Passive Dosing in Chronic Toxicity Tests with the Nematode

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Passive dosing in chronic toxicity tests with the nematode Caenorhabditis elegans Fabian Christoph Fischer, Leonard Böhm, Sebastian Hoess, Christel Möhlenkamp, Evelyn Claus, Rolf-Alexander During, and Sabine Schäfer Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02956 • Publication Date (Web): 05 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016

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Environmental Science & Technology

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Passive dosing in chronic toxicity tests with the

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nematode Caenorhabditis elegans

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Fabian Fischer*,1,2,†, Leonard Böhm2, Sebastian Höss3, Christel Möhlenkamp1, Evelyn Claus1,

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Rolf-Alexander Düring2, Sabine Schäfer1

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German Federal Institute of Hydrology (BfG), Am Mainzer Tor 1, 56068 Koblenz, Germany

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2

Institute of Soil Science and Soil Conservation, Research Center for BioSystems, Land Use,

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and Nutrition (IFZ), Justus Liebig University, Heinrich-Buff-Ring 26, 35392 Giessen, Germany 3

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†

Ecossa, Giselastraße 6, 82319 Starnberg, Germany

Current affiliation: Helmholtz Centre for Environmental Research - UFZ, Department Cell Toxicology, Permoserstraße 15, 04318 Leipzig, Germany

ACS Paragon Plus Environment

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Abstract

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In chronic toxicity tests with Caenorhabditis elegans, it is required to feed with bacteria, which

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reduce the freely dissolved concentration (Cfree) of hydrophobic organic chemicals (HOCs),

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leading to poorly defined exposure with conventional dosing procedures. We examined the

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efficacy of passive dosing of polycyclic aromatic hydrocarbons (PAHs) using silicone O-rings to

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control exposure during C. elegans toxicity testing, and compared the results to those obtained

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with solvent spiking. Solid-phase microextraction and liquid-liquid extraction were used to

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measure Cfree and the chemicals taken up via ingestion.

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During toxicity testing, Cfree decreased by up to 89% after solvent spiking but remained constant

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with passive dosing. This led to a higher apparent toxicity on C. elegans exposed by passive

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dosing than by solvent spiking. With increasing bacterial cell densities, Cfree of solvent spiked

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PAHs decreased while maintained constant with passive dosing. This resulted in lower apparent

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toxicity under solvent spiking, but an increased apparent toxicity with passive dosing, probably

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as a result of the higher chemical uptake rate via food (CUfood). Our results demonstrate the

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utility of passive dosing to control Cfree in routine chronic toxicity testing of HOCs. Moreover,

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both chemical uptake from water or via food ingestion can be controlled, thus enabling to

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discriminate different uptake routes in chronic toxicity studies.


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TOC Art figure

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1. Introduction

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Miniaturized high-throughput toxicity tests using invertebrates are being increasingly employed

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in the ecotoxicity testing of anthropogenic chemicals, both to avoid the ethically questionable

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testing of vertebrates (fish, mammals) and to reduce costs.1-3 Free-living, non-parasitic

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nematodes are ubiquitous, diverse, and ecologically important invertebrates that inhabit soils and

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sediments.4,5 The nematode Caenorhabditis elegans has long been widely used to assess the

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toxicity of environmental pollutants.6-8 Among its advantages are its simple, cost-efficient

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cultivation and short generation time, which allow high-throughput toxicity testing,9 the

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development of a standardized test protocol,10,11 and test systems for assessing the toxicity of

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chemicals on molecular, organismal and population scales.9,12,13

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Defining and controlling the exposure concentrations of hydrophobic organic chemicals (HOCs)

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in aquatic toxicity testing is particularly challenging.14,15 The most common approach for

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introducing HOCs into toxicity tests is by preparing a concentrated stock solution of the test

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compound(s) in a water-miscible organic solvent and then adding a small volume of this solution

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to the test medium (solvent spiking). The observed toxicity is then usually linked to the nominal

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concentration (Cnom), i.e., the amount of chemical per volume of test medium. However, the

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toxicologically effective concentration is defined as the concentration of freely dissolved

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chemical (Cfree) and not Cnom.16-18 In toxicity tests with conventional solvent spiking, Cfree may be

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reduced significantly by sorption to organic matter (e.g., from food19) and the test vessel

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surfaces,20 evaporation, degradation, and even biotransformation and uptake by the test

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organisms,21 all of which can result in a low test sensitivity.22 The sum of Cfree and the sorbed

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concentration is defined as the total concentration (Ctotal) in an aqueous test medium.23,24


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In chronic toxicity tests, organic food particles, such as the Escherichia coli cells used in C.

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elegans toxicity tests, can significantly sorb HOCs and thereby affect the test sensitivity.25,26

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Appropriate standardization of food type and quantity is thus a prerequisite of chronic toxicity

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testing, especially in toxicological assessments of ingested chemicals.27 However, the role of

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food bacteria in altering apparent HOC toxicity on C. elegans is not fully understood. In the

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absence of bacteria, freely dissolved HOCs are the major contributor to chemical uptake in

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nematode tissue.28,29 Toxico-kinetic modeling of phenanthrene (PHE) uptake in C. elegans

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showed that only 9% of the total uptake flux was derived from bacterially-associated PHE,30

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suggesting that, in the presence of bacteria, the Cfree of HOCs is the main determinant of overall

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toxicity on C. elegans. However, minor contributions of food-associated HOCs to the overall

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uptake might increase the observed toxic effect. As rates of bacterial food ingestion by C.

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elegans increase with increasing bacterial density in the medium,31 the chemical uptake rate via

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food (CUfood) may be higher at higher food densities.

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In laboratory test systems, Cfree of HOCs can be controlled and maintained by passive dosing.

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This method makes use of a biocompatible reservoir with a high absorption capacity for HOCs

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that is placed directly into the test medium. During the test, continuous partitioning of

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chemical(s) from the reservoir into the test medium compensates for chemical losses and thus

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maintains constant Cfree.14,32-34 Commercially available silicone O-rings (SRs) are often used as

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reservoirs in passive dosing studies, due to their high practicality and versatility.15,35,36 To date,

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passive dosing has been used successfully to control Cfree during the toxicity testing of single

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HOCs,15,37,38 including acute toxicity testing with C. elegans,39,40 of chemical mixtures with

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defined composition,41,42 and of environmental samples.43-45


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In passive dosing studies, Cfree can be measured after the test either by analyzing the chemical

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concentration in the passive dosing polymer (Cpolymer) and then dividing this value by the

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polymer to water partition ratio (Kpolymer,w),33 or by equilibrating the dosing polymer in a small

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volume of pure water which is then analyzed.46 An alternative approach to measure the Cfree of

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HOCs is passive sampling followed by chemical analysis. In small test systems, solid-phase

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microextraction (SPME) can be applied either in situ (in situ SPME) or via the headspace (hs-

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SPME). When SPME fibers are used in situ, Cfree can be determined by solvent extraction of the

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fiber polymer followed by analysis of the extract, provided that equilibrium partitioning between

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the polymer and the test medium was obtained and sampling was non-depletive.47 Nonetheless,

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in toxicity tests in which the test medium volumes are very small, avoiding chemical depletion

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may be challenging for HOCs. However, using hs-SPME, Cfree can also be measured during the

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test under non-equilibrium conditions. Even in low-volume toxicity tests, sampling with

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negligible depletion can be achieved by limiting the analyte uptake of the SPME fiber to the

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kinetic phase of the extraction, which prevents significant effects on exposure concentrations

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during toxicity testing.48,49

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Although passive dosing has been used in many toxicity studies to control the Cfree of HOCs, its

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utility in chronic toxicity tests that include complex test media containing food has yet to be

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demonstrated. Here, we describe a simple method for the toxicity testing of HOCs applied by

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passive dosing from SRs. It allows chronic toxicity testing with C. elegans as well as

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measurements of Cfree during and after toxicity testing. CUfood is determined based on measured

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E. coli to water partition ratios and C. elegans food ingestion rates, assuming equilibrium

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between E. coli and medium. In this study, Cfree, CUfood, and the apparent toxicity of passively

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dosed polycyclic aromatic hydrocarbons (PAHs) were compared to a conventional solvent


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spiking procedure. We hypothesized that: (i) with conventional solvent spiking, Cfree would

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decrease during toxicity testing, especially due to sorption to food in the test medium, while

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passive dosing would allow the maintenance of a constant Cfree during the test. (ii) Thus, the

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PAH exposure of C. elegans would be higher with passive dosing than with solvent spiking,

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resulting in an increased apparent toxicity. (iii) The food density in the test medium would not

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affect the Cfree of PAHs with passive dosing, whereas CUfood would differ but could be adjusted

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by varying the food density. (iv) A higher CUfood with increasing food density could lead to a

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higher apparent toxicity when Cfree is simultaneously maintained by passive dosing. To test these

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hypotheses, different experiments comparing solvent spiking and passive dosing were performed

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(see Table 1 for overview). Temporal changes in Cfree of PAHs were measured during the entire

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course of C. elegans toxicity testing (Exp. 1). The toxicity of PAHs on C. elegans maximal Cfree

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was measured (Exp. 2) and concentration-response testing was performed (Exp. 3). The effects

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of different food densities on the amount of passively dosed PAHs in the water phase (nfree) and

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sorbed to food (nfood) were examined (Exp. 4). In the test media containing different food

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densities, toxicity was related to Cfree and CUfood (Exp. 5).


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2. Materials & Methods

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2.1 Chemicals

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Naphthalene (NAP), acenaphthene (ACE), fluorene (FLO), phenanthrene (PHE), anthracene

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(ANT), fluoranthene (FLA), pyrene (PYR), benzo[a]anthracene (BaA), chrysene (CHR),

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benzo[a]pyrene (BaP) (≥ 99.5% purity), and tetracycline (99% purity) were purchased from

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Sigma-Aldrich (Munich, Germany). Methanol (MeOH; 99.9% purity, Promochem, LGC

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Standards GmbH, Wesel, Germany), n-heptane (97% purity, Promochem) and acetone (99.8%

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purity, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) were used as organic solvents. PAH

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Mix 33, fluoranthene-d10, and benzo[a]pyrene-d12 were purchased from Dr. Ehrenstorfer GmbH

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(Augsburg, Germany) and served as internal standards.

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Table 1: Experiments performed in this study with respective methods and test substances

Experiment (1) Temporal changes in Cfree during C. elegans toxicity testing (2) Toxicity testing at maximal Cfree (3) Concentrationresponse testing

Exposure Quantification Method

Chemicals Mixture of PHE, ANT, FLA, PYR NAP, ACE, FLO, PHE ANT, FLA PYR, BaA CHR, BaP PHE PYR

Hypothesis tested

Solvent spiking

Passive dosing

Cfree measured by headspace SPME

Cfree measured by headspace SPME

(i)

Cfree modeled by KE.coli,w

Cfree measured by in situ SPME

(ii)

Cfree modeled by KE.coli,w

Cfree measured by (ii) in situ SPME nfree measured by in situ SPME (iii) nfood measured by LLE Cfree measured by in situ SPME (iv) CUfood measured by LLE

(4) nfree and nfood at different food densities

PHE PYR

-

(5) Toxicity at different food densities

PHE PYR

Cfree modeled by KE.coli,w CUfood modeled by KE.coli,w

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2.2 C. elegans toxicity testing

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Caenorhabditis elegans var. Bristol (strain: N2) were cultivated following standard procedures.11

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The C. elegans toxicity test was performed according to ISO 10872, with slight modifications.

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The test medium consisted of Escherichia coli (strain: OP50) suspended in M9-medium (6 g

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Na2HPO4 L-1; 3 g KH2PO4 L-1; 0.25 g MgSO4 × 7H2O L-1, 5 g NaCl L-1) adjusted to a defined E.

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coli cell density of 500 formazine absorbance units (FAU), unless stated otherwise. The test

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medium was amended with 2 mg tetracycline L-1 to inhibit bacterial growth during the

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experiments. This concentration was far below the non-observed effect concentration for C.

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elegans reproduction (NOEC = 10 mg L-1).50 Furthermore, synergistic effects with PAHs were

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expected to be negligible. Ten C. elegans juveniles (J1: first juvenile stage) were transferred to

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the PAH-spiked test medium (Exp. 1: 0.76 mL; Exp. 2-5: 1 mL) in headspace glass vials (hs-

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vials, 46 x 22.5 mm with silicone/polytetrafluoroethylene screw caps, A-Z Analytik-Zubehör

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GmbH, Langen, Germany) or 12-well multidishes (Nalgene Nunc, Rochester, NY, USA). In

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experiment 1, the volume of the test medium had to be reduced to 0.76 mL so that the test design

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was comparable to the passive dosing experiments. The test vials were incubated at 20°C in the

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dark for 96 h, after which the nematodes were heat-killed (15 min at 80°C) and then stained with

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rose bengal. Juvenile offspring of the tested nematodes were counted and the number divided by

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the number of introduced test organisms (reproduction = offspring per test organism). Inhibition

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of reproduction compared to the control was calculated according to Eq. (1):

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% I = 100 − × 100

(1)

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where % IR is the percentage of inhibition, Ri is the reproduction in replicate i, and RC is the

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reproduction in the control treatment.


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2.3 Solvent spiking

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Acetone-based stock solutions with defined concentrations were prepared, to yield the desired

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nominal concentration by adding 5 µL to the respective volume of test medium. For toxicity

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experiments in glass vials and well plates with 1 mL test medium (Exp. 2-5), this resulted in

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0.5% acetone in the test medium, which is below the no-observed-effect-concentration (NOEC =

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0.6%; data not shown). Expectedly, no significant difference was observed between water

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controls and solvent controls in any of the experiments (p > 0.05, Mann-Whitney U-test). The

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test vessels containing the spiked test medium were preincubated on an orbital shaker at 60 rpm

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in the dark for 24 h, after which C. elegans juveniles were added.

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2.4 Passive dosing

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Silicone O-rings (14.00 mm inner diameter, ID: ORS-BS015, density 1.20 g cm-³, Altec Products

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Ltd, Cornwall, UK) were used as passive dosing reservoirs after verifying that their presence had

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no effect on the reproduction of C. elegans (Figure S1). The mean mass of the SRs was 142±2

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mg (n = 20) and their silicone volume was 118±1.7 µL. The SRs were precleaned 3 × 10 min

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with MeOH in an ultrasonic bath. The SRs were loaded with single PAHs (Exp. 2-5) or mixtures

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thereof (Exp. 1), by incubating them in methanolic PAH solutions with defined PAH

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concentrations for at least 48 h at 60 rpm and a temperature of 23±2°C. PAH mixtures below

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their maximal aqueous solubility were tested by incubating the SRs in the methanolic PAH

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solution followed by the addition of water to force partitioning of the PAHs into the silicone

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(Table S1); this method resulted in loading efficiencies of up to 82% (Figure S3). Single PAHs at

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their maximal water solubility were tested by loading the SRs with saturated methanolic PAH

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solutions (Exp. 2); the presence of PAH crystals before and after the loading process was

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checked to assure saturation.14 For concentration-response testing (Exp. 3), PAH dilution series


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in MeOH with defined concentrations were prepared from saturated methanolic PAH solutions

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which were then used to load the SRs.

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The loaded SRs were rinsed 3 × 10 min with bidistilled water and then dried with lint-free tissues

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to remove residual water. Most PAHs reached equilibrium in the test medium within 24 h,

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whereas time to equilibrium increased with increasing molecular weight (Table S2). In all

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passive dosing experiments the SRs were added to test medium and preincubated in the dark on

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an orbital shaker at 60 rpm for 24 h, before the C. elegans juveniles were added. However, for

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the most hydrophobic chemicals tested (BaA, CHR and BaP), equilibrium may not have been

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achieved within the 24 h preequilibration and 96 h test duration, wherefore 1000 rpm agitation

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was alternatively tested during preequilibration for enhanced equilibration kinetics (see S4).

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2.5 Cfree

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We applied different approaches to determine Cfree in the experiments. The Cfree of four PAHs

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over time in the solvent spiked or passively dosed samples were measured using hs-SPME (Exp.

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1). Sampling was carried out directly from the gaseous phase above the test medium. The vials

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were equilibrated at 30°C for 5 min prior to extraction of the samples for 30 min using 100-µm

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PDMS fibers (Fused Silica 23Ga Red, Supelco, Sigma). Cfree was measured during the

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preincubation at 0.5, 4, 8, and 24 h and during C. elegans toxicity testing at 0.5, 4, 8, 24, 48, 72,

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and 96 h (n = 3 samples per time point). PAHs were quantified by means of an external

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calibration series with defined PAH concentrations, and analyzed as described in Section 2.7.

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In toxicity tests with passive dosing (Exp. 2-5), Cfree was measured by SPME fibers

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[polydimethylsiloxane (PDMS)-coated glass core (coating: 30–31 µm, core diameter: 114–108

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µm); Polymicro Technologies Inc., Phoenix, AZ, USA] incubated in situ during the test and then

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removed from the test medium at the end of the experiments. Thereby, non-depletive sampling


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was achieved since analyte losses during sampling were compensated by the SR (see Table S2).

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The SPME fibers were cut to a length of 1 cm, yielding a PDMS volume of 0.136 µL cm-1.52 The

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fibers were precleaned 3 × 10 min with MeOH in an ultrasonic bath and rinsed 3 × 10 min with

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bidistilled water before their transfer to the test medium. For sampling, fibers incubated in the

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test medium were removed, dried using lint-free tissues, and extracted in 200 µL n-heptane for at

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least 24 h. Cfree (µg L-1) was calculated by dividing the concentration in the in situ SPME fiber

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(Cfiber, µg L-1) by the analyte-specific polymer to water partition ratios of the fibers (Kfiber,w, L L-

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1

201

), as described by Eq. (2). !" #

%$Measured C (K , ) = &

(2)

" #$%,'

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Kfiber,w was calculated by multiplying the polymer to AlteSilTM silicone (Altec Products LTD)

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partition ratios (Kfiber,AlteSil, L L-1)53 by the AlteSilTM silicone to water partition ratios (L L-1,

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KAlteSil,w),54 as shown in Eq. (3).

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K , = K ()*+), × K ,()*+)

(3)

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Since non-depletive in situ SPME was not possible when applying solvent spiking, Cfree in the

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solvent-spiked samples was modeled by multiplying Cnom by the free fraction of PAHs in the test

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medium (Exp. 2-5), which was calculated based on E. coli to water partition ratios (KE.coli,w, L

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kg-1), the mass of E. coli bacteria (mE.

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(Vmedium, L), as described in Eq. (4).

211 212 213

coli,

kg dry weight), and the volume of test medium

Modeled C .K /.1234, 5 = 6

7

9 78 :.; × &:.;,' ?9$@ A9

B × C CD

(4)

Log KE.coli,w were calculated as shown in Eq. (5), according to Baughman & Paris:51 log K /.1234, = 0.907 × log K C − 0.361

(5)


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2.6 nfood and CUfood

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In the solvent spiked samples of experiments 2-5, nfood (µg) was modeled using the modeled Cfree

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(µg L-1) from Eq. (4).

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Modeled nCCK .K /.1234, 5 = Modeled C × K /.1234, × m/.1234

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We further determined KE.coli,w for all PAHs on the basis of the measured Cfree and Ctotal in

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passively dosed samples [Eqs. (S4) and (S5)] and compared these with the values obtained from

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Baughman & Paris.50 Determined KE.coli,w were constant for the tested E. coli cell densities

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(Figure S4), and in good agreement with those calculated based on Baughman & Paris,50

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confirming that KE.coli,w can be used to calculate nfood at varying food densities in chronic C.

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elegans toxicity tests. In passively dosed samples, nfood (µg) was thereof quantified by

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multiplying determined KE.coli,w (L kg-1) with measured Cfree (µg L-1) and mE.coli (kg).

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Measured nCCK .K /.1234, 5 = Measured C .MN4OPQ,R 5 × K /.1234, × m/.1234

(6)

(7)

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To compare exposure at different E. coli cell densities in both dosing approaches (Exp. 5), CUfood

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(µg kgC.elegans-1 h-1) was calculated by means of the food ingestion rate of C. elegans at a specific

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cell density (IRa, kgE.coli kgC.elegans-1 h-1),30 while using either modeled nfood in solvent spiking or

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measured nfood in passive dosing samples. U

Modeled/Measured CUCCK = D "VV@ × IRa

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:.;

(8)

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2.7 Chemical analysis

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The PAH concentrations in extracts derived from in situ SPME and LLE were determined by gas

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chromatography-tandem mass spectrometry (GC-MS/MS; 7000 Triple Quadrupole-GC-MS/MS,

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Agilent) with a HP-5MS column (30 m × 250 µm × 0.25 µm, 5% phenyl methyl siloxane,

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Agilent). For hs-SPME measurements, extraction and analysis were performed using an auto-

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sampler equipped with SPME (CTC-Analytics, Combi Pal PAL-System) and by GC-MS (Trace


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GC Ultra and ITQ 900 MS, Thermo Scientific, Waltham, MA, USA) equipped with a

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TraceGOLD TG-XLBMS column (60 m × 250 µm × 0.25 µm, Thermo Scientific). PAHs were

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quantified by external standard calibration. Calibration samples for in situ SPME and LLE

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extracts were prepared at a concentration range of 1-2000 µg L-1 in n-heptane. Those for hs-

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SPME analysis were prepared at a concentration range of 0.1-25 µg L-1 in M9 test medium to

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ensure the comparability of the PAH distribution in the aqueous and gaseous phases.


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3. Results & Discussion

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3.1 Temporal changes in Cfree

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The results of experiment 1 confirmed that Cfree decreased significantly over time with solvent

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spiking, but remained constant during passive dosing after equilibrium partitioning between the

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SR and test medium was achieved [hypothesis (i), Figure 1]. Cfree of each of the four PAHs was

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reduced by 24-53% as early as 30 min after their solvent spiking. Since HOCs sorb to organic

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matter,19,55-57 including bacteria cells,58,59 this reduction probably resulted from sorption to E.

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coli cells. Cfree then remained constant until C. elegans juveniles were added, suggesting that

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equilibrium partitioning had already been attained within 30 min. This finding agrees with those

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of Lunsman and Lick.,59 who reported that the sorption of HOCs to bacteria was in equilibrium

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within a few minutes. As expected, the reduction in Cfree differed between PAHs and generally

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increased with their increasing sorption tendency to E. coli, with analyte losses of 24% for PHE

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(log KE.coli,w: 3.87), 26% for ANT (log KE.coli,w: 3.92), 42% for FLA (log KE.coli,w: 4.40) and 53%

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for PYR (log KE.coli,w: 4.40).


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Figure 1: Cfree during E. coli pre-incubation (0–24 h) and subsequent toxicity testing with C.

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elegans (24–120 h). PAHs were applied by (A) solvent spiking or (B) passive dosing. The blue

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line indicates the Cnom of the four PAHs (25 µg L-1) dosed by solvent spiking at time 0. For

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passive dosing, SRs were loaded with a 4.5 mg L-1 concentrated MeOH solution, yielding

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nominal concentrations between 3.9 (PYR) and 16.3 µg L-1 (PHE), and added to the test medium

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at time 0.

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After the addition of C. elegans J1 juveniles to the test medium (24 h after PAH addition), the

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Cfree of all four PAHs decreased progressively, resulting in analyte losses of up to 89% for PYR

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by the end of the toxicity test (120 h, Figure 1A). A mass balance calculation showed that the

267

sorption of PAHs to growing nematodes did not sufficiently explain the decrease in Cfree, since

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the nematodes accounted for a maximum of 13% of the total lipid biomass in the test medium

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(see Table S3). Metabolic degradation by C. elegans might have contributed to the reduction in

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the Cfree,59 whereas metabolic degradation by E. coli was unlikely, since after the immediate

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decrease due to sorption the Cfree was not further reduced during the preincubation period.

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However, a fungus-like contamination can occasionally occur when performing chronic C.

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elegans toxicity testing. Such an additional biomass might have contributed to the sorption of

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PAHs and thus the continual decrease in the Cfree. Analyte losses due to processes such as


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evaporation, sorption to the test vial plastic walls, and photolysis can be considered as negligible

276

since experiments were performed in closed glass vials in the dark.

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Figure 1B shows that, with passive dosing, equilibrium partitioning of all four PAHs was

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achieved within 24 h such that the Cfree remained constant until the end of the toxicity test (120

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h). This finding is in agreement with earlier studies in which HOCs were passively dosed in

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miniaturized aqueous toxicity tests with Daphnia magna,15 zebrafish embryos,35 and even C.

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elegans.39,40 SRs have served as suitable dosing reservoirs in previous studies,42,61 including

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toxicity testing of single PAHs and mixtures thereof.62,63 However, those were acute toxicity

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tests performed without a food source in the test medium. Our results demonstrate that passive

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dosing using PAH-loaded SRs is also a suitable method to establish constant Cfree of PAHs in

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standardized chronic toxicity tests with C. elegans, in which the nematodes are incubated in a

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complex food medium that acts as a strong sorptive sink for HOCs. This is promising regarding

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the application of passive dosing in other chronic toxicity tests.

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3.2 Influence of the dosing method on the apparent toxicity of PAHs

289

Table 2 shows the half-maximal effect concentrations (EC50) after concentration-response testing

290

of PHE and PYR (Exp. 3). The reproductive toxicity on C. elegans was lower for passively

291

dosed PAHs, whereas solvent spiking of PHE and PYR in the same test vials resulted in EC50

292

values that were 2.6- and 3.8-fold higher, respectively (for the concentration-response curves see

293

Figure S8). These differences probably resulted from the uncertainty associated with using the

294

modeled Cfree as exposure metric, according to Eq. (4), because, as shown here, Cfree actually

295

decreases progressively after C. elegans juveniles are added to the test medium (Figure 1A). In

296

the plastic well plates, the EC50 values were 33- and 12-fold higher than determined in the

297

passive dosing toxicity tests, probably as a result of evaporation (see S9 for results and further


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298

discussion). Although earlier studies showed that passive dosing of HOCs can be performed in

299

well plates,33,40 it is not yet clear whether passive dosing via SRs compensates for the large and

300

rapid evaporative losses in toxicity tests of volatile chemicals in plastic wells containing C.

301

elegans.

302

PAHs mainly act as baseline toxicants, i.e., they impair the integrity and functioning of

303

biological membranes.62 Since the membrane accumulation of PAHs is driven by passive

304

diffusion from the test medium, a higher Cfree of PAHs results in a higher apparent toxicity,46 as

305

observed in earlier studies of acute toxicity using invertebrate species.15,41 In our study, this

306

correlation was illustrated by the activity of the chemicals in the test medium at maximal Cfree

307

(Exp. 2, Figure S7), in accordance with earlier investigations in which the baseline acute toxicity

308

of neutral HOCs was in the 0.01-0.1 range.64 Similar to studies with daphnids, algae, and C.

309

elegans,65,66 our results showed that, when applied by passive dosing, BaA and ANT exhibit

310

chronic toxicity to C. elegans at chemicals activities 0.05), whereas with increasing E. coli cell density nfood increased significantly

319

(Figure 2B, one-way ANOVA: p < 0.05, posthoc Tukey: p < 0.05). The results demonstrate that

320

even for chemicals with high sorption tendency to E. coli, such as PYR (log KE.coli,w: 4.40),

321

passive dosing was able to maintain a constant nfree over a large range of E. coli cell densities

322

[hypothesis (iii)].

323 324

Figure 2: The masses of four PAHs in (A) the water phase (nfree, ng, mean ± standard deviation, n

325

= 3) and (B) sorbed to food (nfood, ng, mean ± standard deviation, n = 3) at different E. coli cell

326

densities (FAU 0, 125, 500 and 2000). The PAH-loaded SR was incubated in the test medium for

327

72 h. Different letters indicate significant differences between treatment groups with different

328

bacterial densities (one-way ANOVA: p < 0.05, posthoc Tukey: p < 0.05).

329

Figure 3 relates the apparent toxicity of PHE to both Cfree and CUfood (Exp. 5). When applying

330

solvent spiking, the apparent toxicity decreased significantly with increasing food density

331

(Figure 3A, one-way ANOVA, p < 0.01, posthoc Tukey: p < 0.01). This likely resulted from the


19


Page 20 of 28

332

decreased chemical uptake via the water phase, since Cfree decreased considerably from 118.1

333

(FAU 125) to 28.5 µg L-1 (FAU 2000). Cfood likewise decreased from 691 (FAU 125) to 167 mg

334

kgE.coli-1 (FAU 2000) as a result of the higher E. coli biomass in the test medium. However,

335

CUfood remained relatively constant due to the increased food ingestion rate of C. elegans at

336

higher food densities (Figure S10). Similar results were observed for PYR (Figure S9). However,

337

these modeled exposure concentrations assume that the chemicals distributed solely between the

338

water phase and the E. coli. The progressive losses that in fact occurred during solvent spiking

339

might have altered Cfree and Cfood even further, thereby impeding the differentiation between

340

uptake routes in chronic toxicity studies.

341

In contrast to solvent spiking, the apparent toxicity of passively dosed PHE increased with

342

increasing bacterial density, with significant differences between FAU 2000 and 500 as well as

343

125 and 500 (Figures 3B and S9, one-way ANOVA: p < 0.01, posthoc Tukey: p < 0.05). These

344

findings could be attributed to the increased CUfood, since the Cfree of the PAHs remained

345

constant over all E. coli cell densities. C. elegans reproduction thereby did not differ

346

significantly in control groups (one-way ANOVA, p = 0.63, Figure S2) and, therefore, it seems

347

unlikely that % IR was affected by the tested food densities, which is a prerequisite when testing

348

toxicity at different food densities. Our results imply that the bacterially sorbed fraction is an

349

important contributor to the overall toxic effect on C. elegans and are in contrast to the

350

conclusion reached by Spann et al.,30 that only 9% of the total uptake flux of C. elegans is

351

induced by bacterially-associated PHE. However, in the experimental set-up of Spann et al.,30

352

both Cfree and CUfood, were consistently altered by varying the bacterial density, since solvent

353

spiking was used; thus, toxicity could not have been unequivocally assigned to a specific

354

chemical fraction (dissolved or dietary). In this study, however, constant Cfree of the PAHs were


20

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355

maintained while simultaneously increasing CUfood with increasing bacterial densities. It may be

356

that, with increasing ingestion, nematodes form more lipid reserves, which would result in higher

357

internal chemical concentrations in the organism—due to the high sorption capacity of lipids for

358

HOCs—and thereof a greater apparent toxicity of PAHs at higher food densities.67 Further

359

studies are needed to investigate the contribution of food-bound chemicals to the overall toxic

360

effect of HOCs on chronic endpoints, both in C. elegans and other invertebrate species.

361 362

Figure 3: Inhibition of C. elegans reproduction (% IR mean ± standard deviation, n = 3) after 96-

363

h exposures to PHE at different E. coli cell densities (FAU 125, 500 and 2000). PHE was

364

provided by (A) solvent spiking or (B) passive dosing. Note, that the apparent toxicity was

365

driven by Cfree with solvent spiking, whereas altered by CUfood while maintaining constant Cfree

366

with passive dosing. The data points were fitted using a logistic model for Cfree (solvent spiking;

367

R2 = 0.85) and CUfood (passive dosing; R2 = 0.99).


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368

3.4 Implementation in toxicity testing of chemicals

369

Our study showed that passive dosing via SRs can eliminate the uncertainties of reduced Cfree

370

caused by sorption of HOCs to food in chronic toxicity tests. Different SPME formats can

371

thereby be used to measure and confirm exposure concentrations. The developed methods can

372

thus improve the comparability and repeatability of the toxicity test, resulting in greater test

373

sensitivity, more reliable toxicity testing, and better comparability of the toxicity data obtained in

374

routine risk assessments of HOCs. A 24-h preincubation of the PAH-loaded SR in the test

375

medium can be easily implemented in standardized nematode toxicity testing. However, higher

376

agitation intensities during preequilibration might be needed prior toxicity testing to accelerate

377

equilibrium partitioning when either very hydrophobic chemicals or high food densities are used.

378

Passive dosing can furthermore be used to control both Cfree and CUfood by loading the passive

379

dosing reservoir with a defined concentration while adjusting the food density in the test

380

medium. Using this approach, we showed that CUfood contributes significantly to the overall

381

reproductive toxicity of PAHs on C. elegans. In future studies, passive dosing can be employed

382

to investigate the role of dietary chemical uptake in the toxicodynamic and toxicokinetic

383

processes of HOCs. With this approach, the differences in measured toxicity can be assigned to a

384

specific uptake route.

385

Corresponding author

386

* Address: Helmholtz Centre for Environmental Research - UFZ, Department Cell Toxicology,

387

Permoserstraße 15, 04318 Leipzig, Germany; Phone: +49 341 235 – 1512; FAX: +49 341 235 –

388

1787; E-Mail address: [email protected]


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389

Acknowledgements

390

This study was financially supported by the German Federal Ministry for the Environment,

391

Nature Conservation, Building and Nuclear Safety. We thank Philipp Mayer for kindly providing

392

the in situ SPME fibers and Patrick Zurek for preliminary experiments. We gratefully

393

acknowledge Julia Bachtin and Marina Ohlig for technical assistance and Benjamin Becker for

394

helpful discussions. We are grateful to Beate Escher for a critical review of and helpful

395

discussion on the manuscript. We thank three anonymous reviewers for helpful comments on the

396

manuscript.

397

Supporting Information Available

398

Information on the methods and results of preliminary experiments performed within method

399

development. Additional details on the experimental results as well as literature data and

400

methods. This information is available free of charge via the Internet at http://pubs.acs.org.

401

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Passive Dosing in Chronic Toxicity Tests with the Nematode

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