PAH toxicity at aqueous solubility in the fish embryo test with Danio rerio using passive dosing

Highlights

• PAH toxicity at aqueous solubility using passive dosing in fish embryo test.

• PDMS silicone elastomer cast in 10-mL vials and 60-mL jars.

• HPLC analysis confirmed constant exposure at the saturation level.

• Feasibility of PDMS silicone oil as passive dosing phase investigated.

• Close relationship between maximum chemical activity and toxicity indicated narcosis.

Abstract

As part of the risk assessment process within REACh, prior to manufacturing and distribution of chemical substances their (eco)toxicological impacts have to be investigated. The fish embryo toxicity test (FET) with the zebrafish Danio rerio has gained a high significance as an in vitro alternative to animal testing in (eco)toxicology. However, for hydrophobic organic chemicals it remains a technical challenge to ensure constant freely dissolved concentration at the maximum exposure level during such biotests.

Passive dosing with PDMS silicone was thus applied to control the freely dissolved concentration of ten PAHs at their saturation level in the FET. The experiments gave repeatable results, with the toxicity of the PAHs generally increasing with the maximum chemical activities of the PAHs. HPLC analysis confirmed constant exposure at the saturation level. In additional experiments, fish embryos without direct contact to the silicone surface showed similar mortalities as those exposed with direct contact to the silicone. Silicone oil overlaying the water phase as a novel passive dosing phase had no observable effects on the development of the fish embryos until hatching.

This study provides further data to support the close relationship between the chemical activity and the toxicity of hydrophobic organic compounds. Passive dosing from PDMS silicone enabled reliable toxicity testing of (highly) hydrophobic substances at aqueous solubility, providing a practical way to control toxicity exactly at the maximum exposure level. This approach is therefore expected to be useful as a cost-effective initial screening of hydrophobic chemicals for potential adverse effects to freshwater vertebrates.

Introduction

Since the 1930s, the worldwide production of chemicals has increased from 1 million to around 300 million tons per year (Schwarzenbach et al., 2006). By 2009, 50 million different chemical compounds were known (CAS, 2009), and these numbers are still increasing. The new EU Chemical Legislation (REACh), which came into force in June 2007, requires that “substances on their own, in preparations or in articles shall not be manufactured in the Community or placed on the market unless they have been registered in accordance with the relevant provisions” of REACh, based on a pre-defined set of data and other information (European Union, 2006). As part of this process, the (eco)toxicological impacts of such compounds have to be investigated using biotest batteries. Since the idea to reduce, refine and replace animal testing by alternative test methods (Russel and Burch, 1959) is an inherent part of REACH, in vitro systems play an increasing role in such test batteries.

One particular biotest that has gained a high significance in recent years is the fish embryo toxicity test (FET) with the zebrafish Danio rerio (Schulte and Nagel, 1994, Nagel, 2002, Braunbeck et al., 2005, Lammer et al., 2009a). D. rerio represents a secondary consumer vertebrate in the limnic ecosystem, making it highly relevant for environmental risk assessment. In a comprehensive analysis of existing fathead minnow (Pimephales promelas) data, McKim (1977) concluded that for the majority of the investigated compounds exposure of developing embryos rather than adult fish resulted in a higher susceptibility to the adverse effects of chemical substances (see also Birge et al., 1979, Nagel and Isberner, 1998, Strmac et al., 2002, Kosmehl et al., 2007). He proposed to use early life stages of fish to establish water-quality criteria and to screen large numbers of chemicals. The fish embryos can also be inspected for sublethal effects, further enhancing the value of the resulting data. Conducting the test in microtiter test plates allows for a higher sample throughput, and dramatically reduces the resources required as compared to the acute fish test. Consequently, the fish embryo assay has already replaced the acute fish test in German legislation on whole effluent testing (DIN, 2001, German Federal Ministry of Justice, 2005), and is the most recent addition to the OECD guidelines for the testing of chemicals regarding effects on biotic systems (OECD, 2013).

However, when spiking with co-solvent, the results of in vitro tests of hydrophobic organic compounds (HOCs) are often biased by poorly defined and declining dissolved exposure concentrations (Eisentraeger et al., 2008, Schreiber et al., 2008, Hinger et al., 2011). This can be due to (1) volatilization of the test substance, (2) sorption to the medium constituents, (3) sorption to plastic material of the microtiter plates or (4) uptake and metabolisation by the test organisms (Schreiber et al., 2008, Smith et al., 2010b). To overcome this, exposure concentrations need to be continuously maintained throughout the test duration. For static systems, one possibility is to regularly re-supply the test substance through renewal of the spiked medium (so-called “static renewal” exposure systems). However, this approach does not completely eliminate uncertainties regarding the actual exposure concentration because of sorption artefacts or fluctuations during the test. Flow-through systems, on the other hand, are not available for all bioassays and are often comparably expensive with regards to the equipment, work involved and the amounts of chemicals needed and afterwards disposed. Most importantly, such systems are particularly difficult to apply at small scales such as in microtiter plates. This holds true for both cell-based assays and small organism assays (Lammer et al., 2009b).

Passive dosing is one promising approach for attaining stable and well-defined exposure concentrations during in vitro biotests (Kramer et al., 2010, Smith et al., 2010b). The freely dissolved concentration of the test substance is controlled by partitioning from a reservoir loaded in a biocompatible polymer. Continual partitioning from the polymer phase compensates for any losses due to volatilization, degradation or sorption (Mayer et al., 1999, Smith et al., 2010a, Turcotte et al., 2011). In this way even biotests conducted in plastic microtiter plates can be reliably used to study the toxicity of HOCs (Kiparissis et al., 2003, Kramer et al., 2010, Smith et al., 2010b).

The fish embryo test with D. rerio has already been conducted with exposure control via passive dosing using silicone coated vials with phenanthrene as a model substance (Butler et al., 2013). For the present study, cast PDMS silicone elastomer saturated with ten PAHs was applied in the fish embryo toxicity test with D. rerio. PAHs are common model substances for the development of PDMS passive dosing approaches (Birch et al., 2010, Smith et al., 2010a, Smith et al., 2010b, Engraff et al., 2011). Since the main aim was to investigating the applicability of passive dosing of PAHs using cast PDMS in the FET, the tests were conducted in various glass vessels. This was decided to avoid confounding effects due to significant substance losses via sorption to the plastic of microtiter plates, and to investigate whether different sizes of test vessels are applicable. Also, one type of vessel was made of brown glass to take into account photodegradation of PAH. In some tests, the embryos were exposed in parallel on stainless-steel meshes, preventing direct contact with the silicone at the bottom of the test vessels. Specifically, the study aimed: (1) to investigate the practicability of passive dosing using PDMS silicone in the fish embryo test with D. rerio in two test vessel formats, (2) to determine the toxicities of 10 PAHs as model HOCs at aqueous solubility, i.e., their maximum exposure, (3) to relate PAH toxicity at aqueous solubility to their properties and maximum chemical activities, (4) to investigate if direct contact of the exposed embryos to the PDMS silicone has an influence on measured toxicity and (5) to elucidate whether silicone oil can serve as an alternative passive dosing phase.