Toxicity assays applied to wastewater treatment

The utility and validity of toxicity tests for monitoring of wastewater treatment have been assessed. The evaluated acute toxicity tests have been Vibrio fischeri, Selenastrum capricornotum and Daphnia magna tests. The validation studies indicated that the acute toxicity tests can be considered as high sensitivity analytical tools to detect common environmental concentrations of the pollutants at concentration levels as low as ng l⁻¹. The toxicity tests showed to have discriminatory ability to distinguish between different degrees of toxicity, and the toxic specificity of the compounds on target organisms. Synergistic, additive or antagonistic effects were evaluated indicating the capacity of the toxicity test to assess the combined effects of chemicals in wastewaters. The reproducibility of these tests, calculated as relative standard deviation, is acceptable in the range of 5-22.3%. The application of multivariate date analysis proved that toxicity and chemical measures are complementary analytical tools for monitoring of wastewaters quality. The toxicity tests are useful analytical tools for screening of chemical analysis and as an early warning system to monitor the treatment of WWTPs. The use of single toxicity test or battery of tests is the best approach to evaluate the risk because they are reliable indices of the toxic impact of effluents in the aquatic environment. The toxicity tests were applied in the quality control of different European WWTPs.     

Comparative evaluation of the effects of pesticides in acute toxicity luminescence bioassays

Acute toxicity of pesticides in water was assessed singly and in mixtures using the responses of the luminescent bacterium Vibrio fischeri (BioTox™), the aquatic invertebrate Daphnia magna (Daphtoxkit™), and the MitoScan™ assay. The latter utilized fragmented mitochondria to enzymatically convert β-nicotinamide adenine dinucleotide (NADH) to its oxidized form, NAD⁺. The rate of the conversion being sensitive to type and concentration of toxicants. The pesticides tested were Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate), Cyromazine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine), Fenamiphos (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate), and Formetanate (3-dimethylaminomethyleneiminophenyl methylcarbamate). The toxicity bioassays were characterized in terms of relative sensitivity, reproducibility, range of the linear response, and the ability to reveal synergistic/antagonistic interactions among toxicants. The D. magna assay was the most sensitive and best able to detect toxic interactions of mixtures. Also, unlike the other assays used, the response of the daphnid system was linear over a 10-fold change in pesticide concentration. Relative to the BioTox™, the MitoScan™ was 2- to 11-fold more sensitive for the compounds and mixtures tested. The EC₅₀ reproducibility of all tests was within ±20% coefficient of variation; however, the lowest observable effect concentration (LOEC) were only reproducible to ±35% on average. Cyromazine was the least toxic of the pesticides tested. To test the predictive value of the concept of concentration addition, toxicities of binary and quaternary mixtures of four different pesticides were analyzed. Synergistic/antagonistic responses were most frequently observed in testing with D. magna. Synergistic/antagonistic effects were seen only in 25 and 50% of the cases with the BioTox™ and the MitoScan™ assays, respectively.     

Chromatography-mass spectrometry and toxicity evaluation of selected contaminants in seawater

Summary In the present work a combined analytical study involving gas and liquid-chromatography and toxicity studies were developed for the determination of various contaminants typically present in sea water samples. The compounds investigated were Diuron, Chlorothalonil, Dichlofluanid, TCMTB (2-thiocyanomethylthiobenzothiazole), lrgarol 1051, Sea nine 211 and MTBE (methyl-tert-butyl ether). The selected compounds are additives of boat paints and gasoline and they can release into the aquatic environment in considerable amounts in areas with intense shipping traffic. The developed analytical protocol consisted on the use of a solid phase extraction procedure with Oasis^™ HLB cartridges followed by GC-El/NCI-MS and LC-ES-MS both in SIM mode. Average recoveries, loading 600 mL of samples with pH=3, varied from 40 to 95% and the detection limits ranged from 1–25 ng L⁻¹. The developed method was applied to real samples from various marinas of Andalusia (Spain).Daphnia magna, Vibrio fischeri andSelenastrum capricornotum toxicity bioassays on individual and mixtures of selected compounds were applied to evaluate EC₅₀ (effective concentration) and LOEC (lowest observable effect concentration) values. Resulting values were in the range of 0.001–720 mg L⁻¹ for EC₅₀ and 0.8.10⁻⁹–30 mg L⁻¹ for LOEC for all contaminants except for MTBE where no toxic effect were found up to 680 mg L⁻¹ onDaphnia magna. Toxicity effects of binary mixtures of the selected compounds showed synergistic effects in the majority of the cases (47.6%) and antagonistic in the lower of the cases (9.5%). Levels of the contaminants found in the seawater samples showed around a 30% of inhibition on tested species, during the summer months considering mixture lrgarol 1051-Diuron. Accelerated toxicity processes were observed in mixtures MTBE-Diuron and MTBE-Dichlofluanid. Presented in the Workshop “GPoll of European Science Foundation” Barcelona, Spain, 8–10 November 2001.     

Determination of antifouling pesticides and their degradation products in marine sediments by means of ultrasonic extraction and HPLC–APCI–MS

A method has been developed for the simultaneous determination of antifouling pesticides and some of their degradation products, e.g. dichlofluanid, diuron, demethyldiuron, 1-(3,4-dichlorophenyl)urea, sea-nine, Irgarol 1051 and one of its metabolites (2-methylthio-4-tert-butylamino-s-triazine) in marine sediments. The determination of these compounds in sediment samples was performed by means of methanolic ultrasonic extraction then clean-up on an Isolute ENV+ solid phase extraction (SPE) cartridge. The resulting extract was then analyzed by reversed-phase high-performance liquid chromatography coupled with atmospheric-pressure chemical-ionization mass spectrometry in negative and positive ion modes (HPLC–APCI–MS). Recovery ranged from 54–109% for the antifouling agents and their degradation products. The determination limits for the different compounds varied between 0.2 and 1.6 μg kg^–1 dry sediment. The analytical procedure was successfully applied to the determination of these pesticides and their degradation products in marine sediment samples from different marinas of the Catalan coast. The compounds detected were: diuron, dichlofluanid, demethyldiuron, sea-nine, and Irgarol 1051. The highest concentrations were those of diuron and Irgarol 1051 – 136 and 88 μg kg^–1, respectively.     

Part-per-trillion level determination of antifouling pesticides and their byproducts in seawater samples by off-line solid-phase extraction followed by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A new method for the simultaneous determination of antifouling pesticides and some of their byproducts such as dichlofluanid, diuron and its byproducts [demethyldiuron and 1-(3,4-dichlorophenyl)urea], (2-thiocyanomethylthio)ben: zothiazole, chlorothalonil, Sea-nine 211, Irgarol 1051 and one of its byproducts (2-methylthio-4-tert.-butylamino-s-triazine) in seawater was developed. The extraction of these compounds from the filtered seawater samples was performed off-line with different solid-phase extraction sorbents using (I) a 500 mg graphitized carbon black cartridge (ENVI-Carb) and (II) 200 mg polymeric cartridges (LiChrolut EN and Isolute ENV+) and passing 500 ml of the sample through these cartridges. The detection was carried out by reversed-phase high-performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry both in the negative and positive ion modes. The recovery ranged from 76 to 96% for the whole antifouling group with the ENVI-Carb cartridges and the detection limit was at the part-per-trillion level except for TCMTB. The method utilizing the polymeric cartridge proved to be very useful, time saving and with good recoveries when only Irgarol and its byproduct, Sea-nine 211 and diuron and its byproducts, have to be analyzed. The different cartridges were applied to the analysis of these pesticides in different marinas of the Catalan coast; diuron, dichlofluanid, Sea-nine 211, Irgarol as well as demethyldiuron and the Irgarol byproduct being the must ubiquitous pollutants. Maximum concentration levels were 2-3.5 microg/l of diuron and Sea-nine 211, respectively.     

Determination of diuron and the antifouling paint biocide irgarol 1051 in Dutch marinas and coastal waters

A sensitive LC–electrospray MS–MS method using off-line solid-phase extraction for the determination of diuron and Irgarol 1051 has been developed, enabling determination of both compounds at sub-ppt levels. Diuron and Irgarol 1051 are used as alternatives for tributyltin in antifouling paints that prevent growth on boats, and an increase in their application is anticipated because of the upcoming ban on tributyltin in 2003. In 2000, a survey was carried out to assess contamination with diuron and Irgarol 1051 of a number of Dutch marinas and coastal waters. Depending on the time of year, both compounds were encountered at levels higher than the maximum permissible concentrations of 430 and 24 ng/l for diuron and Irgarol 1051, respectively. Outside marinas at reference locations, concentrations were much lower, depending on the geographical situation and the nature of the water exchange with the environment related to tidal cycles. A seasonal influence was observed with highest levels in summer, corresponding to the yachting season for both compounds. For diuron, use in agriculture could have contributed to the high concentration encountered in surface waters.     

An evaluation of antifouling booster biocides in Gran Canaria coastal waters using SPE-LC MS/MS

A solid phase extraction (SPE) technique for seawater samples coupled to quantification using liquid chromatography tandem-mass spectrometry (LC-MS/MS) is described to quantify relevant antifouling booster biocides of ecotoxicological concern (Diuron, TCMTB, Irgarol 1051 and Dichlofluanid). The optimised methodology provides a sensitive, easy to use and efficient analytical procedure with detection limits in the range of between 0.1 and 0.2?ng?L^?1 and appropriate reproducibility (with analytical standard deviations of less than 10%). Spiked recoveries for all compounds exceeded 72%. The method was tested through a thorough monitoring regime of commercial port and marinas on the island of Gran Canaria (off the north-west coast of Africa) over a period of several months in 2008. Results provide the first data for antifouling booster biocides in the Canary Islands. Concentrations of Diuron and Irgarol 1051 in samples ranged between 2 and 195?ng?L^?1 and 2 and 146?ng?L^?1, respectively. TCMTB and Dichlofluanid were not detected.     

Application of ring study: water toxicity determinations by bioluminescence assay with Vibrio fischeri

Acceptance of toxicity bioassays as effective analytical tools in environmental areas needs guarantees of standardization but also validation. Ten European laboratories took part in an inter-laboratory study using different commercial devices based on bioluminescence inhibition of bacteria Vibrio fischeri. Reproducibility and stability by short toxicity endpoints, effective concentration that gives 10%, 50% and 80% of inhibition (EC₁₀, EC₅₀ and EC₈₀) is evaluated. Parametric and non-parametric statistic is applied and performance of participant laboratories is addressed by z-scores calculated by non-parametric statistic. z-Score classification was based on harmonised protocol for proficiency testing of analytical laboratories (satisfactory |z| ≤ 2; questionable 2 < |z| ≤ 3; unsatisfactory |z| > 3). Tested samples were phenol, 3,5-dichlorophenol and influent wastewater. Based on z-score classification, more than 70% of the laboratories showed a satisfactory performance for phenol, 3,5-dichlorophenol and influent wastewater (86%, 90% and 70%, respectively). Reproducibility and stability was observed in toxicant references and in wastewater samples. EC₈₀ determination appears to be more robust that EC₁₀ and EC₅₀. EC determinations can be considered favorable at 5 and 15 min of exposition, in particular for EC₈₀. The use of different commercial devices can not be considered an additional source of variation.     

Mathematical model for the analytical signal of an herbicide sensor based on the reaction centre of Rhodobacter sphaeroides

This paper introduces a mathematical model which makes it possible both to determine the concentration of photosynthetic herbicides and to obtain a quantitative parameter in order to compare their activity using a previously described sensing system. The working principle involves the changes in absorption properties at 860 nm of the reaction centre (RC) isolated from the bacteria Rhodobacter sphaeroides when photosynthetic herbicides are present. The method has been used for the determination and activity comparison of five photosynthetic herbicides: diuron, atrazine, terbutryn, terbuthylazine and simazine. Detection limits obtained were 2.2, 0.75, 0.046, 0.25, and 1.4 μM, respectively. The resulting order for the different herbicides according to their action on RC was: terbutryn > terbuthylazine > atrazine > simazine > diuron.     

Radiolysis of selected antibiotics and their toxic effects on various aquatic organisms

This study was conducted to investigate the decomposition of three γ-irradiated antibiotics (e.g., tetracycline, sulfamethazine, and lincomycin) and to compare the toxic effects on Daphnia magna, Vibrio fischeri, and Pseudokirchneriella subcapitata. The median cell growth inhibition concentrations (IC₅₀) of tetracycline, lincomycin, and sulfamethazine for P. subcapitata dramatically increased (e.g., toxicity decreased) after radiolysis. The results demonstrated that γ-radiation treatment was efficient to decompose antibiotics and thereby their toxicity on P. subcaptitata remarkably decreased due to reduced parent compounds.     

Isolation of secondary metabolites from Hortia oreadica (Rutaceae) leaves through high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) with a two-phase solvent system (hexane–ethanol–acetonitrile–water 10:8:1:1, v/v) was applied to examine the leaves of Hortia oreadica, which afforded the known limonoid guyanin (1), the alkaloids rutaecarpin (2) and dictamnine (6), the dihydrocinnamic acid derivatives methyl 5,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran-6-propanoate (3), 5,8-dimethoxy-2,2-dimethyl-2H-1-benzopyran-6-propanoic acid (4), together with the new E-3,4-dimethoxy-α(3-hydroxy-4-carbomethoxyphenyl)cinnamic acid (5). The recovery of compounds 1–6 was determined by comparison with LC-atmospheric pressure chemical ionization MS/MS data: 66.2%, 93.1%, 102.5%, 101.2%, 99.0% and 84.9%, respectively. Compound 3 showed IC₅₀ of 23.6 μM against Plasmodium falciparum and 15.6 μM against Trypanosoma brucei rhodesienses and was not toxic to KB cells (IC₅₀ > 100 μM).     

A sensitive, rapid and inexpensive way to assay pesticide toxicity based on electrochemical biosensor

We reported a rapid toxicity assay method using electrochemical biosensor for pesticides, Escherichia coli (E. coli) was taken as a model microorganism for test. In this method, we adopted ferricyanide instead of natural electron acceptor O₂, and then microbial oxidation was substantially accelerated. Toxicity assays measured the effect of toxic materials on the metabolic activity of microorganisms. The current signal of ferrocyanide produced from the metabolism was proven to be directly related to the toxicity, which could be amplified by ultramicroelectrode array (UMEA). The ratio of the electrochemical signals, recorded in the presence and absence of toxin, provided an index of inhibition. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. 3,5-Dichlorophenol (DCP) was taken as the reference toxicant, its IC50 was estimated to be 8.0 mg/L. Three pesticides were examined using this method. IC50 values of 6.5 mg/L for Ametryn, 22 mg/L for Fenamiphos and 5.7 mg/L for Endosulfan were determined and in line with EC50 values reported in the literature. Atomic force microscopy (AFM) was also used for morphology characterization of E. coli induced by three pesticides. These results confirmed the present electrochemical method used is reliable. In addition, the electrochemical method is a sensitive, rapid and inexpensive way for toxicity assays of pesticides.     

A comparison study between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples

In the present study, a comparison between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples was made.In order to realize this study, a disposable electrochemical DNA biosensor has been reported. The DNA biosensor is assembled by immobilizing double stranded Calf Thymus DNA onto the surface of a disposable carbon screen-printed electrode. The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal to detect the DNA damage; the presence of low molecular weight compounds with affinity for nucleic acids is measured by their effect on the guanine oxidation peak.Wastewater samples provided during First European Interlaboratory Exercise on water toxicity in the course of the project SWIFT-WFD were analyzed, and biosensor results were compared with a currently used toxicity test ToxAlert^®100 based on the bioluminescence inhibition of Vibrio fischeri. This test have been used because is rapid, easy handling and cost effectively responses for the toxicity assessment in real water samples.The results showed a promising correlation between two tests used for the detection of toxic compounds in water samples.     

Analytical method development for the determination of four biocides used in antifouling paints in estuarine waters and sediments by gas chromatography-mass spectrometry

Summary Irgarol 1051, chlorothalonil, dichlofluanid and diuron are biocides utilised in antifouling paints as alternatives to organotin compounds, after restrictions imposed in the use of tributyltin in 1987. Effective analytical methods for the simultaneous determination of these four biocides in waters and sediments have been developed and method performance data are presented. Quantification is by gas chromatography—electron ionisation mass spectrometry (GC-EI-MS). The method involves the simultaneous extraction of the biocides from water with dichloromethane or from sediments with dichloromethane and acetone. For the determination of diuron the extract was methylated prior to GC analysis to avoid thermal degradation in the instrument. The method was applied to water and sediment samples spiked with the biocides. The highest detection sensitivities were achieved in the selected-ion monitoring (SIM) mode. Recovery studies were performed at 20, 2 and 1 ppb for all biocides in water and 400, 40 and 20 ppb in sediments. The percentage recoveries ranged between 90 and 100% for waters and 80 and 90% for sediments. Seven determinations were made at each concentration level along with a procedural blank. The quantification limit of the method was around 0.2 ppb for water and 5 ppb for sediments depending on the individual compound.     

Spirostaphylotrichin W,a spirocyclic γ-lactam isolated from liquid culture of Pyrenophora semeniperda, a potential mycoherbicide for cheatgrass (Bromus tectorum) biocontrol

A novel spirocyclic γ-lactam, named spirostaphylotrichin W (1), was isolated together with the well known and closely related spirostaphylotrichins A, C, D, R and V, as well as triticone E, from the liquid cultures of Pyrenophora semeniperda (anamorph: Drechslera), a seed pathogen proposed for cheatgrass (Bromus tectorum) biocontrol. Spirostaphylotrichin W was characterized as (3S*,4S*,5S*,6S*,9Z,10Z)-4,6-dihydroxy-2,3-dimethoxy-3-methyl-10-propyliden-2-azaspiro [4.5]dec-8-ene-1,7-dione, by spectroscopic and chemical methods. The relative stereochemistry of spirostaphylotrichin W was assigned using NOESY experiments and in comparison to those of spirostaphylotrichin V (2) and triticone E (6). In fact, the relative stereochemistry at C-3 was the same of that of 2, while that at C-4 and C-6 was inverted in respect to that reported, respectively, for 2 and 6. In a B. tectorum coleoptile bioassay at concentration of 10⁻³, spirostaphylotrichin A proved to be the most active compound, followed by spirostaphylotrichins C and D. Spirostaphylotrichin W and V showed mild toxicity while spirostaphylotrichin R and triticone E were not active. When tested on host and non-host plants by leaf puncture bioassay, spirostaphylotrichins A, C and D caused the appearance of necrotic spots while the other compounds were inactive.     

Synthesis, structural and larvicidal studies of some triorganotin 2-(p-chlorophenyl)-3-methylbutyrates

A series of triorganotin 2-(p-chlorophenyl)-3-methylbutyrates, (R₃SnO₂CCH(CH(CH₃)₂)C₆H₄Cl-4), where R = methyl, ethyl, n-propyl, n-butyl, phenyl and cyclo-hexyl, have been synthesized. Elemental analyses, Mössbauer, Infrared and NMR spectroscopies have been used to characterize their structures. Based on the spectroscopic results, all the complexes with the exception of the tricyclohexyl compound were found to be five-coordinated in the solid state while the tricyclohexyltin derivative was determined to be four-coordinated. Structural assignments based on spectroscopic data are supported by the crystallographic results of four of the triorganotin butyrates (trimethyl-, tri-n-propyl-, tri-n-butyl- and tricyclohexyltin 2-(p-chlorophenyl)-3-methylbutyrate). Multinuclear NMR spectroscopy studies indicated that all the complexes were tetrahedral in solution. Larvicidal activities of the complexes were evaluated against the 2nd instar stage of the Anopheles stephensi, Aedes aegypti and Culex pipiens quinquefasciatus mosquitoes. The toxicity data indicate that there does not appear to be any significant differences of the compounds towards the different mosquito species based on their averaged toxicity values. In addition, the toxicity of the triorganotin compounds towards the mosquito larvae was concluded to be dependent on both the compound and the species of mosquito larvae.     

Separation and determination of nitroaniline isomers by capillary zone electrophoresis with amperometric detection

In this paper, capillary zone electrophoresis with amperometric detection (CZE-AD) was firstly applied to the simultaneous separation and determination of nitroaniline positional isomers. The three analytes could be perfectly analyzed by using the buffer of extreme pH. The effects of several important factors were investigated to find optimum conditions. A carbon-disk electrode was used as working electrode. The optimal conditions were 40 mmol/L tartaric acid-sodium tartrate (pH 1.2) as running buffer, 17 kV as separation voltage and 1.10 V (versus saturated calomel reference electrode, SCE) as detection potential. Under the optimum conditions, o-, m- and p-nitroaniline were separated successfully and good linearity, reproducibility and recovery results were obtained. The detection limit for m-nitroaniline was as low as at 9.06 × 10⁻⁹ mol/L. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 1.8% for migration time and 1.1% for peak areas. The utility of this method was demonstrated by monitoring dyestuff wastewater and the assay results were satisfactory.     

Assessment of the acute toxicity of triclosan and methyl triclosan in wastewater based on the bioluminescence inhibition of Vibrio fischeri

In this work, the contributions of triclosan and its metabolite methyl triclosan to the overall acute toxicity of wastewater were studied using Vibrio fischeri. The protocol used in this paper involved various steps. First, the aquatic toxicities of triclosan and methyl triclosan were determined for standard substances, and the 50% effective concentrations (EC₅₀) were determined for these compounds. Second, the toxic responses to different mixtures of triclosan, methyl triclosan, and surfactants were studied in different water matrices, i.e., Milli-Q water, groundwater and wastewater, in order to evaluate (i) the antagonistic or synergistic effects, and (ii) the influence of the water matrices. Finally, chemical analysis was used in conjunction with the toxicity results in order to assess the aquatic toxicities of triclosan and its derivative in wastewaters. In this study, the toxicities of 45 real samples corresponding to the influents and effluents from eight wastewater treatment works (WWTW) were analyzed. Thirty-one samples were from a wastewater treatment plant (WWTP) equipped with two pilot-scale membrane bioreactors (MBR), and the influent and the effluent samples after various treatments were characterized via different chromatographic approaches, including solid-phase extraction (SPE), liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), and SPE coupled to gas chromatography–mass spectrometry (GC–MS). The toxicity was determined by measuring the bioluminescence inhibition of Vibrio fischeri. In order to complete the study and to extrapolate the results to different WWTPs, the toxicity to V. fischeri of samples from seven more plants was analyzed, as were their triclosan and methyl triclosan concentrations. Good agreement was established between the overall toxicity values and concentrations of the biocides, indicating that triclosan is one of the major toxic organic pollutants currently found in domestic wastewaters.     

Comparison of laser ablation-inductively coupled plasma-mass spectrometry and micro-X-ray fluorescence spectrometry for elemental imaging in Daphnia magna

Visualization of elemental distributions in thin sections of biological tissue is gaining importance in many disciplines of biological and medical research. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and scanning micro-X-ray fluorescence spectrometry (micro-XRF) are two widely used microanalytical techniques for elemental mapping. This article compares the capabilities of the two techniques for imaging the distribution of selected elements in the model organism Daphnia magna in terms of detection power and spatial resolution. Sections with a thickness of 10 and 20 μm of the fresh water crustacean Daphnia magna were subjected to LA-ICP-MS and micro-XRF analysis. The elemental distributions obtained for Ca, P, S and Zn allow element-to-tissue correlation. LA-ICP-MS and micro-XRF offer similar limits of detection for the elements Ca and P and thus, allow a cross-validation of the imaging results. LA-ICP-MS was particularly sensitive for determining Zn (LOD 20 μg g⁻¹, 15 μm spot size) in Daphnia magna, while the detection power of micro-XRF was insufficient in this context. However, LA-ICP-MS was inadequate for the measurement of the S distributions, which could be better visualized with micro-XRF (LOD 160 μg g⁻¹, 5 s live time). Both techniques are thus complementary in providing an exhaustive chemical profiling of tissue samples.