Validation of an FT-IR microscopy method for the determination of microplastic particles in surface waters.

For analysis of microplastic (MP) particles in aquatic or solid compartments, standardized methods are required, yet data obtained by current methods are of limited comparability. Current methods include Fourier-transform Infrared (FT-IR) microscopy, Raman microscopy or thermo-analytical methods and attempts to compare data-sets from these methods have largely failed. Only little quality data based on validated methods and appropriate quality standards is available. Thus, reports of presence and numbers of MP still vary significantly from each other without a reliable indicator which of the reported data fulfils data acceptable quality requirements. A methodology for the determination of MP via FT-IR microscopy is introduced and critically discussed regarding mandatory validation parameters and applicability. Furthermore, advantages and challenges of this method are put into relation to other spectroscopic and spectrometric techniques.

Identification of potentially mobile and persistent transformation products of REACH-registered chemicals and their occurrence in surface waters.

Transformation of industrial chemicals might be a significant source of hitherto unknown persistent and mobile organic contaminants (PMOC, PM chemicals) present in the aquatic environment. Herein we depicted a three-step strategy consisting of (I) the prioritization of potential PMOC precursors among REACH-registered chemicals, (II) their lab scale transformation through hydrolysis, photolysis, MnO2 oxidation, and biotransformation and subsequent structural elucidation of derived transformation products, and finally (III) the assessment of their environmental relevance. The proposed procedure was utilized to investigate eleven chemicals, for nine of which a concentration reduction was observed. For six of these chemicals transformation products were at least tentatively identified and partially confirmed with a commercially available reference standard. Retrospective assessment of high-performance liquid chromatography - high-resolution mass spectrometry data as well as a target screening method for the identified TPs and some of the prioritized REACH chemicals revealed the widespread presence of the following chemicals in the environment: 2-pyrrolidone (hydrolysis product of vinylpyrrolidone), TP 216 (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-acetic acid, biotransformation product of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-ethanol), and 1,3-diphenylguanidine (prioritized chemical with experimental evidence of environmental stability). 2-Pyrrolidone was detected in 23/25 investigated surface water samples and present in concentrations of up to 400 ng/L. TP 216 was detected in 20/25 surface water samples and an additional sampling of a waste water treatment plant and the receiving surface water confirmed that TP 216 is formed in waste water treatment plants. The vulcanisation agent 1,3-diphenylguanidine was present in all investigated samples. A leaching experiment with a tire suggested that tires and thus tire wear particles are a potential source of 1,3-diphenylguanidine. With these data the depicted approach was proven successful and suitable for true unknowns like TP 216, and thus an alternative to non-target screenings or suspect-screenings with predicted TPs to identify environmentally relevant transformation products.

Behavior of perfluorinated compounds in soils during leaching experiments.

Perfluorinated compounds (PFCs) can be detected worldwide in both, soil and water. In order to study the leaching behavior of this heterogeneous group of compounds in soil, flow-through column experiments have been conducted. Ten perfluoro carboxylates and four perfluoro sulfonates ranging from C4 to C14 in chain length, and contaminated sewage sludge, have been used to spike a standard soil. The aqueous column effluent was analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) with direct injection. The observed percolation velocity seems to be strongly correlated with the length of the perfluorinated chain. Other factors that additionally contribute to the leaching behavior are the functional group of the PFC, the organic carbon content of the soil and the presence of other adsorbates. A mass balance calculation showed that perfluorobutanoic acid can adsorb strongly to the soil, when no PFC with longer carbon chain are present. Only about 60% of the added perfluorobutanoic acid could be detected in the percolate water. The missing amount started to elute again when longer chain PFC or stearate were added to the soil. Thus it would appear that larger and more lipophilic molecules can displace shorter PFC from their binding sites in the soil. The results of a monitoring study using 32 surface water samples and 150 groundwater samples confirm that the PFC with the highest concentrations in groundwater are the short chain PFC with less than 7 (fluorinated) carbon atoms. The dominating PFC in surface waters are perfluorooctanoic acid and perfluorooctane sulfonic acid.

Synthesis and analytical follow-up of the mineralization of a new fluorosurfactant prototype.

Fluorinated surfactants have become essential in numerous technical applications due to their unparalleled effectiveness and efficiency. The environmental persistence of the non-biodegradable perfluorinated alkyl moiety has become a matter of concern. Therefore, it was searched for new molecules with chemically stable fluorinated end groups which can be microbially transformed into labile fluorinated substances. One prototype substance, 10-(trifluoromethoxy)decane-1-sulfonate, has shown biomineralization. Monitoring the formation of metabolites over time elucidated the mechanism of biotransformation. Analysis was performed utilizing liquid chromatography-single quadrupole mass spectrometry (LC-MS) and quadrupole-time of flight tandem mass spectrometry (QqTOF-MS). It was possible to distinguish between two major degradation pathways of the fluorinated alkylsulfonate derivative: (i) a desulfonation and subsequent oxidation and degradation of the alkyl chain being predominant and (ii) an insertion of oxygen with a subsequent cleavage and degradation of the molecule. The utilized trifluoromethoxy-endgroup resulted in instable trifluoromethanol after degradation of the alkyl chain, which led to a high degree of mineralization of the molecule.

First interlaboratory exercise on non-steroidal anti-inflammatory drugs analysis in environmental samples.

Comparability of monitoring data are essential for any meaningful assessment and for the management of environmental risks of emerging pollutants. The reliability and comparability of data at European level is often limited, because analytical methods for emerging pollutants are often not fully validated, not harmonized or not suitable for all relevant matrices. This paper describes a collaborative interlaboratory exercise for the analysis of non-steroidal anti-inflammatory drugs (NSAIDs) residues in freshwater and wastewater, held in the framework of the EU project "Network of reference laboratories for monitoring of emerging environmental pollutants" (NORMAN). The NSAID compounds selected in this study were ketoprofen, naproxen, ibuprofen and diclofenac. Thirteen laboratories distributed along nine European Countries (Austria, France, Germany, Greece, Italy, Slovak Republic, Slovenia, Spain, and Switzerland) took part in this exercise, 126 samples were analyzed and a total number of 473 values in duplicate were collected. Samples selected in this study include environmental water (river water and waste water) and artificial water (fortified environmental and distilled water) with different ranges of complexity. Two analytical methods were proposed by the organiser; one is based on the use of solid phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the second one is based on SPE followed by gas-chromatography-mass spectrometry (GC-MS), however, in the first round some different approaches were also admitted. The main goals of this interlaboratory comparison were to evaluate the available analytical schemes for NSAID analysis in natural waters, to evaluate the repeatability (r) and reproducibility (R) between participating laboratories, and to evaluate the influence of the analytical method and sample matrices on the results.

Removal of persistent polar pollutants through improved treatment of wastewater effluents (P-THREE).

The EU-project P-THREE started with the establishment of analytical methods for persistent polar pollutants (P3) and quality assurance, followed by screening of P3 in influents and effluents of known wastewater (WW) treatment plants (TP), receiving waters and tap water produced thereof in several European countries. A final selection of analytes for further studies has been performed. Model MBR reactors have been constructed and an optimisation on synthetic wastewater spiked with P3 (linear alkylbenzene sulfonates (LAS), naphthalene sulfonates) has been performed. An initial dynamic modelling of treatment processes has also started. Advanced oxidation process (AOP) treatment has been done in groundwater with isoproturon and nonylphenol ethoxylates (NPEO). The analysis of individual P3 and potential degradation products was also performed. An integrated systeme analysis of the WW treatment processes has also been initiated.

Comparison of linear alkylbenzene sulfonates removal in conventional activated sludge systems and membrane bioreactors.

The potential of a membrane bioreactor (MBR) and a conventional activated sludge (CAS) system to remove polar micropollutants was evaluated using linear alkylbenzene sulfonates (LAS) as model components. Removal efficiencies over 97% were achieved in both reactor systems. The appearance of biological breakdown metabolites and the respirometric response of the sludges to LAS addition indicated that LAS removal was due to biodegradation, rather than sorption phenomena. The effect of operational variables, such as hydraulic retention time, LAS composition and hydrophobicity of the membrane used in the MBR, was negligible in the range tested. A stepwise increase in LAS influent concentration resulted in higher residual effluent concentrations but did not change the procentual removal efficiency. Because an increase in LAS and SPC effluent concentration occurred to a larger extent in the CAS than in the MBR under similar operating conditions, MBRs may turn out to be be more robust with respect to biological degradation of micropollutants than CAS.

Analysis and fate of insect repellents.

During summer months the insect repellent N,N-diethyl-m-toluamide (DEET) could always be detected in influents and effluents of wastewater treatment plants (WWTP) and anthropogenically influenced surface waters. In Germany the concentrations have decreased constantly since 1999, when DEET was substituted by Bayrepel. DEET can be degraded in WWTP, but only after an adaptation period and values in the influent above the threshold value. Since the year 2000 Bayrepel could also be detected during the summer months in the influents of WWTP, whereas in the effluents Bayrepel was not present.

Parvibaculum lavamentivorans converts linear alkylbenzenesulphonate surfactant to sulphophenylcarboxylates, alpha,beta-unsaturated sulphophenylcarboxylates and sulphophenyldicarboxylates, which are degraded in communities.

AIMS: The aims were to test whether Parvibaculum lavamentivoransT degraded commercial linear alkylbenzenesulphonate (LAS) surfactant via omega-oxygenation and beta-oxidation to sulphophenylcarboxylates (SPCs), whether the organism was widespread and reisolable, and whether the degradative community used the 4-sulphocatechol 1,2-dioxygenase to cleave the aromatic ring from LAS. METHODS AND RESULTS: Heterotrophic P. lavamentivoransT converted LAS (side chain length C10-C13) to SPCs (C4-C13), alpha,beta-unsaturated SPCs (C4-C13) and sulphophenyldicarboxylates (SPdCs) (at least C8-C12). Identifications came from high performance liquid chromatography (HPLC) separation, an electrospray interface and mass spectrometry. No evidence for other paths was found. The degradation of LAS in trickling filters inoculated with environmental samples always showed transient SPC intermediates (HPLC) and the presence of the P. lavamentivorans morphotype in the community. One new isolate was obtained. A community able to mineralize LAS contained 4-sulphocatechol-1,2-dioxygenase at high specific activity. CONCLUSIONS: Parvibaculum lavamentivoransT degrades commercial LAS via omega-oxygenation, oxidation and chain shortening through beta-oxidation to yield a wide range of SPCs. The latter are degraded in bacterial communities which contain organisms like P. lavamentivorans, and which utilize sulphocatechol dioxygenase for ring cleavage. SIGNIFICANCE AND IMPACT OF THE STUDY: There is one widespread pathway to degrade LAS. Any traces of LAS and larger amounts of SPCs in the effluent from sewage works are exposed to degradative organisms in acclimated and pristine environments. These degradative reactions can now be studied in pure cultures.

Utilisation of electrospray time-of-flight mass spectrometry for solving complex fragmentation patterns: application to benzoxazinone derivatives.

In this paper we describe the application of electrospray time-of-flight mass spectrometry (ESI-TOFMS) to structural elucidation of the fragment ions formed from a range of natural and synthetic allelochemical derivatives. The extensive mass spectrometric characterisation of ten non-glucosylated benzoxazinone derivatives using this method is described here for the first time. The analytes include six naturally occurring 1,4-benzoxazin-3(4H)-one derivatives, including the hydroxamic acids DIMBOA [2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] and DIBOA [2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one], lactams HBOA [2-hydroxy-2H-1,4-benzoxazin-3(4H)-one] and HMBOA [2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one], benzoxazolinones BOA [benzoxazolin-2(3H)-one] and MBOA [6-methoxy-benzoxazolin-2(3H)-one] and four synthetic variations, 2'H-DIBOA [4-hydroxy-2H-1,4-benzoxazin-3(4H)-one], 2'OMe-DIBOA [2-methoxy-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one], 2'H-HBOA [2H-1,4-benzoxazin-3(4H)-one] and 2'OMe-HBOA [2-methoxy-2H-1,4-benzoxazin-3(4H)-one]. Assignments of the mass spectral fragments were aided by elemental composition calculation results, comparison of structural analogues and background literature, and acquired knowledge regarding feasible structures for the compounds. The influence of substituents on the chemical reactivity of the compounds with respect to the observed MS behaviour over varying nozzle potentials is addressed and, through comparison of the structural analogues, generic fragmentation patterns have also been identified.

Electrospray ionization mass spectrometric studies on the amphoteric surfactant cocamidopropylbetaine.

After liquid chromatographic (LC) separation, electrospray ionization mass spectrometry (ESI-MS) was investigated for the determination of the amphoteric surfactant cocamidopropylbetaine (CAPB). In the positive ion mode the molecule formed the adduct ions [M + H](+), [M + Na](+) and [M + K](+). Adducts of these cations were also detected with decreasing abundance as dimer and trimer clusters. Additionally, doubly charged molecular ions with different combinations of cations were identified. It was noticed that the relative abundances of individual cation adducts were not reproducible, apparently owing to varying contents of alkali metal ions originating from the solvent and the sample. Under negative ionization, the major molecular ion was [M - H](-). Higher clusters formed by two and three surfactant molecules, i.e. [2M - H](-) and [3M - H](-) were likewise registered. The tendency to form clusters in both positive and negative ion modes, even at 0.1 mg l(-1) levels, was attributed to strong electrostatic interactions between the zwitterionic head groups. Further evidence for this assumption was provided by the detection of a fragment formed from [2M - H](-) which contained the two charged head groups. Studies were undertaken in the negative ion mode on the concentration- and orifice voltage-dependent monomer, dimer and trimer formation of C(12)-CAPB in order to evaluate potential issues in using the ion [M - H](-) mode for quantitative analysis. Finally, the established (-)-LC/ESI-MS method was applied to follow up the primary degradation of CAPB in a laboratory-scale fixed-bed bioreactor (FBBR) spiked with a test concentration of 10 mg l(-1). Direct analysis without sample pretreatment revealed that higher alkyl homologues were more prone to adsorption. Primary biodegradation of all alkyl homologues was completed after a period of 4 days. Selected lyophilized FBBR samples were examined for the presence of transient or stable degradation intermediates, but no metabolite could be identified.

Aerobic biodegradation studies of nonylphenol ethoxylates in river water using liquid chromatography-electrospray tandem mass spectrometry.

The aerobic biodegradation of nonylphenol ethoxylates (A9PEO) was kinetically investigated in a laboratory-scale bioreactor filled with riverwater, spiked at a concentration of 10 mg L(-1) nonionic surfactants. Analyses of the samples applying liquid chromatography-electrospray mass spectrometry (LC-ES-MS) after solid-phase enrichment revealed a relatively fast primary degradation of A9PEO with >99% degradation observed after 4 days. Contrary to the generally proposed degradation pathway of EO chain shortening, it could be shown that the initiating step of the degradation is omega-carboxylation of the individual ethoxylate chains: metabolites with long carboxylated EO chains are identified (A9PEC). Further degradation proceeds gradually into short-chain carboxylated EO with the most abundant species being AgPE2C. The oxidation of the nonyl chain proceeds concomitantly with this degradation, leading to metabolites having both a carboxylated ethoxylate and an alkyl chain of varying lengths (CAPEC). The identity of the CAPEC metabolites was confirmed by the fragmentation pattern obtained with LC-ES-MS/MS. Both A9PEC and CAPEC metabolites are still present in the bioreactor after 31 days. In the aerobic degradation pathway, A9PEO2 is formed only to a minor extent and is even further degraded in several days. The endocrine disruptor nonylphenol was not found as a metabolite in this study.

Recalcitrance of poly(vinylpyrrolidone): evidence through matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

The aerobic biodegradability of an extensively used synthetic polymer was monitored the first time on a laboratory-scale fixed-bed bioreactor (FBBR) applying matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Polymeric poly(vinylpyrrolidone) (PVP) was spiked at concentrations of 10 mg l(-1) onto the FBBR run with river water and the biodegradation monitored after lyophilization of aliquots of the test liquor applying MALDI-TOF-MS. The latter proved to be a powerful tool for qualitative screening purposes of PVP in a molecular mass range <20 kDa in particularly yielding a high sensitivity and shot-to-shot reproducibility. The sample-to-sample reproducibility was enhanced applying the anchor target device. Post-source decay-MALDI-TOF-MS fragmentation investigations determined the unknown end groups of PVP unambiguously. Poor biodegradability of PVP can be assumed, since even after 30 days, no oxidation of the terminal groups and no difference in the repeating units was observed. A decrease in the molecular mass distribution can be drawn back rather to adsorption of PVP in the FBBR other than to biodegradation. This was further investigated performing an adsorption experiment with sewage sludge as solid matrix and analyses of the aqueous phase and sludge samples. Extrapolating these results to the situation in wastewater treatment plants, it is highly likely that PVP is eliminated from the dissolved phase by adsorption onto sludge particles.

Inter-laboratory comparison of liquid chromatographic techniques and enzyme-linked immunosorbent assay for the determination of surfactants in wastewaters.

Seven laboratories participated in an inter-laboratory comparison exercise within the framework of the PRISTINE, SANDRINE and INEXsPORT European Union Projects. Solid-phase extraction (SPE) methodologies were used for the extraction of target analytes from wastewaters. The analytical strategies were based on liquid chromatography (LC) coupled to mass spectrometric (MS) or to fluorescent (FL) detection in all cases with the exception of one laboratory using a test-tube enzyme-linked immunosorbent assay kit. Samples were spiked with the surfactants nonylphenolpolyglycol ether, coconut diethanolamide, linear alkylbenzene sulfonate, nonylphenolpolyglycol ether sulfate, alkylpolyglycol ether and secondary alkane sulfonate. After enrichment on previously conditioned SPE cartridges, the SPE cartridges were distributed among the participating laboratories without the information about the amount of spiked surfactants. In addition, SPE cartridges loaded with a real-world environmental sample containing a tannery wastewater were also analyzed. The results of the programme showed that SPE followed by LC-MS techniques are reliable for the surfactants determination at submicrogram to microgram per liter levels in wastewaters. Inter-laboratory precision values were calculated as the reproducibility relative standard deviation (RSD(R)) which was determined from the reproducibility standard deviation (sR) and the average concentration at a particular concentration level. When data from all laboratories were pooled, the RSD(R) values ranged from 5.1 to 28.3% for the determination of target analytes. The most accurate result corresponded to that given for linear alkylbenzene sulfonates. Taking into account that different methodologies were used (including non-chromatographic techniques) and the complexity of the samples analyzed, it can be considered that acceptable reproducibility values were obtained in this inter-laboratory study.

Determination of linear alkylbenzenesulfonates in wastewater treatment plants and coastal waters by automated solid-phase extraction followed by capillary electrophoresis-UV detection and confirmation by capillary electrophoresis-mass spectrometry.

Linear alkylbenzenesulfonates (LASs) were determined in wastewaters and coastal waters by solid-phase extraction, using two different sample preparation protocols depending on the sample treated, followed by capillary electrophoresis and ultraviolet detection (CE-UV). The linear range of the proposed method varied from 3 to 53 and from 25 to 495 microg/l, depending on the compound, with a limit of detection of 1 microg/l when 250 ml of coastal water was preconcentrated. [M-H]- ions were used for CE-MS confirmation after quantification by CE-UV. CE-MS diagnostic ions were the same ones used in LC-electrospray (ESI) MS and corresponded to m/z 297, 311, 325 and 339 for C10, C11, C12 and C13 LASs, respectively. LASs were determined in wastewater samples of the influent and effluent of three wastewater treatment plants (WWTPs), two of them using biological treatment with secondary settlement and receiving mainly domestic wastewaters whereas one of the plants was operated with physicochemical treatment and received mainly industrial wastewaters. LASs were also analyzed in two samples from coastal waters of the bay of Cadiz (Spain) receiving untreated domestic effluents. All samples were also analyzed by LC-ESI-MS and the results are compared with the CE-UV method developed in this work. The concentration levels of total LASs varied from 988 to 1309 microg/l in the influents of WWTPs, whereas in the effluents the concentrations varied from 136 to 197 microg/l. The levels of LASs in coastal wastewaters of the bay of Cadiz varied from 739 to 911 microg/l, indicating that the wastewaters discharged into the bay did not undergo any treatment at all.

Unknown bisethylisooctanollactone isomers in industrial waste water. Isolation, identification and occurrence in surface water.

Unknown bisethylisooctanollactone isomers (BIOL isomers) which are chemical by-products of butyraldehyde synthesis, were isolated from industrial waste water applying various purification methods with subsequent semi-preparative high-performance liquid chromatography. Through interpretation of mass spectra after gas chromatographic separation the individual BIOL isomers were identified as stereoisomers of 2,4-diethyl-3-n-propyl-delta-valerolactone. Thus, it was possible for the first time to quantify the BIOL isomers in the river Rhine, Germany, with a mean sum concentration of 1.6 microg l(-1). A regular analysis performed over a period of almost two years of the river Rhine always gave the same ratio among the individual isomers. Drinking water production out of such water was studied, revealing that activated carbon filtration led to a 95% reduction of the BIOL concentration. Additional subsoil passage and a subsequent slowsand filtration led to a total elimination due to microbial degradation. Even if the BIOL isomers proved not to be relevant to drinking water, their behavior in the aquatic environment needs to be more thoroughly investigated since these compounds have been discharged for many years in high amounts into the river Rhine.

Investigations on the metabolism of alkyl polyglucosides and their determination in waste water by means of liquid chromatography-electrospray mass spectrometry.

Alkyl polyglucosides (APGs) were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry with electrospray ionization. Analytes were separated according to the chain length of the alkyl homologues, whereas the separation of isomeric forms of the glucose moiety was achieved partially. Depending on the structure of the glucose ring the alkyl monoglucosides show a distinct affinity in terms of the formation of sodium and ammonium adduct ions. Metabolism of isomer pure alkyl monoglucosides was studied on a testfilter device to gather information about the degradation behavior and to obtain eventually poorly degradable metabolites. In spite of unsuccessful detection of any metabolites such as "polyglucoside alcanoic acids", a degradation pathway was proposed including the cleavage of the glucosidic bond as initial step. In addition, a method for the determination of APGs in municipal waste water effluent was developed using solid-phase extraction on reversed-phase material. Recovery rates were in the range of 66 to 98% for three spiked alkyl monoglucosides and a quantitation limit of 0.2 microg l(-1) was achieved.

Analysis of polar organic micropollutants in water with ion chromatography-electrospray mass spectrometry.

The coupling of ion chromatography (IC) with electrospray mass spectrometry (ES-MS) opens new ways for the determination of polar organic micropollutants in water samples. The technique of conductivity suppression has been found to reduce the background signal in the range of about two orders of magnitude leading to a significant increase in sensitivity. In addition, the formation of salt adducts has been avoided. The usefulness of this method was proven on several polar and environmentally relevant micropollutants such as the herbicide glyphosate and its metabolite aminomethylphosphonic acid (AMPA), the chelating agent ethylenediamine tetraacetate (EDTA) and diacetonketogulonic acid (DAG). This present study has shown that IC-ES-MS is a simple, sensitive and quick method for the determination of these polar organic traces in water samples after separation on an anion-exchange column without any derivatization. In this work, several possibilities of applications of IC-ES-MS (with varying conditions) are presented. Analysis of glyphosate, AMPA, DAG and EDTA in ground and surface water has been achieved by IC-ES-MS without additional sample preparation at a concentration level of 1 microgram/l.

Determination of the glycosylation patterns, disulfide linkages, and protein heterogeneities of baculovirus-expressed mouse interleukin-3 by mass spectrometry.

The primary structure of mouse interleukin-3 (IL-3) expressed by recombinant baculovirus-infected silkworm (Bombyx mori) larvae was analyzed by subjecting isolated IL-3 derived peptides to liquid secondary ion mass spectrometry. Two species of IL-3 were isolated from the silkworm hemolymph by reverse-phase high-pressure liquid chromatography. The major component has M(r)20-22 x 10(3) as determined by SDS-PAGE. Liquid secondary ion mass spectrometric analysis was carried out on the reduced tryptic and endopeptidase lysyl-C peptides of glycosylated and deglycosylated IL-3. These studies provided evidence that (1) Asn-16 is heterogeneously glycosylated with four different oligosaccharides, (2) Asn-86 is either nonglycosylated or has attached to it one oligosaccharide, (3) the N-glycosylation sites Asn-44 and Asn-51 are not glycosylated, and (4) there is no O-glycosylation. Liquid secondary ion mass spectrometric analysis of the unreduced tryptic peptides provided evidence for disulfide linkages between Cys-140 and Cys-79 or Cys-80 and between Cys-17 and Cys-79 or Cys-80. In comparison to the major component, a minor IL-3 species (M(r) 17-19 x 10(3) by SDS-PAGE) isolated from the hemolymph showed no difference with respect to the glycosylation pattern or the disulfide linkages, but it was cleaved between Ala-127 and Ser-128, and only a disulfide linkage between Cys-140 and Cys-79 or Cys-80 held the molecule together.(ABSTRACT TRUNCATED AT 250 WORDS)