study and recent atmospheric studies indicate that they are largely used as replacements to previously used BDE mixtures. A variety of Dechloranes, or derivatives of Mirex and Dechlorane Plus, were measured. Although, measured at lesser concentrations, environmental behavior of these compounds may be similar to mirex and warrant future research in aquatic species. Concentrations of OPs in nestling plasma were two to three orders of magnitude greater than all other groups of flame retardants. In addition to chemical indicators, bald eagles have also been proposed as indicators to identify ecological stressors using population measures that are tied to the fitness of individuals and populations. Using mortality as a population vitality rate, vehicle collisions were found to be the main source of mortality with a greater incidence for females during white-tailed deer (Odocoileus virginianus) hunting months and spring snow-melt. Lead poisoning was the second greatest source of mortality, with sources likely due to unretrieved hunter-killed, white-tailed deer carcasses, and possibly exacerbated by density-dependent effects due to the growing population in Michigan.

Assessing the safety of Pseudomonas putida introduction in the environment: an overview of ecotoxicological tests.

Risk assessment guidelines for the environmental release of microbial agents are performed in a tiered sequence which includes evaluation of exposure effects on non target organisms. However, it becomes important to verify whether environmental risk assessment from temperate studies is applicable to tropical countries, as Brazil. Pseudomonas putida is a bacteria showing potential to be used for environmental applications as bioremediation and plant disease control. This study investigates the effects of this bacteria exposure on rodents and aquatic organisms (Daphnia similes) that are recommended to be used as non-target organism in environmental risk assessments. Also, the microbial activity in three different soils under P. putida exposure was evaluated. Rats did not show clinical alterations, although the agent was recovered 16 h after the exposure in lung homogenates. The bacteria did not reduce significantly the reproduction and survival of D. similis. The soil enzymatic activities presented fluctuating values after inoculation with bacteria. The measurement of perturbations in soil biochemical characteristics is presented as an alternative way of monitoring the overall effects of the microbial agent to be introduced even in first stage (Tier I) of the risk assessment in tropical ecosystems.

Multilocus genetic analyses provide insight into speciation and hybridization in aquatic grasses, genus Ruppia

Aquatic plants of the genus Ruppia inhabit some of the most threatened habitats in the world, such as coastal lagoons and inland saline to brackish waters where their meadows play several key roles. The evolutionary history of this genus has been affected by the processes of hybridization, polyploidization, and vicariance, which have resulted in uncertainty regarding the number of species. In the present study, we apply microsatellite markers for the identification, genetic characterization, and detection of hybridization events among populations of putative Ruppia species found in the southern Iberian Peninsula, with the exception of a clearly distinct species, the diploid Ruppia maritima. Microsatellite markers group the populations into genetically distinct entities that are not coincident with geographical location and contain unique diagnostic alleles. These results support the interpretation of these entities as distinct species: designated here as (1) Ruppia drepanensis, (2) Ruppia cf. maritima, and (3) Ruppia cirrhosa. A fourth distinct genetic entity was identified as a putative hybrid between R. cf. maritima and R. cirrhosa because it contained a mixture of microsatellite alleles that are otherwise unique to these putative species. Hence, our analyses were able to discriminate among different genetic entities of Ruppia and, by adding multilocus nuclear markers, we confirm hybridization as an important process of speciation within the genus. In addition, careful taxonomic curation of the samples enabled us to determine the genotypic and genetic diversity and differentiation among populations of each putative Ruppia species. This will be important for identifying diversity hotspots and evaluating patterns of population genetic connectivity. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 00, 000–000.

Selenite and Selenate Effects on Mercury (Hg2+) Uptake and Distribution in Tilapia, Oreochromis niloticus L., Assessed by Chronic Bioassay

Aquatic organisms are considered excellent biomarkers of mercury (Hg) occurrence in the environment. Selenium (Se) acts in antagonism to this metal, stimulating its elimination, and reducing its toxicity. In this paper, tilapia (Oreochromis niloticus) were chronically acclimated in sub-lethal Hg2+, Hg2+ + Se4+ and Hg2+ + Se6+ concentrations. Distribution and bioaccumulation of both elements were evaluated in fish tissues. The kidney was the main target of the Hg and Se uptake, and the presence of Hg induced the Se hepatic elimination. The Hg bioaccumulation in the gill, spleen and heart were higher in the presence of Se6+ than in the presence of Se4+.

Effect of Urucu oil (Brazilian Amazon) on the biomass of the aquatic macrophyte Eichhornia crassipes (Mart.) Solms (Pontederiaceae)

Os rios e lagos de várzea da província petrolífera de Urucu, na Amazônia Central, são amplamente colonizados por macrófitas aquáticas, que podem ser afetadas por acidentes durante a exploração e o transporte de petróleo. Entre as macrófitas, a espécie flutuante Eichhornia crassipes (aguapé) ocorre abundantemente na região; OBJETIVO: O objetivo desse estudo foi verificar o efeito de diferentes dosagens do petróleo de Urucu (0; 0,5; 1,5 e 3,0 L.m-2) na biomassa viva e morta de E. crassipes e em algumas características físicas e químicas da água; MÉTODOS: O experimento teve oitenta e quatro dias de duração. A cada sete dias foi determinada a biomassa (viva e morta) de E. crassipes e os valores de temperatura, pH, condutividade elétrica e oxigênio dissolvido da água; RESULTADOS: A dosagem de 0,5 L.m-2 foi suficiente para causar mortalidade parcial (48%) em E. crassipes após trinta e cinco dias de exposição ao petróleo. A dosagem de 3,0 L.m-2 causou mortalidade total (100%) em E. crassipes em oitenta e quatro dias de exposição. A decomposição do petróleo e da biomassa morta de E. crassipes provocam a redução do oxigênio dissolvido e do pH, e aumento da condutividade elétrica e de fósforo total na água; CONCLUSÕES: Nós concluímos que um derramamento de petróleo pode provocar mortalidade total em uma população de uma espécie de macrófita, mas não em uma outra. Isto pode alterar a diversidade de espécies de macrófitas na região impactada. No caso de Eichhornia crassipes e Pistia stratiotes, um derramamento de petróleo de Urucu pode favorecer E. crassipes, a espécie menos sensível ao petróleo.

APPLICATION OF REMOTE SENSING IN AQUATIC ECOSYSTEMS

I utilized state the art remote sensing and GIS (Geographical Information System) techniques to study large scale biological, physical and ecological processes of coastal, nearshore, and offshore waters of Lake Michigan and Lake Superior. These processes ranged from chlorophyll a and primary production time series analysies in Lake Michigan to coastal stamp sand threats on Buffalo Reef in Lake Superior. I used SeaWiFS (Sea-viewing Wide Field-of-view Sensor) satellite imagery to trace various biological, chemical and optical water properties of Lake Michigan during the past decade and to investigate the collapse of early spring primary production. Using spatial analysis techniques, I was able to connect these changes to some important biological processes of the lake (quagga mussels filtration). In a separate study on Lake Superior, using LiDAR (Light Detection and Ranging) and aerial photos, we examined natural coastal erosion in Grand Traverse Bay, Michigan, and discussed a variety of geological features that influence general sediment accumulation patterns and interactions with migrating tailings from legacy mining. These sediments are moving southwesterly towards Buffalo Reef, creating a threat to the lake trout and lake whitefish breeding ground.

Environmental Dynamics of Dissolved Black Carbon in Aquatic Ecosystems

Black carbon (BC), the incomplete combustion product from biomass and fossil fuel burning, is ubiquitously found in soils, sediments, ice, water and atmosphere. Because of its polyaromatic molecular characteristic, BC is believed to contribute significantly to the global carbon budget as a slow-cycling, refractory carbon pool. However, the mass balance between global BC generation and accumulation does not match, suggesting a removal mechanism of BC to the active carbon pool, most probable in a dissolved form. The presence of BC in waters as part of the dissolved organic matter (DOM) pool was recently confirmed via ultrahigh resolution mass spectrometry, and dissolved black carbon (DBC), a degradation product of charcoal, was found in marine and coastal environments. However, information on the loadings of DBC in freshwater environments and its global riverine flux from terrestrial systems to the oceans remained unclear. The main objectives of this study were to quantify DBC in diverse aquatic ecosystems and to determine its environmental dynamics. Surface water samples were collected from aquatic environments with a spatially significant global distribution, and DBC concentrations were determined by a chemical oxidation method coupled with HPLC detection. While it was clear that biomass burning was the main sources of BC, the translocation mechanism of BC to the dissolved phase was not well understood. Data from the regional studies and the developed global model revealed a strong positive correlation between DBC and dissolved organic carbon (DOC) dynamics, indicating a co-generation and co-translocation between soil OC and BC. In addition, a DOC-assistant DBC translocation mechanism was identified. Taking advantage of the DOC-DBC correlation model, a global riverine DBC flux to oceans on the order of 26.5 Mt C yr-1 (1 Mt = 1012 g) was determined, accounting for 10.6% of the global DOC flux. The results not only indicated that DOC was an important environmental intermediate for BC transfer and storage, but also provided an estimate of a major missing link in the global BC budget. The ever increasing DBC export caused by global warming will change the marine DOM quality and may have important consequences for carbon cycling in marine ecosystem.