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When sunscreens reach the soil: Impacts of a UV filter on the life cycle of earthworms
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When sunscreens reach the soil: Impacts of a UV filter on the life cycle of earthworms Silvia Casqueroa, Dolores Trigoa, Jose L. Martínez Guitarteb, Marta Novoa,b,* a b
Biodiversity, Ecology and Evolution Department, Faculty of Biology, Complutense University of Madrid, Spain Environmental Toxicology and Biology Group, Sciences Faculty, UNED, Spain
ARTICLE INFO
ABSTRACT
Keywords: Ecotoxicology Survival Reproduction Xenobiotics Endocrine disruptors 4-hydroxybenzophenone
4-hydroxybenzophenone (4-OHBP) is a UV filter used in sunscreens, perfumery and containers for food products that may end up in soil. The impacts of 4-OHBP in earthworms (Eisenia fetida and Dendrobaena veneta) were investigated. To prove the consequence of its direct application on earthworms' epidermis, a contact test (48 h) was performed. Significant mortality was already observed at 4-OHBP concentrations of 2 mg ml−1 at 24 h, and juveniles showed higher mortality than matures at 0.2 and 0.02 mg ml−1. In order to investigate the effects in their natural habitat, a soil test was performed with mature earthworms. Results showed that reproduction success of E. fetida was impacted by the toxicant. The number of hatched juveniles (EC50 = 152.68 mg kg−1) and the number (EC50 = 94.05 mg kg−1) and weight of unhatched cocoons decreased with an increasing concentration of 4-OHBP. The LC50 for E. fetida at 28 days was 1800.12 mg kg−1, but LC50 and EC50 values could not be calculated for D. veneta adults because of lack of effect. For the latter, the soil test was continued with juveniles in a transgenerational study showing that their mortality increased with 4-OHBP concentrations above 10 mg kg−1 (LC50 = 72.02 mg kg−1 at 7 days and 19.49 mg kg−1 at 14 days). Results showed that 4-OHBP is harmful for both species, by causing reproduction decrease for E. fetida adults (F0) and mortality increase for D. veneta juveniles (F1). However, concentrations at which 4-OHBP affects earthworm populations are much higher than those reported in the environment, and therefore, there seems to be no risk for them, except in case of accidental spill.
1. Introduction Ultraviolet (UV) filters are a group of synthetic compounds that act as barriers against UV radiation. Their use in cosmetics and creams has been intensified in the last years because of the increase of UV radiation and associated health damages. UV filters are not only added to sunscreens, but also to food packaging and perfumery products in order to avoid their odour and colour degradation (Lewis, 2016; Wypych, 2015). They can be divided into two types depending on their properties. Physical filters, such as zinc oxide and titanium oxide, work by diffraction and dispersion of the rays. Chemical filters work by absorbing radiation, and they present one or various aromatic structures with double bonds and/or conjugated with carbonyl radicals, which makes them highly lipophilic (Ozaez et al., 2013). The latter are classified in different families based on their molecular structure, such as pamino benzoates, camphors or benzophenones, among others (Chisvert and Salvador, 2007). Benzophenones are present in a diversity of products, from plastic containers to perfumes, but they are mainly part of
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the widely used sunscreens (Chisvert and Salvador, 2007; Urbach, 2001). Benzophenones are a family of aromatic ketones with the capacity of absorbing UV radiation (λ = 280–400 nm) and dissipate it as heat (Iribarne, 2016). A growing number of studies, mainly focused on vertebrates, prove that certain benzophenones act as endocrine disruptors, evidencing the importance of investigating the effects of these compounds in living beings. For example, Weisbrod et al. (2007) showed that 2-benzophenone (2-BP) inhibited the development of gametes in fathead minnows (Pimephales promelas) and caused a decrease in egg production and cessation of spawning activity. Egg production was also decreased for Japanese medaka (Oryzias latipes) after exposure to 3-benzophenone (3BP) (Kim et al., 2014). Fewer studies have shown the effects of chemical UV filters in invertebrates and sediments. Schmitt et al. (2008) tested 3benzylidene-camphor (3-BC) and 4-MBC in the freshwater oligochaete Lumbriculus variegatus that showed a decrease in reproduction after 28 days (EC50 of 1.43 mg kg−1 for 3-BC) and in the snail Potamopyrgus antipodarum, whose mortality increased after 56 days at 32 mg kg−1 but
Corresponding author at: Biodiversity, Ecology and Evolution Department, Faculty of Biology, Complutense University of Madrid, Spain. E-mail address: [email protected] (M. Novo).
https://doi.org/10.1016/j.apsoil.2019.09.004 Received 16 March 2019; Received in revised form 6 September 2019; Accepted 8 September 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Silvia Casquero, et al., Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.09.004
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also showed an increase in embryo production at lower concentrations (6–7 mg kg−1). Kaiser et al. (2012) showed that the filter EHMC caused a toxic reproduction effect in two snails, P. antipodarum (at 0.4 mg kg−1 after 56 days) and Melanoides tuberculata (at 10 mg kg−1 after 28 days), but no effect was found for the annelid L. variegatus (tested concentrations up to 50 mg kg−1 after 28 days) or other tested UV filters. Unfortunately, there is a lack of studies on the effects of organic UV filters in soil-dwelling animals. Most of these studies have been carried out in aquatic ecosystems because of their connection with sunscreens, since the aquatic leisure activities are relevant in UV filters distribution. On the other hand, sewage sludge generated in the sewage treatment plants is used for the fertilisation of farmlands after being processed. UV filters, being quite novel xenobiotics, are not properly removed during the waste treatments (Harrison et al., 2006). In fact, a study done in 15 sewage treatment plants of Catalonia (Spain) proved the presence of several UV filters in the local sludge, although at low concentrations (0.04–9.17 mg kg−1; Gago-Ferrero et al., 2011). Another way to enter the edaphic ecosystems would be through irrigation water, since the use of treated sewage water in farmlands is habitual in many countries. Studies on concentrations of UV filters in soils are scarce. Jeon et al. (2006) showed that concentrations of UV filters in soil ranged from 500 to 18,380 ng kg−1, while in water, they ranged from 27 to 204 ng L−1, in Korea. However, there is not much information on the biodegradation or accumulation potential of these compounds in soil. Earthworms constitute up to 80% of the zoomass in soil, reaching 5000 kg/ha (Diogene et al., 1997; Yasmin and Souza, 2010). They represent the main engineers of the edaphic system by improving percolation, aeration and aggregate stabilisation and favouring the processing of organic matter in the surface layer, thus increasing fertility (Domínguez et al., 2009). Moreover, they are an important part of the food chain, so both their extinction in contaminated areas and the accumulation of toxicants in their bodies may have fatal consequences for ecosystems. Earthworms can be impacted by the harmful substances present in soil, either by the contact through their epidermis or by ingestion (Laycock et al., 2016; Phipps et al., 1993). Different species sensitivity to toxicants has been suggested for earthworms, even within the same ecological category (e.g. Ma and Bodt, 1993; Verdu et al., 2018), which makes interesting, the use of more than one species for tests in order to make robust conclusions. Given the increasing use and release of emergent xenobiotics, such as organic UV filters in the environment, it is extremely important to understand how they affect different organisms, especially those that are ecologically relevant, such as earthworms. The obtained results will shed some light on the impacts that they may have on earthworm populations. The present study aims to assess the effects of the UV filter 4OHBP on two epigeic earthworm species: Dendrobaena veneta and Eisenia fetida. They usually live close to the surface and feed on the organic remains present in the most superficial layers of the soil. Moreover, their reproductive cycles are short when compared to other earthworm species. The specific objectives were: 1) to study the effects of acute direct exposure to 4-OHBP through a contact test; 2) to evaluate life history traits, such as mortality, growth and reproduction of earthworms living in soil contaminated by 4-OHBP through a soil test; 3) to compare the response of adult and juvenile earthworms and 4) to assess the sensitivity of different earthworm species to 4-OHBP under similar conditions.
21 ± 0.5 °C and food ad libitum (untreated horse manure defaunated by freezing and subsequent drying). 4-Hydroxibenzophenone (4-OHBP) was purchased from SIGMAALDRICH® (purity > 98%). This compound has a water solubility of 145 mg L−1 at 20 °C, a log Kow of 3.07 at 25 °C and a molecular weight of 198.22 (CAS No. 1137-42-4) Absolute ethanol was used to dissolve it previous to the addition of water (see below). All the experiments were protected from light by aluminium foil wrapping in order to prevent its hypothetical degradation by light, since transparent glass jars and Petri dishes were used for the experiments. 2.2. Contact toxicity test We studied the mortality effects of 4-OHBP when in direct contact with the epidermis of earthworms. For that purpose, we performed contact tests with adult and juvenile earthworms from our acclimated stocks, following the methodology proposed by the OECD (1984) and modified as described in Verdu et al. (2018). Weight ranges were 0.37–1.03 g and 0.022–0.14 g for D. veneta and 0.21–0.68 g and 0.08–0.2 g for E. fetida adults and juveniles, respectively. Two pieces of 9 cm diameter filter paper were placed in Petri dishes of the same size, and 1 ml of solution, including the corresponding concentration of 4OHBP solved in ethanol, was pipetted in (only ethanol in the case of negative control). Three concentrations of 4-OHBP were tested: 0.02, 0.2 and 2 mg ml−1, based on experience from Verdu et al. (2018). After the ethanol was evaporated in dark conditions, the filter papers were remoistened with 2 ml of distilled water, and one earthworm was introduced between the two papers per plate. Dishes were wrapped with aluminium foil and were introduced in a controlled temperature chamber at 21 °C in darkness. Mortality of earthworms was checked at 24 and 48 h. We included 10 replicates (one earthworm per replicate) with mature individuals and 10 replicates with juveniles per concentration and species. 2.3. Soil toxicity test We evaluated 4-OHBP effects on growth and reproduction of earthworms through a Soil Toxicity Test, following the methodology described in Verdu et al. (2018), modified from the OECD Soil Toxicity Test (OECD, 2016), in order to reach the most natural conditions possible, by adding 4-OHBP not only to the soil but also to the manure. The added manure served as food for the earthworms in all the described experiments. Each experimental unit (glass jar of 750 ml capacity) included 320 g of artificial soil mixture including 10% sphagnum peat, 20% kaolinite (white clay), 70% quartz sand and 1% Calcium Carbonate (CaCO3). The chemistry composition of this soil was 3.93% of organic C, 0.0863% of total organic N and 6.56 of pH. Furthermore, 30 g of untreated horse manure (dry weight), with a composition of 22.27% of organic C, 1.267% of N and 8.01 of pH, was added to the surface of the microcosms, thus simulating the real conditions in agricultural soils (Verdu et al., 2018). The powdered 4-OHBP was dissolved in 2 ml of ethanol, which was subsequently added to water, mixed and finally added to the dry soil mix described above (20% moisture). The same concentrations of 4-OHBP were added to the manure but dissolved in 500 μl of ethanol and mixed afterwards with water, in order to moisten this organic component at 70%. Once the jars were prepared, earthworms were introduced. Note that in order to avoid the increase in temperature caused by the activation of the bacterial flora with the possible lethal effects for earthworms (Díaz Cosín et al., 1996), the manure was moistened with half of the water for 48 h before preparing the experimental units. We tested five different concentrations (1, 10, 100, 1000 and 2000 mg of 4-OHBP per kg of soil), plus one negative control without the toxicant but including the same quantities of ethanol. It has been previously shown that the small quantities of ethanol used do not impact the earthworms (Verdu et al., 2018). Test concentrations were also selected following Verdu et al. (2018), which
2. Material and methods 2.1. Earthworms and UV filter Two species of epigeic earthworms, Eisenia fetida and Dendrobaena veneta, were used. Earthworms were obtained from our own laboratory cultures, previously genotyped (Verdu et al., 2018) to ensure genetic homogeneity. Before starting the experiment, all earthworms were kept in culture chambers under controlled temperature conditions of 2
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is one of the few studies where the effects of endocrine disruptors in earthworms have been tested. Six replicates per concentration were performed with each species (36 jars per species). Earthworms were previously acclimatized in control soil for two weeks, with a similar composition as in the control conditions of the experiment, including the manure on the surface. Six mature earthworms, previously weighed (D. veneta = 1.18 ± 0.10 g and E. fetida = 0.22 ± 0.02 g), were introduced per jar and kept in a controlled temperature chamber at 21 °C and in darkness. At 14 days, mortality was recorded and earthworms were weighed and reintroduced. At 28 days, the process was repeated, and adult earthworms were removed from the jars, leaving juveniles and cocoons inside. At day 56 (28 days from the adults' extraction), cocoons and juveniles were counted and weighed. The jars were exposed to bain-marie at 60 °C, over 30 min, so that the juveniles went out to the surface, and they could be extracted (OECD, 2016). Unhatched cocoons were searched with the help of a 2 mm mesh sieve and water. In the case of D. veneta, given that the reproduction of adults was not affected by the 4-OHBP, a subset of juveniles was exposed to the same concentrations of 4-OHBP as their progenitors in order to test long-term effects on their populations. Therefore, we firstly manually chose 10 juveniles with similar weights (0.025 ± 0.004 g) from each jar to be used in the juvenile experiment. Once those 10 juveniles were extracted, OECD protocol for juvenile extraction was followed, as explained above. New jars of 120 ml capacity were prepared with 40 g of dry soil and 3.75 g of manure in the same conditions as the previous experiment. The concentrations of 4-OHBP were the same as those used with the progenitors (i.e., the juveniles obtained from the earthworms exposed to 1 mg kg−1 were exposed to 1 mg kg−1 and so on), and the experiment was maintained for 56 days. The animals were weighed every 7 days.
parametric Kruskal-Wallis was carried out. For those contrasts showing significant differences (p-value < 0.05), pairwise comparisons were tested with a post hoc Conover test for multiple comparisons, which corrects the type I error with the Holm method. Statistical analyses were performed in SPSS v.24. Median lethal concentration (LC50) values for survival were calculated using probit analysis, and EC50 values were calculated using ATT Bioquest Quest Graph™ E50 calculator (https://www.aatbio.com/tools/ec50-calculator). 3. Results 3.1. Contact toxicity test All the individuals were dead at 24 h when exposed to the highest concentration of 4-OHBP (2 mg ml−1, Table 1). The second highest concentration tested (0.2 mg ml−1) showed mortality effects as well, statistically significant only for D. veneta juveniles (p < 0.05, Table 1). No statistically significant differences were found for the lowest concentration tested (0.02 mg ml−1, Table 1), although ca. 50% of juveniles were dead at 48 h. In fact, when analyses were performed by grouping Juveniles and Adults from both species, statistically significant differences (p < 0.05) were found between both stages at this concentration, at 24 and 48 h, indicating a higher mortality of juveniles. Moreover, juveniles showed statistically significant (p < 0.05) higher mortality for 0.2 mg ml−1 at 48 h. No significant differences were found between both species. 3.2. Soil toxicity test Regarding mortality (Fig. 1), no statistically significant differences were found for any of the species at days 14 or 28. However, for E. fetida, the mean mortality increased with the increase in 4-OHBP concentration (Fig. 1D, E, F). Statistically significant differences were found for this species in the second period of the test (14d–28d, Fig. 1E, KW = 12.831, d.f. = 5, p < 0.05). The Conover test indicated that the concentration of 2000 mg kg−1 provoked a significantly higher mortality than concentrations 0, 1, 10 and 100 mg kg−1, with no mortality. The concentration of 1000 mg kg−1 caused certain mortality but was not significantly different from any of the two groups (Fig. 1E). Nevertheless, and probably due to the high variability of the data, those differences were not detected at day 28, and therefore, we cannot conclude that 4-OHBP affected survival at the concentrations tested. The LC50 for E. fetida at 28 days was 1800.12 (95% CI: 93.94, > 10,000) mg kg−1. It was not possible to calculate the LC50 for D. veneta due to the lack of effect of 4-OHBP on the mortality of this species. Regarding the individual weight increase (Fig. 2), we found a weak but positive relation with 4-OHBP concentration for D. veneta at day 28 (Table 2, Fig. 2C). Statistically significant differences were found at that time point (ANOVA, F5,30 = 5.46, p < 0.01), and the highest concentration tested (2000 mg kg−1) implied a weight increase of D. veneta adults when compared to the control (mean weight increase of 60,9% and 40.96% respectively) Regarding reproductive success (Figs. 3 and
2.4. Statistical analyses For the contact test data, mortality was recorded at 24 and 48 h, and χ2 tests were performed. Tests were carried out for each combination of species (D. veneta and E. fetida) and stage (juveniles and adults) separately, while also comparing global numbers of juveniles vs. adults and both species responses. For the soil toxicity test we calculated the percentage of mortality and increase of weight per earthworm in each jar, in three different periods of time for adults (1–14 days, 14–28 days and 1–28 days) and each week for juveniles of D. veneta. The number of juveniles and unhatched cocoons produced at 56 days, as well as their weights, were analysed as variables for reproduction success. Normality and homoscedasticity of variables were checked with the Shapiro-Wilk and Levene tests, respectively. When those assumptions were not fulfilled by the data, transformations (log(x + 1)) were performed and checked. Other data transformations were checked but resulted unsuccessful. For those variables that met the assumptions, lineal regressions were performed. Moreover, in order to understand the differences in those variables among tested concentrations, one-way ANOVA was carried out with the concentration of toxicant as the independent factor. A Tukey test was used for post hoc comparisons when pvalue < 0.05. When data or transformations were not normal, a non-
Table 1 Results of contact toxicity test after acute direct exposure to 4-OHBP. Percentage of dead earthworms is indicated for juveniles and adults of both species at 24 and 48 h. Tested concentrations of 4-OHBP were 0 (control), 0.02, 0.2 and 2 mg ml−1. a/b/ab (within category) indicate significantly different categories according to χ2 test (p < 0.05). 24 h Control D. veneta E. fetida
Juveniles (N10) Adults (N10) Juveniles (N10) Adults (N10)
a
0 0a 10a 10a
0.02 a
10 0a 30a 0a
48 h 0.2 b
70 50ab 50ab 10a
3
2
Control b
100 100b 100b 100b
a
10 0a 20a 10a
0.02 ab
50 10a 40a 0a
0.2 b
80 50ab 70ab 30a
2 100b 100b 100b 100b
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Fig. 1. Box plots representing mortality percentage of Dendrobaena veneta (A, B, C) and Eisenia fetida (D, E, F), for each level of concentration (mg kg−1) of 4-OHBP. A/D: From day 0 to day 14 of experiment. B/E: From day 14 to day 28 of experiment. C/F: From day 0 to day 28 of experiment. Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisk indicates significant result. Letters (a, b) indicate homogeneous groups according to Conover test.
Fig. 2. Box plots representing weight percentage increase per earthworm of Dendrobaena veneta (A, B, C) and Eisenia fetida (D, E, F), for each level of concentration (mg kg−1) of 4-OHBP. A/D: From day 0 to day 14 of experiment. B/E: From day 14 to day 28 of experiment. C/F: From day 0 to day 28 of experiment. Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisk indicates significant result. Letters (a, b) indicate homogeneous groups according to Tukey test.
4
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Table 2 Significant linear regression models (p < 0.05) for Dendrobaena veneta and Eisenia fetida variables measured in the soil toxicity test. Independent variable is concentration of 4-OHBP: [cc].
D. veneta E. fetida
Linear regression model
p-Value
% R2
β
%Δweight/earthworm 28 days = 41.141 + 0.006*[cc] Number of juveniles = 72.291–0.029[cc] Number of cocoons = 14.818–0.006[cc] log (weight per cocoon +1) = 0.002–0.0000008[cc]
0.043 0.001 < 0.001 0.001
11.5 28.9 31.2 28.2
0.339 −0.538 −0.558 −0.531
4), for E. fetida the number of juveniles and cocoons at the 56th day of the experiment showed strong negative relationships with 4-OHBP concentration (Fig. 3C, D), as shown by linear regression analyses (Table 2). Calculated EC50 values were 152.68 mg kg−1 (number of juveniles), 94.05 mg kg−1 (number of unhatched cocoons) and 145.52 mg kg−1 (total production: number of juveniles plus number of unhatched cocoons). Statistically significant differences were found among treatments in the number of juveniles (Fig. 3C: ANOVA, F5,30 = 9.38, p < 0.001) and the number of unhatched cocoons (Fig. 3D: ANOVA, F5,30 = 6.71, p < 0.001) that were significantly lower for the concentrations of 1000 and 2000 mg kg−1. Moreover, a negative relationship was detected by linear regression analysis between weight per cocoon and 4-OHBP concentration (Table 2), and significant differences among treatments were found (Fig. 4D: ANOVA, F5,30 = 18,27, p < 0.001). Concentrations above 10 mg kg−1 implied a lower weight per cocoon. No effect of 4-OHBP concentration on weight per juvenile was detected (Fig. 4C). For D. veneta, no significant relationships or differences were found in reproductive success measurements (Figs. 3A, B, 4 A, B), and no EC50 values could be calculated. For the latter, a subset of juveniles was exposed to the same concentrations of 4-OHBP as their progenitors. Fig. 5A represents the percentage of accumulated mortality during this experiment, showing
that differences could already be detected in the first week. From day 14, all the individuals exposed to the highest concentrations (100, 1000 and 2000 mg kg−1) were dead, except one single earthworm at 2000 mg kg−1 that died at 21 days. Kruskal-Wallis results from the time points of 7 days (Fig. 5C) and 14 days indicate that there is a significantly higher mortality of D. veneta juveniles at the three highest concentrations of 4-OHBP (7 days: KW = 17.6303, d.f. = 5, p < 0.005; 14 days: KW = 26.064, d.f. = 5, p < 0.001). According to the Conover test, the concentration of 10 mg kg−1 also shows a difference with the control at the 7th day (Fig. 5C). The LC50 for D. veneta juveniles after 7 days of exposure was 72.02 (95% CI: 13.67, 379.49) mg kg−1, and after 14 days, it was 19.49 (95% CI: 2.9, 131.1) mg kg−1. Results of the growth for exposed juveniles of D. veneta are shown in Fig. 4B, represented as the percentage of accumulated weight per earthworm. Kruskal Wallis analyses did not show significant results at any of the time points (see results at day 7 in Fig. 4D). 4. Discussion In this study, we have evaluated the impact of the UV filter 4-OHBP on edaphic fauna, through the assessment of mortality, growth, reproduction and juvenile performance for two epigeic earthworms. Our
Fig. 3. Box plots representing the number of juveniles (A, C) and cocoons (B, D) for both species Dendrobaena veneta (A, B) and Eisenia fetida (C, D), for each concentration of 4-OHBP (mg kg−1). Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisks indicate significant results. Letters (a, b, c) indicate homogeneous groups according to Tukey test. 5
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Fig. 4. Box plots representing the weight per juvenile (A, C) and weight per unhatched cocoon (B, D) for both species Dendrobaena veneta (A, B) and Eisenia fetida (C, D), for each concentration of 4-OHBP (mg kg−1). Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisk indicates significant result. Letters (a, b, c, d) indicate homogeneous groups according to Tukey test (ANOVA performed with log (x + 1)).
results indicate that the acute exposure through a direct contact of 4OHBP with the epidermis of earthworms causes their mortality at concentrations of 0.2–2 mg ml−1, suggesting that this substance is harmful for these animals (Table 1).The juveniles were shown to be slightly more sensitive to 4-OHBP in the direct contact test. A higher sensitivity of juveniles has been observed in many works of earthworms exposed to other compounds (e.g., Lowe and Butt, 2005; Pelosi et al., 2013; Spurgeon et al., 2004; Zhou et al., 2008). Immature individuals may not have fully developed their detoxification mechanisms as suggested by biomarker responses after exposure to insecticides (Booth and O’Halloran, 2001). Another possible reason accounting for the higher sensitivity of juveniles may be their smaller body size. Due to their higher surface-area-to-volume ratio, juveniles probably assimilate a higher relative amount of the xenobiotic. Some studies prove that the body size is one of the factors explaining differences of sensitivity in earthworms (Łaszczyca et al., 2004; Schreck et al., 2008). A faster metabolism and higher cellular turnover of juveniles may be another reason that cannot be discarded. For the soil tests, which are more representative of the natural environment, much higher concentrations of 4-OHBP were needed in order to see impacts, as expected from previous studies (Verdu et al., 2018; Wang et al., 2012a, 2012b). The potential adsorption of the toxicant to the soil colloids reduces the direct exposure of the earthworms and attributes a buffering effect to the soil (Miguel and Martí, 2011). Our results for exposures of mature earthworms indicate that D. veneta adults are more resistant to 4-OHBP, whereas E. fetida adults show a higher sensitivity (Figs. 1–4). This is demonstrated by the fact that there were no negative impacts for any of the D. veneta recordings, whereas reproduction success of E. fetida was impacted (see below). Newman et al. (2000) and Dittbrenner et al. (2011), already highlighted the importance of evaluating exposures in different species of the same group without relying on the results from a single species,
because the impacts of the same substance may differ even within the same ecological category as our results show. A difference of sensitivity to toxicants between these two species has been already proposed by Verdu et al. (2018) who found D. veneta to be more resistant to Bisphenol A than E. fetida, similar to our results. Sensitivity can vary between close species, since they may present slightly different metabolic rates, detoxification mechanisms or abilities to bind xenobiotics (Fitzgerald et al., 1996; Givaudan et al., 2014; Ma and Bodt, 1993; Pelosi et al., 2013). Moreover, as mentioned above referring to the higher sensitivity of juveniles, the difference in size could be another explanation, since individuals of D. veneta (1.18 + −0.1 g) were slightly bigger than E. fetida (0.22 + −0.02 g). We found significantly higher mortality for E. fetida during the second period of the test (14–28 days) at the highest concentration tested (2000 mg kg−1, Fig. 1). However, this difference was not statistically significant at the end of the test's 28 days, probably due to the high variability of the data. Nevertheless this could indicate that if longer tests were carried out those differences may be reflected in the end. Regarding reproductive success, our results suggest that there are negative relationships between the concentration of 4-OHBP and the number of juveniles (significantly lower from 1000 mg kg−1, Fig. 3C), the number of unhatched cocoons (significantly lower from 100 mg kg−1, Fig. 3D) and the weight per cocoon (significantly lower from 10 mg kg−1, Fig. 4D) of E. fetida. A lighter weight of cocoons produced by earthworms exposed to contaminants has been previously found (Dominguez et al., 2016) and discussed as detrimental, since they may be associated to weaker juveniles. Schmitt et al. (2008) also observed a negative effect of a similar UV filter (BP-3) in the reproduction of the Californian blackworm (L. variegatus). Unfortunately, there is a lack of references on the effect of benzophenones in earthworms with which to compare. Meanwhile D. veneta adults (F0) were unaffected by the 4-OHBP 6
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Fig. 5. Mortality and weight data for juvenile individuals (transgenerational study) of Dendrobaena veneta after exposure to different concentrations of 4-OHBP (0, 1, 10, 100, 1000 and 2000 mg kg−1). A: Accumulated mortality (%) during 8 weeks of soil test. B: Accumulated weight increase per earthworm (%) during 8 weeks of soil test; jars with 50% or higher mortality were removed from B graph, since growth presented high variability in comparison with the rest within their treatment. C: Box plot of accumulated mortality (%) at day 7. D: Box plot of increase of weight (%) at day 7. Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisk indicates significant result. Letters (a, b, c) indicate homogeneous groups according to Conover test.
All these values are far from the 500 to 18,380 ng kg−1reported in Korean soils by Jeon et al. (2006). Nevertheless, more studies are needed for assessing the concentrations of UV filters in soils, since the information is still scarce and biased. There is also a gap in the existing literature in relation to the adsorption of 4-OHBP to the soils, degradation or bioaccumulation. Moreover, further studies in more natural conditions could shed some light on the environmentally relevant effects of 4-OHBP. Earthworm species of different ecological categories may show different reactions because they feed differently, and the organic matter content and microorganism diversity may have an effect on the bioavailability of the toxicant. The toxicity of any chemical substance in the soil is closely related to environmental variables and physicochemical properties (principally organic matter and clay content, Miguel and Martí, 2011). There is not much data on biotic degradation, but given the log Kow value of 4-OHBP (3.07), organic matter content may not have a great influence on its availability because of the low adsorption expected. It is worth mentioning that the feeding conditions the OECD recommends do not include, in our experience, enough organic matter for the well-being of the earthworms. Starvation has been linked to higher chemical toxicity (Holmstrup et al., 2010; Warne et al., 2001), and the effect of the amount of organic matter on toxicity to earthworms has been discussed (Irizar et al., 2015). Therefore, even though the amount of organic matter may not
exposed juveniles (F1) of the same species, shown to be impacted by this UV filter, showing a higher mortality at 10 mg kg−1 and higher concentrations (Fig. 5C) and therefore, showing more sensitivity than adults of both species. The potential causes, such as possible differences in detoxification mechanisms, extensive cell division during growth or differences in size were already mentioned above. Moreover, the exposure of adults (F0) as well as cocoons could have also affected this second generation of earthworms. We demonstrate here, the importance of transgenerational studies for understanding the long term true impact of toxicants for the populations, since the adult (F0) test could give a false impression. It needs to be noted that manipulation of juveniles every week for weighting purposes may have caused some mortality, as observed, for example, in the control after the 4th week. However, the mortality observed at the highest concentrations of the UV filter occurred the first two weeks, when the control worms seemed to be almost unaffected. Even though we found some effects of 4-OHBP on the studied earthworms, the potentially harmful concentrations are much higher than those reported in the environment. For E. fetida, the LC50 at 28 days was 1800.12 mg kg−1 and the EC50s were 152.68 mg kg−1 (juveniles) and 94.05 mg kg−1 (unhatched cocoons). The first generation of D. veneta was unaffected and the LC50 for second generation juveniles was 72.02 mg kg−1 at 7 days and19.49 mg kg−1 at 14 days. 7
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modify availability of 4-OHBP, it could be expected that a lower organic matter content, as suggested by the OECD, may produce a higher toxicity due to starvation effects. All said, and basing on the currently available information, we must assume that 4-OHBP does not represent a risk or has very low toxicity for earthworm populations. Nevertheless, additional research, including longer term transgenerational studies, will be needed to confirm it.
Irizar, A., Rodriguez, M.P., Izquierdo, A., Cancio, I., Marigomez, I., Soto, M., 2015. Effects of soil organic matter content on cadmium toxicity in Eisenia fetida: implications for the use of biomarkers and standard toxicity tests. Arch. Environ. Con. Tox. 68, 181–192. Jeon, H.K., Chung, Y., Ryu, J.C., 2006. Simultaneous determination of benzophenonetype UV filters in water and soil by gas chromatography-mass spectrometry. J. Chromatogr. A 1131, 192–202. Kaiser, D., Sieratowicz, A., Zielke, H., Oetken, M., Hollert, H., Oehlmann, J., 2012. Ecotoxicological effect characterisation of widely used organic UV filters. Environ. Pollut. 163, 84–90. Kim, S., Jung, D., Kho, Y., Choi, K., 2014. Effects of benzophenone-3 exposure on endocrine disruption and reproduction of Japanese medaka (Oryzias latipes)-a two generation exposure study. Aquat. Toxicol. 155, 244–252. Łaszczyca, P., Augustyniak, M., Babczyńska, A., Bednarska, K., Kafel, A., Migula, P., Wilczek, G., Witas, I., 2004. Profiles of enzymatic activity in earthworms from zinc, lead and cadmium polluted areas near Olkusz (Poland). Environ. Int. 30, 901–910. Laycock, A., Diez-Ortiz, M., Larner, F., Dybowska, A., Spurgeon, D., Valsami-Jones, E., Rehkamper, M., Svendsen, C., 2016. Earthworm uptake routes and rates of ionic Zn and ZnO nanoparticles at realistic concentrations, traced using stable isotope labeling. Environ. Sci. Technol. 50, 412–419. Lewis, P.R., 2016. Forensic Polymer Engineering: Why Polymer Products Fail in Service, 2 ed. Woodhead Publishing. Lowe, C.N., Butt, K.R., 2005. Culture techniques for soil dwelling earthworms: a review. Pedobiologia 49, 401–413. Ma, W.c., Bodt, J., 1993. Differences in toxicity of the insecticide chlorpyrifos to six species of earthworms (Oligochaeta, Lumbricidae) in standardized soil tests. Bull. Environ. Contam. Toxicol. 50, 864–870. Miguel, A., Martí, C., 2011. Principios de ecotoxicología: Diagnóstico, tratamiento y gestión del medio ambiente. (Editorial Tébar). Newman, M.C., Ownby, D.R., Mezin, L.C.A., Powell, D.C., Christensen, T.R.L., Lerberg, S.B., Anderson, B.A., 2000. Applying species-sensitivity distributions in ecological risk assessment: assumptions of distribution type and sufficient numbers of species. Environ. Toxicol. Chem. 19, 508–515. OECD, 1984. OECD 207 - Earthworm, Acute Toxicity Tests. OECD Guideline for Testing of Chemicals. OECD, 2016. OECD 222- Earthworm Reproduction Test (Eisenia Fetida/Eisenia Andrei), OECD Guidelines for the Testing of Chemicals. Ozaez, I., Martinez-Guitarte, J.L., Morcillo, G., 2013. Effects of in vivo exposure to UV filters (4-MBC, OMC, BP-3, 4-HB, OC, OD-PABA) on endocrine signaling genes in the insect Chironomus riparius. Sci. Total Environ. 456, 120–126. Pelosi, C., Joimel, S., Makowski, D., 2013. Searching for a more sensitive earthworm species to be used in pesticide homologation tests – a meta-analysis. Chemosphere 90, 895–900. Phipps, G.L., Ankley, G.T., Benoit, D.A., Mattson, V.R., 1993. Use of the aquatic oligochaete Lumbriculus variegatus for assessing the toxicity and bioaccumulation of sediment-associated contaminants. Environ. Toxicol. Chem. 12, 269–279. Schmitt, C., Oetken, M., Dittberner, O., Wagner, M., Oehlmann, J., 2008. Endocrine modulation and toxic effects of two commonly used UV screens on the aquatic invertebrates Potamopyrgus antipodarum and Lumbriculus variegatus. Environ. Pollut. 152, 322–329. Schreck, E., Geret, F., Gontier, L., Treilhou, M., 2008. Neurotoxic effect and metabolic responses induced by a mixture of six pesticides on the earthworm Aporrectodea caliginosa nocturna. Chemosphere 71, 1832–1839. Spurgeon, D.J., Svendsen, C., Kille, P., Morgan, A.J., Weeks, J.M., 2004. Responses of earthworms (Lumbricus rubellus) to copper and cadmium as determined by measurement of juvenile traits in a specifically designed test system. Ecotoxicol. Environ. Saf. 57, 54–64. Urbach, F., 2001. The historical aspects of sunscreens. J. Photochem. Photobiol. B 64, 99–104. Verdu, I., Trigo, D., Martinez-Guitarte, J.L., Novo, M., 2018. Bisphenol A in artificial soil: effects on growth, reproduction and immunity in earthworms. Chemosphere 190, 287–295. Wang, Y., Cang, T., Zhao, X., Yu, R., Chen, L., Wu, C., Wang, Q., 2012a. Comparative acute toxicity of twenty-four insecticides to earthworm, Eisenia fetida. Ecotoxicol. Environ. Saf. 79, 122–128. Wang, Y., Wu, S., Chen, L., Wu, C., Yu, R., Wang, Q., Zhao, X., 2012b. Toxicity assessment of 45 pesticides to the epigeic earthworm Eisenia fetida. Chemosphere 88, 484–491. Warne, M.A., Lenz, E.M., Osborn, D., Weeks, J.M., Nicholson, J.K., 2001. Comparative biochemistry and short-term starvation effects on the earthworms Eisenia veneta and Lumbricus terrestris studied by H-1 NMR spectroscopy and pattern recognition. Soil Biol. Biochem. 33, 1171–1180. Weisbrod, C.J., Kunz, P.Y., Zenker, A.K., Fent, K., 2007. Effects of the UV filter benzophenone-2 on reproduction in fish. Toxicol. Appl. Pharmacol. 225, 255–266. Wypych, G., 2015. Handbook of UV Degradation and Stabilization, Second Edition. ChemTech Publishing. Yasmin, S., Souza, D., 2010. Effects of pesticides on the growth and reproduction of earthworm: a review. Appl. Environ. Soil Sci. 2010:(JD 678360). Zhou, S., Duan, C., Wang, X., Michelle, W.H.G., Yu, Z., Fu, H., 2008. Assessing cypermethrin-contaminated soil with three different earthworm test methods. J. Environ. Sci. 20, 1381–1385.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We are grateful to Irene Verdú for guidance on the experiments, Jorge Domínguez for providing earthworms, to the team from the Soil Zoology Group from Complutense University of Madrid (UCM), for their support in the lab and to Antón Álvarez for providing help in the statistical analyses. MN was supported by a Postdoctoral Fellowship (FPDI-2016-16407) from the Spanish Government and a UCM Postdoctoral Fellowship. This study was funded by the grants: CTM2015-64913-R and CGL2013-42908-P from the Spanish Government. References Booth, L.H., O’Halloran, K., 2001. A comparison of biomarker responses in the earthwormAporrectodea caliginosato the organophosphorus insecticides diazinon and chlorpyrifos. Environ Toxicol Chem. 20 (11), 2494–2502. https://doi.org/10. 1002/etc.5620201115. Chisvert, A., Salvador, A., 2007. UV filters in sunscreens and other cosmetics. Regulatory aspects and analytical methods. In: Salvador, A., Chisvert, A. (Eds.), Analysis of Cosmetic Products. Elsevier, Amsterdam, pp. 83–120. Díaz Cosín, D.J., Moro, R.P., Valle, J.V., Garvín, M.H., Trigo, D., Jesús, J.B., 1996. Producciín de heces de Hormogaster elisae úlvarez, 1977 (Oligochaeta, Hormogastridae) en diferentes tipos de cultivos en laboratorio. Bol. R. Soc. Espaóola Hist. Nat. (Biol.) 92, 177–184. Diogene, J., Dufour, M., Poirier, G.G., Nadeau, D., 1997. Extrusion of earthworm coelomocytes: comparison of the cell populations recovered from the species Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum. Lab. Anim. Uk 31, 326–336. Dittbrenner, N., Schmitt, H., Capowiez, Y., Triebskorn, R., 2011. Sensitivity of Eisenia fetida in comparison to Aporrectodea caliginosa and Lumbricus terrestris after imidacloprid exposure. Body mass change and histopathology. J. Soils Sediments 11, 1000–1010. Domínguez, J., Aira, M., Gómez Brandón, M., 2009. El papel de las lombrices de tierra en la descomposición de la materia orgánica y el ciclo de nutrientes. Ecosistemas 18, 20–31. Dominguez, A., Brown, G.G., Sautter, K.D., de Oliveira, C.M., de Vasconcelos, E.C., Niva, C.C., Bartz, M.L., Bedano, J.C., 2016. Toxicity of AMPA to the earthworm Eisenia andrei bouche, 1972 in tropical artificial soil. Sci. Rep. 6, 19731. Fitzgerald, D.G., Warner, K.A., Lanno, R.P., Dixon, D.G., 1996. Assessing the effects of modifying factors on pentachlorophenol toxicity to earthworms: applications of body residues. Environ. Toxicol. Chem. 15, 2299–2304. Gago-Ferrero, P., Diaz-Cruz, M.S., Barcelo, D., 2011. Occurrence of multiclass UV filters in treated sewage sludge from wastewater treatment plants. Chemosphere 84, 1158–1165. Givaudan, N., Binet, F., Le Bot, B., Wiegand, C., 2014. Earthworm tolerance to residual agricultural pesticide contamination: field and experimental assessment of detoxification capabilities. Environ. Pollut. 192, 9–18. Harrison, E.Z., Oakes, S.R., Hysell, M., Hay, A., 2006. Organic chemicals in sewage sludges. Sci. Total Environ. 367, 481–497. Holmstrup, M., Bindesbol, A.M., Oostingh, G.J., Duschl, A., Scheil, V., Kohler, H.R., Loureiro, S., Soares, A.M.V.M., Ferreira, A.L.G., Kienle, C., Gerhardt, A., Laskowski, R., Kramarz, P.E., Bayley, M., Svendsen, C., Spurgeon, D.J., 2010. Interactions between effects of environmental chemicals and natural stressors: a review. Sci. Total Environ. 408, 3746–3762. Iribarne, L.M., 2016. Cuantificación de contaminantes con actividad disruptora endocrina en leche materna: puesta a punto de la metodología analítica y resultados preliminares. Universidad de Granada, Spain, pp. 99.
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When sunscreens reach the soil: Impacts of a UV filter on the life cycle of earthworms Silvia Casqueroa, Dolores Trigoa, Jose L. Martínez Guitarteb, Marta Novoa,b,* a b
Biodiversity, Ecology and Evolution Department, Faculty of Biology, Complutense University of Madrid, Spain Environmental Toxicology and Biology Group, Sciences Faculty, UNED, Spain
ARTICLE INFO
ABSTRACT
Keywords: Ecotoxicology Survival Reproduction Xenobiotics Endocrine disruptors 4-hydroxybenzophenone
4-hydroxybenzophenone (4-OHBP) is a UV filter used in sunscreens, perfumery and containers for food products that may end up in soil. The impacts of 4-OHBP in earthworms (Eisenia fetida and Dendrobaena veneta) were investigated. To prove the consequence of its direct application on earthworms' epidermis, a contact test (48 h) was performed. Significant mortality was already observed at 4-OHBP concentrations of 2 mg ml−1 at 24 h, and juveniles showed higher mortality than matures at 0.2 and 0.02 mg ml−1. In order to investigate the effects in their natural habitat, a soil test was performed with mature earthworms. Results showed that reproduction success of E. fetida was impacted by the toxicant. The number of hatched juveniles (EC50 = 152.68 mg kg−1) and the number (EC50 = 94.05 mg kg−1) and weight of unhatched cocoons decreased with an increasing concentration of 4-OHBP. The LC50 for E. fetida at 28 days was 1800.12 mg kg−1, but LC50 and EC50 values could not be calculated for D. veneta adults because of lack of effect. For the latter, the soil test was continued with juveniles in a transgenerational study showing that their mortality increased with 4-OHBP concentrations above 10 mg kg−1 (LC50 = 72.02 mg kg−1 at 7 days and 19.49 mg kg−1 at 14 days). Results showed that 4-OHBP is harmful for both species, by causing reproduction decrease for E. fetida adults (F0) and mortality increase for D. veneta juveniles (F1). However, concentrations at which 4-OHBP affects earthworm populations are much higher than those reported in the environment, and therefore, there seems to be no risk for them, except in case of accidental spill.
1. Introduction Ultraviolet (UV) filters are a group of synthetic compounds that act as barriers against UV radiation. Their use in cosmetics and creams has been intensified in the last years because of the increase of UV radiation and associated health damages. UV filters are not only added to sunscreens, but also to food packaging and perfumery products in order to avoid their odour and colour degradation (Lewis, 2016; Wypych, 2015). They can be divided into two types depending on their properties. Physical filters, such as zinc oxide and titanium oxide, work by diffraction and dispersion of the rays. Chemical filters work by absorbing radiation, and they present one or various aromatic structures with double bonds and/or conjugated with carbonyl radicals, which makes them highly lipophilic (Ozaez et al., 2013). The latter are classified in different families based on their molecular structure, such as pamino benzoates, camphors or benzophenones, among others (Chisvert and Salvador, 2007). Benzophenones are present in a diversity of products, from plastic containers to perfumes, but they are mainly part of
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the widely used sunscreens (Chisvert and Salvador, 2007; Urbach, 2001). Benzophenones are a family of aromatic ketones with the capacity of absorbing UV radiation (λ = 280–400 nm) and dissipate it as heat (Iribarne, 2016). A growing number of studies, mainly focused on vertebrates, prove that certain benzophenones act as endocrine disruptors, evidencing the importance of investigating the effects of these compounds in living beings. For example, Weisbrod et al. (2007) showed that 2-benzophenone (2-BP) inhibited the development of gametes in fathead minnows (Pimephales promelas) and caused a decrease in egg production and cessation of spawning activity. Egg production was also decreased for Japanese medaka (Oryzias latipes) after exposure to 3-benzophenone (3BP) (Kim et al., 2014). Fewer studies have shown the effects of chemical UV filters in invertebrates and sediments. Schmitt et al. (2008) tested 3benzylidene-camphor (3-BC) and 4-MBC in the freshwater oligochaete Lumbriculus variegatus that showed a decrease in reproduction after 28 days (EC50 of 1.43 mg kg−1 for 3-BC) and in the snail Potamopyrgus antipodarum, whose mortality increased after 56 days at 32 mg kg−1 but
Corresponding author at: Biodiversity, Ecology and Evolution Department, Faculty of Biology, Complutense University of Madrid, Spain. E-mail address: [email protected] (M. Novo).
https://doi.org/10.1016/j.apsoil.2019.09.004 Received 16 March 2019; Received in revised form 6 September 2019; Accepted 8 September 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Silvia Casquero, et al., Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2019.09.004
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also showed an increase in embryo production at lower concentrations (6–7 mg kg−1). Kaiser et al. (2012) showed that the filter EHMC caused a toxic reproduction effect in two snails, P. antipodarum (at 0.4 mg kg−1 after 56 days) and Melanoides tuberculata (at 10 mg kg−1 after 28 days), but no effect was found for the annelid L. variegatus (tested concentrations up to 50 mg kg−1 after 28 days) or other tested UV filters. Unfortunately, there is a lack of studies on the effects of organic UV filters in soil-dwelling animals. Most of these studies have been carried out in aquatic ecosystems because of their connection with sunscreens, since the aquatic leisure activities are relevant in UV filters distribution. On the other hand, sewage sludge generated in the sewage treatment plants is used for the fertilisation of farmlands after being processed. UV filters, being quite novel xenobiotics, are not properly removed during the waste treatments (Harrison et al., 2006). In fact, a study done in 15 sewage treatment plants of Catalonia (Spain) proved the presence of several UV filters in the local sludge, although at low concentrations (0.04–9.17 mg kg−1; Gago-Ferrero et al., 2011). Another way to enter the edaphic ecosystems would be through irrigation water, since the use of treated sewage water in farmlands is habitual in many countries. Studies on concentrations of UV filters in soils are scarce. Jeon et al. (2006) showed that concentrations of UV filters in soil ranged from 500 to 18,380 ng kg−1, while in water, they ranged from 27 to 204 ng L−1, in Korea. However, there is not much information on the biodegradation or accumulation potential of these compounds in soil. Earthworms constitute up to 80% of the zoomass in soil, reaching 5000 kg/ha (Diogene et al., 1997; Yasmin and Souza, 2010). They represent the main engineers of the edaphic system by improving percolation, aeration and aggregate stabilisation and favouring the processing of organic matter in the surface layer, thus increasing fertility (Domínguez et al., 2009). Moreover, they are an important part of the food chain, so both their extinction in contaminated areas and the accumulation of toxicants in their bodies may have fatal consequences for ecosystems. Earthworms can be impacted by the harmful substances present in soil, either by the contact through their epidermis or by ingestion (Laycock et al., 2016; Phipps et al., 1993). Different species sensitivity to toxicants has been suggested for earthworms, even within the same ecological category (e.g. Ma and Bodt, 1993; Verdu et al., 2018), which makes interesting, the use of more than one species for tests in order to make robust conclusions. Given the increasing use and release of emergent xenobiotics, such as organic UV filters in the environment, it is extremely important to understand how they affect different organisms, especially those that are ecologically relevant, such as earthworms. The obtained results will shed some light on the impacts that they may have on earthworm populations. The present study aims to assess the effects of the UV filter 4OHBP on two epigeic earthworm species: Dendrobaena veneta and Eisenia fetida. They usually live close to the surface and feed on the organic remains present in the most superficial layers of the soil. Moreover, their reproductive cycles are short when compared to other earthworm species. The specific objectives were: 1) to study the effects of acute direct exposure to 4-OHBP through a contact test; 2) to evaluate life history traits, such as mortality, growth and reproduction of earthworms living in soil contaminated by 4-OHBP through a soil test; 3) to compare the response of adult and juvenile earthworms and 4) to assess the sensitivity of different earthworm species to 4-OHBP under similar conditions.
21 ± 0.5 °C and food ad libitum (untreated horse manure defaunated by freezing and subsequent drying). 4-Hydroxibenzophenone (4-OHBP) was purchased from SIGMAALDRICH® (purity > 98%). This compound has a water solubility of 145 mg L−1 at 20 °C, a log Kow of 3.07 at 25 °C and a molecular weight of 198.22 (CAS No. 1137-42-4) Absolute ethanol was used to dissolve it previous to the addition of water (see below). All the experiments were protected from light by aluminium foil wrapping in order to prevent its hypothetical degradation by light, since transparent glass jars and Petri dishes were used for the experiments. 2.2. Contact toxicity test We studied the mortality effects of 4-OHBP when in direct contact with the epidermis of earthworms. For that purpose, we performed contact tests with adult and juvenile earthworms from our acclimated stocks, following the methodology proposed by the OECD (1984) and modified as described in Verdu et al. (2018). Weight ranges were 0.37–1.03 g and 0.022–0.14 g for D. veneta and 0.21–0.68 g and 0.08–0.2 g for E. fetida adults and juveniles, respectively. Two pieces of 9 cm diameter filter paper were placed in Petri dishes of the same size, and 1 ml of solution, including the corresponding concentration of 4OHBP solved in ethanol, was pipetted in (only ethanol in the case of negative control). Three concentrations of 4-OHBP were tested: 0.02, 0.2 and 2 mg ml−1, based on experience from Verdu et al. (2018). After the ethanol was evaporated in dark conditions, the filter papers were remoistened with 2 ml of distilled water, and one earthworm was introduced between the two papers per plate. Dishes were wrapped with aluminium foil and were introduced in a controlled temperature chamber at 21 °C in darkness. Mortality of earthworms was checked at 24 and 48 h. We included 10 replicates (one earthworm per replicate) with mature individuals and 10 replicates with juveniles per concentration and species. 2.3. Soil toxicity test We evaluated 4-OHBP effects on growth and reproduction of earthworms through a Soil Toxicity Test, following the methodology described in Verdu et al. (2018), modified from the OECD Soil Toxicity Test (OECD, 2016), in order to reach the most natural conditions possible, by adding 4-OHBP not only to the soil but also to the manure. The added manure served as food for the earthworms in all the described experiments. Each experimental unit (glass jar of 750 ml capacity) included 320 g of artificial soil mixture including 10% sphagnum peat, 20% kaolinite (white clay), 70% quartz sand and 1% Calcium Carbonate (CaCO3). The chemistry composition of this soil was 3.93% of organic C, 0.0863% of total organic N and 6.56 of pH. Furthermore, 30 g of untreated horse manure (dry weight), with a composition of 22.27% of organic C, 1.267% of N and 8.01 of pH, was added to the surface of the microcosms, thus simulating the real conditions in agricultural soils (Verdu et al., 2018). The powdered 4-OHBP was dissolved in 2 ml of ethanol, which was subsequently added to water, mixed and finally added to the dry soil mix described above (20% moisture). The same concentrations of 4-OHBP were added to the manure but dissolved in 500 μl of ethanol and mixed afterwards with water, in order to moisten this organic component at 70%. Once the jars were prepared, earthworms were introduced. Note that in order to avoid the increase in temperature caused by the activation of the bacterial flora with the possible lethal effects for earthworms (Díaz Cosín et al., 1996), the manure was moistened with half of the water for 48 h before preparing the experimental units. We tested five different concentrations (1, 10, 100, 1000 and 2000 mg of 4-OHBP per kg of soil), plus one negative control without the toxicant but including the same quantities of ethanol. It has been previously shown that the small quantities of ethanol used do not impact the earthworms (Verdu et al., 2018). Test concentrations were also selected following Verdu et al. (2018), which
2. Material and methods 2.1. Earthworms and UV filter Two species of epigeic earthworms, Eisenia fetida and Dendrobaena veneta, were used. Earthworms were obtained from our own laboratory cultures, previously genotyped (Verdu et al., 2018) to ensure genetic homogeneity. Before starting the experiment, all earthworms were kept in culture chambers under controlled temperature conditions of 2
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is one of the few studies where the effects of endocrine disruptors in earthworms have been tested. Six replicates per concentration were performed with each species (36 jars per species). Earthworms were previously acclimatized in control soil for two weeks, with a similar composition as in the control conditions of the experiment, including the manure on the surface. Six mature earthworms, previously weighed (D. veneta = 1.18 ± 0.10 g and E. fetida = 0.22 ± 0.02 g), were introduced per jar and kept in a controlled temperature chamber at 21 °C and in darkness. At 14 days, mortality was recorded and earthworms were weighed and reintroduced. At 28 days, the process was repeated, and adult earthworms were removed from the jars, leaving juveniles and cocoons inside. At day 56 (28 days from the adults' extraction), cocoons and juveniles were counted and weighed. The jars were exposed to bain-marie at 60 °C, over 30 min, so that the juveniles went out to the surface, and they could be extracted (OECD, 2016). Unhatched cocoons were searched with the help of a 2 mm mesh sieve and water. In the case of D. veneta, given that the reproduction of adults was not affected by the 4-OHBP, a subset of juveniles was exposed to the same concentrations of 4-OHBP as their progenitors in order to test long-term effects on their populations. Therefore, we firstly manually chose 10 juveniles with similar weights (0.025 ± 0.004 g) from each jar to be used in the juvenile experiment. Once those 10 juveniles were extracted, OECD protocol for juvenile extraction was followed, as explained above. New jars of 120 ml capacity were prepared with 40 g of dry soil and 3.75 g of manure in the same conditions as the previous experiment. The concentrations of 4-OHBP were the same as those used with the progenitors (i.e., the juveniles obtained from the earthworms exposed to 1 mg kg−1 were exposed to 1 mg kg−1 and so on), and the experiment was maintained for 56 days. The animals were weighed every 7 days.
parametric Kruskal-Wallis was carried out. For those contrasts showing significant differences (p-value < 0.05), pairwise comparisons were tested with a post hoc Conover test for multiple comparisons, which corrects the type I error with the Holm method. Statistical analyses were performed in SPSS v.24. Median lethal concentration (LC50) values for survival were calculated using probit analysis, and EC50 values were calculated using ATT Bioquest Quest Graph™ E50 calculator (https://www.aatbio.com/tools/ec50-calculator). 3. Results 3.1. Contact toxicity test All the individuals were dead at 24 h when exposed to the highest concentration of 4-OHBP (2 mg ml−1, Table 1). The second highest concentration tested (0.2 mg ml−1) showed mortality effects as well, statistically significant only for D. veneta juveniles (p < 0.05, Table 1). No statistically significant differences were found for the lowest concentration tested (0.02 mg ml−1, Table 1), although ca. 50% of juveniles were dead at 48 h. In fact, when analyses were performed by grouping Juveniles and Adults from both species, statistically significant differences (p < 0.05) were found between both stages at this concentration, at 24 and 48 h, indicating a higher mortality of juveniles. Moreover, juveniles showed statistically significant (p < 0.05) higher mortality for 0.2 mg ml−1 at 48 h. No significant differences were found between both species. 3.2. Soil toxicity test Regarding mortality (Fig. 1), no statistically significant differences were found for any of the species at days 14 or 28. However, for E. fetida, the mean mortality increased with the increase in 4-OHBP concentration (Fig. 1D, E, F). Statistically significant differences were found for this species in the second period of the test (14d–28d, Fig. 1E, KW = 12.831, d.f. = 5, p < 0.05). The Conover test indicated that the concentration of 2000 mg kg−1 provoked a significantly higher mortality than concentrations 0, 1, 10 and 100 mg kg−1, with no mortality. The concentration of 1000 mg kg−1 caused certain mortality but was not significantly different from any of the two groups (Fig. 1E). Nevertheless, and probably due to the high variability of the data, those differences were not detected at day 28, and therefore, we cannot conclude that 4-OHBP affected survival at the concentrations tested. The LC50 for E. fetida at 28 days was 1800.12 (95% CI: 93.94, > 10,000) mg kg−1. It was not possible to calculate the LC50 for D. veneta due to the lack of effect of 4-OHBP on the mortality of this species. Regarding the individual weight increase (Fig. 2), we found a weak but positive relation with 4-OHBP concentration for D. veneta at day 28 (Table 2, Fig. 2C). Statistically significant differences were found at that time point (ANOVA, F5,30 = 5.46, p < 0.01), and the highest concentration tested (2000 mg kg−1) implied a weight increase of D. veneta adults when compared to the control (mean weight increase of 60,9% and 40.96% respectively) Regarding reproductive success (Figs. 3 and
2.4. Statistical analyses For the contact test data, mortality was recorded at 24 and 48 h, and χ2 tests were performed. Tests were carried out for each combination of species (D. veneta and E. fetida) and stage (juveniles and adults) separately, while also comparing global numbers of juveniles vs. adults and both species responses. For the soil toxicity test we calculated the percentage of mortality and increase of weight per earthworm in each jar, in three different periods of time for adults (1–14 days, 14–28 days and 1–28 days) and each week for juveniles of D. veneta. The number of juveniles and unhatched cocoons produced at 56 days, as well as their weights, were analysed as variables for reproduction success. Normality and homoscedasticity of variables were checked with the Shapiro-Wilk and Levene tests, respectively. When those assumptions were not fulfilled by the data, transformations (log(x + 1)) were performed and checked. Other data transformations were checked but resulted unsuccessful. For those variables that met the assumptions, lineal regressions were performed. Moreover, in order to understand the differences in those variables among tested concentrations, one-way ANOVA was carried out with the concentration of toxicant as the independent factor. A Tukey test was used for post hoc comparisons when pvalue < 0.05. When data or transformations were not normal, a non-
Table 1 Results of contact toxicity test after acute direct exposure to 4-OHBP. Percentage of dead earthworms is indicated for juveniles and adults of both species at 24 and 48 h. Tested concentrations of 4-OHBP were 0 (control), 0.02, 0.2 and 2 mg ml−1. a/b/ab (within category) indicate significantly different categories according to χ2 test (p < 0.05). 24 h Control D. veneta E. fetida
Juveniles (N10) Adults (N10) Juveniles (N10) Adults (N10)
a
0 0a 10a 10a
0.02 a
10 0a 30a 0a
48 h 0.2 b
70 50ab 50ab 10a
3
2
Control b
100 100b 100b 100b
a
10 0a 20a 10a
0.02 ab
50 10a 40a 0a
0.2 b
80 50ab 70ab 30a
2 100b 100b 100b 100b
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Fig. 1. Box plots representing mortality percentage of Dendrobaena veneta (A, B, C) and Eisenia fetida (D, E, F), for each level of concentration (mg kg−1) of 4-OHBP. A/D: From day 0 to day 14 of experiment. B/E: From day 14 to day 28 of experiment. C/F: From day 0 to day 28 of experiment. Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisk indicates significant result. Letters (a, b) indicate homogeneous groups according to Conover test.
Fig. 2. Box plots representing weight percentage increase per earthworm of Dendrobaena veneta (A, B, C) and Eisenia fetida (D, E, F), for each level of concentration (mg kg−1) of 4-OHBP. A/D: From day 0 to day 14 of experiment. B/E: From day 14 to day 28 of experiment. C/F: From day 0 to day 28 of experiment. Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisk indicates significant result. Letters (a, b) indicate homogeneous groups according to Tukey test.
4
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Table 2 Significant linear regression models (p < 0.05) for Dendrobaena veneta and Eisenia fetida variables measured in the soil toxicity test. Independent variable is concentration of 4-OHBP: [cc].
D. veneta E. fetida
Linear regression model
p-Value
% R2
β
%Δweight/earthworm 28 days = 41.141 + 0.006*[cc] Number of juveniles = 72.291–0.029[cc] Number of cocoons = 14.818–0.006[cc] log (weight per cocoon +1) = 0.002–0.0000008[cc]
0.043 0.001 < 0.001 0.001
11.5 28.9 31.2 28.2
0.339 −0.538 −0.558 −0.531
4), for E. fetida the number of juveniles and cocoons at the 56th day of the experiment showed strong negative relationships with 4-OHBP concentration (Fig. 3C, D), as shown by linear regression analyses (Table 2). Calculated EC50 values were 152.68 mg kg−1 (number of juveniles), 94.05 mg kg−1 (number of unhatched cocoons) and 145.52 mg kg−1 (total production: number of juveniles plus number of unhatched cocoons). Statistically significant differences were found among treatments in the number of juveniles (Fig. 3C: ANOVA, F5,30 = 9.38, p < 0.001) and the number of unhatched cocoons (Fig. 3D: ANOVA, F5,30 = 6.71, p < 0.001) that were significantly lower for the concentrations of 1000 and 2000 mg kg−1. Moreover, a negative relationship was detected by linear regression analysis between weight per cocoon and 4-OHBP concentration (Table 2), and significant differences among treatments were found (Fig. 4D: ANOVA, F5,30 = 18,27, p < 0.001). Concentrations above 10 mg kg−1 implied a lower weight per cocoon. No effect of 4-OHBP concentration on weight per juvenile was detected (Fig. 4C). For D. veneta, no significant relationships or differences were found in reproductive success measurements (Figs. 3A, B, 4 A, B), and no EC50 values could be calculated. For the latter, a subset of juveniles was exposed to the same concentrations of 4-OHBP as their progenitors. Fig. 5A represents the percentage of accumulated mortality during this experiment, showing
that differences could already be detected in the first week. From day 14, all the individuals exposed to the highest concentrations (100, 1000 and 2000 mg kg−1) were dead, except one single earthworm at 2000 mg kg−1 that died at 21 days. Kruskal-Wallis results from the time points of 7 days (Fig. 5C) and 14 days indicate that there is a significantly higher mortality of D. veneta juveniles at the three highest concentrations of 4-OHBP (7 days: KW = 17.6303, d.f. = 5, p < 0.005; 14 days: KW = 26.064, d.f. = 5, p < 0.001). According to the Conover test, the concentration of 10 mg kg−1 also shows a difference with the control at the 7th day (Fig. 5C). The LC50 for D. veneta juveniles after 7 days of exposure was 72.02 (95% CI: 13.67, 379.49) mg kg−1, and after 14 days, it was 19.49 (95% CI: 2.9, 131.1) mg kg−1. Results of the growth for exposed juveniles of D. veneta are shown in Fig. 4B, represented as the percentage of accumulated weight per earthworm. Kruskal Wallis analyses did not show significant results at any of the time points (see results at day 7 in Fig. 4D). 4. Discussion In this study, we have evaluated the impact of the UV filter 4-OHBP on edaphic fauna, through the assessment of mortality, growth, reproduction and juvenile performance for two epigeic earthworms. Our
Fig. 3. Box plots representing the number of juveniles (A, C) and cocoons (B, D) for both species Dendrobaena veneta (A, B) and Eisenia fetida (C, D), for each concentration of 4-OHBP (mg kg−1). Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisks indicate significant results. Letters (a, b, c) indicate homogeneous groups according to Tukey test. 5
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Fig. 4. Box plots representing the weight per juvenile (A, C) and weight per unhatched cocoon (B, D) for both species Dendrobaena veneta (A, B) and Eisenia fetida (C, D), for each concentration of 4-OHBP (mg kg−1). Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisk indicates significant result. Letters (a, b, c, d) indicate homogeneous groups according to Tukey test (ANOVA performed with log (x + 1)).
results indicate that the acute exposure through a direct contact of 4OHBP with the epidermis of earthworms causes their mortality at concentrations of 0.2–2 mg ml−1, suggesting that this substance is harmful for these animals (Table 1).The juveniles were shown to be slightly more sensitive to 4-OHBP in the direct contact test. A higher sensitivity of juveniles has been observed in many works of earthworms exposed to other compounds (e.g., Lowe and Butt, 2005; Pelosi et al., 2013; Spurgeon et al., 2004; Zhou et al., 2008). Immature individuals may not have fully developed their detoxification mechanisms as suggested by biomarker responses after exposure to insecticides (Booth and O’Halloran, 2001). Another possible reason accounting for the higher sensitivity of juveniles may be their smaller body size. Due to their higher surface-area-to-volume ratio, juveniles probably assimilate a higher relative amount of the xenobiotic. Some studies prove that the body size is one of the factors explaining differences of sensitivity in earthworms (Łaszczyca et al., 2004; Schreck et al., 2008). A faster metabolism and higher cellular turnover of juveniles may be another reason that cannot be discarded. For the soil tests, which are more representative of the natural environment, much higher concentrations of 4-OHBP were needed in order to see impacts, as expected from previous studies (Verdu et al., 2018; Wang et al., 2012a, 2012b). The potential adsorption of the toxicant to the soil colloids reduces the direct exposure of the earthworms and attributes a buffering effect to the soil (Miguel and Martí, 2011). Our results for exposures of mature earthworms indicate that D. veneta adults are more resistant to 4-OHBP, whereas E. fetida adults show a higher sensitivity (Figs. 1–4). This is demonstrated by the fact that there were no negative impacts for any of the D. veneta recordings, whereas reproduction success of E. fetida was impacted (see below). Newman et al. (2000) and Dittbrenner et al. (2011), already highlighted the importance of evaluating exposures in different species of the same group without relying on the results from a single species,
because the impacts of the same substance may differ even within the same ecological category as our results show. A difference of sensitivity to toxicants between these two species has been already proposed by Verdu et al. (2018) who found D. veneta to be more resistant to Bisphenol A than E. fetida, similar to our results. Sensitivity can vary between close species, since they may present slightly different metabolic rates, detoxification mechanisms or abilities to bind xenobiotics (Fitzgerald et al., 1996; Givaudan et al., 2014; Ma and Bodt, 1993; Pelosi et al., 2013). Moreover, as mentioned above referring to the higher sensitivity of juveniles, the difference in size could be another explanation, since individuals of D. veneta (1.18 + −0.1 g) were slightly bigger than E. fetida (0.22 + −0.02 g). We found significantly higher mortality for E. fetida during the second period of the test (14–28 days) at the highest concentration tested (2000 mg kg−1, Fig. 1). However, this difference was not statistically significant at the end of the test's 28 days, probably due to the high variability of the data. Nevertheless this could indicate that if longer tests were carried out those differences may be reflected in the end. Regarding reproductive success, our results suggest that there are negative relationships between the concentration of 4-OHBP and the number of juveniles (significantly lower from 1000 mg kg−1, Fig. 3C), the number of unhatched cocoons (significantly lower from 100 mg kg−1, Fig. 3D) and the weight per cocoon (significantly lower from 10 mg kg−1, Fig. 4D) of E. fetida. A lighter weight of cocoons produced by earthworms exposed to contaminants has been previously found (Dominguez et al., 2016) and discussed as detrimental, since they may be associated to weaker juveniles. Schmitt et al. (2008) also observed a negative effect of a similar UV filter (BP-3) in the reproduction of the Californian blackworm (L. variegatus). Unfortunately, there is a lack of references on the effect of benzophenones in earthworms with which to compare. Meanwhile D. veneta adults (F0) were unaffected by the 4-OHBP 6
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Fig. 5. Mortality and weight data for juvenile individuals (transgenerational study) of Dendrobaena veneta after exposure to different concentrations of 4-OHBP (0, 1, 10, 100, 1000 and 2000 mg kg−1). A: Accumulated mortality (%) during 8 weeks of soil test. B: Accumulated weight increase per earthworm (%) during 8 weeks of soil test; jars with 50% or higher mortality were removed from B graph, since growth presented high variability in comparison with the rest within their treatment. C: Box plot of accumulated mortality (%) at day 7. D: Box plot of increase of weight (%) at day 7. Boxes represent the 50% of data around the median, which is represented as a horizontal line. Closed circles represent the mean and open circles represent the outliers. Asterisk indicates significant result. Letters (a, b, c) indicate homogeneous groups according to Conover test.
All these values are far from the 500 to 18,380 ng kg−1reported in Korean soils by Jeon et al. (2006). Nevertheless, more studies are needed for assessing the concentrations of UV filters in soils, since the information is still scarce and biased. There is also a gap in the existing literature in relation to the adsorption of 4-OHBP to the soils, degradation or bioaccumulation. Moreover, further studies in more natural conditions could shed some light on the environmentally relevant effects of 4-OHBP. Earthworm species of different ecological categories may show different reactions because they feed differently, and the organic matter content and microorganism diversity may have an effect on the bioavailability of the toxicant. The toxicity of any chemical substance in the soil is closely related to environmental variables and physicochemical properties (principally organic matter and clay content, Miguel and Martí, 2011). There is not much data on biotic degradation, but given the log Kow value of 4-OHBP (3.07), organic matter content may not have a great influence on its availability because of the low adsorption expected. It is worth mentioning that the feeding conditions the OECD recommends do not include, in our experience, enough organic matter for the well-being of the earthworms. Starvation has been linked to higher chemical toxicity (Holmstrup et al., 2010; Warne et al., 2001), and the effect of the amount of organic matter on toxicity to earthworms has been discussed (Irizar et al., 2015). Therefore, even though the amount of organic matter may not
exposed juveniles (F1) of the same species, shown to be impacted by this UV filter, showing a higher mortality at 10 mg kg−1 and higher concentrations (Fig. 5C) and therefore, showing more sensitivity than adults of both species. The potential causes, such as possible differences in detoxification mechanisms, extensive cell division during growth or differences in size were already mentioned above. Moreover, the exposure of adults (F0) as well as cocoons could have also affected this second generation of earthworms. We demonstrate here, the importance of transgenerational studies for understanding the long term true impact of toxicants for the populations, since the adult (F0) test could give a false impression. It needs to be noted that manipulation of juveniles every week for weighting purposes may have caused some mortality, as observed, for example, in the control after the 4th week. However, the mortality observed at the highest concentrations of the UV filter occurred the first two weeks, when the control worms seemed to be almost unaffected. Even though we found some effects of 4-OHBP on the studied earthworms, the potentially harmful concentrations are much higher than those reported in the environment. For E. fetida, the LC50 at 28 days was 1800.12 mg kg−1 and the EC50s were 152.68 mg kg−1 (juveniles) and 94.05 mg kg−1 (unhatched cocoons). The first generation of D. veneta was unaffected and the LC50 for second generation juveniles was 72.02 mg kg−1 at 7 days and19.49 mg kg−1 at 14 days. 7
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modify availability of 4-OHBP, it could be expected that a lower organic matter content, as suggested by the OECD, may produce a higher toxicity due to starvation effects. All said, and basing on the currently available information, we must assume that 4-OHBP does not represent a risk or has very low toxicity for earthworm populations. Nevertheless, additional research, including longer term transgenerational studies, will be needed to confirm it.
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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We are grateful to Irene Verdú for guidance on the experiments, Jorge Domínguez for providing earthworms, to the team from the Soil Zoology Group from Complutense University of Madrid (UCM), for their support in the lab and to Antón Álvarez for providing help in the statistical analyses. MN was supported by a Postdoctoral Fellowship (FPDI-2016-16407) from the Spanish Government and a UCM Postdoctoral Fellowship. This study was funded by the grants: CTM2015-64913-R and CGL2013-42908-P from the Spanish Government. References Booth, L.H., O’Halloran, K., 2001. A comparison of biomarker responses in the earthwormAporrectodea caliginosato the organophosphorus insecticides diazinon and chlorpyrifos. Environ Toxicol Chem. 20 (11), 2494–2502. https://doi.org/10. 1002/etc.5620201115. Chisvert, A., Salvador, A., 2007. UV filters in sunscreens and other cosmetics. Regulatory aspects and analytical methods. In: Salvador, A., Chisvert, A. (Eds.), Analysis of Cosmetic Products. Elsevier, Amsterdam, pp. 83–120. Díaz Cosín, D.J., Moro, R.P., Valle, J.V., Garvín, M.H., Trigo, D., Jesús, J.B., 1996. Producciín de heces de Hormogaster elisae úlvarez, 1977 (Oligochaeta, Hormogastridae) en diferentes tipos de cultivos en laboratorio. Bol. R. Soc. Espaóola Hist. Nat. (Biol.) 92, 177–184. Diogene, J., Dufour, M., Poirier, G.G., Nadeau, D., 1997. Extrusion of earthworm coelomocytes: comparison of the cell populations recovered from the species Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum. Lab. Anim. Uk 31, 326–336. Dittbrenner, N., Schmitt, H., Capowiez, Y., Triebskorn, R., 2011. Sensitivity of Eisenia fetida in comparison to Aporrectodea caliginosa and Lumbricus terrestris after imidacloprid exposure. Body mass change and histopathology. J. Soils Sediments 11, 1000–1010. Domínguez, J., Aira, M., Gómez Brandón, M., 2009. El papel de las lombrices de tierra en la descomposición de la materia orgánica y el ciclo de nutrientes. Ecosistemas 18, 20–31. Dominguez, A., Brown, G.G., Sautter, K.D., de Oliveira, C.M., de Vasconcelos, E.C., Niva, C.C., Bartz, M.L., Bedano, J.C., 2016. Toxicity of AMPA to the earthworm Eisenia andrei bouche, 1972 in tropical artificial soil. Sci. Rep. 6, 19731. Fitzgerald, D.G., Warner, K.A., Lanno, R.P., Dixon, D.G., 1996. Assessing the effects of modifying factors on pentachlorophenol toxicity to earthworms: applications of body residues. Environ. Toxicol. Chem. 15, 2299–2304. Gago-Ferrero, P., Diaz-Cruz, M.S., Barcelo, D., 2011. Occurrence of multiclass UV filters in treated sewage sludge from wastewater treatment plants. Chemosphere 84, 1158–1165. Givaudan, N., Binet, F., Le Bot, B., Wiegand, C., 2014. Earthworm tolerance to residual agricultural pesticide contamination: field and experimental assessment of detoxification capabilities. Environ. Pollut. 192, 9–18. Harrison, E.Z., Oakes, S.R., Hysell, M., Hay, A., 2006. Organic chemicals in sewage sludges. Sci. Total Environ. 367, 481–497. Holmstrup, M., Bindesbol, A.M., Oostingh, G.J., Duschl, A., Scheil, V., Kohler, H.R., Loureiro, S., Soares, A.M.V.M., Ferreira, A.L.G., Kienle, C., Gerhardt, A., Laskowski, R., Kramarz, P.E., Bayley, M., Svendsen, C., Spurgeon, D.J., 2010. Interactions between effects of environmental chemicals and natural stressors: a review. Sci. Total Environ. 408, 3746–3762. Iribarne, L.M., 2016. Cuantificación de contaminantes con actividad disruptora endocrina en leche materna: puesta a punto de la metodología analítica y resultados preliminares. Universidad de Granada, Spain, pp. 99.
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