The data obtained in this study report the levels capable to induce acute toxicity of four UV-filters substances in three distinct aquatic organisms and evidenced multigenerational effects in D. magna exposed to environmental concentrations of these chemicals.
3.1 Acute toxicity assays
The toxicity tests were validated by the absence of significant effects for the control and DMSO 0.003% organisms, as well as by the high coefficients of linear regression (r² > 0.93), obtained for the tested concentrations versus the responses observed for the three different organisms (Table 1). The EC50 values found for A. salina were: AVO = 2.22, BP-3 = 2.53, EHMC = 0.37 and OC = 2.97 mg/L (Detailed description in the Supplementary Material as Figure S1).
Table 1
Acute toxicity of UV filters to different indicator organisms.
SUBSTANCE
|
EC50(48h) (mg L−1)
|
EC50(72h) (mg L−1)
|
EC50(48h) (mg L−1)
|
|
A. salina
|
D. subpicatus
|
D. magna
|
EHMC
|
0.37
|
0.37
|
0.5
|
OC
|
2.97
|
1.54
|
2.57
|
AVO
|
2.22
|
1.07
|
1.89
|
BP-3
|
2.53
|
0.82
|
1.72
|
Legend: EHMC = 2-ethylhexyl, 4-methoxycinnamate; BP3 = benzophenone-3; OC = octocrylene; AVO = avobenzone.
While the EC50 values determined for AVO, BP-3 and OC were similar, greater toxicity was observed for A. salina exposed to EHMC, which showed a lower EC50 of 0.37 mg/L. Thorel et al (2020) evaluated the toxic effects of ten UV filters for A. salina. Results indicated that among the different UV filters tested, OC was the most toxic (EC50 =0.6 mg/L), followed by AVO (EC50 =1.8 mg/L). For BP-3, no toxicity was observed even at the highest concentration. It is noteworthy that this is the first study reporting the EC50 concentration of EHMC in A. salina, highlighting the importance of investigating the potential toxicity of these chemicals in different aquatic organisms.
Due to the high liposolubility, these molecules tend to bioaccumulate and thus increase potential effects over time (Blüthgen et al. 2014; Manová et al. 2015; Necasová et al. 2016). The bioaccumulation and biomagnification capacity of UV filters in brine shrimp were demonstrated by Li et al. (2018). Second-generation (F1) of Danio rerio embryos exhibited toxic effects after trophic exposure of F0 fed with contaminated nauplii with BP-3, EHMC, and OC. UV filters have a high capacity to contaminate marine environments, especially during the summer, when there is a significant consumption increase in recreational activities on the coasts (Sharifanet al. 2016).
The EC50 values found for D. subspicatus were even lower than those for A. salina: EHMC = 0.37, AVO = 1.07, BP-3 = 0.82 and OC = 1.54 mg/L (Table 1). Similarly, it was observed that EHMC demonstrated the greatest toxic potential of the analyzed UV filters, significantly inhibiting D. subpicatus growth at concentrations below 0.5 mg/L. Sieratowicz et al. (2011) determined the EC50 for BP-3 using D. subspicatus and obtained similar concentrations (0.96 mg/L) from this study. They also obtained the EC10 value of 0.61 mg/L as an inhibitory concentration of 10% for microalgae proliferation. However, for the other molecules of interest, no available studies were found for comparison, only for other species of microalgae and under different exposure conditions. Rodil et al. (2009b), reported an EC50 value for Desmodesmus vacuolatus exposed to 0.19 and 0.36 mg/L of EHMC and BP-3, respectively (Table 2).
Table 2
Comparison of acute toxicity values for A. salina, D. subspicatus and D. magna with the literature.
|
A. salina
|
D. subpicatus
|
D. magna
|
SUBSTANCE
|
EC50(72h) (mg L−1)
|
Literature (mg L−1)
|
EC50(72h) (mg L−1)
|
Literature (mg L−1)
|
EC50(48h) (mg L−1)
|
Literature (mg L−1)
|
EHMC
|
0.37
|
---
|
0.37/0.02
|
D. subspicatus: EC10(72h) = 0,07b
D. vacuolatus: EC50(24h) = 0,19c
|
0.5
|
0.57b
|
OC
|
2.97
|
0.6a
|
1.54/0.46
|
D. vacuolatus: EC50(24h) = 0,05c
|
2.57
|
3.18d
|
AVO
|
2.22
|
1.8a
|
1.07/0.18
|
---
|
1.89
|
1.95d
|
BP-3
|
2.53
|
NTa
|
0.82/0.28
|
D. subspicatus: EC50(72h) = 0,96b
D. vacuolatus: EC50(24h) = 0,36c
|
1.72
|
1.67a/1.9e
|
References: a = Thorel et al, 2020; b = Sieratowicz et al., 2011; c = Rodil et al., 2009b; d = Park et al., 2017; e = Fent et al., 2010
Legend: EHMC = 2-ethylhexyl, 4-methoxycinnamate; BP3 = benzophenone-3; OC = octocrylene; AVO = avobenzone.
For Daphnia magna, the EC50 values showed a similar pattern than for other organisms, that is, the EHMC was the most toxic (Table 1, Figure S2). While AVO, BP-3, and OC showed related toxicity potential, EHMC presented the lowest and very similar EC50 values (48h) for the three different aquatic organisms (Table 2). This effect could be partially explained by the relatively high liposolubility of the four molecules, which increases the ability to penetrate cell membranes and bioaccumulate in tissues (Fent et al. 2010; Pestotnik et al. 2014; Park et al. 2017). Fent et al. (2010), reported that D. magna exposed to different organic UV-filters (BP-3, BP-4, 4-methyl-benzylidene camphor, and EHMC) showed increased toxicity with greater hydrophobicity of the compound. On the other hand, for the current study, the substance with higher hydrophobicity (OC, logKow = 7.35), had the lowest toxic potential (EC50 = 2.57 mg/L), while EHMC (logKow= 5.43) presented the highest degree of toxicity (EC50 = 0.37 mg/L), even though it was less lipophilic.
According to Kaiser et al. (2012), more lipophilic molecules do not necessarily have greater toxicity, since only EHMC caused significant effects on benthic organisms exposed to the same four UV-filters from this study. The authors discussed that this could be explained by the lower bioavailability of AVO and OC, which can interact more easily with sediments, particulate matter, and present low absorption capacity when ingested. In general, the results obtained herein corroborated with the previous descriptions of the scientific literature (Table 2), confirming the reproducibility and reliability of the experiments conducted in this study.
3.4 Daphnia magna multigenerational exposure
This is the first study evaluating D. magna longevity, reproductive parameters, and biochemical biomarkers in more than one generation exposed to ambient concentrations of AVO, EHMC, BP-3, and OC in the mixture. It is important to emphasize that these UV filters represent the main filters used in personal care products worldwide (Kwon and Choi 2020) (Table 3).
Table 3
Effects observed after chronic multigenerational exposure of Daphnia magna to AVO, BP-3, EHMC. OC and the mixture.
Sample
|
Longevity F0 (%)
|
Longevity F1 (%)
|
First offspring F0 (days)
|
First offspring F1 (days)
|
Reproduction rate F0
|
Reproduction rate F1
|
Control
|
91.66
|
100
|
8.60 ± 0.84
|
8.25 ± 1.06
|
47.20 ± 6.37
|
46.00 ± 10.33
|
DMSO
|
100
|
100
|
8.83 ± 0.94
|
8.67 ± 1.78
|
41.08 ± 7.15
|
41.33 ± 11.83
|
AVO
|
91.66
|
100
|
9.17 ± 1.19
|
10.33 ± 0.89*
|
46.18 ± 7.64
|
34.50 ± 8.07*
|
BP-3
|
91.66
|
100
|
8.73 ± 1.10
|
10.08 ± 1.00*
|
44.10 ± 5.00
|
31.92 ± 7.27*
|
EHMC
|
100
|
100
|
8.50 ± 1.08
|
9.25 ± 1.48
|
47.40 ± 7.44
|
35.17 ± 6.63*
|
OC
|
100
|
91.66
|
8.83 ± 094
|
8.73 ± 1.27
|
49.73 ± 8.31
|
39.45 ± 9.65
|
MIX
|
100
|
91.66
|
8.08 ± 0.90
|
10.09 ± 1.38*
|
46.58 ± 6.80
|
35.18 ± 6.54*
|
Legend: (DMSO) dimethylsulfoxide 0.003%; (AVO) avobenzone 4.4 µg L-1; (BP-3) benzophenone-3 0.17 µg L-1; (EHMC): 2-ethylhexyl, 4-methoxycinnamate 0.2 µg L-1; (OC) octocrylene 4.4 µg L-1; (MIX) mixture. (*) Significant difference to control in ANOVA + Dunnett, with p<0.05.
In longevity analysis no significant differences were found for the F0 and F1 groups, demonstrating no lethality for D. magna at the exposure conditions tested. The absence of effects at this endpoint was expected, considering the low concentrations of UV filters (ng/L) used in this study, which are lower than the concentrations capable of causing mortality, even for longer exposure tests. As confirmed in this study, EC50 values for AVO, BP-3, EHMC, and OC in exposures of 48h have been described in mg/L range (Rodil et al. 2009b; Fent et al. 2010; Sieratowicz et al. 2011; Kaiser et al. 2012; Park et al. 2017) and no observed effect concentrations (NOEC) in µg/L (Sieratowicz et al. 2011; Lambert et al, 2021).
Regarding reproductive endpoints, no significant delay was observed for the period to produce the first offspring in F0, as well as no differences in reproduction rates, were found when comparing the treatment groups with the control group. On the other hand, for F1 generation, AVO (10.33 d), BP-3 (10.08 d) and MIX (10.09 d) groups showed a longer period to produce the first offspring than F1 control (8.2 d). Again, in the F1 generation, an inhibitory effect after 21d of exposure was found in groups exposed to AVO (34.50 neonates/replicate), BP-3 (31.92 neonates/replicate), EHMC, and MIX (35.17 neonates/replicate) when compared to the F0 (47.20 neonates/replicate) and F1 controls (46.0 neonates/replicate). For all analyzed parameters, in both generations, there were no significant differences between negative control and solvent control groups, confirming the absence of effects for the DMSO solvent at the tested concentrations.
Despite no significant effects were observed for the first generation exposed to the UV-filters, they were evident only in the F1 generation, proving that multigenerational studies represent a more realistic approach considering longer-term effects through the life cycle and generations of D. magna. In some cases, contaminants can affect exposed organisms and their offspring, with toxic effects being aggravated in consecutive generations (Barata et al. 2016; Campos et al. 2016).
According to Lambert et al. (2021), significant delays in the first offspring and reduction in the total number of neonates of D. magna were observed after exposure to BP-3 (166 µg/L) in a traditional chronic 21d trial. The same significant effects were not observed after exposure to EHMC (75 µg/L) by these authors. These data corroborate with the results observed for the F1 generation of BP-3 and AVO in the current study. Even though exposed to lower concentrations, these two molecules demonstrate potential for reproductive-endocrine interference, as well as for EHMC (Morohoshi et al. 2005; Ozáez et al. 2016; Wang et al. 2016). The benzophenones group is constantly related to multiple estrogenic and androgenic changes in fish, rats, and humans. The most common effects are inhibition of estradiol, activation of estrogen receptors, induction of vitellogenin production in fish, and inhibition of the action of testosterone in rats. Endocrine regulation is directly linked to the normal functioning of an organism. If affected, can trigger dysfunctions, for example, delayed reproductive development, decreased or complete inhibition of reproductive rates (Morohoshi et al. 2005; Campos et al. 2016; Ozáez et al. 2016; Wang et al. 2016). Additionally, the biotransformation of these compounds may potentiate the disrupting effects, since multiple metabolites can be generated (Morohoshi et al. 2005; Molina-Molina et al. 2008; Watanabe et al. 2015).
UV filters are molecules of low solubility in water and high lipophilicity. EHMC has a solubility of 0.156 mg/L and log Kow = 5.80, BP-3 with 68.6 mg/L and log Kow = 3.79, OC with 0.004 mg/L and log Kow = 6.88 and AVO with log Kow = 6.88 (Rodil et al. 2009b; Kaiser et al. 2012). Therefore, possible bioaccumulation effects can also be suggested, since exposures over generations may cause higher levels of organic compounds in the tissues of organisms, especially in generations following the parental. This may happen because the organisms remain exposed to the contaminants from embryonic stages until adult life. In addition, when a substance is not metabolized and excreted at the same rate at which it is absorbed, bioaccumulation and biomagnification may occur, depending on the trophic level and form of exposure. Bioaccumulation of organic contaminants can compromise cell metabolism functions, generate oxidative stress, cause damage to lipid membranes, interfere with cellular permeability, and hinder the transport of nutrients (Fent et al. 2010; Pestotnik et al. 2014; Gago-Ferrero et al. 2015).
When F1 offspring was exposed to a MIX sample (AVO 4.4; BP-3 0.17: EHMC 0.2; and OC 4.4 µg/L), reproductive effects were similar to those observed for the isolated compounds. According to Park et al. (2017), the interaction between AVO, EHMC, and OC is antagonistic, showing that the mixture of the three molecules is less toxic than when isolated. Despite this effect was not verified in the MIX results, it is important to consider the presence of BP-3 in the mixture, which hinders to predict their interactions (Pablos et al. 2015).
The reproductive effects observed for the F1 generation exposed to environmental concentrations of UV filters should be considered of ecological relevance, since a reproductive delay and a decreased reproductive rate may potentially impact at the populational level. Thus, different levels of the biological organization can be affected and cause chain effects in future populations, as continuous exposure to contaminants directly affects the health of new individuals (Campos et al. 2016; Silva et al. 2019).
Recent studies have also evaluated the chronic effects of UV filters on D. magna, but in general they differed from the filters chosen, concentrations tested, the endpoints, and the methodology, when compared to this current study. Lambert et al (2021) investigated the sublethal effects in D. magna by chronic exposure to three organic UV filters, 4-methylbenzylidene camphor (4MBC), OC, and BP-3, with particular emphasis on molting and development. They demonstrated that 4MBC, OC, and BP-3 affect development and long-term health in neonates of exposed parents at concentrations of 130, 75, and 166 µg/L, respectively. Additionally, the expression of endocrine-related genes is significantly altered by 4MBC and BP-3 exposure, which may be related to their developmental toxicity.
Boyd et al (2021) evaluated the acute and chronic effects of AVO, BP-3 and OC, isolated and mixed, in D. magna at environmentally realistic concentrations. The main results observed were: delayed mortality up to seven days post-exposure at 200 µg/L of AVO and OC; 21d chronic exposure to 7.5 µg/L OC yielded complete mortality within 7d, while sublethal chronic exposure to AVO increased D. magna reproductive output and decreased metabolic rate. BP-3 (2 µg/L) induced a 25% increase in metabolic rate of adult daphnids, and otherwise no toxic effects at this dose.
3.5 Biochemicals biomarkers
After exposure to the UV filters, the F0 and F1 generations showed different activities for the catalase (CAT) but not for the glutathione-S-transferase (GST) enzyme (Figure 1).
With the results obtained for CAT activity, no significant differences were observed for the groups exposed in the first generation (F0). However, the F1 organisms exposed to BP-3 and EHMC had significantly increased the CAT activity, compared to F1 Control. On the other hand, activity of GST did not change significantly between the treatment groups compared to the control group, both for F0 and F1. However, even without statistical significance, it was observed, for F1, a tendency of increased activity in the samples of AVO, BP-3 and MIX.
The increase in CAT activity in F1 may suggest an induction of the antioxidant defense system by the exposure of BP-3 and EHMC. The antioxidant system present in eukaryotic cells allows protection against reactive oxygen species (ROS) that are generated in the detoxification of xenobiotics. The enzymes CAT and GST exert important functions in the degradation of ROS. In peroxisomes, xenobiotics undergo oxidation with the generation of hydrogen peroxide (H2O2) as a by-product which is degraded by CAT (Boelsterli 2003; Oost et al. 2003). Organic molecules having aromatic rings and hydroxyls in their chemical structures, as in the case of BP-3 and EHMC, may undergo biotransformation during cellular metabolism and generate reactive intermediates increasing the production of ROS and consequently the activity of detoxifying enzymes, such as CAT, GST and SOD (Boelsterli 2003; Watanabe et al. 2015). In fact, oxidative stress in fish (Carssius auratus) liver, was triggered by 0.5 mg/L of BP-3 exposure (Li et al., 2015), with the production of ROS and altered SOD, GST and CAT activities. Increase in CAT activity in Tetrahymena thermophyla caused by 1 µg/L of BP-3 was also reported by Gao et al. (2013). Regarding Spearman's correlation analysis, there was no significant correlation between any of the analyzed parameters.
In general, EHMC followed by BP-3 showed the highest toxic potential for the acute and chronic tests, respectively, among the analyzed UV-filter molecules. For the chronic exposure, both compounds were, capable to reduce reproduction rates and induce the catalase activity in F1, even at the lowest concentrations (0.17 and 0.2 µg/L). The acute toxicity responses corroborated this result, showing that EHMC followed by BP-3 induced higher toxicity at lower doses. Although NOEC levels were still above those found in aquatic environments, this study evidenced the toxic potential of EHMC and BP-3 associated with prolonged exposure to environmental concentrations of these chemicals. More sensitive responses, such as biochemical biomarkers, are suitable for showing earlier effects that may predict others biological responses of higher ecological relevance, such as reproductive endpoints and population impact. Therefore, additional studies considering different levels of biological responses are necessary to better understand the real impacts of environmental chemicals, such as UV filters, in aquatic organisms at concentrations of environmental relevance.