Evidence for High Concentrations and Maternal Transfer of

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Evidence for high concentrations and maternal transfer of substituted diphenylamines in European eels analyzed by GCxGC-ToF MS and GC-FTICR-MS Roxana Sühring, Xavier Ortiz, Miren Pena Abaurrea, Karl J. Jobst, Marko Freese, Jan-Dag Pohlmann, Lasse Marohn, Ralf Ebinghaus, Sean M. Backus, Reinhold Hanel, and Eric J Reiner Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04382 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 30, 2016

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Environmental Science & Technology

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Graphical Abstract

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Evidence for high concentrations and maternal transfer of substituted diphenylamines in European eels analyzed by GCxGC-ToF MS and GC-FTICR-MS

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Roxana Sühring*1,2, Xavier Ortiz3, Miren Pena-Abaurrea3, Karl J. Jobst3, Marko Freese4, Jan-Dag Pohlmann4, Lasse Marohn4, Ralf Ebinghaus1, Sean Backus5, Reinhold Hanel4, Eric J. Reiner3 1

Helmholtz-Zentrum Geesthacht, Centre for Materials and Coastal Research, Max-Planck-Str. 1, 21502 Geesthacht, Germany 2 Centre for Environment, Fisheries and Aquaculture Science (Cefas), Lowestoft, Suffolk NR33 0HT, United Kingdom 3 Ontario Ministry of the Environment and Climate Change, 125 Resources Road, Toronto, Ontario M9P 3V6, Canada 4 Thünen Institute of Fisheries Ecology, Palmaille 9, 22767 Hamburg, Germany 5 Canada Centre for Inland Waters, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6, Canada

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ACS Paragon Plus Environment


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Abstract

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Chemical pollution is hypothesized to be one of the factors driving the strong decline of the

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critically endangered European eel population. Specifically, the impact of contaminants on

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the quality of spawning eels and subsequent embryo survival and development has been

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discussed as crucial investigation point. However, so far only very limited information on

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potential negative effects of contaminants on the reproduction of eels is available.

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Through the combination of non-targeted ultra-high resolution mass spectrometry and

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multidimensional gas chromatography, combined with more conventional targeted analytical

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approaches and multimedia mass-balance modelling, compounds of particular relevance and

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their maternal transfer in artificially matured European eels from the German river Ems have

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been identified.

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Substituted diphenylamines were, unexpectedly, found to be the primary organic

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contaminants in the eel samples, with concentrations in the µg g-1 ww range. Furthermore, it

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could be shown that these contaminants, as well as polychlorinated biphenyls (PCBs),

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organochlorine pesticides and polyaromatic hydrocarbons (PAHs), are not merely stored in

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lipid rich tissue of eels, but maternally transferred into gonads and eggs.

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The results of this study provide unpreceded information on both the fate and behavior of

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substituted diphenylamines in the environment as well as their relevance as contaminants in

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European eels.

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1. Introduction

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The European eel (Anguilla anguilla) is regarded as a critically endangered species (1, 2). Scientists

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agree that the “quality of spawners” is a vital factor for the survival of the species (1) and that its

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impairment might be one of the reasons for the strong decline of juvenile eel (glass eel) recruitment

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during the last three decades. “Quality of spawners” is, in this context, defined as the health status of

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mature silver eels migrating back to their spawning ground in the Sargasso Sea and their ability to

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produce healthy offspring (1).

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Halogenated contaminants have been postulated as potential compounds of high concern for the

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quality of spawning eels (3). They are suspected to affect the eel’s lipid metabolism, to decrease its

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ability to reproduce or affect the viability of offspring (3).

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Our recent study (4) reported that the majority of brominated and chlorinated flame retardants were

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maternally transferred into gonads and eggs of artificially matured silver eels.

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The targeted analysis of the maternal transfer of potentially hazardous compounds in European eels

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has been a unique opportunity to gather more information on the potential impact of contaminants

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on the quality of spawning eels and their developing gonads and eggs. However, the approach of

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targeted analysis, only provides information on a selection of compounds, which means, that

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important, not-targeted, compounds might be missed.

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To gather the necessary understanding on the importance of different organic contaminants in the

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contamination and maternal transfer of European eels, selected samples of artificially matured eels

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through hormonal treatment and a non-treated comparison group from the same habitat were

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analyzed by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

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(GCxGC-ToF MS) and gas chromatography Fourier transform ion cyclotron resonance mass

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spectrometry (GC-FTICR-MS). Selected identified compounds were subsequently analyzed by gas

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chromatography tandem mass spectrometry (GC-MS/MS), to quantify levels in samples.

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The focus of all analysis steps was on non-polar compounds, because eels have been reported to

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accumulate lipophilic contaminants especially during their continental life phase (5, 6) due to their

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high body fat content (up to 40% of total body weight (7)) and their longevity. 3



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2. Materials and Methods

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2.1. Sampling and sample preparation

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Sampling and sample preparation as well as the artificial maturation process have been described in

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Sühring et al. (4). For the non-target analysis, three female eels from the German river Ems, caught at

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the onset of migration and treated with salmon pituitary extract (SPE) to induce maturation, were

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used. Additionally, two female eels without hormone treatment from the same habitat were sampled

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to assess the initial contamination situation in the habitat, control for potential contamination

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through the hormone treatment process, and gather information on the potential impact of

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maturation on the contaminant distribution within the eel. Muscle and gonad tissue (3 g each), as

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well as 5 g eggs (in case of artificially matured eels) were sampled in duplicate and stored at -20°C

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until analysis. For targeted analysis three additional female eels were sampled to increase the

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comparison group.

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Extraction and clean-up methods are described in Sühring et al. (8). The frozen samples were

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homogenized with anhydrous sodium sulfate and extracted by pressurized liquid extraction (ASE-200,

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Dionex, Sunnyvale, USA) using dichloromethane (DCM, ROTH, Karlsruhe, Germany) at 100°C and 120

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bar. All samples were spiked with 13C isotope labeled polychlorinated biphenyl standards (PCB- 77, -

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81, -105, -114, -118, -126, -156, -157, -167, -169, -189, -170, -180 clean-up standard (13C12, 99%),

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Cambridge Isotope Laboratories (Tewksbury, USA)) prior to extraction. Clean-up consisted of a gel

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permeation chromatography with hexane:DCM (1:1, v/v) and 10% deactivated silica gel column clean-

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up with hexane. The lipid content of samples was determined gravimetrically from separate aliquots

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following a method described in Sühring et al. (8).

98 99

2.2. Instrumentation

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GCxGC-ToF MS

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Samples and blanks were analyzed in accordance with the method reported in Pena-Abaurrea et al.

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(9), using a Pegasus 4D (Leco Corp., St. Joseph, MI, USA) consisting of a modified GC×GC Agilent 6890

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chromatograph and a ToF MS with electron ionization (EI) mode, fitted with a Rtx-5MS × BPX-50 4


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column set (30 m × 0.25 mm internal diameter (i.d.) × 0.25 μm film thickness and 1.6 m × 0.15 mm

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i.d. × 0.15 μm film thickness, respectively). A nitrogen quad-jet dual-stage modulator was used for

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sample focusing and reinjection in the secondary column.

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FTICR-MS

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Samples and blanks were analyzed in accordance with the method reported in Ortiz et al. (10), using

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a Varian GC-QQQ-FTICR MS (Varian Inc., Walnut Creek, CA). The GC was fitted with a DB5-MS capillary

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column (40 m x 0.18 mm i.d. x 0.18 μm film thickness) from Agilent (Santa Clara, CA). Samples were

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injected (1 μL) in splitless mode with helium as carrier gas. The MS was operated in the EI mode (70

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eV) and was set to pass all ions. The FTICR MS was operated at a resolving power of 100,000 to

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150,000 (fwhm). Mass spectra were obtained using arbitrary waveform excitation and broadband

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detection from m/z 75 to 650. Detection and cycle times were set at 524 ms and 1.5 s, respectively.

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External mass calibration was performed using perfluorotributylamine, and internal mass calibration

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was performed using protonated diisononyl phthalate (background ion) at m/z 419.315 60.

117 118

GC-MS/MS

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Samples and blanks were analyzed by gas chromatography with tandem-mass spectrometry GC-

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MS/MS (Agilent QQQ 7010) in EI mode. The instrument was fitted with a HP-5MS column (30 m x

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0.25 mm i.d. x 0.25 µm film thickness, J&W Scientific) with Helium (purity 99.999%) as carrier gas and

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Nitrogen as collision gas. The instrument was operated in multiple reactions monitoring mode (MRM)

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at 70 eV ionization energy. A technical benzamine, n-phenyl-, reaction products with styrene and

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2,4,4-trimethylpentene (BNST) standard (AK Scientific Inc. (Union City, CA, USA), lot TC36296) was

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used to

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quantify substituted diphenylamines in the eel samples with 13C isotope labelled PCB standards as an

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indication how much analyte was lost during extraction and clean-up.

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Nine diphenylamine reaction products with styrene and 2,4,4-trimethylpentene were analyzed (Table

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1).

130 5



131 132 133 134 135

MRM RT Compound Formula MW Log KOW Log KAW transitions [min] 16.8 Diphenylamine (DPA) C12H11N 169 3.5 -3.9 169->167 23.34 Monostyrenated-DPA 1 C20H19N 273 5.5 -5.3 273->180 23.42 Monooctyl-DPA C20H27N 281 7.3 -2.2 281->210 25.46 Monostyrenated-DPA 2 C20H19N 273 5.5 -5.3 273->258 27.21 Monooctyl-monostyrenated -DPA 1 C28H35N 385 9.2 -4.4 385->314 28.20 Monooctyl-monostyrenated -DPA 2 C28H35N 385 9.2 -4.4 385->314 28.47 Dioctyl-DPA C28H43N 393 11 393->322 30.25 Monooctyl-monostyrenated -DPA 3 C28H35N 385 9.2 -4.4 385->314 31.15 Dioctyl-monostyrenated -DPA C36H51N 497 13 497->496 Table 1: Analyzed substituted diphenylamines. The summary includes compound name, structural formula, molecular weight (MW) octanol-water partitioning coefficient (Log KOW), air-water partitioning coefficient (Log KAW), MRM transitions and retention time (RT) [min]. Partitioning coefficients were estimated using EPISuite KOWWIN v1.67 and HENRYWIN v3.10 (13).

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Recoveries for the 13C PCB standards ranged from 57 ± 26 % for PCB 81 to 96 ± 34 % for PCB 169.

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However, due to the impurity of the technical standard and the lack of accurate isotope labelled

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reference standards, only the order of magnitude (i.e. pg g-1, ng g-1 or µg g-1) could be determined.

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2.3. QA/QC

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Extraction and clean-up were conducted in a clean lab (class 10000). A blank test, using Na2SO4

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treated similar to real samples, was conducted with every extraction batch (five samples). Solvent

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blanks were run between experimental runs to ensure no carryover between samples.

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2.4. Data analysis

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Analysis of GCxGC-ToF MS results was performed using ChromaToF software (version 4.50). FTICR

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spectra were analyzed using Varian Omega (version 9.1.21). Elemental compositions were assigned

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through a combination of custom macros for Excel (GCxGC-ToF MS) described in Pena-Abaurrea et al.

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(9), analysis through Kendrick mass defect plots (11), as well as Elemental Composition Calculator

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(Varian Inc.) and subsequent library matching (similarity).

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Kendrick mass defect plots utilize the convention by the International Union of Pure and Applied

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Chemistry (IUPAC) who defined a mass scale based on the carbon exact mass (C = 12.000 00 Da). By

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using a mass scale based on CH2 (i.e., the Kendrick mass scale, where CH2 = 14.000 00 Da), 6


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homologous series of hydrocarbons can be grouped based on their difference between exact mass

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and nominal mass (mass defect) (11). This principle can be applied similarly to chlorinated analogue

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series. The resulting Kendrick mass defect can then be plotted against the nominal mass, resulting in

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simplified visualizations of large data sets where compounds containing halogens are aligned

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horizontally (12).

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GC-MS/MS data was analyzed using Agilent Technologies MassHunter Workstation Software

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Quantitative Analysis B.06.00. Further data analysis was performed with Microsoft Office Excel 2010.

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Physical-chemical properties of substituted diphenylamines, namely octanol- water and air-water

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partitioning coefficients (Log KOW, Log KAW) were estimated using EPISuite KOWWIN v1.67 and

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HENRYWIN v3.10 (13). Distribution of substituted diphenylamines into different environmental media

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was estimated using the Level III multimedia mass balance model (fugacity model) by Mackay et al.

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(14).

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3. Results and Discussion

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3.1 From “usual suspects” to unexpected findings

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An analysis of the GCxGC-ToF MS results and Kendrick plots of the FTICR-MS data first returned a

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compound spectrum of what could be called “usual suspects” in the analysis of organic contaminants

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in eels. Both Kendrick Plots and GCxGC-ToF MS chromatograms were dominated by polychlorinated

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biphenyls (PCBs) (Figure 1).

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Figure 1: Kendrick plot displaying results of a muscle sample of a hormone treated eel from river Ems. The mass defect (y-axis) is plotted as a function of nominal mass (x-axis) in a mass scale based on the exact and IUPAC mass of Cl, as defined in Taguchi et al. (12).

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Furthermore,

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dichlorodiphenyldichloroethylene (DDE) and the fungicide hexachlorobenzene (HCB) could be

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detected in every sample (Table 2). The polyaromatic hydrocarbons (PAH) pyrene and fluoranthene,

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as well as phenylethene (styrene) and 1,8-dichloronaphtalene (PCN-9) were detected in the majority

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of samples using the custom macros for Excel (GCxGC-ToF MS) described in Pena-Abaurrea et al. (9)

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(Table 2). The brominated flame retardant tribromophenole (TBP) as well as its transformation

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product tribromoanisole (TBA) were identified during FTICR-MS analysis and could primarily be

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detected in muscle tissue (Figure 1). The presence of these compounds in eels from Europe as well as

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North America has been reported previously (3, 5, 6, 15-19). However, the detection of all of these

8

the

dichlorodiphenyltrichloroethane

(DDT)


transformation

product

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contaminants in gonads and eggs of eels further underlined the necessity to control and monitor

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these hazardous chemicals. Detection frequency [%] Retention time [s] m/z 1D 2D Muscle (5) Gonads (5) Eggs (3) Phenylethene 178.15 852 2.34 100 100 100 1,8-dichloronaphthalene 197.06 750 1.86 100 100 83 Pyrene 202.25 1014 3.37 100 100 100 Fluorenthene 202.26 1062 3.78 100 100 100 Hexachlorobenzene 284.80 822 1.75 100 100 100 Dichlorodiphenyldichloroethylene 318.02 1092 2.86 100 100 100 Table 2: Summary of commonly detected contaminants in muscle, gonads and eggs (number of samples) of hormone treated eels analyzed with GCxGC-ToF MS; including retention time [s] in 1D and 2D dimension, detection frequency [%] Compounds

189 190 191 192 193 194

A closer analysis of the compound pattern found in gonads of artificially matured eels revealed a new picture of chemicals of potential concern for the quality of spawning eels:

195 196

3.2 Detection and characterization of substituted diphenylamines

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Unsuspectedly, high abundance compared to other detected compounds of substituted

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diphenylamines was found in GCxGC-ToF MS measurements of gonads of artificially matured eels for

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non-halogenated compounds with primary fragments at m/z 210 and m/z 393, respectively using the

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elemental composition calculator and library matching (similarity) (Figures 2, S1).

9



201 202 203 204 205

Figure 2: GCxGC chromatogram (top) and mass-spectrum for the peak marked by the red circle (bottom) of non-halogenated compound (m/z 393) with high abundance in GCxGC-ToF MS analysis of gonads of artificially matured eels.

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GC-FTICR-MS measurements confirmed high abundance of non-halogenated compounds at the

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respective m/z fragments. Through elemental composition assignment, using a combination of

208

custom macros for Excel, the Elemental Composition Calculator (Varian Inc.) and library matching

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(Figure S1), it was possible to identify the two compounds as monooctyl-DPA (m/z 281 and m/z 210)

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and dioctyl-DPA (m/z 393 and m/z 323) with a delta m/z < 2 ppm for the elemental composition

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search (Figure S1). Both of these compounds are integral parts of benzamine, n-phenyl-, reaction

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products with styrene and 2,4,4-trimethylpentene (BNST). BNST (CAS 68921-45-9) is a high volume

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production chemical with production and import of over 10 000 tons yearly in 2006 in Canada (20) 10


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and 100 – 1000 tons per year in the European Union (21). It is used as additive antioxidant in vehicle

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engine oil and in some commercial and industrial lubricants (22).

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A final screening assessment report by Environment Canada (23) concluded that BNST is highly

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persistent and bioaccumulative and “… is entering or may be entering the environment in a quantity

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or a concentration or under conditions that have or may have an immediate or long-term harmful

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effect on the environment or its biological diversity.” ((22), section 1.2, first paragraph). This

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assessment led to the addition of BNST to the Prohibition of Certain Toxic Substances Regulations

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(24), leading to prohibitions of its use, sale and offer for sale in Canada by March 2013 (25).

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Exemptions include the use in vehicle engine oils, commercial and industrial lubricants until March

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2015, as well as manufacture, sale, use and import as additive in rubber (except tires) as well as

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products such as vehicle engine oil intended for personal use only (25). In the European Union BNST

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has been registered under REACH, classifying it as “very persistent” (vP), but lacking evidence for

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bioaccumulation or toxicity (21).

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One of the key missing factors, at present, is the missing experimental data on BNST in biota. The

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detection in European eels could therefore significantly increase and impact the information

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regarding environmental fate and behavior of BNST.

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To verify the presence of BNST and related substituted diphenylamines in the eel samples, and

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minimize potential errors due to blank contamination or carry-over, and get information on the

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magnitude of substituted diphenylamines present in different tissue types of eels, all samples and

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laboratory blanks were analyzed with GC-MS/MS using the technical substituted diphenylamines

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mixture for identification and calibration.

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Considering the impurity of the standard and the lack of isotope labeled reference standards, it was

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only possible to determine the relative order of magnitude (i.e. pg g-1, ng g-1 or µg g-1) of substituted

237

diphenylamines in the analyzed samples. Highest levels were detected in gonads of the artificially

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matured eels with concentrations in the µg g-1 wet weight range for dioctyl-DPA and monooctyl-DPA.

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In the gonads and muscle tissue of non-hormone treated eels, as well as eggs, dioctyl-DPA and

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monooctyl-DPA concentrations were in the ng g-1 wet weight range, with the highest concentrations 11



241

in gonads followed by muscle tissue and eggs. Other BNST components were in the >10 ng g-1 wet

242

weight range for most tissue types. Exemptions were monooctyl-monostyrenated -DPA 3 and dioctyl-

243

monostyrenated -DPA that were detected in concentrations below 1 ng g-1 wet weight for the

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majority of tissue types.

245

Even with the limitations regarding quantitative analysis using a technical mixture as standard, the

246

magnitude of substituted diphenylamines in the European eel samples exceeded concentrations

247

reported for halogenated contaminants such as flame retardants (HFRs) and polychlorinated

248

biphenyls (PCBs) (19). PCB concentrations measured in the same samples were in the ng g-1 wet

249

weight range. This implies that concentrations of substituted diphenylamines in eels from the

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comparably small and remote river Ems did not only exceed the PCB concentrations in the same

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samples, but are in the same range or even higher than concentrations of PCBs and organochlorine

252

pesticides reported in eels form the known highly polluted river Scheldt in Belgium (14) or Lake

253

Ontario in Canada (16).

254 255

3.3 Patterns and tissue distribution of the detected substituted diphenylamines

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The pattern of substituted diphenylamines differed strongly between the composition of the

257

technical mixture analyzed via GC-MS/MS, the hormone treated eels and in the comparison group, as

258

well as among the analyzed tissue types.

259

The technical mixture was found to primarily contain monooctyl-DPA (approx. 30%), dioctyl-DPA

260

(approx. 19%), dioctyl-monostyrenated-DPA (approx. 15%), monooctyl- monostyrenated-DPA (approx.

261

10%), as well as smaller amounts (5 -10% each) of monostyrenated-DPA and the monooctyl-

262

monostyrenated-DPA isomers (Table 3).

263

Observed patterns in eel tissue, however, did not resemble the composition of the technical mixture,

264

but rather showed strong accumulation of individual compounds (Table 3).

265

In muscle and gonads of hormone treated eels dioctyl-DPA was predominant with relative

266

contributions of 97 ± 23% and 83 ± 15%, respectively. In eggs, on the other hand, monostyrenated-

267

DPA was predominant with 48 ± 28% contribution, followed by dioctyl-DPA (19 ± 11%) and 12


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monooctyl-monostyrenated-DPA (13 ± 14%). Patterns in muscle samples from the comparison group

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also primarily displayed contamination with monostyrenated-DPA (71 ± 59%), followed by dioctyl-DPA

270

(17 ± 34%). Gonad tissue of comparison group eels was the only tissue type with a significant

271

contribution of DPA (33 ± 25%). Apart from DPA it displayed patterns close to muscle and gonads of

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hormone treated eels, with 45 ± 15% dioctyl-DPA, followed by monostyrenated-DPA (15 ± 6%) (Table

273

3).

Hormone treated eel

Comparison group

Technical mixture

Muscle (n=9)

Gonad (n=9)

Egg (n=9)

Muscle (n=10)

Gonad (n=10)

DPA Monostyrenated-DPA 1,2

n.a. 9

1.3 ± 0.096 1.2 ± 21

0.24 ± 0.67 0.76 ± 17

8.8 ± 6.0 48 ± 28

8.1 ± 24 71 ± 59

33 ± 25 15 ± 6

Monooctyl-DPA Monooctyl-monostyrenated DPA 1-3 Dioctyl-DPA

30

0.075 ± 1.4

16 ± 8.9

7.7 ± 0.022

2.2 ± 2.1

7.6 ± 4

8-10 19

0.019 ± 0.34 97 ± 23

0.039 ± 0.020 83 ± 15

13 ± 14 19 ± 11

1.8 ± 1.4 17 ± 34

n.d. 45 ± 15

Dioctyl-monostyrenated -DPA

15

n.d.

0.0090 ± 0.0052

2.4 ± 2.2

n.d.

n.d.

274 275 276 277 278

Table 3: Overall summary of relative contribution [%] (average ± standard deviation) of compounds of the BNST mixture in the technical mix. Each three samples of muscle, gonads and eggs of from three hormone treated eels, as well as each two samples of muscle and gonads of the five comparison group eels. The total number of analyzed samples (including replicates) is depicted as “n”.

279

These differences in patterns could indicate different persistence of the substituted diphenylamines

280

in the environment, transformation processes during uptake and distribution in the eel’s body,

281

transformation or re-distribution processes during the maturation process as well as different uptake

282

and distribution processes depending on the properties of the compound.

283

Physical-chemical properties of the analyzed substituted diphenylamines varied strongly with

284

estimated octanol-water partitioning coefficients (Log KOW) between 3.5 (DPA) and 13 (dioctyl-

285

monostyrenated-DPA) and estimated air-water partitioning coefficient (Log KAW) between -5.3

286

(monostyrenated-DPA) and -2.2 (monooctyl-DPA) (Table 1).

287

This work used the Fugacity III model developed by Mackay et al. (14) to estimate distribution of

288

substituted diphenylamines in air, water, soil or sediments assuming emissions into air (scenario 1) or

289

water (scenario 2). This distribution into different environmental media could provide indications of 13



290

potential sources of substituted diphenylamines for eels and could furthermore give indications on

291

partitioning behavior of the tested compounds into non-polar matrices (e.g. sediment and lipid-rich

292

tissue) or primarily aqueous matrices (e.g. water and blood).

293

The first scenario (emission into air) showed that most substituted diphenylamines would partition

294

into soil if emitted into air and would not, or only in limited amounts, reach the water-phase (Table

295

S1). It could therefore be concluded that emission into air and subsequent deposition was not likely

296

the primary source of BNST contamination in the analyzed eels. The second scenario (emission into

297

water), however, showed strong differences in the predicted partitioning of different substituted

298

diphenylamines (Table 4, Figure S2). Monooctyl-DPA and monooctyl-monostyrenated-DPA were

299

predicted to primarily partition into sediments, while all other substituted diphenylamines were

300

predicted to primarily partition into the water-phase.

301

302 303

Compound % air % water % soil % sediment DPA 0 96 0 4 Monostyrenated-DPA 1,2 0 95 0 5 Monooctyl-DPA 0 4 0 96 Monooctyl-monostyrenated- DPA 1-3 0 2 0 98 Dioctyl-DPA 0 94 0 6 Dioctyl-monostyrenated-DPA 0 97 0 3 Table 4: Overview of the fugacity III model predictions of partitioning [%] of substituted diphenylamines into air, water, soil and sediment assuming 100 % emission into water.

304

Correlating the contamination pattern found in different tissue types of hormone treated eels and

305

comparison group with patterns of the technical mixture and these predicted partitioning into

306

sediments and water provided indications on potential tissue distribution and sources of substituted

307

diphenylamines (Table 5). Predicted partitioning into sediments could indicate an affinity of the

308

substances to partition into non-polar matrices such as lipids in biota, whereas a predicted

309

partitioning into water would be an indication for the substances to be present in blood of the eels.

310

The results of the correlation could only provide a first indication, mainly due to the limited available

311

samples. The apparent correlations with a regression coefficient (r) above 0.5 have been marked with

312

“+” (see table 5). Nevertheless, this analysis only contains three tissue types from three hormone 14


Page 14 of 19

Page 15 of 19


313

treated fish, and two tissue types from five reference fish. Therefore, these results should be treated

314

with caution as these results will have limited statistical validity.

315

Patterns of substituted diphenylamines in muscle and gonad tissue of hormone treated eels as well as

316

the comparison group indicated correlations with the patterns for substituted diphenylamines that

317

were predicted to partition into sediments (r = 0.7 – 0.99). This could be an indication that uptake

318

from sediments might be a relevant source for substituted diphenylamine contamination in these

319

eels. It, furthermore, indicated that uptake and tissue distribution into muscle and gonads could be

320

related to the lipid distribution and lipid re-distribution during the maturation of eels, as previously

321

reported for halogenated flame retardants (HFRs) in eels (4), zebrafish (26), as well as

322

organochlorines in oviparous organisms (27) and walleye (28). Contamination patterns in eel eggs, on

323

the other hand, were dominated by compounds predicted to partition into the water-phase,

324

indicating that the maternal transfer into eggs might not solely be related to the lipid transfer, as

325

described for HFRs, but by transfer via primarily aqueous media such as e.g. blood and water (Table

326

5). The comparably high contribution of DPA in gonads of eels from the comparison group could be

327

an indication for transformation or metabolism processes. Previous research on the maternal transfer

328

of halogenated flame retardants, evidence suggests a significant increase of metabolites in gonad

329

tissue compared to muscle tissue (4).

330 331 332 333 334 335 336 337 338 339 15



Page 16 of 19

Hormone treated eels

340 341 342 343 344

Technical mixture

Sediment

Water

Muscle (n = 3)

Gonads (n = 3)

Eggs (n = 3)

Muscle (n = 5)

Gonads (n = 5)

-

0.35

-0.39

0.09

0.45

-0.28

0.02

0.29

+

0.15 0.81

Sediment

0.35

-

-0.24

0.86

0.99

Water

-0.39

-0.24

-

0.23

-0.25

Hormone treated eels

+

Muscle

0.09

0.86

+

0.23

-

0.86

Gonads

0.45

0.99

+

-0.25

0.86

Eggs

-0.28

0.15

Comparison group

Technical mixture

Comparison group

Muscle

0.02

0.67

Gonads

0.29

0.99

0.81

+

+

0.63

0.67 +

+

0.50 0.70

+

-

0.21

0.49

+

0.21

-

0.65

+

0.49

0.65

0.63

+

0.50

0.70

+

-0.24

0.88

+

0.98

+

0.21

+

+

+

0.88 0.98

+

0.52 +

-

3.4 Implications

346

The results of this study have shown the benefits and necessity of combining targeted and non-

347

targeted analytical approaches. These mechanisms will help to comprehensively assess chemicals

348

with potential negative impact on the quality of spawning eels. Without the non-target analysis, the

349

high contribution and potential relevance of substituted diphenylamines as contaminants in eels

350

would not have come to our attention.

351

Our results showed that substituted diphenylamines were taken up and accumulated by eels to

352

concentrations similar or even exceeding those of pesticides and PCBs and were several orders of

353

magnitude higher than concentrations of halogenated flame retardants in the same samples (4).

354

Despite the limited number of samples and analyzed habitats, the results of this study, clearly require

355

further research. Particularly, on the environmental fate, behavior and, especially, uptake and impacts

356

on biota of substituted diphenylamines.

357

Furthermore, it could be observed that the majority of detected organic contaminants, including

358

legacy POPs such as PCBs, organochlorine pesticides and DDT transformation products, as well as

359

PAHs and substituted diphenylamines, are not merely stored in lipid rich tissue of adult eels, but 16


+ +

0.21

Table 5: Summary of Pearson correlation of substituted diphenylamine patterns in the technical mixture, sediment, water, muscle, and eel samples. All apparent correlations with r > 0.5 were marked with +, it was not possible to determine statistical significance of the correlations due to the limited sample size (n).

345

+

-0.24

0.52

0.99

+

Page 17 of 19


360

maternally transferred into gonads and eggs, making them a potential threat to the quality of

361

spawners and recovery of the European eel stock. Model predictions by e.g. recently developed

362

physiologically based toxic-kinetic model for the European eel (29) could provide valuable

363

information on the kinetics and tissue distribution of contaminants, especially considering the limited

364

availability of samples.

365 366

1. Acknowledgments

367

We would like to thank Mehran Alaee for establishing the contact that led to the cooperation of this

368

project. We would like to thank Sebastien Samson for helping with developing the concept design of

369

the graphical abstract.

370 371

2. Acknowledgments

372

We would like to thank Mehran Alaee for establishing the contact that led to the cooperation for this

373

project and Sebastien Samson for the concept design of the graphical abstract.

374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394

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