3.1. Effect of Chorion Status on Bioactivity
In teleosts, the chorion is an acellular, semipermeable membrane surrounding a developing embryo. It provides some degree of mechanical protection from environmental disturbances and facilitates gas and ion exchange. Guidelines within the OECD FET test address the potential for the chorion to serve as a chemical barrier, though it does not make recommendations for mechanical or enzymatic removal of the chorion [19
]. For biomedical discovery research, our group has promoted the removal of the chorion to facilitate high-throughput screening for two main reasons. First, we were concerned that the chorion may present a barrier for chemical uptake. We reasoned that if some chemicals or nanomaterials are not able to reach the developing embryo for the first 48 h encompassing the sensitive period of primary organogenesis, screening may result in an unacceptable false negative rate [8
]. In zebrafish, the chorion surrounds the embryo for the first 48 hpf [31
] and may also serve as a barrier to some chemicals, though its permeability dynamics are not straightforward or well-understood. Recent studies have indicated increased sensitivity to chemicals with the removal of the chorion, including some organophosphate flame retardants and nanomaterials [32
Our second motivation for advocating the removal of the chorion is to facilitate the measurements of endpoints and to reduce the potential confounding effects of chemical-mediated hatching failure. Non-invasive microscopic imaging of embryonic structures is simply easier when the developing embryo is not encased in a round rolling configuration, as dechorionated embryos generally lay flat and are easily manipulated for consistent orientation for imaging. Additionally, since the EPR assay captures contralateral tail bends, the presence of the chorion may reduce the measurable activity, resulting in a decrease in sensitivity [27
]. It is well established by ecotoxicological studies that some chemical exposures affect hatching, and the lack of hatching produces secondary adverse effects that include edemas, body axis curvature, and skeletal deformities. In this study, relative to the Tanguay laboratory’s standard (dechorionated) condition, leaving the chorion intact until natural hatchout increased the incidence of abnormal morphology associated with abamectin, chlorpyrifos, and permethrin exposures, and decreased the incidence of abnormal morphology associated with retene (Figure 2
). For each chemical that showed altered responses with an intact chorion, the specific endpoints affected did not change, though the concentration required to elicit a significant response was altered. The exception is mortality, which was significantly reduced for retene and MWCNTs at both time points with intact chorions compared to dechorionated embryos. Figure 3
shows concentration–response curves for select chemical–condition combinations that displayed the range of responses (see Figure S1
for concentration–response for all combinations).
An intact chorion influenced early life stage behavior responses at both 24 and 120 hpf. At 24 hpf, the presence of the chorion eliminated E-phase hypoactivity for estradiol, pyrene, and retene and induced hypoactivity for MWCNTs in the E phase (EPR). The observed lack of response with the chorion on further supports the recommendation of conducting bioactivity studies with dechorionated embryos. At 120 hpf, chlorpyrifos and estradiol induced hypoactivity in the D phase, while pyrene induced hyperactivity in the L phase and permethrin induced hyperactivity in both the D and L phases (LPR) with chorionated embryos (Figure 4
). Figures S2 and S3
display EPR and LPR responses, respectively, for all test agent–condition combinations.
While an EC50
could not be calculated for the MWCNT treatment under any condition, when compared to the standard exposure regime, the presence of the chorion during exposure completely eliminated incidences of abnormal morphology as displayed in Figure 3
. Figure S1
displays all endpoints for MWCNTs exposure under each altered condition, showing significant mortality at both 24 and 120 hpf in the standard condition and no significant effects with an intact chorion. The chorion contains pores that allow for water, gas, and ion exchange. By the gastrula stage (5.25–10 hpf [9
]) when exposures were initiated in this study, the pores have diameters 0.5–0.7 µM [35
], much larger than the chemicals tested, though small enough to prohibit diffusion of large polymers or agglomerated nanomaterials [36
]. This may explain the decreased bioactivity of multi-walled carbon nanotubes toward chorion-intact embryos.
This study illustrates that chorion status can significantly affect a chemical or nanomaterial’s bioactivity, and that, for traditional small molecules, the effect is more complex than simple size exclusion. The exact mechanism is unknown and likely different for each substance, as indicated by increased bioactivity for some chemicals and decreased bioactivity for others with intact chorions.
Some evidence suggests the chorion pores increase in size throughout development, potentially altering chemical uptake at critical developmental periods [38
]. Others suggest that the plasma membrane and syncytial layers between the developing embryo and the chorion may play a role in reducing the uptake of chemical agents into embryo tissue [39
]. Many chemicals have also been shown to affect hatching ability, including 2,3,7,8-tetrachlorodibenzo-p
-dioxin (TCDD), benzene [40
], trichloroethylene [41
], and diethylnitrosamine [42
], potentially by weakening or paralyzing the fish, or by prohibiting the production of choriolytic enzymes naturally produced to degrade the chorion. This delay or inhibition of hatching could explain malformations later in development for some compounds. In this study, abamectin altered hatching, with 79.4% of viable larvae still in their chorions at 120 hpf at 1 µM (Figure 5
). The hatching rate was measured at 120 hpf by visual observation of live larvae remaining inside an intact chorion. No other test agents induced hatching failure at 120 hpf. Multiple mechanisms of chemical protection by the chorion are likely operant, as might be expected from an evolutionarily costly but successful adaptation. As this study shows, its presence can affect the calculated concentration response profile.
3.2. Effect of Light Status on Bioactivity
Some laboratories have expressed concern about the need to conduct chemical exposures under a light:dark (LD) cycle [21
] to maintain circadian rhythms during embryonic development. The circadian clock in zebrafish is linked to cell cycle regulation, locomotor activity, and xenobiotic metabolism [43
]. In zebrafish, the retina, pineal gland, and many peripheral tissues maintain rhythmic cycles of gene expression and cell proliferation concordant with the LD cycle of their environment. In constant darkness, these tissues maintain their cyclical pattern independently of one another but require light stimulus to synchronize the expression oscillations of these tissues to the environmental light cycle [47
]. While light exposure is necessary to synchronize circadian rhythms in zebrafish, a constant LD cycle may not be required to maintain them. Carr and Whitmore demonstrated that a single light pulse can trigger the synchronization of cellular clocks for several days [49
In this study, chemical exposures under a 14 h light: 10 h dark cycle potentiated the incidence of morphological effects associated with abamectin and chlorpyrifos and inhibited the incidence of morphological effects associated with permethrin. The profiles of specific morphological effects for each chemical were not greatly altered under a LD cycle as compared to the standard (dark) exposure condition, though the concentration eliciting a response was altered for some endpoints under this treatment. See Figure S1
for concentration–response data for each individual endpoint. Larval photomotor response was altered under this condition for chlorpyrifos by eliminating hyperactivity in the D phase, for pyrene by eliminating hypoactivity in the D phase, and for MWCNTs by inducing hyperactivity in the D phase (Figure 4
Chemical photodegradation of labile compounds could be accelerated when using light:dark cycling during screening, which could confound data interpretation. If a chemical rapidly degrades to less toxic intermediates due to co-exposure with light, its bioactivity may be underestimated. Alternatively, if the photo intermediates are more bioactive than the parent compound, its bioactivity may be overestimated. In this study, the incidence of morphological effects was altered for abamectin, chlorpyrifos, and permethrin, all of which are known to be photolabile. Specifically, the EC50
for incidence of morphological effects of chlorpyrifos was dramatically lowered from 64.4 µM under dark conditions to 34.1 µM under an LD cycle (Figure 2
and Figure 3
). A previous study identified that low concentrations of a metabolite, chlorpyrifos-oxon, but not the parent compound itself, induced developmental toxicity in zebrafish [50
]. As we did not measure the chlorpyrifos:chlorpyrifos-oxon ratio in the medium, we cannot be sure that exposure to chlorpyrifos under a LD cycle produced the -oxon form in this study. However, these results indicate that potentiation of bioactivity under a light co-exposure regimen was readily detectable.
3.3. Effect of Exposure Regimen on Bioactivity
Exposure techniques such as static, daily renewal, and flow-through exposures each present pros and cons in early life stage chemical bioactivity screening. Static exposures are less labor-intensive, require less test chemicals, and avoid repeated disturbance of the developing organism, a potential but unquantified stressor on development. However, static exposures face limitations from poor chemical solubility, lability, and rapid metabolism, which may each dramatically curtail the intended exposure period. Without a priori knowledge of these chemical limitations, generally the case in high-throughput library screens, static exposures achieve higher throughput and lower cost at the potential expense of fewer bioactivity hits. Daily renewal exposures may reduce throughput, certainly increase costs, but also maximize the likelihood of hit detection.
In this study, daily renewal of the test solution potentiated the incidence of morphological effects associated with abamectin, chlorpyrifos, permethrin, pyrene, and retene. There were no chemicals for which the daily renewal condition reduced the associated bioactivity relative to the standard condition (Figure 2
and Figure 3
). For each chemical with potentiated effects, daily chemical renewal increased sensitivity of multiple morphological endpoints, most dramatically craniofacial effects (eye, snout, jaw) and edemas (yolk sac edema, pericardial edema). There were very few differences in the specific endpoints affected by this treatment versus static exposure, though the effective concentration was significantly lowered for the five chemicals listed above. See Figure S1
for all concentration–response data for endpoints. Daily renewal affected larval photomotor response for abamectin by inducing D phase hyperactivity, for estradiol by inducing D phase hyperactivity, and for pyrene by eliminating D phase hypoactivity (Figure 4
The potential for metabolism and bioaccumulation should be considered when deciding on an exposure regimen. The bioactivity of a chemical that accumulates in the body will likely be potentiated by a renewal regimen while a chemical that is readily metabolized early in the exposure period may no longer be available in the solution to induce effects on later-developing tissues, therefore lowering perceived bioactivity in static exposures. Of the nine test agents, five exhibited greater incidence of morphological effects with chemical renewal. Each of these chemicals has a log KOW
value above 4.4 (Table 1
), which may indicate potential for bioaccumulation with repeated chemical renewal.
Most chemical screening studies report nominal water concentrations and do not analyze concentrations of parent chemicals or their metabolites in the exposure medium or in the organism’s body. The OECD FET test makes recommendations that exposure regimen may be adapted for volatile, easily degradable, or adsorptive substances and that exposure concentrations should be verified at the beginning and end of the test for static exposures and at the beginning and end of each exposure interval when using a renewal regimen [19
]. While this would be the ideal method to ensure consistent concentration throughout the exposure, it is not feasible to collect and analyze these additional samples when conducting high-throughput screening, so physiochemical properties like log KOW
may be useful to consider when choosing an exposure regimen.