In vitro biotransformation assays using fish liver cells: Comparing rainbow trout and carp hepatocytes.

Biotransformation assays using primary hepatocytes from rainbow trout, Oncorhynchus mykiss, were validated as a reliable in vitro tool to predict in vivo bioconcentration factors (BCF) of chemicals in fish. Given the pronounced interspecies differences of chemical biotransformation, the present study aimed to compare biotransformation rate values and BCF predictions obtained with hepatocytes from the cold-water species, rainbow trout, to data obtained with hepatocytes of the warm-water species, common carp (Cyprinus carpio). In a first step, we adapted the protocol for the trout hepatocyte assay, including the cryopreservation method, to carp hepatocytes. The successful adaptation serves as proof of principle that the in vitro hepatocyte biotransformation assays can be technically transferred across fish species. In a second step, we compared the in vitro intrinsic clearance rates (CLin vitro, int) of two model xenobiotics, benzo[a]pyrene (BaP) and methoxychlor (MXC), in trout and carp hepatocytes. The in vitro data were used to predict in vivo biotransformation rate constants (kB) and BCFs, which were then compared to measured in vivo kB and BCF values. The CLin vitro, int values of BaP and MXC did not differ significantly between trout and carp hepatocytes, but the predicted BCF values were significantly higher in trout than in carp. In contrast, the measured in vivo BCF values did not differ significantly between the two species. A possible explanation of this discrepancy is that the existing in vitro-in vivo prediction models are parameterized only for trout but not for carp. Therefore, future research needs to develop species-specific extrapolation models.

Uptake of perfluorinated alkyl acids by crops: results from a field study.

Four crops with different edible plant parts (radish, lettuce, pea and maize) were grown in outdoor lysimeters on soil spiked with 13 perfluorinated alkyl acids (PFAAs) at 4 different levels. PFAA concentrations were measured in soil, soil pore water, and different plant parts at harvest. Edible part/soil concentration factors ranged over seven orders of magnitude and decreased strongly with increasing PFAA chain length, by a factor of 10 for each additional fluorinated carbon (nCF) for pea. Three processes were responsible for most of the variability. The first was sorption to soil; calculating whole plant concentration factors on the basis of concentration in pore water instead of soil reduced the variability from five orders of magnitude to two. Second, the journey of the PFAAs with the transpiration stream to the leaves was hindered by retention in the roots driven by sorption; root retention factors increased by a factor 1.7 for each nCF. Third, transfer of PFAAs from the leaves to the fruit via the phloem flow was also hindered - presumably by sorption; fruit/leaf concentration factors decreased by a factor 2.5 for each nCF. A simple mathematical model based on the above principles described the measured concentrations in roots, leaves, fruits and radish bulbs within a factor 4 in most cases. This indicates that the great diversity in PFAA transfer from soil to crops can be largely described with simple concepts for four markedly different species.

Influence of soil on the uptake of perfluoroalkyl acids by lettuce: A comparison between a hydroponic study and a field study.

This study explores whether mechanistic understanding of plant uptake of perfluoroalkyl acids (PFAAs) derived from hydroponic experiments can be applied to soil systems. Lettuces (Lactuca sativa) were grown in outdoor lysimeters in soil spiked with 4 different concentrations of 13 PFAAs. PFAA concentrations were measured in soil, soil pore water, lettuce roots, and foliage. The PFAA uptake by the lettuce was compared with uptake measured in a hydroponic study. The foliage:pore water concentration ratios in the lysimeter were similar to the foliage:water concentration ratios from the hydroponic experiment. In contrast, the root:pore water concentration ratios in the lysimeter were 1-2 orders of magnitude lower than in the hydroponic study for PFAAs with 6 or more perfluorinated carbons. Hence, hydroponic studies can be expected to provide a good quantitative measure of PFAA transfer from soil to foliage if one accounts for soil:pore water partitioning and differences in transpiration rate. However, hydroponic studies will be of little value for estimating PFAA transfer from soil to roots because sorption to the root surface is greatly enhanced under hydroponic conditions.

First evidence of anticoagulant rodenticides in fish and suspended particulate matter: spatial and temporal distribution in German freshwater aquatic systems.

Anticoagulant rodenticides (ARs) have been used for decades for rodent control worldwide. Research on the exposure of the environment and accumulation of these active substances in biota has been focused on terrestrial food webs, but few data are available on the impact of ARs on aquatic systems and water organisms. To fill this gap, we analyzed liver samples of bream (Abramis brama) and co-located suspended particulate matter (SPM) from the German Environmental Specimen Bank (ESB). An appropriate method was developed for the determination of eight different ARs, including first- and second-generation ARs, in fish liver and SPM. Applying this method to bream liver samples from 17 and 18 sampling locations of the years 2011 and 2015, respectively, five ARs were found at levels above limits of quantifications (LOQs, 0.2 to 2 µg kg-1). For 2015, brodifacoum was detected in 88% of the samples with a maximum concentration of 12.5 µg kg-1. Moreover, difenacoum, bromadiolone, difethialone, and flocoumafen were detected in some samples above LOQ. In contrast, no first generation AR was detected in the ESB samples. In SPM, only bromadiolone could be detected in 56% of the samples at levels up to 9.24 µg kg-1. A temporal trend analysis of bream liver from two sampling locations over a period of up to 23 years revealed a significant trend for brodifacoum at one of the sampling locations.

Detection of tetrabromobisphenol A and its mono- and dimethyl derivatives in fish, sediment and suspended particulate matter from European freshwaters and estuaries.

An analytical method was developed for the determination of tetrabromobisphenol A (TBBPA), 3,3',5,5'-tetrabromobisphenol-A-monomethyl ether (MM-TBBPA) and 3,3',5,5'-tetrabromobisphenol-A-dimethyl ether (DM-TBBPA), and its valid application on fish muscle matrix (bream and sole), suspended particulate matter (SPM) and surface sediment layer samples, using only 0.5 g sample material, is demonstrated. Here, for the first time, DM-TBBPA could be determined by an LC-MS/MS-based method applying atmospheric pressure photoionization (APPI), using the same sample extracts for all three analytes. Samplings covered freshwater fish (bream; annually, period 2007-2013) and SPM or sediment (every second year in the period 2008-2014) at selected European sites (rivers: Tees/UK, Mersey/UK, Western Scheldt/NL, Götaälv/SE, Rhône/FR; Lake Belau/DE). TBBPA could be quantified in 13 of 36 bream samples (range about 0.5-1.2 µg kg-1 ww) and 7 of 7 sole muscle samples (range about 0.5-0.7 µg kg-1 ww). Further, it could be quantified in 11 of the 14 SPM samples (range about 0.5-9.4 µg kg-1 dw) and in both of the surface sediment layer samples (2.3-2.6 µg kg-1 dw). MM-TBBPA could be quantified in 12 of 36 bream and 4 of 7 sole muscle samples (range about 0.8-1.8 µg kg-1 ww). Further, it could be quantified in 10 of the 14 river SPM samples (range about 2.3-4.5 µg kg-1 dw) and in both lake surface sediment layer samples (5.2-5.5 µg kg-1 dw). DM-TBBPA was rarely detectable and could not be quantified above the limit of quantification in any sample.

Perfluoroalkyl and polyfluoroalkyl substances in consumer products.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are used in a wide range of products of all day life. Due to their toxicological potential, an emerging focus is directed towards their exposure to humans. This study investigated the PFAS load of consumer products in a broad perspective. Perfluoroalkyl sulfonic acids (C4, C6-C8, C10-PFSA), carboxylic acids (C4-C14-PFCA) and fluorotelomer alcohols (4:2, 6:2; 8:2 and 10:2 FTOH) were analysed in 115 random samples of consumer products including textiles (outdoor materials), carpets, cleaning and impregnating agents, leather samples, baking and sandwich papers, paper baking forms and ski waxes. PFCA and PFSA were analysed by HPLC-MS/MS, whereas FTOH were detected by GC/CI-MS. Consumer products such as cleaning agents or some baking and sandwich papers show low or negligible PFSA and PFCA contents. On the other hand, high PFAS levels were identified in ski waxes (up to about 2000 µg/kg PFOA), leather samples (up to about 200 µg/kg PFBA and 120 µg/kg PFBS), outdoor textiles (up to 19 µg/m(2) PFOA) and some other baking papers (up to 15 µg/m(2) PFOA). Moreover, some test samples like carpet and leather samples and outdoor materials exceeded the EU regulatory threshold value for PFOS (1 µg/m(2)). A diverse mixture of PFASs can be found in consumer products for all fields of daily use in varying concentrations. This study proves the importance of screening and monitoring of consumer products for PFAS loads and the necessity for an action to regulate the use of PFASs, especially PFOA, in consumer products.

Retrospective monitoring of perfluorocarboxylates and perfluorosulfonates in human plasma archived by the German Environmental Specimen Bank.

Due to the increased awareness of the ubiquitous contamination of all environmental compartments and of human beings with perfluoroalkyl substances (PFAS), voluntary withdrawals and shifts in products and manufacturing technologies, as well as in regulatory measures, have been made. To investigate whether these activities are reflected in the human exposure to PFASS, we examined human blood archived by the German Environmental Specimen Bank. Plasma samples (n=258, age range 20-29 years) covering the observation period 1982-2010 were analyzed for eleven perfluoroalkylcarboxylates (C4-C14) and five perfluoroalkylsulfonates (C4-C10) by HPLC-MS-MS. We detected perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and perfluorohexanesulfonate (PFHxS) most often of all PFASs. Following a sharp increase from 1982 to 1986, median PFOS concentrations remained in the range of 20-24ng/mL until the end of the 1990s. Between 2001 and 2010, PFOS concentrations decreased steadily to 4ng/mL in plasma. Except for a similar strong increase from 1982 to 1986, we observed PFOA concentrations fluctuating between 4.8 and 6.3ng/mL in the following years. Since 2008, ESB data suggest a decreasing trend of PFOA. PFHxS concentrations increased continuously between 1982 and 2001 from about 1-2ng/mL. After nearly unchanged concentrations until 2005, a downward trend of PFHxS in plasma became apparent and in 2010 resulted in levels which were about 20% lower than those observed in the early 1980s. In the case of shorter and longer chained PFASs, quantification frequencies were between 0 and 60% and we found no indication of any temporal trends in human plasma concentrations.

Survey of patterns, levels, and trends of perfluorinated compounds in aquatic organisms and bird eggs from representative German ecosystems.

PURPOSE: Samples from the German Environmental Specimen Bank (ESB) covering particularly the years 1994-1996, 2000-2002, and 2006-2009 were analyzed for perfluorinated compounds (PFC; mainly C4-C13 carboxylic and sulfonic acids) to gain an overview on current PFC levels and patterns in marine, limnetic, and terrestrial biota; to assess their concentrations in different trophic levels; and to investigate whether risk management measures for PFC are successful. METHODS: Specimens, either standardized annual pooled samples (blue mussels, eelpout liver, bream liver, pigeon eggs) or individual single samples (cormorant eggs, rook eggs), were collected for the German ESB program from representative sampling sites according to documented guidelines. After appropriate extraction, PFC were quantified under ISO/IEC 17025 accreditation by HPLC/MS-MS with isotopically labeled internal standards. Limits of quantification (LOQs) were 0.2-0.5 ng/g. Data are reported on a wet weight basis. RESULTS AND DISCUSSION: In most samples the predominant PFC was perfluorooctane sulfonic acid (PFOS). However, in marine mussels from North and Baltic Seas, PFOS levels were mostly below the LOQ, but low residues of PFOS amide were found which declined in recent years. Livers of eelpout showed maximum concentrations of 15-25 ng/g PFOS in the period 2000-2002 and low amounts of perfluoropentanoate in all years. Beside PFOS (median 48 ng/g) several PFC could be determined in cormorant eggs sampled in 2009 from a Baltic Sea site. For a freshwater ecosystem, current PFC burdens for cormorant eggs were even higher (median 400 ng/g PFOS). Livers of bream from rivers showed concentrations of 130-260 ng/g PFOS, but for bream from a reference lake levels were only about 6 ng/g. In contrast to cormorants, eggs of rook and feral pigeon from terrestrial ecosystems displayed only low PFC burdens (up to 6 ng/g PFOS). CONCLUSIONS: Generally, PFC levels were lower in marine than in freshwater biota. PFC burdens were higher in biota from the ESB-North Sea sites than in Baltic Sea organisms. Levels of PFC were quite high especially in top predators of both limnetic and marine ecosystems. Only low PFC levels were detected in eggs of terrestrial birds. A decrease of PFOS levels from maximum values around the year 2000 observed at least in North Sea biota may be a result of a production cease and shifts in marketing pattern.

Determination of sulfonylurea herbicides in water and food samples using sol-gel glass-based immunoaffinity extraction and liquid chromatography-ultraviolet/diode array detection or liquid chromatography-tandem mass spectrometry.

Immunoaffinity supports (IAS) were prepared using broad specific polyclonal anti-sulfonylurea (SU) antibodies immobilized in sol-gel glass. Two different kinds of supports were applied, crushed sol-gel monoliths and sol-gel-coated highly porous silica particles. Both were used for the quantitative enrichment of SUs in natural water and food samples followed by high-performance liquid chromatography-ultraviolet/diode array detection (HPLC-UV/DAD) and tandem mass spectrometry (LC-MS/MS), respectively. Loading, washing, and elution conditions of IAS were optimized. The capacity of supports was determined for 30 SUs and compared with the cross-reactivity pattern of the direct competitive enzyme-linked immunosorbent assay. The capacities correlated well with the affinity to individual SU compounds. Even analytes to which the polyclonal antibodies showed only a lower cross-reactivity could be enriched to a certain degree, if a sufficient capacity of IAS was provided. The IAS could be reused at least 10 times without a loss of effectiveness. Recovery of 16 selected SUs extracted from spiked water and food samples was dependent on the affinity of both immobilized antibodies to single compounds and matrix interferences. In water, 13 SUs showed recoveries higher than 80% when immunoaffinity extraction was used in combination with LC-UV/DAD. On the basis of the enrichment of 200 mL of aqueous sample, corresponding limit of detection (LOD) values ranged between 20 and 100 ng/L. The recoveries of 10 SUs, which were extracted from 10 g of potato spiked at a 10 microg/kg level, were higher than 75%. For grain samples, recoveries were at the same order for at least five SU herbicides. The LOD of LC-MS/MS measurements was about 1 order of magnitude higher, i.e., gave LODs between 1.1 and 6.9 microg/kg of food sample, depending on the compound and extraction procedure. These LODs provide evidence that the main advantage of the prepared IAS is their high selectivity for group specific recognition of SUs as compared to other nonspecific solid phase extraction materials.