Supplementary materials
CHAPITRE 4 ACCUMULATION ET EFFETS SUB-LETAUX DU TRICLOSAN
ET DE SON PRODUIT DE TRANSFORMATION LE METHYL-TRICLOSAN SUR LE VERS DE TERRE EISENIA ANDREI, EXPOSES A DES SOLS ARTIFICIELS CONTAMINES A DES CONCENTRATIONS REALISTES
Résumé :
Dans ce deuxième article, les mêmes protocoles expérimentaux que précédemment sont appliqués dans le cas d’une exposition à long-terme (56 jours) des vers E. andrei à une seule molécule, le triclosan (TCS), ajouté à une concentration de 50 ng.g-1 (masse sèche) à un sol artificiel. Les effets sur la croissance, la survie et la reproduction des vers de terre sont déterminés, ainsi que l’endommagement de l’ADN des vers adultes (essai comètes). L’augmentation du nombre de cocons et de juvéniles associées à la réduction de leur poids moyens secs après exposition au TCS comparé aux contrôles peut traduire tant un changement dans la stratégie de reproduction des vers, afin d’assurer la survie de l’espèce, qu’un effet direct du TCS. L’accumulation du TCS par les vers de terre adultes est modérée (BAF ~ 1) et similaire lors d’un ajout de la molécule à un sol artificiel ou lors de son ajout via une boue contaminée. La formation du methyl-triclosan (MeTCS) est favorisée par la présence de boue. Il est plus fortement bioaccumulé dans les tissus des vers de terre que le TCS, bien que la concentration en MeTCS dans les sols restent au seuil de quantification. Nos résultats mettent en évidence l’accumulation du TCS et MeTCS par E. andrei donc la possible contamination des échelons supérieurs du réseau trophique, notamment si un phénomène de bioamplification a lieu. Le MeTCS paraît présenter un risque plus important pour les vers que le TCS au regard des résultats d’analyses chimiques, bien que des études complémentaires soient nécessaires pour confirmer ou infirmer cette hypothèse.
Contribution de l’auteur :
L’ensemble des manipulations, ainsi que la mise au point et l’application des méthodes d’analyses chimiques aux vers de terre et aux sols ont été faites par l’auteur principal (F. Chevillot), aidée de sa stagiaire Mélanie Guyot. Les co-auteurs Mélanie Desrosiers, Nicole Cadoret, Éloïse Veilleux, Hubert Cabana et Jean-Philippe Bellenger ont participé au bon déroulement des essais et analyses (chimiques et biochimiques) et contribué à l’interprétation des résultats. Ils ont également participé à la correction du présent manuscrit dont la soumission est prévue au printemps 2017 dans le
77 journal Environmental Chemistry and Toxicology.
78
Accumulation and sub-lethal effects of triclosan and its degradation product
1
methyl-triclosan in earthworm Esenia andrei exposed to environmental
2
concentrations in an artificial soil
3
Fanny Chevillot 1, Mélanie Guyot 1, Mélanie Desrosiers 2, Nicole Cadoret 2, Éloïse 4
Veilleux 2, Hubert Cabana 3, Jean-Philippe Bellenger 1, * 5
1 Centre Sève, Department of Chemistry, Université de Sherbrooke, Faculty of Sciences,
6
J1K2R1 QC, Canada
7
2 Centre d'expertise en analyse environnementale du Québec, ministère du Développement
8
durable de l'Environnement et de la Lutte contre les changements climatiques, Quebec city,
9
G1P3W8 QC, Canada
10
3 Department of Civil Engineering, Université de Sherbrooke, Faculty of Engineering,
11 J1K2R1 QC, Canada 12 13 * Corresponding author: 14 Jean-Philippe Bellenger 15
2500 boul. de l'Université, Sherbrooke, Québec, J1K 2R1, Canada 16
E-mail address: jean-philippe.bellenger@usherbrooke.ca 17
18
79
Abstract
20
This study determined sub-lethal effects of triclosan (TCS) on the earthworm Eisenia andrei 21
exposed to environmentally relevant concentration (< 50 ng.g-1 dry weight) in an artificial soil. The
22
long-term exposure (56-days) experimental design used allowed us to evaluate the impact of TCS 23
on survival, growth and reproduction of earthworms. DNA damage was also determined on adult 24
earthworm coelomocytes using the comet assay. Bioaccumulation study (21-days) was also made 25
on earthworms exposed to biosolids-born TCS. Chemical analysis of TCS and methyl-triclosan 26
(MeTCS) were realized on soil and earthworms tissue sampled in the two sets of experiment. Under 27
the tested conditions, the only significant effects observed on adult earthworms were on 28
reproduction parameters, with a significant increase in the number of cocoons and the number of 29
juveniles (p < 0.05) and a decrease on the dry weight of juveniles. The bioaccumulation of TCS, 30
expressed as a factor of bioaccumulation (BAF), was higher in juvenile earthworm tissues (BAF > 31
2.5) than in adult earthworm tissue (BAF ~ 1). MeTCS was quantified in earthworm tissue when 32
they were exposed to biosolids-amended soil only and reached higher concentration than TCS in 33
all samples. This accumulation of TCS and MeTCS in earthworms could represent a vector of 34
contamination of higher levels of the food chain. The higher sensitivity of juveniles than adults in 35
TCS toxicity might be attribute to the apparition of MeTCS in soil. This highlight the need of 36
chemical analysis of both parent and degradation product(s) to better understand potential 37
ecotoxicological effect observed. 38
K
EYWORDS:
Triclosan, Methyl-triclosan, Earthworms, Reproduction assay, Bioaccumulation 3940 41
80
Introduction
42
The use of biosolids from municipal wastewater treatment plants (WWTP) as fertilizing agent is a 43
common agricultural practice in several countries around the world [1]. This revalorization of 44
residual materials is also supported by governments as an alternative to the incineration of biosolids 45
emitting greenhouse gas [2,3] as well as landfilling. The efficiency of wastewater treatment 46
processes toward pharmaceuticals and personal care products (PPCPs) varies greatly with the 47
technology employed and the target compound [4–6]. Removal efficiency is mostly evaluated from 48
a treated water stand point. It is generally calculated as the ratio between effluent and influent 49
concentrations. Thus, the different elimination processes occurring in WWTP often includes the 50
transfer of PPCPs to biosolids. Indeed, the contamination of biosolids from WWTP by PPCPs and 51
other chemical of concern has been reported in many studies [7–10]. Thus, despite the benefit of 52
the use of WWTP biosolids as fertilizer, this practice could be a way of entry of organic 53
contaminants (i.e. PPCPs) to the environment [11]. 54
Organic compounds with low polarity are easily adsorbed by solids in suspension and are 55
predisposed to accumulate in biosolids during wastewater treatment. For instance, triclosan (TCS), 56
an antibacterial agent ubiquitously used in personal care product such as soap, antimicrobial 57
product or toothpaste, is characterized by a relatively low solubility (≤ 1 mg.L-1) and low polarity
58
(log Kow ~ 4.8) [12]. Consequently, it has been found in large amount in municipal biosolids across 59
the world, with reported concentrations ranging from 0.1 to 32.9 µg.g-1 (dry weight, dw) [13,14].
60
In soil, reported concentration of TCS are in the low ng.g-1 range [14,15]. Due to its potential effect
61
on human health and the environment [16], the use of TCS has be restricted in the US [17], in 62
Canada [18] and in Europe [19]. However, the use of TCS is not prohibited and is still unregulated 63
in many countries. In addition, the main degradation product of TCS in biosolids, namely methyl- 64
triclosan (MeTCS) [20–23], is known to be more persistent in soil than TCS [24]. Thus, while 65
actions are being taken to limit the consumption of TCS, at least in some countries, biosolid 66
81 amended agricultural soils have been and will continue to be exposed to TCS and its transformation 67
product MeTCS. 68
Earthworms are important organisms of the soil ecosystem. They represent a large part of the soil 69
biomass and are involved in regulation of major soil processes such as modification of soil porosity 70
by burrowing or stimulation of microbial activities [25,26]. Earthworms are directly exposed to soil 71
pollutants by their skin or their feeding [27]. Thus, they are highly relevant to assess the 72
bioavailability and toxicity of organic contaminants to soil biotas. 73
Environmental toxicity of TCS to aquatic organisms is well documented [16,28,29]. However, to 74
our knowledge, few TCS toxicological and bioaccumulation data existed for earthworm. Lin et al. 75
(2010) reported that exposure to TCS resulted in DNA damages at concentration as low as 76
6.25 µg.g-1 in Eisenia fetida [30]. The inhibition of catalase and glutathione-S-transferase, involved
77
in defense mechanisms against free radicals and xenobiotic compounds respectively, was observed 78
for exposure of TCS in concentration higher than 50 µg.g-1 [30]. In 2014, the same group reported
79
the effect of TCS on E. fetida reproduction (production of cocoons and juveniles) under similar 80
experimental conditions (1 to 300 µg.g-1 TCS) [31]. The bioaccumulation of TCS in earthworm has
81
been studied in both laboratory test (spiked soil) and field collected samples [32,33]. Reported data 82
on bioaccumulation of TCS are very variable and no clear mechanism for TCS accumulation in 83
earthworm has been proposed. In a study on native earthworms from a biosolids-amended soil, 84
MeTCS concentration in earthworm tissues were higher than the concentration of its parent 85
molecule, TCS [24]. However, no information is available in the literature regarding the effect of 86
MeTCS on earthworm physiology and reproduction. 87
One of the major flaws of previous studies [30,31] is the unrealistic concentration at which 88
experiments where performed. Indeed, experiments were conducted at concentration several order 89
of magnitude higher than those encountered in real ecosystems [33]. This makes it hard to transpose 90
results to real ecosystems, with the risk of overestimating the toxicity of this antibacterial agent. 91
82 Experiments under more realistic concentrations are needed to draw a more comprehensive picture 92
of the potential impact of TCS (and MeTCS) in soil biotas, especially earthworms. 93
In this study, we filled this gap by evaluating the effects of TCS exposure at environmentally 94
relevant concentration (50 ng.g-1 dw) on the physiology and reproduction of the model earthworm
95
E. andrei. The tested concentration was selected based on direct measurement of TCS in biosolids
96
and by considering its approximated dilution in agricultural soil (see further details below). We 97
monitored the effects of TCS on adult survival and DNA damages, using a cytotoxicity test (comet 98
assay) [34], as well as the production of cocoons and juveniles in a standardized 56-days 99
reproduction test [35]. We also monitored the accumulation of TCS in adult and juvenile earthworm 100
tissues, the formation of TCS degradation product MeTCS and its accumulation in earthworms in 101
an artificial soil spiked with TCS. The bioaccumulation of biosolids-born TCS and MeTCS in 102
earthworms was also monitored using a standardized bioaccumulation test [36] in an artificial soil 103
amended with biosolids from a local WWTP. 104
105