Elsevier

Aquatic Toxicology

Volume 157, December 2014, Pages 134-140
Aquatic Toxicology

Influence of the perivitelline space on the quantification of internal concentrations of chemicals in eggs of zebrafish embryos (Danio rerio)

https://doi.org/10.1016/j.aquatox.2014.10.008Get rights and content

Highlights

  • Internal concentrations of polar compounds are overestimated in unhatched zebrafish embryos.

  • Dechorionation before sample preparation is proposed for the quantification of true internal concentrations.

  • Short time exposure experiments provide a reasonable estimate of the substance concentration in the perivitelline space.

  • For the polar substances tested, the chorion of the zebrafish embryo does not represent a transport barrier.

Abstract

The chorion and the perivitelline space which surround unhatched zebrafish embryos (ZFE, Danio rerio) may affect the determination of internal concentrations of study compounds taken up in early life-stages of ZFE. Internal concentration-time profiles were gathered for benzocaine, caffeine, clofibric acid, metribuzin and phenacetin as study compounds over 96 h of exposure starting with ZFE at 4 h post-fertilization. Liquid chromatography coupled to tandem-mass spectrometry (LC–MS/MS) was used to determine the concentration of the study compounds from intact (i.e. unhatched), dechorionated and from hatched ZFE. The mass of the study compounds per ZFE was 5–30 ng higher for intact ZFE compared to dechorionated ones. Thus, internal concentrations were overestimated if only intact ZFE were analyzed. Dechorionation of unhatched ZFE after their exposure is proposed to determine the true internal concentration in the embryo. For the compounds studied here the mass of the study compounds determined in unhatched ZFE after a short term (5 min) exposure provided a reasonable estimate of the mass taken up by the chorion and the PVS. This mass can be subtracted from the total mass found in unhatched ZFE to calculate the true internal mass. Estimating the mass in the chorion and the PVS from the concentration of the study compound in the external exposure medium and the volume of the PVS provided no reasonable results.

Introduction

Since the fish embryo toxicity test (FET) has become a potential alternative to acute fish toxicity testing (Lammer et al., 2009, OECD, 2014), the zebrafish embryo (ZFE) is gaining popularity in hazard assessment. In high throughput studies effects of different chemicals on the test organism are investigated in order to collect toxicity data concerning lethal or effect concentrations (LC50 or EC50 values) (Ali et al., 2012, Carlsson et al., 2013, Padilla et al., 2012). The observed effects are often compared to external concentrations but for a better understanding of toxic effects the internal concentrations should be known (Escher and Hermens, 2004). We have recently published an analytical method to determine internal concentrations in ZFE (Brox et al., 2014).

In routine toxicity tests ZFE are usually exposed to chemicals in very early life-stages. But exposure experiments for the analytical investigation of internal concentrations quite often start with ZFE of an older age of 72 h post-fertilization (hpf) (El-Amrani et al., 2012, El-Amrani et al., 2013, Gonzalo-Lumbreras et al., 2012, Jones et al., 2012). If experiments are performed at early life-stages of the embryo the first samples are often taken after 72 hpf (Bluthgen et al., 2012, Van den Bulck et al., 2011). However, it would be worthwhile to cover also very early life-stages of ZFE (<72 hpf) to learn about the uptake and metabolism of different study compounds in these early stages of development, in which the ZFE did not hatch. A toxicokinetic study requires a frequent sampling that covers these life-stages. The constitution of the ZFE is an important aspect that has to be considered in this context.

In its early life-stages the unhatched ZFE is surrounded by the so called chorion and the aqueous perivitelline space (PVS) between the chorion and the ZFE. The chorion is a permeable membrane of 1.5–10 μm thickness with circular pores of a diameter of 0.5–1.5 μm with a centre-to-centre distance of 1.5–3 μm (Bonsignorio et al., 1996, Cheng et al., 2007, Laale, 1977, Lee et al., 2005, Rawson et al., 2000). The chorion is assumed to protect the embryo in early life-stages. Penetration of water and electrolytes from the surrounding exposure medium via the chorion into the PVS is possible through the pores (Laale, 1977). The macromolecular structure of the chorion consists of partly N-linked glycoproteins (Bonsignorio et al., 1996, Lee et al., 2005). Between 48 and 72 hpf the embryo hatches.

The hardness of the chorion increases within the first hour of development and it was assumed that this turns the chorion into a barrier against the uptake of pollutants (Gellert and Heinrichsdorff, 2001). Therefore standard fish embryo test guidelines require to expose ZFE very early in their development before the hardening of the chorion (OECD, 2014). In the last decade this barrier function has been questioned. It may only be effective for large molecules of a size >3 kDa (Creton, 2004). Chemicals of low molecular weight such as atrazine have been shown to pass through the chorion into the perivitelline space within a few seconds (Wiegand et al., 2000). In general the barrier function seems to increase for more lipophilic compounds (Braunbeck et al., 2005). Additionally commonly used solvents may change the permeability of the chorion (Kais et al., 2013). Until now only a limited number of studies investigate the uptake of chemicals in early life-stages of the ZFE in combination with (Brox et al., 2014, Stanley et al., 2009) or without (Kühnert et al., 2013, Petersen and Kristensen, 1998) dechorionation. The dechorionation of early life-stages of the ZFE is proposed for exposure experiments starting at 24 hpf (Henn and Braunbeck, 2011). But any test start before 24 hpf (which is a standard procedure in risk assessment with ZFE) includes the chorion and the PVS. If these two have an influence on the determined analyte concentration of the whole ZFE, results would be under- or overestimated.

To fill this gap of knowledge and diminish the uncertainty, the influence of the chorion and the PVS on the determination and interpretation of internal concentration-time profiles in ZFE is investigated starting at early life-stages at 4 hpf. Compounds with a high to moderate polarity (log KOW −0.04 to 2.58) and low molecular weight (165–215 Da) were used since these compounds have been less studied and effects on the ZFE can be observed (Ali et al., 2012, Padilla et al., 2012).

Section snippets

Chemicals and reagents

Benzocaine (CAS RN 94-09-7), caffeine (CAS RN 58-08-2), metribuzin (CAS RN 21087-64-9) and phenacetin (CAS RN 62-44-2) were purchased from Sigma Aldrich (Munich, Germany). Clofibric acid (CAS RN 882-09-7) was obtained from ICN Biomedicals (Eschwege, Germany). Calcium chloride dehydrate (CAS RN 10035-04-8), magnesium sulfate heptahydrate (CAS RN 10034-99-8), sodium hydrogen carbonate (CAS RN 144-55-8) and potassium chloride (CAS RN 7447-40-7) for the preparation of the standard embryo water

Results and discussion

Considering the different compartments of the ZFE (Fig. 1) it is assumed that the total mass (mtot) of a study compound determined from an unhatched ZFE is the sum of the mass of four compartments: the mass in the PVS (mpvs), the mass adsorbed to the chorion (mc), the mass adsorbed to or partitioned into the ZFE membrane (mm) and the ‘true’ internal mass (mZFE) (Eq. (1)):mtot=mpvs+mc+mZFE+mmmtot=(Vpvs×cpvs)+mc+(VZFE×cZFE)+mm

With Vpvs, volume of the PVS; cpvs, concentration in the PVS; VZFE,

Conclusion

The presented data highlight the need to differentiate between total mass and internal mass of a study compound in exposure experiments with unhatched ZFE. If this is not done, the internal concentration in unhatched ZFE can be severely overestimated and temporal trends become biased, at least for polar analytes of the types used here.

Of the three approaches tested in this study to account for the contribution of the chorion and the PVS the most robust but also the most laborious one is the

Acknowledgements

This work is part of the research topic “Chemicals in the Environment – Chemicals and Active Transport” (CITE-CAT) within the research programme of the Helmholtz Centre for Environmental Research-UFZ. We thank C. Petzold for her support in laboratory work as well as the colleagues involved in the CAT project for helpful discussions.

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