Developmental Toxicity of Detergent Surfactants on Zebrafish: A Review of Current Evidence and Future Directions

1. INTRODUCTION 

1.1 Environmental occurrence of detergent surfactants 

Amphiphilic compounds (detergent surfactants) have both a hydrophilic and a hydrophobic part that are able to adsorb at interfaces, reduce surface tension, and promote mobilization of oils and particulate soils in water [1]. The relevance of these properties to household detergents, cleaning formulations and products, and hygiene practices, such as hand washing, where products can help reduce the transmission of viruses via the respiratory system. It is a key example of the use of cleaning products in a public-health context [2]. Most of these products are used in water, and thus the discharge of wastewater is a potential pathway for surfactant contamination of aquatic ecosystems. However, exposure levels will vary depending on the composition of the formulation, environmental transformations and the efficiency of WWT, and nominal exposure concentrations used in laboratory experiments should be differentiated from environmental concentrations. Surfactants are traditionally classified into different families based on the nature of the head-group charge: anionic, cationic, non-ionic and amphoteric. The fate of a surfactant after discharge is part of the question of exposure to the surfactant, and may generate new materials that differ in toxicity from the parent. 

1.2 Potential developmental toxicity of detergent surfactants 

Exposure at this stage could disrupt the normal development process of embryogenesis. Although exposure levels may not be lethal, they could disrupt the normal process of tightly coordinated cell division, migration and differentiation. Therefore, both lethal and sublethal effects must be taken into account, and endpoints that are typically evaluated include developmental delay, abnormal morphology, changes in hatching and changes in larval behavior. Stretching beyond the exposure window, early-life perturbation can have consequences, as seen in the zebrafish models of early life stress and its effect on brain and behavioral development [3]. An automatic transfer of toxicity from one surfactant to another will not happen, as it depends on the identity of the surfactant, the level of exposure and the duration of the exposure. 

1.3 Rationale for zebrafish embryo-based assessment 

Zebrafish (Danio rerio) are one of the most popular vertebrate models for toxicology studies [4]. Their embryos grow outside of their mothers, can be examined optically, are very small and suitable for multi-well screening, and morphology/heart function/hatching can be assessed directly in the living animal, which can be coupled with molecular and behavioral assessment. The high percentage of shared genes between zebrafish and humans renders them interesting for translation [5]. The genetic conservation is not, however, a conservation of human developmental risk, and zebrafish results are not automatically translated to human developmental risks, but rather to the risk of hazard during aquatic development. It is also a model for practical reasons: many embryos can be exposed under controlled conditions, effects can be followed from fertilization to the larval stage, and concentration-response designs can be performed in days. 

1.4 Scope and objectives of the review 

This review reports only work in zebrafish embryos and larvae that employed detergents, or detergents and surfactants, as the surfactant. It aims to consider the evidence of developmental toxicity, examine the benefits and limitations of the model, and identify mechanistic uncertainties that suggest future research needs. It first begins with premises for the model (Section 3). Then a survey of the characteristics of the surfactant classes (Section 4) was followed by analyses of reported developmental evidence (Section 5), suggested mechanisms 

(Section 6), environmental significance (Section 7) and research needs (Section 8). Wherever possible, direct evidence from zebrafish embryos to support the text is provided; supporting evidence from other life stages, species or chemical groups is included. 

2. REVIEW METHODOLOGY 

2.1 Literature databases and search strategy 

This is a narrative (critical review) and not a systematic review. Studies pertaining to the use of zebrafish embryos and the effects of surfactants in zebrafish embryos (or the effects of detergents in zebrafish embryos) and the developmental toxicity of detergents in zebrafish embryos (embryogenesis and zebrafish development) were identified through relevant scholarly databases with additional web searching using combinations of the terms. Boolean operators were used, and search terms were alternated to allow the inclusion of individual surfactant names in addition to classes and to allow for the identification of studies describing a specific surfactant not included in a title using the word "surfactant. 

2.2 Study inclusion and exclusion criteria 

Peer-reviewed original research that used zebrafish embryos or larvae and was exposed to identified surfactants was emphasized. In studies that were eligible, mortality, developmental abnormalities, molecular changes or behavior were reported. Those studies that did not include sufficient information about exposure or outcomes were excluded, as were those that were studies of unrelated chemicals [6]. Only data from adult zebrafish, other species, biocides in combination with surfactant compounds and fluorinated compounds were used for purposes of mechanistic discussion. Therefore, evidence was classified as direct (direct exposure of zebrafish embryo or larva to a surfactant), supporting (adult zebrafish, other organisms, related biocides and fluorinated compounds) and contextual (methodological and review articles) and classified accordingly in the following sections. Also, syntheses of zebrafish toxicity have focused primarily on per- and polyfluoroalkyl substances (PFAS) so far and not on detergent surfactants, which makes it important to consider only that literature as supporting information [7]. 

2.3 Data extraction and critical synthesis 

From each study, the name of the compound, class of the surfactant, concentration and length of exposure, developmental stage, endpoints and main findings were obtained. Strategies have been used to compare the study designs and outcomes, taking note of any differences in experimental conditions and contradictions, methodological limitations, and gaps in evidence. Critical appraisal included statements and justification of exposure concentrations, as well as stating and reporting controls and replication, event definition of endpoints, and consistency between the data presented and the conclusions. No formal risk of bias instrument was used; the appraisal was qualitative and is to be read more as an informed judgement and not graded. No quantitative pooling was attempted as the designs of the exposures and the endpoints vary widely. 

3. ZEBRAFISH  EMBRYOS  AS A MODEL  FOR DEVELOPMENTAL TOXICITY ASSESSMENT 

3.1 Biological characteristics of zebrafish embryos 

The fertilization of the zebrafish is external, meaning that development occurs outside of the body of the mother and can, therefore, be readily seen starting at the first cell division. The embryo can be optically accessed until early organogenesis, permitting studies of undissected early development of organs like the heart, eyes, notochord and somites. Multiple embryos can be obtained from a single spawn to allow for many concentrations, replicates and controls to be conducted on the same spawn. The effects of exposure at a particular event are crucial to interpretation, since the same chemical might have different effects when exposed during cleavage, gastrulation or organogenesis; and embryonic protective barriers vary with level of development. Embryos are small, which means a minimal amount of test substance, and they can be exposed in standard multi-well plates, but this also means that evaporation, plastic adsorption and the amount of exposure medium can change the concentration that embryos are exposed to. 

3.2 Major stages of zebrafish embryogenesis 

Development starts with cleavage and the development of the blastula and continues with gastrulation, in which cell movements result in the formation of the germ layers and body axis. Segmentation gives rise to somites, and organogenesis gives rise to the heart, brain, eyes, etc and hatching takes place and gives rise to the free-swimming larva. Thus, gastrulation disruption could result in defects of axis formation and of head and face development, while disruption at later stages is more likely to cause defects in organ function. As a result, the phenotypes attained may vary depending on the developmental timing of application of the surfactant. A general surfactant action may, for instance, result in different phenotypes if applied during gastrulation and organogenesis, respectively. In such studies, in which embryos are exposed only for a set period of time after fertilization, or observation of the embryos is only started after hatching, it may be possible to miss out on early effects entirely. Morphological effects are dependent on exposure time and concentration, and developmental stage should be noted along with chronological time of exposure [8]. 

3.3 Advantages of the zebrafish embryo model 

The model is amenable to microscopy, behavioral assays and molecular techniques and morphological changes can be scored in living embryos. It can be used for concentrationresponse assessment and comparative screening because of these features, for example, to screen the developmental toxicity of large numbers of fluorinated compounds [9]. Embryo assays can also supplement additional aquatic toxicity assays by offering information on the level of development of the tested species. Algal, invertebrate and juvenile fish assays may also be conducted simultaneously with embryo assays. Therefore, developmental sensitivity must also be taken into consideration along with the other trophic levels, and screening in embryos can also be used to prioritize compounds for more expensive and demanding assays [10]. The model has been applied to assessing oral care products containing surfactants [11]. 

3.4 Limitations of the zebrafish embryo model 

Results obtained are species-specific and extrapolation to other vertebrates is limited. Chorion properties, chemical bioavailability, and water conditions affect exposures; an example of one chemical being more unstable in a common vehicle and misrepresenting the nominal concentration shows how this can influence exposures [12]. Acute embryo toxicity is to be differentiated from chronic ecological effects, and laboratory endpoints may not necessarily translate into ecological effects. There are also issues in interpretation, as numerous abnormalities are also manifestations of general distress and therefore do not speak of a particular mode of action [13]. 

4. CLASSIFICATION AND  CHARACTERISTICS  OF  DETERGENT SURFACTANTS 

4.1 Anionic surfactants 

Anionic surfactants are those with a sulfate or sulfonate negative electrical charge on the head group, and examples are sodium dodecyl sulfate (SDS) and linear alkylbenzene sulfonates (LAS). Their various application in personal-care, laundry and dishwashing products is due to their very good detergency and foaming qualities. Their effects are concentration-dependent, and it is generally believed that their effects are due to interactions with membrane lipids and proteins [14]. Biocompatibility of sodium lauryl sulphate has been tested in developing zebrafish embryos and the acute toxicity of LAS has been reported together with chromium, as the life stage of the zebrafish should be verified before considering this study as embryonic evidence [15]. 

4.2 Cationic surfactants 

Cationic surfactants, usually represented as quaternary ammoniums (e.g., benzalkonium chloride and cetylpyridinium chloride), have both detergent and antimicrobial properties and have a positive charge that naturally associates with the negative charge of cell membranes [16]. Benzalkonium chloride is known to affect hematopoietic stem cells and to be immunotoxin to zebrafish larvae, although the toxicity of this compound has been studied in the context of the use of disinfectants [17]. The association of reproductive and developmental toxicity of the quaternary ammonium compounds has been reviewed in different species, and there is a call for studies at environmentally relevant doses [18]. Although there is no evidence of any areas where people intentionally release cationic detergents, they are used for preservation of aquatic environmental DNA [19]. Ranking of universal toxicity is not possible on the basis of other classes and is dependent on compound, endpoint, and exposure. 

4.3 Nonionic surfactants 

The non-ionic surfactants are nonionized and are made of a polyoxyethylene-derived hydrophilic group. Fatty alcohol ethoxylates are very frequently used in cleaning formulations. Both ethoxylate AEO-7 and acetyl alcohol polyethoxylated have been found to cause severe cardiac, liver and locomotor damage in zebrafish embryos and metabolic health disruption in zebrafish developed through exposure in the embryo [20, 21]. These results indicate that nonionic surfactants may have some effect on development, yet the effect observed for one compound should not be extrapolated to the class since there are variations in ethoxylate chain length and hydrophobic structure. 

4.4 Amphoteric surfactants and commercial mixtures 

The developmental toxicity of a commercial detergent cannot be predicted using its main surfactant because these detergents include other ingredients such as builders, enzymes, preservatives and fragrances. Sodium lauryl sulphate (SLS) and linear alkylbenzene sulphonate had a higher acute toxicity than the commercial detergents in zebrafish of unknown age [22]. It can also be valuable to compare the performance of the products and their contained surfactants in parallel, and in zebrafish embryos, an SLS-containing product and SLS alone caused disruption of oxidant-antioxidant balance, but a detergent-free product did not [23, 24]. 

In addition, non-surfactant disinfectants may be in need of separate embryo assessment. 

4.5 Chemical and exposure factors affecting toxicity 

In zebrafish embryos, concentration, exposure duration, and developmental window are all important in whether a surfactant disrupts embryogenesis, all three of which should be reported. There is also a consideration of chemical structure because the adipose-promoting activity of nonylphenol polyethoxylates in developmentally exposed zebrafish varied with ethoxylate chain length [25]. The hardness of the water affected the toxicity of fatty acid salts on Daphnia magna [26]. Temperature also altered the effects of sodium lauryl sulfate in mussels, indicating that media in which embryos are tested might also impact results [27]. The obtained concentrations, which are available for cationic surfactants by analytical methods, are preferable to nominal concentrations [28]. 

5. CURRENT EVIDENCE OF DEVELOPMENTAL TOXICITY 

IN ZEBRAFISH EMBRYOS 

5.1 Evidence from comparative surfactant studies 

The prospective comparative study compared the effects of SDS, the cationic surfactant dodecyl dimethyl benzyl ammonium chloride (compound 1227) and a non-ionic fatty alcohol polyoxyethylene ether (AEO) on zebrafish embryos and larvae [29]. The embryos were exposed from the one-cell stage at a dose of 0.2 or 1 µg/mL and observed at 120 hpf, and the larval behavior was recorded upon exposure to 0.1 to 1 µg/mL. All three surfactants had a concentration-dependent delay in development; the AEO surfactant had the greatest impact, and the cationic and non-ionic surfactants had a greater impact than SDS on locomotor behavior of larvae. This trend allows for a compound-specific evaluation and not for a general evaluation of all the surfactants, as only one compound from each class was studied. 

5.2 Concentration-response relationships 

Concentration-response data are required to establish thresholds of effects, and such data were reported in the comparative study described above that showed effects increase with exposure. In a study of acetyl-pyridinium chloride, embryos and larvae were exposed to doses between 4 and 1,200 µg/L for the first 120 hours; high doses of this substance reduced the number of survivors, movement, and heart rate, whereas the low doses increased embryo activity [30]. The non-monotonic behaviour responses indicate that lethal concentrations and sub-lethal effect concentrations need to be treated separately. These are not used for comparing numerical toxicity values since durations of exposure, compounds and test conditions vary among these studies. 

5.3 Effects on embryonic development and morphology 

The comparative study showed that 1 µg/mL AEO caused reduced eye size and reduced body length at 28 hours post-fertilization and that reduced eye size, reduced head size and reduced body length were all present at 48 hours. Some of the effects were transient, as the effects at 72 hours decreased and were no longer significant at 96 and 120 hours. The results indicated slight, but not significant, changes in the sizes of the eyes, head and body by SDS and the cationic compound from 28 to 120 hours; the authors' discussion, however, said they had decreased results for all three compounds, with AEO being the most severe. Persistency of morphological changes cannot therefore be assumed after these periods, and only the endpoints which were reported in the studies selected are considered. 

5.4 Survival, hatching, and acute toxicity 

The survival is to be viewed together with the time of exposure. The comparative study showed more than 35 % mortality at 8.5 hours and 53 % mortality at 10 hours after fertilization with 1 µg/mL AEO compared to less than 10 % with SDS and compound 1227. A multi-model profile of sodium lauryl sulfate highlighted cytotoxic, cardiotoxic, developmental and antimicrobial effects [31]. Nonylphenol ethoxylates are degraded to nanomolar nonylphenol, which was associated with swim bladder abnormalities and altered gene expression – sublethal outcomes accompany the effects on survival [32]. This survival itself, thus, cannot define a profile of developmental toxicity. 

5.5 Molecular and behavioral evidence 

DNA markers complement other methods of observation as morphology, while comparative study revealed that surfactant-exposed embryos have a decrease in expression of the hindbrain marker krox20, and altered expression of ntl (a notochord and axial mesoderm marker). The authors conclude that the decreases in krox20 are responsible for smaller head size, and that altered ntl expression is likely responsible for impaired migration and growth of cells. These associations indicate but are not by themselves proof of causality. Behavioral evidence involves fewer rest bouts at night following exposure to compound 1227 and a shift in the rest-wake pattern following AEO, but not SDS [33]. In addition, developmental exposure to benzalkonium chloride has also influenced mechanosensory hair cells and nociceptive responses, and time-series behavioral analysis has been developed using cetylpyridinium chloride in male zebrafish, which is supporting and not embryonic [34]. Behavioral endpoints are used in conjunction with morphology and will confirm functional change when the structure is normal. 

5.6 Evidence beyond the initial comparative studies 

Dioctyl sodium sulfosuccinate (DOSS) is not reported for zebrafish embryo studies built upon in this review, and is considered an evidence gap. The organ-specific toxicity of stearamidopropyl dimethylamine (SAPDMA), a softener active for fabrics, has been tested in zebrafish embryos [35]. A known ecotoxic silicone quaternium-22 surfactant for anticorrosion has been tested with embryos as well [36]. Histological results in liver and kidney postexposure to sodium dodecylbenzene sulfonate (DBS) should not be compared to embryonic results since this is organ-level injury in fish [37]. In all of these studies, endpoints and designs vary, and there are limited numbers of compounds per class and no test conditions overlap significantly. 

6. POTENTIAL MECHANISMS UNDERLYING  SURFACTANT-INDUCED DEVELOPMENTAL TOXICITY 

6.1 Membrane interactions and cellular injury 

The most likely events to initiate change in the membrane integrity and permeability are the incorporation of the amphiphilic structure of the surfactants into the membranes. Loss of barrier function could disrupt ion balance, cell signaling and survival in fast-proliferating embryonic cells and impede. Few of the studies reviewed directly measured damage to membranes; rather, most studies infer damage to membranes from their measured chemical properties or from downstream consequences of membrane damage, such as cell death. Thus, damage to membranes should be considered a plausible mechanism, but not an established one. This could be tested by direct measurements, such as measuring membrane permeability dyes, measuring lipid peroxidation products, or cell-viability markers in intact embryos. An effective process is also not likely to be uniformly distributed across classes, as the anionic, cationic and non-ionic head groups differ in their interactions with charged as well as neutral constituents of cell membranes. 

6.2 Oxidative stress and antioxidant responses 

Oxidative stress can trigger lipid peroxidation and apoptosis and occurs when the production of reactive oxygen species is more numerous than antioxidant systems. A method combining a biocide, chloroxylenol (CX) with a surfactant caused cardiovascular toxicity with oxidative stress, inflammation and apoptosis in Zebrafish embryos, while a biosurfactant-microplastic hybrid resulted in oxidative stress and apoptosis and lowered coordinated toxicity in zebrafish embryos [38, 39]. The adult-fish findings are supplementary, but experiments directly demonstrating oxidative stress in the embryos upon exposure to surfactant agents, such as antioxidant rescue experiments, remain to be performed. A non-specific response can also be oxidative stress, which can occur with almost any cellular damage, making it impossible to see the cause of the damage. 

6.3 Developmental signaling and gene expression 

The findings of the comparative study, the krox20 and ntl results, are important since they have found a link between the smaller heads and the change in genes and an axial change, but association does not mean causation. Using developmental gene expression can be useful in interpreting morphology, as it is important to note that a specific abnormality should not be considered evidence of a pathway unless there is causal evidence for it. Changes in expression at one time point could also be due to changes in tissue composition or to differences in development time [40]. Thus, embryos that are behind schedule, will appear to have less expression of markers even if they have not changed at all. Therefore, exposed embryos should be compared with control embryos at the same stage of development, not just the same age in hours, before any difference in gene expression is taken as evidence that a surfactant has disrupted a signaling pathway [41]. Defined signaling mechanisms that involve thyroid hormone include disruption of MAPK signaling in a hematopoietic cell-specific manner after chloroxylenol, disruption of thyroid hormone receptor signaling by ethoxylated non-ionic surfactant, and decrease in thyroid hormone after early-life triclosan exposure [42]. 

6.4 Cardiac, neural, and organ-system effects 

Many zebrafish embryos are used to study cardiac and neural development. It has been observed that common antimicrobials were able to affect early zebrafish development via immune-cardiac signaling, while chloroxylenol and benzethonium chloride resulted in changes in spinal neuron development and behavior [43, 44]. CPs with hormonal activity have also been evaluated with regard to developmental Neurotoxins [45]. Proposed mechanisms of phenotypes might be related to cellular injury, which could plausibly adversely affect organ development. A decrease in heart rate, changes in the pericardium and a change in locomotion could result from toxicity to heart tissue and the nervous system, either directly or indirectly from reduced overall development or from disturbances in osmoregulation. The separation of these possibilities is achieved by using tissue-specific reporters as well as functional measurements and exposure windows to separate between early and late effects. 

6.5 Integrated interpretation of mechanisms 

The careful working theory suggests that the membrane perturbation is the primary event, followed by oxidative stress, signaling and apoptosis abnormalities, based on which morphological and behavioral changes were observed. It has been found in one study that the perfluorinated sulfonate used is a basis for developmental toxicity, in comparison to another perfluorinated sulfonate: perfluorooctane sulfonate (PFOS) [46]. There are several pathways that can be involved, and the sequence offered is a hypothesis-generating pathway and is not known as a universally accepted mechanism (Figure 1). 

7. ENVIRONMENTAL RELEVANCE AND LIMITATIONS OF  CURRENT EVIDENCE 

7.1 Laboratory concentrations and environmental exposure 

Laboratory exposure relevance relates to measured exposure in the environment. The concentrations tested were much higher than those found in freshwater or seawater, and might not reflect environmental levels. It is therefore necessary to differentiate concentrations of wastewater and receiving-water concentrations, and at what realistic levels the most extreme effects observed are reported has not been clearly resolved. The difference is important to interpretation: a strong effect at a concentration order of magnitude higher than one being monitored shows hazard and mechanism, but very little in terms of risk, while an effect closer to the measured concentration would carry much greater regulatory weight. Concentrations of the different surfactants also depend on site, season, population density and the technology used for treatment, and no one concentration can usually be used as a benchmark. 

7.2 Environmental mixtures and wastewater treatment 

Environmental exposures are complex with the presence of mixtures of compounds, cooccurring contaminants, degradation products and treatment effects, none of which are seen in a single-compound test [47]. A time- and concentration-resolved analysis of mixture effects has demonstrated that mixture effects can be studied in the zebrafish embryo, and co-exposure with cetylpyridinium chloride affected the toxicity of zeolitic imidazolate framework nanoparticles [48]. The toxicity of mixtures cannot be reliably predicted based on the toxicity of the individual compounds, and little evidence is available for detergent mixtures. The presence of transformation products, due to biodegradation, which may lead to lower concentrations of the parent compound but to higher concentrations of transformation products, and the process of sorption to sludge and sediment, leading to a shift of exposure from the water column to sludge and sediment, adds another layer of complexity in wastewater treatment. This should be recognized when results are extended to receiving waters because this is not reflected in embryo assays performed in clean waters of these laboratories. 

7.3 Limitations of current zebrafish surfactant studies 

The exposure period, test concentrations, endpoint and water variables are all different in the studies, and the number of compounds and classes studied is limited. However, acute testing does not necessarily equal in-depth environmental risk assessment and the results in zebrafish alone cannot be used to determine risks for human health (Table 2). However, this should serve as a lesson about fluorinated surfactant replacements, and the plausible assumption of similar benefits for new replacement compounds should not be made without proper analysis and study [49]. 

8. FUTURE DIRECTIONS FOR ZEBRAFISH-BASED  SURFACTANT TOXICITY ASSESSMENT 

8.1 Standardization of experimental methods 

Developmental stage, duration of exposure, concentration, controls and water chemistry should be clearly reported, and the same morphological and functional endpoints should be used in studies [50]. Historical control data can be used by investigators to assess whether an effect is above background fluctuations, while also implementing procedural improvements in the assay to decrease variability [51]. 

8.2 Environmentally relevant exposure assessment 

The concentrations for tests should be based on environmental monitoring data – repeat or longer exposures should be made as appropriate. Use representative mixtures and detergent formulations for comparison to individual surfactants, and measure exposure concentrations, if practical. Chronic or repeated designs would indicate if the effects observed with short embryo exposures are able to persist and accumulate through later development, or if compensation occurs, and formulation-level tests would demonstrate if the co-formulants change the response towards the active surfactant. 

8.3 Mechanistic and high-throughput approaches 

The molecular, biochemical and behavioral endpoints should accompany the morphological endpoints. Here are a couple of examples of how imaging together with automated tracking and transcriptomics can be of great value. For example, multi-endpoint profiling using behavior, morphology and gene expression has been able to distinguish closely related fluorinated compounds based on behavior [52]. These should be used to investigate mechanistic hypotheses, and the occurrence of molecular change should not be taken as evidence of a toxicity pathway. High-throughput formats, along with automated image analysis, will enable multiple surfactants and concentrations to be screened in a single design with appropriate controls and stage matching and exposure verification. 

8.4 Evidence gaps and research priorities 

Embryo-level assessments of other understudy classes like amphoteric surfactants, DOSS and bio-based materials, including orange-peel-derived surfactants, are needed. Embryo assays, other aquatic models, and monitoring information should be compared and delayed effects and environmentally relevant mixtures should be given the highest priority [53]. This should be done in a manner that is reproducible based on evidence of interpretation. Coordinated studies using a common protocol with representative anionic, cationic, non-ionic and amphoteric compounds would be more useful for elucidating class differences than more single-compound report papers, with open, free sharing of raw data to facilitate re-analysis. 

9. CONCLUSION 

Zebrafish embryos provide a platform for the evaluation of effects of surfactant exposure on survival, developmental advancement, morphology, and specific functional and molecular end points. Depending on the compounds and exposure circumstances, the effects differ, and there is no evidence that all surfactants have the same effect. The direct evidence of embryonic effects has focused on a few compounds, and oxidative stress, gene expression and membrane damage can only be seen as hypotheses and possible mechanisms, not as direct causal links. The emphasis will be on standard protocols, real-world and actual measured concentrations, and direct validation of mechanisms. The zebrafish embryo test should be applied along with other ecotoxicological data and the use of developmental responses from the test will help to understand environmental risk. In this way, the zebrafish embryo can be used as an effective "triage" tool for identifying surfactants which may need to be further evaluated in the environment, alongside a weight-of-evidence strategy. 

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