Development and validation of a rapid method for the determination and confirmation of 10 nitroimidazoles in animal plasma using liquid chromatography tandem mass spectrometry

A rapid LC–MS/MS method has been developed and validated for the simultaneous identification, confirmation and quantitation of 10 nitroimidazoles in plasma. The method validated in accordance with Commission Decision (CD) 2002/657/EC is capable of analysing for metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ) and ternidazole (TRZ) which are rarely analysed by modern methods. MNZ, DMZ and RNZ have a recommended level (RL) of 3 ng mL^?1 which this method is easily able to detect for all the nitroimidazole compounds. Plasma samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid–liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS). The decision limits (CCα) range from 0.5 to 1.6 ng mL^?1 and the detection capabilities (CCβ), range from 0.8 to 2.6 ng mL^?1. The results of the inter-assay study, which was performed by fortifying bovine plasma samples (n = 18) on three separate days, show the accuracy calculated for the various analytes range between 101% and 108%. The precision of the method, expressed as CV% values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 ng mL^?1), ranged between 4.9% and 15.2%. A day 4 analysis was carried out to examine species variances in animals such as avian, ovine, porcine and equine.     

High performance liquid chromatography determination of sulphachloropyrazine residues in broiler and turkey edible tissues

A HPLC method to determine and quantify sulphachloropyrazine residues from broilers and turkeys is reported. This procedure permitted sulphachloropyrazine to be separated from muscle tissue, liver, kidneys and fat with skin after extraction with dichloromethane under slightly acidic conditions. The analytical methodology showed a high specificity and sensitivity and an adequate precision and accuracy with a limit of quantification of 56 ng mL^?1. The peak area showed a linear relationship with a concentration over the range 50–750 ng mL^?1 for sulphachloropyrazine standard solutions. Recovery dates were also satisfactory with values between 69.7 and 77.5%.     

Yessotoxins production during the culture of Protoceratium reticulatum strains isolated from Galician Rias Baixas (NW Spain)

Yessotoxins (YTXs) production along the culture growth of three strains of the dinoflagellate Protoceratium reticulatum isolated from seawater of Galician Rias Baixas, Spain was investigated. Quantification and toxin profile determination in both cells and culture medium along the growth curve were performed by liquid chromatography–mass spectrometry (LC–MS³) analysis. The YTX profile was very similar among strains, the three algal strains produce mainly YTX and also some YTX analogs. Among the strains the maximum toxin production ranged between 416 and 576 ng mL⁻¹. This is the first report about YTX production by P. reticulatum isolated in Galician coast, NW Spain.     

The measurement of ecstasy in human hair by triple phase directly suspended droplet microextraction prior to HPLC-DAD analysis

New pre-concentration technique, triple phase suspended droplet microextraction (SD-LPME) and liquid chromatography-photodiode array detection was applied to determine ecstasy, MDMA (3,4-methylendioxy-N-methylamphetamine) in hair samples. In this research MDMA in hair was digested and after treatment extracted. The effective parameters were investigated and method was evaluated. Under the optimal conditions, the MDMA was enriched by factor 98.11. Linearity (r = 0.9921), was obtained in the range of 10–15,000 ng mL^?1 and detection limit was 0.1 ng mL^?1.     

Determination of phenothiazine derivatives in human urine by using ionic liquid-based dynamic liquid-phase microextraction coupled with liquid chromatography

A simple and rapid method for the determination of seven phenothiazines derivatives (chlorpromazine, promethazine, levomepromazine, prochlorperazine, trifluoperazine, fluphenazine and thioridazine) in human urine samples is presented. The analytes are extracted from the sample in 50 μL of the ionic liquid 1-butyl-3-methyl-imidazolium hexafluorophosphate working in an automatic flow system under dynamic conditions. The chemical affinity between the extractant and the analytes allows a good isolation of the drugs from the sample matrix achieving at the same time their preconcentration. The separation and detection of the extracted compounds is accomplished by liquid chromatography and UV detection. The proposed method is a valuable alternative for the analysis of these drugs in urine within the concentration range 0.07–10 μg mL^?1. Limits of detection were in the range from 21 ng mL^?1 (thioridazine) to 60 ng mL^?1 (levomepromazine). The repeatability of the proposed method expressed as RSD (n = 5) varied between 2.2% (levomepromazine) and 3.9% (chlorpromazine).     

Determination of metoclopramide in human plasma by LC–ESI-MS and its application to bioequivalance studies

An LC–MS method for the determination of metoclopramide in human plasma was developed and validated. Sample preparation involved extraction with ethyl acetate. Chromatographic separation was performed on a Thermo Hypersil-Hypurity C₁₈ (150 mm × 2.1 mm, 5 μm) with the mobile phase consisting of 40 mM ammonium acetate–methanol–acetonitrile. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H]⁺ ions at m/z 300 for metoclopramide and at m/z 384 for the internal standard (prazosin). The method was validated over 0.78–50.00 ng mL^?1 for metoclopramide. The recovery was 67.8–83.1%, and the limit of quantitation (LOQ) detection was 0.78 ng mL^?1 for metoclopramide. The intra- and inter-day precision of the method at three concentrations was 5.0–13.6% with accuracy of 99.2–104.0%. Stability of compounds was established in a battery of stability studies. The method was successfully applied to bioequivalence studies of metoclopramide hydrochloride tablets to obtain the pharmacokinetic parameters.     

Determination of selenium and its compounds in marine organisms

In this study, we investigate the type and quantity of selenium compounds in fish and marine organisms, using ion-pair reversed phase LC–ICP-MS, developed and applied for the analysis of Atlantic cod, Atlantic salmon, Greenland halibut, Atlantic herring, blue mussel, common crab, scallop, calanus, and Euphasia super. Of the samples examined, the lowest level of selenium was found in farmed Atlantic salmon (0.17 mg Se kg⁻¹ dm). The total selenium extraction efficiency by phosphate buffer was 2.5 times higher in sea plankton and shellfish samples than in fish samples. Analysis of Se species in each hydrolysate obtained by proteolysis showed the presence of selenomethionine, which constituted 41.5% of the selenium compounds detected in hydrolysates of Atlantic herring and 98.4% of those in extracts of Atlantic salmon. Inorganic compounds, such as selenates and selenites, were detected mainly in sea plankton and shellfish samples (<0.13 mg Se kg⁻¹ wm), although no correlation was found between the presence of inorganic compounds and total selenium concentration. The accuracy of the total selenium determination was validated using a certified reference material (oyster tissue (NIST 1566b)). A lyophilised powder of cod (Gadus morhua) was used to validate speciation analysis, enzymatic hydrolysis of lyophilised powder of cod recovered 54 ± 6% of total selenium, and SeMet constituted 83.5 ± 5.28% of selenium detected in hydrolysates. The chromatographic detection limits were, respectively, 0.30 ng mL⁻¹, 0.43 ng mL⁻¹, 0.54 ng mL⁻¹, 0.55 ng mL⁻¹, 0.57 ng mL⁻¹ and 0.72 ng mL⁻¹ for selenate, selenomethionine, selenite, Se-methyl-selenocysteine, selenocystine and selenomethionine selenoxide.The data on selenium concentrations and speciation presented here could be useful in estimating levels of selenium intake by seafood consumption.     

HPLC method for determination of fluorescence derivatives of cortisol,cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids

11β-Hydroxysteroid dehydrogenase isoform 2 (11β-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11β-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard – prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C₁₈ cartridges. The enzymatic hydrolysis of conjugated steroids was provided using β-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0–1000.0 ng mL^?1, for allo-THF and THE + allo-THE 10.0–1000.0 ng mL^?1. LOD (S/N = 3:1) for all analytes amounted 3.0 ng mL^?1. Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0–12.1% and 9.2–14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0–174.5 ng mL^?1 and 17.4–35.9 ng mL^?1, respectively. Free urinary steroids were in the ranges: 12.0–54.1 μg/24 h (UFF) and 37.8–76.2 μg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11β-HSD2 activity in man.     

Analysis of cyclosporine A and its metabolites in rat urine and feces by liquid chromatography–tandem mass spectrometry

A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of cyclosporine A (CyA) and the identification of its metabolites in rat urine and feces. The analytes were extracted from waste samples via liquid–liquid extraction. A Turboionspray source was used as a detector. It was operated in a positive ion mode with transitions of m/z 1225 → m/z 1112 for CyA and in a selected multiple reactions monitoring (MRM) mode with transitions of m/z 1239 → m/z 1099 for the internal standard (cyclosporine D, CyD). Linear calibration curves were obtained for CyA concentration ranges of 12.5–250 ng mL^?1 in urine and 2.5–375 ng mg^?1 in feces. The intra- and inter-day precision values (relative standard deviation) obtained were less than 8%, and the accuracy was within ±15% for each of the analytes. Extraction recoveries of CyA and CyD were both over 80%. The identification of the metabolites and elucidation of their structure were performed on the basis of their retention times and mass spectrometry fragmentation behaviors. A total of seven metabolites in rat feces were identified as dimethyl CyA, hydroxy CyA, and dihydroxy CyA after the oral administration of cyclosporine A-Eudragit^® S100 nanoparticles (CyA-NP). Six of these metabolites were also detected in rat urine. A possible metabolic pathway was also proposed. The newly developed method was proven to be sensitive, simple, reproducible, and suitable for the rapid determination of CyA. It was successfully employed to study the excretion of CyA in rats and could be used to better understand the in vivo metabolism of CyA-NP, a potentially effective nanoparticle system.     

Simultaneous determination of quinolones for veterinary use by high-performance liquid chromatography with electrochemical detection

A selective method based on high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed to enable simultaneous determination of three fluoroquinolones (FQs), namely danofloxacin (DANO), difloxacin (DIFLO) and sarafloxacin (SARA). The fluoroquinolones are separated on a Novapack C-18 column and detected in a high sensitivity amperometric cell at a potential of +0.8 V. Solid-phase extraction was used for the extraction of the analytes in real samples. The range of concentration examined varied from 10 to 150 ng g^?1 for danofloxacin, from 25 to 100 ng g^?1 for sarafloxacin and from 50 to 315 ng g^?1 for difloxacin, respectively. The method presents detection limits under 10 ng g^?1 and recoveries around 90% for the three analytes have been obtained in the experiments with fortified samples. This HPLC-ECD approach can be useful in the routine analysis of antibacterial residues being less expensive and less complicated than other more powerful tools as hyphenated techniques.     

Capillary zone electrophoresis with diode-array detection for analysis of local anaesthetics and opium alkaloids in urine samples

The present study describes the simultaneous determination of four drugs, two local anaesthetics (lidocaine and bupivacaine) and two opium alkaloids (noscapine and papaverine) by capillary zone electrophoresis (CZE) with solid-phase extraction (SPE) procedure using Oasis HLB cartridges. Their recoveries ranged from 81 to 107% at the target concentrations of 2.0, 5.0 and 8.0 μg mL^?1 in spiked urine samples. Coefficients of variation of the recoveries ranged from 2.1 to 11.3% at these concentrations. The quantitation limits of the method were approximately 300 ng mL^?1 for the different compounds studied. The assay is very specific for these compounds and requires a short sample preparation procedure prior to the electrophoretic analysis.     

Heterotrimeric G protein α-subunit is localized in the plasma membrane of Pinus bungeana pollen tubes

Heterotrimeric G proteins are involved in a variety of cellular responses, but relatively little is known about their function and biochemistry in plant pollen. In this paper, we establish the presence of a G protein associated with the plasma membranes of Pinus bungeana pollen tube. A 40 kDa polypeptide is detected and immunolocalized predominantly in pollen tube plasma membranes by polyclonal antisera directed against conserved peptides of mammalian Gα-subunit during pollen tube development. Cholera and pertussis toxins exhibited biphasic actions on tube growth, that is to say, inhibited pollen tube growth and result in rupture of tubes at concentrations less than 400 ng mL⁻¹, whereas stimulated pollen tube growth at concentration over 500 ng mL⁻¹. Fourier transform-infrared (FT-IR) spectra showed that the two toxins at concentrations of 400 ng mL⁻¹ resulted in enhanced synthesis of phenolics and reduced synthesis of cellulose, hemicellulose, and xylan of pollen tube wall, which may account for incidental rupture of pollen tubes at the concentration. These results suggest that the two toxins possibly affect pollen tube growth via downstream pertussis or cholera toxin-sensitive functional proteins, which regulate tube wall biosynthesis than at the Gα-subunit in P. bungeana tube growth.     

An o-aminobenzoic acid film-based immunoelectrode for detection of the cardiac troponin T in human serum

An amperometric immunosensor for cardiac troponin T detection in human serum troponin T, a marker considered as “gold standard” for acute myocardial infarction diagnosis, is described. A stable carboxylic film to covalently bind antibodies against cTnT onto electrode surface was achieved with electropolymerization of the o-aminobenzoic acid. A fractional factorial study was performed to optimize the electropolymerization parameters. Cyclic voltammetry assays were carried out for characterize steps of the modified electrode surface. The obtained calibration curve at −0.05 V by amperometry presented a good linear response range from 0.05 to 5.0 ng mL⁻¹ cTnT with a correlation coefficient of 0.992 (n = 6) and 0.016 ng mL⁻¹ detection limit. The electrodes showed a good stability upon the analytical responses retaining 91.6% of its initial response after 18 days. This sensor showed outgoing results regarding sensitivity allowing reliable measurements of the cTnT at levels of clinical significance for acute myocardial infarctions diagnosis.     

Synthesis and structure–activity relationships of 2-phenyl-1-[(pyridinyl- and piperidinylmethyl)amino]-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents

Continuous efforts on the synthesis and structure–activity relationships (SARs) studies of modified 1-benzylamino-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as antifungal agents, allowed identification of new 1-[(pyridinyl- and piperidinylmethyl)amino] derivatives with MIC₈₀ values ranging from 1410.0 to 23.0 ng mL^?1 on Candida albicans. These results confirmed both the importance of π–π stacking and hydrogen bonding interactions in the active site of CYP51-C. albicans.     

Mixotrophy in the newly described dinoflagellate Yihiella yeosuensis: A small,fast dinoflagellate predator that grows mixotrophically,but not autotrophically

To investigate tropical roles of the newly described Yihiella yeosuensis (ca. 8 μm in cell size), one of the smallest phototrophic dinoflagellates in marine ecosystems, its trophic mode and the types of prey species that Y. yeosuensis can feed upon were explored. Growth and ingestion rates of Y. yeosuensis on its optimal prey, Pyramimonas sp. (Prasinophyceae), as a function of prey concentration were measured. Additionally, growth and ingestion rates of Y. yeosuensis on the other edible prey, Teleaulax sp. (Cryptophyceae), were also determined for a single prey concentration at which both these rates of Y. yeosuensis on Pyramimonas sp. were saturated. Among bacteria and diverse algal prey tested, Y. yeosuensis fed only on small Pyramimonas sp. and Teleaulax sp. (both cell sizes = 5.6 μm). With increasing mean prey concentrations, both specific growth and ingestion rates of Y. yeosuensis increased rapidly before saturating at a mean Pyramimonas concentration of 109 ng C mL⁻¹ (2725 cells mL⁻¹). The maximum growth rate (mixotrophic growth) of Y. yeosuensis fed with Pyramimonas sp. at 20 °C under a 14:10-h light-dark cycle of 20 μE m⁻² s⁻¹ was 1.32 d⁻¹, whereas the growth rate of Y. yeosuensis without added prey was 0.026 d⁻¹. The maximum ingestion rate of Y. yeosuensis fed with Pyramimonas sp. was 0.37 ng C predator⁻¹ d⁻¹ (9.3 cells predator⁻¹ d⁻¹). At a Teleaulax concentration of 1130 ng C mL⁻¹ (66,240 cells mL⁻¹), growth and ingestion rates of Y. yeosuensis fed with Teleaulax sp. were 1.285 d⁻¹ and 0.38 ng C predator⁻¹ d⁻¹ (22.4 cells predator⁻¹ d⁻¹), respectively. Thus, Y. yeosuensis rarely grows without mixotrophy, and mixotrophy supports high growth rates in Y. yeosuensis. Y. yeosuensis has the highest maximum mixotrophic growth rate with the exception of Ansanella graniferaamong engulfment feeding mixotrophic dinoflagellates. However, the high swimming speed of Y. yeosuensis (1572 μm s⁻¹), almost the highest among phototrophic dinoflagellates, may prevent autotrophic growth. This evidence suggests that Y. yeosuensis may be an effective mixotrophic dinoflagellate predator on Pyramimonas and Teleaulax, and occurs abundantly during or after blooms of these two prey species.     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Design of new antifungal agents: synthesis and evaluation of 1-[(1H-indol-5-ylmethyl)amino]-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols

We previously reported on the design and synthesis of 1-[((hetero)aryl- or piperidinylmethyl)amino]-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propan-2-ols showing various degrees of antifungal activity against Candida albicans and Aspergillus fumigatus strains. Now we have identified a series of 1-[(1H-indol-5-ylmethyl)amino] derivatives which exhibited potent MICs (<65 ng mL^?1) against C. albicans strain. The synthesis and SAR behind the indole scaffold will be discussed.     

Salicylic acid-induced resistance to Fusarium oxysporum f. sp. lycopersici in tomato

We demonstrated that exogenous application of 200 μM salicylic acid through root feeding and foliar spray could induce resistance against Fusarium oxysporum f. sp. Lycopersici (Fol) in tomato. Endogenous accumulation of free salicylic acid in tomato roots was detected by HPLC and identification was confirmed by LC–MS/MS analysis. At 168 h of salicylic acid treatment through roots, the endogenous salicylic acid level in the roots increased to 1477 ng g^?1 FW which was 10 times higher than control plants. Similarly, the salicylic acid content was 1001 ng g^?1 FW at 168 h of treatment by foliar spray, which was 8.7 times higher than control plants. The activities of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and peroxidase (POD, EC 1.11.1.7) were 5.9 and 4.7 times higher, respectively than the control plants at 168 h of salicylic acid feeding through the roots. The increase in PAL and POD activities was 3.7 and 3.3 times higher, respectively at 168 h of salicylic acid treatments through foliar spray than control plants. The salicylic acid-treated tomato plants challenged with Fol exhibited significantly reduced vascular browning and leaf yellowing wilting. The mycelial growth of Fol was not significantly affected by salicylic acid. Significant increase in basal level of salicylic acid in noninoculated plants indicated that tomato root system might have the capacity to assimilate and distribute salicylic acid throughout the plant. The results indicated that the induced resistance observed in tomato against Fol might be a case of salicylic acid-dependent systemic acquired resistance.     

Interaction between mosquito-larvicidal Lysinibacillus sphaericus binary toxin components: Analysis of complex formation

The two components (BinA and BinB) of Lysinibacillus sphaericus binary toxin together are highly toxic to Culex and Anopheles mosquito larvae, and have been employed world-wide to control mosquito borne diseases. Upon binding to the membrane receptor an oligomeric form (BinA2.BinB2) of the binary toxin is expected to play role in pore formation. It is not clear if these two proteins interact in solution as well, in the absence of receptor. The interactions between active forms of BinA and BinB polypeptides were probed in solution using size-exclusion chromatography, pull-down assay, surface plasmon resonance, circular dichroism, and by chemically crosslinking BinA and BinB components. We demonstrate that the two proteins interact weakly with first association and dissociation rate constants of 4.5 × 10³ M^?1 s^?1 and 0.8 s^?1, resulting in conformational change, most likely, in toxic BinA protein that could kinetically favor membrane translocation of the active oligomer. The weak interactions between the two toxin components could be stabilized by glutaraldehyde crosslinking. The cross-linked complex, interestingly, showed maximal Culex larvicidal activity (LC₅₀ value of 1.59 ng mL^?1) reported so far for combination of BinA/BinB components, and thus is an attractive option for development of new bio-pesticides for control of mosquito borne vector diseases.     

Marine snow formation by the toxin-producing diatom,Pseudo-nitzschia australis

The formation of marine snow (MS) by the toxic diatom Pseudo-nitschia australis was simulated using a roller table experiment. Concentrations of particulate and dissolved domoic acid (pDA and dDA) differed significantly among exponential phase and MS formation under simulated near surface conditions (16 °C/12:12-dark:light cycle) and also differed compared to subsequent particle decomposition at 4 °C in the dark, mimicking conditions in deeper waters. Particulate DA was first detected at the onset of exponential growth, reached maximum levels associated with MS aggregates (1.21 ± 0.24 ng mL⁻¹) and declined at an average loss rate of ∼1.2% pDA day⁻¹ during particle decomposition. Dissolved DA concentrations increased throughout the experiment and reached a maximum of ∼20 ng mL⁻¹ at final sampling on day 88. The succession by P. australis from active growth to aggregation resulted in increasing MS toxicity and based on DA loading of particles and known in situ sinking speeds, a significant amount of toxin could have easily reached the deeper ocean or seafloor. MS formation was further associated with significant dDA accumulation at a ratio of pDA: dDA: cumulative dDA of approximately 1:10:100. Overall, this study confirms that MS functions as a major vector for toxin flux to depth, that Pseudo-nitzschia-derived aggregates should be considered ‘toxic snow’ for MS-associated organisms, and that effects of MS toxicity on interactions with aggregate-associated microbes and zooplankton consumers warrant further consideration.