The exposure of zebrafish embryos to selected PCPs in this study resulted in effects that were compound and concentration-specific, i.e., development abnormalities, decrease of heartbeat rate, among others. In addition, sea urchin embryos showed interruption and delay of development, decrease in larval length and morphological abnormalities. These effects are likely to impact survival by affecting locomotion, behavior, and the ability to escape from predation.
3.1. 4-MBC: An Ultra Violet Filter
The use of UV filters has increased during the past decade due to public concern for the effects of UV radiation. The consequences of UV filters for aquatic organisms remain poorly studied, due to the limited and fragmentary ecotoxicological studies available, which do not contribute to a reliable risk assessment of these compounds on aquatic ecosystems. However, several reports point to UV filters’ estrogenic activity at a range from 10−5
of those of estradiol, and similar to other xenoestrogens [14
]. In addition, binding studies reported that the 4-MBC can bind to both ERβ and ERα isoform [14
A seasonal variation of UV filter concentration in aquatic ecosystems seems to occur. This is related to recreational activities such as bathing and swimming that leads to UV filters washing off from the skin [18
]. Although its use as a UV sunscreen component is not allowed in the USA [26
], 4-MBC is still frequently used in sunscreens despite evidence of endocrine disruption effects [27
Although a few studies have reported the impact of 4-MBC in aquatic organisms, there is a lack of information about the effects in initial embryonic development stages [30
]. In zebrafish embryos exposed to high 4-MBC levels, 0.5 and 5 mg/L, a significant increase in abnormal involuntary muscular contractions was observed. In addition, exposure to 4-MBC at concentrations equal to or higher than 5 mg/L, induced development delay and an abnormal development in embryos, affecting heartbeats and delaying the hatching time.
In a previous study performed by Li et al. with dechorinated zebrafish embryos [30
], an increase in mortality was reported at high levels (between 3.82 and 6.36 mg/L). Here, 4-MBC at 2.54 mg/L also increased the percentage of embryos with axial curvature. In the present study, no significant differences in cumulative mortality rate were observed at the concentration range reported in the Li et al. study. This may be associated with differences in the genetic background of the fish stock or the experimental design. In the Li et al. study, zebrafish embryos were exposed to 4-MBC after dechorination [30
]. This may explain the high mortality rate reported. This example also supports the hypothesis that the lower sensitivity of zebrafish embryos to some chemicals, in comparison to sea urchin embryos, may be associated with the protection provided by the chorion. In the former study, 4-MBC-treated embryos showed an impact in the swimming capacity and no response to tactile stimulations, which can be partly attributed to abnormal axial formation [30
]. Based on that study, the altered axial curvature and shorter body observed are likely related to failure in the notochord differentiation process. Moreover, in that study it was also reported that 4-MBC inhibited acetylcholinesterase, which could lead to an accumulation of acetylcholine and inactivation of their receptors, resulting in defects in axonogenesis and muscle formation. Thus, in the present study, the increase in the percentage of zebrafish embryos with abnormal involuntary muscular contractions may be related to muscular dysfunctions from acetylcholinesterase inhibition. These effects in locomotion can hypothetically compromise survival.
In a recent study, using a similar test design (48 h exposure of P. lividus
embryos to 4-MBC) an 50%-Effective Concentration (EC50) of 854 µg/L and NOEC and Lowest Observed Effect Concentration (LOEC) values of 300 and 600 µg/L for larval length were reported [26
]. In our study, exposure of sea urchin embryos to 4-MBC resulted in a significant decrease of larval length at a much lower 4-MBC concentration (LOEC = 2 µg/L), which is in the range of environmental concentrations (Table 2
Moreover, according to the results of both endpoints for 4-MBC exposure, the lower LOEC reported in our study (0.8 µg/L) for P. lividus
shows a clear effect at concentrations lower than those already reported for this specie and at the same range of concentrations detected in coastal areas (Table 2
). The reason for these contrasting results is unclear. Genetic or sensitivity differences between organisms may explain these results, which emphasizes the importance of performing more studies to understand the potential toxicological effects and environmental impacts, in order to achieve more reliable risk assessments of UV filters.
Using larvae length as the endpoint of the sea urchin bioassay has numerous advantages in terms of continuous response, observer-independence and feasibility of using software to perform the measurements. These advantages not only speed up test readings but also result in a more than two-fold increase in sensitivity when compared to the classical morphological endpoints [32
]. Larval abnormalities require a higher sample size and there are observer-dependent endpoints that may result in subjective microscopial inspection when considering the presence and the degree of the different abnormalities [32
]. However, according to our results in Ribeiro et al., the classical endpoints reveal a higher sensitivity for the pharmaceuticals propranolol, simvastatin and sertraline [33
]. This may be related to the bioactivity of pharmaceuticals or their behavior and mode of in non-target organisms. Hence, we considered that for a robust and more informative toxicity assessment, both endpoints (larval abnormalities and larvae length) should be used in sea urchin embryo bioassays.
An increase in 4-MBC concentration in intertidal zones is expected during the summer due to the use of the greatest amount of sunscreens during recreational beach activities. On the other hand, bathing season corresponds to the spawning period of many organisms, including sea urchin. Some studies report 4-MBC environmental concentrations similar to those that impaired sea urchin embryo development in our study [23
]. As sea urchin is present in both intertidal and subtidal zones, we can not rule out the hypothesis that actual concentrations of 4-MBC and/or other UV filters can affect embryonic development of wild populations, compromising the development of this species and perhaps as well as of other sensitive taxa
, and of other organisms of the trophic web that depend on them, since P. lividus
is a key stone organism in many rocky shore ecosystems.
3.2. Propylparaben: A Preservative
Propylparaben is widely used in cosmetic formulations and often detected in aquatic environments [20
]. Despite this, only a few studies evaluated long-term effects of parabens in aquatic organisms [11
]. Parabens with longer hydrocarbon chains can induce more adverse acute effects and are in general more persistent in the environment. In fact, whereas methyl- and ethylparaben are rapidly transformed, propyl- and butylparaben require more time to biodegrade [20
Some parabens such as benzyl-, butyl- and propylparaben have been found to induce low-level estrogenic effects in aquatic organisms [11
] Propylparaben was reported to increase vitellogenin (VTG) levels in plasma and upregulate the transcription of VTG genes in male medaka. Intraperitoneal injections of propylparaben in rainbow trout also induced estrogenic responses with induction of VTG in male fish [31
]. Moreover, propylparaben can also influence sexual differentiation, as reported in Mikula et al. in a juvenile zebrafish 45-days bioassay, resulting in a significant increase of the female:male sex ratio [35
In the present study, all zebrafish embryos exposed to concentrations equal to or higher than 3.5 mg/L displayed abnormalities at the end of the assay. In a recent study, González-Doncel et al. exposed Oryzias latipes
(Temminck and Schlegel, 1846) embryos to several concentrations of propylparaben and examined physiological and anatomical abnormalities in embryos, eleutheroembryos (13 days post-fertilization—dpf) and larvae (42 dpf) [36
]. No early or late toxic effects were observed at concentrations bellow 1 mg/L propylparaben. Exposure to 4 mg/L consistently induced significant deleterious effects during medaka embryonic, eleuteroembryonic and larval development. Exposure to concentrations equal to or higher than 0.4 mg/L resulted in a significant increase in the mortality rate during the larval stage. These results are comparable to those reported in the present study for zebrafish and sea urchin embryos, revealing a greater sensitivity of these species than Leuciscus idus
(Linnaeus, 1758) (NOEC 48 h = 5 mg/L) [37
], Pimephales promelas
(Rafinesque, 1820) (LOEC 7 d growth and reproduction = 2.5 mg/L) [34
], Dapnhia magna
(Straus, 1820) (EC50 48 h immobilization = 15.4 mg/L) [38
] and Pseudokirchneriella subcapitata
(Hindák, 1990) (EC50 48 h growth = 15 mg/L) [38
]. Actual environmental concentrations of propylparaben [20
] (Table 2
) are lower than the effective concentrations observed in this study, and, therefore, no effects are expected upon acute exposure of embryos of these species in the environment. However, possible adverse effects cannot be disregarded after long-term exposure to propylparaben due to its potential to bioaccumulate in organisms and act as an endocrine disrupting compound [39
3.3. Triclocarban: Bactericide and Antifungal Agent
There are only a few studies reporting triclocarban toxicity in aquatic organisms. However, recent short- and long-term studies for aquatic invertebrates and fish indicate a slightly higher toxicity of triclocarban compared to triclosan, the most commonly detected and studied disinfectant [6
]. Benthic invertebrates could be affected by triclocarban given that this compound has the potential to sorption to sediment [11
]. In addition, triclocarban bioaccumulates in aquatic organisms, which can lead to biomagnification through the food chain [22
]. Moreover, triclocarban exhibits significant persistence in the environment and has been frequently detected in WWTP effluents and surface water over the last years at concentrations higher than triclosan (TCS) and its methyl derivate methyl-triclosan [11
Recent studies indicate that triclocarban can behave as an endocrine disrupting chemical, affecting the transcription of steroid sex hormones in human cell lines [41
]. It was also shown that a diet containing a mixture of triclocarban and testosterone, fed to male castrated rats, resulted in synergetic effects and increase of gonad weight, in comparison with the control diets or single compound diets [41
]. Triclocarban does not seem to act as a ligand of androgen receptor but shows amplification of androgen’s activity both in in vivo and in vitro studies, suggesting that the mode of action does not involve modulation at the receptor level [31
]. Furthermore, triclocarban affected the transcription of genes associated with thyroid hormone signaling in frog and rat cells [40
]. Exposure of freshwater mudsnails, Potamopyrgus antipodarum
(Gray, 1843), to triclocarban promoted a significant increase in the number of embryos produced per female at concentrations above 0.2 µg/L. Moreover, a significant increase was also reported in the number of unshelled embryos at concentrations equal to or higher than 1.6 µg/L when compared to controls [42
]. In a study performed by Schultz and Bartell, a decrease in aggression was reported in P. promelas
adult males exposed to triclocarban (1.6 µg/L) or to mixtures (560 ng/L TCS + 179 ng/L TCC and 1.6 µg/L TCS + 450 ng/L TCC). This effect was observed up to four days after the end of exposure, and may lead to a decrease in defense ability and reproduction success [43
]. The effects reported in previous studies were observed at concentrations that impacted sea urchin embryos development in the present study.
Zebrafish embryos were affected by triclocarban at concentrations above environmental relevance. However, the impact in sea urchin larval length was observed at environmentally relevant concentrations (LOEC = 0.64 µg/L). Taken together with the available data in the literature, it strongly indicates that triclocarban is likely to impact the most sensitive taxa at environmentally relevant concentrations.