Effects of Roundup formulations on biochemical biomarkers and male sperm quality of the livebearing Jenynsia multidentata

Accepted Manuscript Effects of Roundup formulations on biochemical biomarkers and male sperm quality of the livebearing Jenynsia multidentata Jessica Andrea Albañil Sánchez, Antonio Sergio Varela, Junior, Carine Dahl Corcini, Janaina Camacho da Silva, Ednei Gilberto Primel, Sergiane Caldas, Roberta Daniele Klein, Camila De Martinez Gaspar Martins PII:

S0045-6535(17)30336-3

DOI:

10.1016/j.chemosphere.2017.02.147

Reference:

CHEM 18910

To appear in:

ECSN

Received Date: 17 November 2016 Revised Date:

22 February 2017

Accepted Date: 27 February 2017

Please cite this article as: Sánchez, J.A.A., Varela Junior., , A.S., Corcini, C.D., da Silva, J.C., Primel, E.G., Caldas, S., Klein, R.D., Martins, C.D.M.G., Effects of Roundup formulations on biochemical biomarkers and male sperm quality of the livebearing Jenynsia multidentata, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.02.147. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Lipoperoxidative damage

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Effects of Roundup formulations on biochemical biomarkers and male sperm quality of

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the livebearing Jenynsia multidentata

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Jessica Andrea Albañil Sáncheza, Antonio Sergio Varela Juniora, b, Carine Dahl Corcinid,

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Janaina Camacho da Silvab, Ednei Gilberto Primelc, Sergiane Caldasc, Roberta Daniele

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Kleinb, Camila De Martinez Gaspar Martinsa,b*

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a

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Ciências Biológicas, Universidade Federal do Rio Grande, Av. Itália km 8, 96203-900, Rio

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Programa de Pós-Graduação em Biologia de Ambientes Aquáticos Continentais, Instituto de

Grande, RS, Brazil.

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b

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96203-900, Rio Grande, RS, Brazil.

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c

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96203-900, Rio Grande, RS, Brazil.

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d

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Pelotas. Caixa Postal 354, 96001-970, Pelotas, RS, Brazil.

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Escola de Química e Alimentos, Universidade Federal do Rio Grande, Av. Itália km 8,

Departamento de Patologia Animal, Faculdade de Veterinária. Universidade Federal de

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Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Av. Itália km 8,

Corresponding author: Camila De Martinez Gaspar Martins

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Universidade Federal do Rio Grande - FURG Instituto de Ciências Biológicas Av. Itália km 8 – Campus Carreiros

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96.203-900 – Rio Grande – RS – Brazil

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Phone: + 55 53 393-5162

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FAX: + 55 53 3233-6848

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E-mail:[email protected]

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Abstract

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Roundup® formulations are the most consumed glyphosate-based herbicides in the

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world. When applied, they may reach water bodies and exert toxicity toward non-target

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species. This study evaluated and compared the effects of different variations of Roundup on

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biochemical biomarkers as oxidative parameters and Acethylchorinesterase (AChE) activity,

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and sperm quality of the livebearing Jenynsia multidentata. Fish were acutely (96 h) exposed

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to Roundup Original® (RO), Roundup Transorb® (RT) and Roundup WG® (RWG) at 0.0, 0.5,

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1 and 5 mg L-1 of nominal glyphosate. The highest mortality (60%) was observed for fish

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exposed to RT at the highest concentration tested and at 0.5 mg L-1 non-mortality was

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observed, so this concentration was chosen for the experiments. Fish exposed to RO and RT

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(24 and 96 h) presented a state of oxidative imbalance, which caused lipid peroxidation

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(LPO) in their livers. Oxidative stress was more severe in RO treatment, which may be

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resulted in the highest hepathosomatic index at 96 h. However, fish exposed to RT presented

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a marked inhibition of AChE activity from membrane cells of muscle and brain tissues.

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Sperm quality was investigated in livebearing exposed (24 and 96 h) to the three

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formulations. Spermatozoa motility and concentration were affected by all formulations.

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Overall, Roundup formulations are harmful to the fish J. multidentata at 0.5 mg L-1 of

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glyphosate; however, mechanisms and potential of toxicity are different between

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formulations. The J. multidentata also represents a sensitive species and a good regional bio-

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monitor.

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Key-words: glyphosate; oxidatve stress; acethylchorinesterase; sperm quality; biomarker;

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fish.

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Introduction

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Glyphosate (N-phosphonomethylglycine) is a broad-spectrum, post emergent, systemic and non-selective herbicide. It inhibits plant growth via interference with the

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shikimate pathway, thereby reducing the aromatic acids phenylalanine, tyrosine, and

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tryptophan production, which are required for protein synthesis (Duke, 2011). Glyphosate is

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presented under different names and commercial formulations, with the Roundup

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formulations (produced by Monsanto) representing the most commercialized herbicide in the

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world (James, 2011and IBGE, 2015). According to the Brazilian National Health

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Surveillance Agency (ANVISA) (2010), Brazil is the largest consumer of agrochemicals in

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the world. Further, glyphosate represented 62.4 % of commercialized agrochemicals in Brazil

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in 2015 (IBGE, 2015).

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Roundup formulations present high water solubility, varying from 10,000 to 15,700 mg L-1 at 25 oC. Roundup contains glyphosate as the active ingredient, along with other

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compounds described as “inert” in the package. It is known that the polyethoxylene amine

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(POEA), a nonionic surfactant, is part of these “inert compounds” in some formulations

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(William et al., 2000, Burger and Fernandez, 2004, Braush and Smith, 2007 and Tsui and

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Chu, 2013). The added surfactant increases the efficiency of Roundup formulations by

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promoting penetration of the herbicide through plant cuticles (Brausch and Smith, 2007).

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Aquatic ecosystems can be contaminated with glyphosate-herbicides by leaching, run-

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off, or through spraying (World Health Organization, 2005). Herbicide products that enter

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water bodies are potentially harmful to fish populations and other forms of aquatic life that

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are considered non-target organisms (Giesy et al., 2000, Harayashiki et al., 2013 and Sandrini

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et al., 2013). Pure glyphosate may be relatively non-toxic to animals (WHO, 2005), however

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its formulations are often toxic to aquatic organisms due to the addition of the surfactant that

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is used to improve its penetration into plants (Tsui and Chu, 2004). In fact, acute toxicity tests

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(96 h) with several teleost fish species have shown that the median lethal concentration

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(LC50) of pure glyphosate is about 10 times higher when compared to the LC50 of Roundup

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formulations (Giesy et al., 2000). Studies regarding exposures of fish species to glyphosate (0.2 to 10 mg L-1) and

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Roundup (1 to 10 mg L-1) have demonstrated that they may cause biochemical alterations

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such as oxidative damage (Glusczak et al., 2006, 2007 and Modesto and Martinez, 2010a,

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2010b), as well as inhibition of acethylcholinesterase enzyme (AChE) activity (Salbego et al.,

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2010 and Sandrini et al., 2013). Additionally, studies focused on fish reproduction have

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shown that pure glyphosate at 5 and 10 mg L-1 and the Roundup Original® formulation at 0.7

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and 1.3 mg L-1 of glyphosate can cause negative effects on sperm cell quality of the zebrafish

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Danio rerio and of the guppy Poecilia vivipara, respectively (Harayashiki et al., 2013 and

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Lopes et al., 2014). Roundup (Max Granular formulation at 0.5 mg L-1) has also been shown

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to affect the ability of the male livebearing Jenynsia multidentata to copulate with associated

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females (Hued et al., 2012).

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Concentrations of glyphosate used in laboratory tests to determine its biological

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effects are generally higher than those found in the environment. Battaglin et al. (2014)

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compiled results regarding the occurrence of glyphosate and its major degradation product

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aminomethylphosphonic acid (AMPA) in environmental samples (3,732 samples of water

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and sediments) of 38 states in the U.S., collected over 10 years. Glyphosate was detected in

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39.4 % of all samples, with the maximum concentration found as 0.476 mg L-1 or mg kg-1;

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while AMPA were detected in 55 % of samples and its maximum concentration was 0.397

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mg L-1 or mg kg-1. However, the median concentration found for both elements was < 0.02

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µg L-1 or µg kg-1, below the levels of concern for humans or wildlife. In Europe, a Monsanto

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report (Horth, 2012) shows that glyphosate and AMPA have been frequently detected in

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AMPA. In Southern America, data about presence of glyphosate and AMPA in

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environmental samples are relatively rare. Peruzzo et al. (2008) detected levels of glyphosate

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in the water (0.1 to 0.7 mg L-1), sediment (1.15 to 1.38 mg L-1) and soil (0.53 to 4.45 mg L-1)

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sampled from transgenic soybean cultivations in north pampasic region of Argentina. On the

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other hand, Aparicio et al. (2013) determined significant levels of glyphosate ranging

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between 0.035 to 1.502 mg L-1 in soils of agricultural basins in the province of Buenos

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Aires/Ar; and Ronco et al. (2016), measured about 0.6 mg L-1 of glyphosate in sediment of

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Paraguay and Parana rivers, and also in Argentina. In Brazil, there is a measurement of 1.48

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mg L-1 of glyphosate in water samples from a stream in Arapoti city, which is located close to

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transgenic soy plantations. This measurement was made in water samples collected two days

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after glyphosate spraying followed by rain in the surrounding plantations (Tzaskos et al.,

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2012).

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Most existing reports regarding the exposure and effects of glyphosate-herbicides on

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fish have tested pure glyphosate or only one formulation and, to date, only one has compared

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the effects of different formulations used in agriculture (Murussini et al., 2016). Thus, the aim

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of this study was to evaluate and compare the effects of Roundup formulations on oxidative

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parameters, AChE activity and sperm quality of the livebearing Jenynsia multidentata in

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order to elucidate whether they present different toxic effects to fish species.

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The neotropical fish species Jenynsia multidentata (Anablepidae,

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Cyprinodontiformes), used as a biological model in this work, presents a wide distribution in

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an extensive area of South America (Malabarba et al., 1998) and can be found in pristine as

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well as severely degraded habitat (Hued and Bistoni, 2005). In Brazil, J. multidentata often

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lives associated with rice plantations, and as such is susceptible to the action of agrotoxics.

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This fish species has also been suggested as a regional bioindicator model to evaluate the

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effects of different chemicals on biological processes (Hued et al., 2006, Ballesteros et al.,

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2007 and Pinto et al., 2015), and because it is a sexually dimorphic, it may also be used as an

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excellent model to evaluate these effects on reproductive features.

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Materials and Methods

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Fish collection and acclimation

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Adult specimens of livebearing Jenynsia multidentata (males: 0.54 ± 0.03 g of weight, 2.90 ± 0.06 cm of length and females: 0.69 ± 0.03 g of weight, 3.05 ± 0.05 cm of length)

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were collected (SISBIO 37129-2) in streams from Cassino Beach (Rio Grande RS, Southern

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Brazil) using a hand net. Collected fish were transferred to an animal care room in the

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Institute of Biological Sciences, Federal University of Rio Grande, where they were

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acclimated for at least 15 days in aquariums filled with water at salinity of 5 ppt (natural

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seawater diluted with dechlorinated tap water) continuously aerated. The tanks were also

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equipped with a biofilter to ensure water quality. Photoperiod was fixed at 12L: 12D and

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temperature was 20 oC. Fish were fed twice a day with commercial fish food (Tetra

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ColorBits®). A proportion of 1g of fish per 1L of water was maintained during acclimation.

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Fish were arranged in a ratio of 3:1 female to male in the aquaria to stimulate sexual activity

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in the males.

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Herbicides

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Three commercial formulations of glyphosate: Roundup Original® (RO), Roundup Transorb® (RT) and Roundup WG® (RWG) were used in this work. Concentrations of

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ACCEPTED MANUSCRIPT glyphosate used in the experiments were calculated based on the concentration of glyphosate

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acid (mg of GlyAE /L) existing in each formulation: 360, 480, 720 g L-1 of glyphosate in RO

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and RT and RWG, respectively. The form of glyphosate salt present in RO and RT

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formulations is the glyphosate isopropylamine salt (IPA), and in the RWG is the glyphosate

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ammonium salt. Monsanto produces these herbicides, which are variable in the proportion of

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glyphosate and “inert compounds” present. It is known that the surfactant MON 0818,

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containing POEA, integrates the RO formulation. The MON 0818 (a code of Monsanto for

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designation for preparation of POEA) is a mixture of polyethoxylated long-chain alkylamines

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synthesized from animal-derived fatty acids and is added to facilitate glyphosate penetration

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into the plants. Due to risk assessment, the surfactant generally accounts for 15 % or less in a

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formulation (Giesy et al., 2000). The producer does not reveal the composition of “inert

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compounds” for the other two formulations (RT and RWG).

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Fish exposure to herbicides

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A mortality test was performed in order to determine a non-lethal concentration of glyphosate to enable evaluation of the effects of the three herbicides in the livebearing

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Jenynsia multidentata. After acclimation, the fish (males and females) were randomly

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distributed in aquaria filled with seawater at 5 ppt, at a proportion of 1g of fish weight per 1L

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of water. Fish were acutely exposed (96 h) to RO, RT and RWG at nominal concentrations of

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0.5, 1 and 5 mg L-1 of GlyAE (n = 5 fish per treatment). A control group was used in the test.

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Herbicides from stock solutions of 3.6 g L-1, 4.8 g L-1 and 7.2 g L-1 of nominal GlyAE (RO,

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RT and RWG, respectively) were directly added to the water at the beginning of the

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experiment only, and the medium was not renewed. Feeding was stopped 24 h prior to the

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beginning of experiment, such that fish were fasting during the experimental period. Fish

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without respiratory action and insensitive to tactile stimuli were considered dead and were

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removed from the aquarium. Fish that remained alive until the end of the test were euthanized

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by immersion in 500 mg L-1 benzocaine (Sigma-Aldrich).

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Data of mortality tests are presented below in Results section. Because no mortality was observed only at 0.5 mg L-1 glyphosate among the three formulations RO, RT and RWG,

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this concentration was chosen for analyses of hepatossomatic index, biochemical biomarkers,

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and sperm quality.

Biochemical biomarkers and hepatossomatic index were analyzed in fish (n = 48, in a

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ratio of 2:1 female to male) acutely exposed (24 or 96 h) to 0.5 mg L-1 of glyphosate in the

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RO and RT formulations. A control group was maintained throughout the experiment. Water

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samples (5 mL) were daily collected for glyphosate monitoring. Fish were euthanized with

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benzocaine (500 mg L-1) at 24 h (n = 24) and 96 h (n = 24), weighed, and their tissues (liver,

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brain and muscle) were dissected. Livers were weighed for analysis of hepatossomatic index,

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and then they were used for the assays of reactive oxygen species (ROS), total antioxidant

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capacity against peroxyl radicals (ACAP), glutathione (GSH), glutathione-S-transferase

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(GST) activity and lipid peroxidation (LPO) (pool of 2 livers). Brain and muscle tissues

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(from pool of two animals) were used for the dosage of acetylcholinesterase (AChE) activity.

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Analysis of sperm quality was performed in acclimated males of J. multidentata (n = 20) acutely exposed (24 and 96 h) to 0.5 mg L-1 of glyphosate in the RO, RT and RWG

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formulations. A control group was also maintained during the experiment. Water samples (5

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mL) were daily collected for glyphosate measurements. Fish were euthanized (500 mg L-1 of

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benzocaine) at 24 and 96 h of exposure to glyphosate. Testis were then dissected and

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collected into 1.5 mL conical tubes filled with Hanks balanced solution with specific

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composition (pH 7.4; 320 mOSm; 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44

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mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 e 4.2 mM NaHCO3) for J. multidentata sperm

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cells, like described in Da Silva et al. (2015).

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Experimental conditions regarding water physic-chemistry, addition of herbicides to the water and fish starvation were consistent with the three experiments described above

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(experiments of mortality, biochemical biomarkers and sperm quality analysis). Water

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physicochemical parameters were measured daily in experiments as follows: salinity 5 ppt,

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temperature 21.4 ± 0.69 °C, pH 7.1 ± 0.10, dissolved oxygen 6.7 ± 1.37 mg L-1, nitrite

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ranging from 0.0 to 0.25 µg L-1 and total ammonia ranging from 0.0 to 2.0 between the

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beginning and the end of experiments. Glyphosate was measured in stock solutions and in

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waters sampled from experimental media by ion chromatograph (IC Compact 881, Metrohm,

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Herisau,Switzerland) following procedure described in Harayashiki et al., 2013.

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The Ethics Committee for Animal Use of the Federal University of Rio Grande (CEUA – FURG; reference Pq015/2013) approved the protocols performed in this study.

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Hepatosomatic index (HI)

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Hepatosomatic index was determined using the weights of fish liver and of the whole animal in the formula described by Thomé et al. (2005), as follows:

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 (%) =  ( )/ ( ) 100

Where, HI is hepatosomatic index; BW and LW are body weight and liver weight, respectively.

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Biochemical assays

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A portion of dissected livers (n = 6, pools of 2 animals) was homogenized (9:1 w/v) in a buffer solution (100 mM Tris HCl, 2 mM EDTA and 5 mM MgCl2.6H2O, pH 7.75) and

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divided into aliquots to determine ROS, ACAP and GSH concentrations and GST activity.

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The remaining liver portion (n = 6, pools of 2 animals) was homogenized in a buffer

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containing 1.15 % KCl and 35 µM BHT, for LPO assay. ROS was quantified following

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LeBel et al. (1996) method. The 2’,7’dichlorodihidrofluorescein diacetate (H2DCFDA,

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Invitrogen) was used, which generates a fluorochrome in the presence of ROS. ROS

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concentration was calculated as the relative area of fluorescence and standardized by the

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protein content in the tissue homogenate. ACAP was determined according to Amado et al.

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(2009). The method is also based on the fluorimetric detection of ROS using H2DCFDA

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(Invitrogen) as substrate. However, in this case, a replication of each tissue extract is exposed

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to peroxyl radicals produced by the thermal decomposition (35 °C) of 2,2'-azobis (2-

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methylpropionamidine) dihydrochloride (ABAP, 4 mM). ACAP is calculated by the

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difference in ROS concentration in the samples with and without ABAP divided by the ROS

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concentration recorded without ABAP. For both ROS and ACAP, fluorimetric readings were

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detected at a wavelength of 488 and 525 nm of excitation and emission, respectively, in a

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fluorometer with microplate reader (Victor2 ™ Multilabel Counter model 1420-051. Turku,

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Finland). Glutathione (GSH) concentration were determined via the reaction of 2,3-

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naphthalenedicarboxaldehyde (NDA, Sigma-Aldrich) with GSH, according to the method

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described by White et al. (2003). NDA-GSH reaction is used to measure GSH with high

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specificity. This reaction generate cyclic products that were measured by fluorescence,

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excitation: 472 nm; emission: 528 nm using the microplate fluorescence reader and expressed

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as nmol of GSH/mg protein. GST activity was determined by the rate of complexation of

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reduced glutathione (GSH) with CDNB at 340 nm, and expressed as nmol of conjugated

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ACCEPTED MANUSCRIPT CNDB/min/mg protein as described by Keen et al., 1976). LPO was determined based on the

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reaction between malondialdehyde (MDA), a product of lipid degradation by ROS, and 2-

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thiobarbituric acid (TBA), which under high temperature and acidity conditions yield a

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chromogen. The chromogen is detected by spectrofluorometry (515 and 553 nm of excitation

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and emission, respectively) (Oakes and Van Der Kraak, 2003). Data were normalized by the

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the protein content in the tissue homogenate. LPO was expressed as nmol MDA/min/mg

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protein.

The activity of acetylcholinesterase enzyme (AChE) was determined in brain (n = 6,

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pools of 2 animals) and muscle tissues (n = 12). Samples were homogenized in phosphate

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buffer (50 mM) containing 20 % of glycerol and pH adjusted to 7.4. Then, they were

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centrifuged at 9000 g for 20 min at 4 °C and the resulting supernatants were considered as the

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S9 soluble fraction. The pellets were resuspended in the same buffer with added Triton X-100

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(0.5 %) and stirred for 30 min at room temperature. Subsequently, the samples were

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centrifuged at 9000 g for 30 min at 4 °C and the resulting supernatants were considered the

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TX S9 membrane fraction (Sandrini et al., 2013). Enzyme activity was determined using 5,5-

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dithiobis-2-nitrobenzoic acid (DTNB) and acetylthiocholine iodide (0.75 mM) as substrates

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(Ellman et al, 1961). The yellow color yielded by thiocholine after its reaction with DTNB

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was monitored by spectrophotometer at 412 nm. The results were expressed as nmols of

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acetylcholine iodide hydrolyzed/min/mg of protein in the homogenate.

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Protein content in all assays was determined using a commercial reagent kit for

protein based on the Biuret method (Doles Regantes Ltda., Goiânia, Brazil).

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Sperm quality

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espermatozeugmatas (sperm bundles). The sperm were then released by rupture of

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espermatozeugmatas using a 10µL pipette tip. The subsequently suspended sperm were used

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for the following analysis: sperm motility and concentration, sperm mitochondrial

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functionality and integrity of membrane and DNA.

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Sperm motility was estimated using a microscope at 200 x magnification (Olympus America BX 51, Inc., São Paulo, SP, Brazil). The result was expressed as percentage of cells

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actively moving forward (Sun et al., 2010). Sperm concentration was determined using a

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Neubauer chamber (Varela Junior et al., 2012).

Mitochondrial functionality was assessed according to the methodology of He and

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Woods (2004) and adapted by Varela Jr. et al. (2012) using the dye Rhodamine 123 (Rh123)

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(≥ 96 %, Sigma-Aldrich). Cells exhibiting green fluorescence were classified as presenting

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functional mitochondria, while sperm showing no fluorescence represented cells with

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dysfunctional mitochondria. A total of 200 cells were counted in this analysis using an

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epifluorescence microscope at 400 x magnification. Mitochondrial functionality was

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expressed as the proportion of cells producing fluorescence compared with total number of

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sperm analyzed.

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Sperm membrane integrity was assessed using two fluorescent probes:

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carboxyfluorescein diacetate (CFDA) and propidium iodide (PI). Intact membranes

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accumulate CFDA, which is hydrolysed in carboxyfluorescein and yields a green

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fluorescence. Damaged cell membranes absorb PI and emit a red color. For quantitative

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analysis of membrane integrity, 200 cells were counted and classified according to their color

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(Harrison and Vickers, 1990). Cells were counted using an epifluorescence microscope at 400

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x magnification.

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Integrity of sperm cell DNA was measured using the method described by Tejada et al. (1984). Cells were stained with Acridine Orange (AO) solution (0.2 g L-1), such that

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sperm with green fluorescence represented those with normal DNA while cells displaying

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red, orange or yellow fluorescence represented cells with damaged DNA. A total of 200 cells

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were used for this analysis, which were also counted under 400 x magnification in an

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epifluorescence microscope. The epifluoresecence microscope used in the analyses was an

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Olympus America BX 51, Inc., São Paulo, SP, Brazil.

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The assays described above that were used to assess sperm quality are further detailed in Harayashiki et al. (2013).

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Glyphosate concentration measurements

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Glyphosate concentration in stock solutions and water samples was determined via ionic chromatograph (IC Compact 881, Meltrohm, Herisau, Switzerland) with

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conductometric detector, using an ion exchange column (Metrosep A Supp 5 150/4.0) and

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chemical suppressor. The mobile phase was made with 9.6 mmol L-1 of sodium bicarbonate.

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Calibration curves were prepared to determine the concentration of glyphosate in stock

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solutions (0.05 to 10.0 mg L-1 of glyphosate) and water samples (0.05 to 5.0 mg L-1 of

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glyphosate). All injections were performed with a 20 µL injection cycle. The lower limit of

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quantification and detection was 0.01 and 0.05 mg L-1, respectively. The collection and

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processing of data was performed using the software MagicNet 2.3 (Metrohm, Herisau,

318

Switzerland) (Harayashiki et al., 2013).

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Statistical analysis

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ACCEPTED MANUSCRIPT Data were expressed as mean ± standard error (SEM). Comparisons between the mean

322

values were made by analysis of variance (ANOVA - two way) followed by Tukey test. Data

324

normality distribution and homoscedasticity were previously tested. Significance level of

325

95% (p <0.05) was used in all analyses. Statistical analyses were performed using the

326

software Sigma-Plot 11.0.

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327 328

Results

330

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Percentage of mortality of J. multidentata exposed (96 h) to 0.5, 1 and 5 mg L-1 of nominal glyphosate were 0, 40 and 60 % when they were submitted to RT and 0, 20 and 20

332

% under RWG exposure, respectively. RO did not cause mortality in any concentration

333

employed in this work. The concentration of 0.5 mg L-1 of glyphosate was non-lethal,

334

regardless of the formulation tested. This concentration was then used to evaluate

335

biochemical biomarkers and sperm quality. The measurements of glyphosate in the media

336

indicate that herbicide concentrations did not vary over the 96 h of test for all treatments and

337

all experiments performed here. Mean values of glyphosate concentrations were 3.6 g L-1, 5.1

338

g L-1 and 6.8 g L-1 in stock solutions and 0.53 ± 0.01, 0.59 ± 0.02 and 0.93 ± 0.09 mg L-1

339

(mean ± standard error) in water for the formulations RO, RT and RWG, respectively.

340

Glyphosate was not detected in the water of control groups. Thus, if there were any

341

glyphosate in the water of control groups, it would be in concentrations lower than the 0.05

342

mg L-1, which is the limit of detection of the equipment (see methods).

343

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With respect to biochemical measurements, Figure 1 shows a significant increase in

344

the quantity of ROS in the livers of fish exposed to the RT formulation. In fact, in RT

345

treatments, the amount of ROS was significantly reduced within 24 h of experiment when

346

compared with its respective control. At 96 h ROS increased about 1000 fold in comparison

14

ACCEPTED MANUSCRIPT to both control at 96 h and RT at 24 h. The RO formulation did not influence ROS

348

production. On the other hand, ACAP concentration did not vary significantly in the livers of

349

animals exposed to RT, neither in relation to control nor over the sample time. There was a

350

tendency towards an increase in ACAP in the livers of control fish from 24 to 96 h, but it was

351

not significant (Figure 2). ACAP was significantly higher in the livers from animals

352

submitted to RO treatment at 96 h when compared to those exposed to RO for 24 h. Despite

353

that, ACAP in fish exposed to RO were not different from control animals or those exposed

354

to RT (Figure 2).

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Insert Figure 1

357

Insert Figure 2

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Results from Figure 3 shows that GSH levels were preserved in the livers of control

360

group animals during the exposure period. However, GSH concentration reduced over time

361

(reduction at 96 h when compared to 24 h of test) in the livers of fish exposed to RO and RT.

362

There was no difference between control group and exposed fish. The GST activity did not

363

change by the presence of glyphosate formulations, but it increased over time in the livers of

364

control animals.

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Insert Figure 3 Insert Figure 4

368 369

LPO measurements are displayed in Figure 5. At 24 h, LPO was higher in the livers of

370

control fish and those exposed to RT when compared to fish exposed to RO. However, from

371

24 to 96 h the levels LPO decreased in livers of control fish, remained constant in the livers

15

ACCEPTED MANUSCRIPT 372

of RT exposed fish, and increased in the livers of RO exposed fish. Indeed, at 96 h the levels

373

of LPO in livers of Roundup exposed fish were significantly higher than in the control fish.

374

Thus, oxidative damage was promoted by exposure to both formulations at 96 h, with RO the

375

most damaging formulation.

377

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Insert Figure 5

378

Mean values of HI from fish sampled at 24 h were: 1488,6 % ± 165,6, 2487,8 % ±

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529 and 1534,6 % ± 100 in control, RO and RT treatment, respectively. For the fish sampled

381

at 96 h, the HI was 890,1 % ± 72,1, 2095 % ± 518,5 and 1568,6 % ± 179,4 in control, RO and

382

RT group, respectively. These data indicate a significant effect of RO (p = 0.03) that

383

augments the values of HI for fish exposed to this formulation for 96 h.

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Figure 6 shows AChE activity in different fractions (cytosolic and membrane,

385

respectively) of the brain (Figures 6a and 6b) and muscle (Figures 6c and 6d) tissues of fish

386

exposed to the formulations. Overall, AChE activity in the tissues of fish from control groups

387

changed between sampling periods. In the cytosolic and membrane fraction of brain tissue,

388

AChE activity increased at 96 h when compared with 24 h. In muscle tissue, AChE activity

389

decreased at 96 h in the cytosolic fraction, but did not change in the membrane fraction

390

between 24 and 96 h of test. A marked effect from the herbicides could be observed at each

391

experimental time when comparing AChE activity in the fish from control and exposed

392

groups. This effect was more evident in fish exposed to RT, as a strong inhibition of AChE in

393

both tissues was observed. Additionally, this inhibition was apparent in the membrane

394

fraction of the tissues (Fig. 6b and 6d).

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Insert Figure 6

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ACCEPTED MANUSCRIPT 397 398

Figure 7 displays the sperm quality analysis following exposure to RO, RT and RWG. There were significant differences in sperm motility and sperm concentration (Figure 7a and

400

7b), with reduction of these two parameters in all herbicide treatments. Sperm motility was

401

reduced at 24 h in relation to control and remained reduced after 96 h of exposure. Sperm

402

concentration from exposed fish decreased at 24 h but recovered at 96 h. For the other

403

parameters as sperm mitochondrial functionality and sperm integrity of membrane and sperm

404

integrity of DNA (Figure 7 b, c and d, respectively), no differences between the formulations

405

were observed.

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Discussion

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The present study shows that Roundup formulations at 0.5 mg L-1 of glyphosate are toxic to the livebearing J. multidentata through mechanisms including: the promotion of

413

oxidative stress, inhibition of AChE activity and decreases in male sperm quality. However,

414

the toxic potential or specific mechanism of action of the Roundup formulations (RO, RT and

415

RWG) varied significantly. Among the formulations tested here, the most toxic was RT,

416

which induced 60 % mortality at 5 mg L-1 of nominal glyphosate, followed by RWG, which

417

induced 20 % mortality at 1 and 5 mg L-1 of nominal glyphosate, an then by RO that did not

418

cause mortality in any concentration employed here. At this point, it is important to

419

remember that glyphosate were not measured in the experimental media of mortality test, but

420

only in the media from the experiments performed for biochemical and sperm quality

421

analysis. In these last experiments, the concentration of glyphosate in RO and RT exposure

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ACCEPTED MANUSCRIPT media was similar and about 0.5 mg L-1. However, the concentration of glyphosate in RWG

423

exposure media was almost twofold higher (0.93 ± 0.09 mg L-1) in the last experiment. A

424

condition that might have influenced this difference in glyphosate concentrations would be

425

the way in which each formulation is presented. The RO and RT herbicides are liquid

426

formulations, while RWG is granular and needs to be dissolved before using.

427

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As mentioned before, the RO and RT formulations are composed of IPA, differing from the RWG formulation that contains glyphosate ammonium salt. The glyphosate

429

ammonium salt stimulates ATPase activity by increasing the adhesion of the herbicide to

430

plant leaves, promoting a powerful absorption of the product. In the case of RT, translocation

431

is faster but primarily concentrated in the roots. According to the labels, RO, RT and RWG

432

formulations vary from each other in percentage of glyphosate salts, glyphosate acids, and

433

"inert" ingredients.

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In spite of Roundup used as an herbicide, many studies have shown that glyphosate and its formulations are toxic to fish (Giesy et al., 2000, Glusczak et al., 2007, Modesto and

436

Martinez, 2010a, 2010b, Hued et al., 2012 and Murussi et al., 2015). LC50 (96h) values of

437

Roundup® vary widely among species with results ranging from 2 to 50 mg L-1 (Folmar et al,

438

1979; Mitchell et al., 1987, WHO, 1994, Abdelghani et al., 1997, Giesy et al., 2000,

439

Jiraungkoorskul et al., 2002 and Hued et al., 2012). According to Hued et al. (2012), the LC50

440

(96h) of Roundup Max Granular®, is calculated as 19.02 mg L-1 for the livebearing J.

441

multidentata, which classifies this species as moderately sensitive to Roundup. Surfactants

442

added to the formulations to facilitate penetration of the herbicide into the plants (Williams et

443

al., 2000) and represented by the “inert” ingredients in the package are in fact one of the

444

agents that cause toxicity to non-target species. The POEA (synthesized from animal derived

445

fatty acids) preset in some of Roundup® formulations is an example of non-inert surfactant.

446

Tsui and Chu (2003) have shown that the LC50 of POEA and Roundup® formulations for the

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ACCEPTED MANUSCRIPT microcrustaceans Ceriodaphnia dubia and Acartia tonsa is 40 fold higher than LC50 of

448

GlyAE or IPA. The authors reported that POEA is responsible for more than 86% of the

449

toxicity from Roundup® in these organisms, while also indicating that the toxicity of POEA is

450

species-dependent. In teleost fish, variations between the LC50 (96h) of glyphosate,

451

Roundup® and surfactants have been observed. Giesy et al. (2000) has reported that each of

452

the LC50 (96h) of glyphosate, Roundup® and POEA were calculated as 24, 5.8 and 1.3 mg L-

453

1

, respectively, for bluegill fish Lepomis macrochirus, and 30 mg L-1 for glyphosate, 20 mg L-

454

1

for Roundup® and 2.8 mg L-1 for POEA for the chinook salmon Oncorhynchus tshawytscha.

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In order to comparatively analyze differences between the RO, RT and RWG

456

formulations regarding potential toxicity to the native fish J. multidentata, an acute test with

457

non-lethal concentration of glyphosate for this species was used. Mortality tests were

458

conducted in order to define this non-lethal concentration, and 0.5 mg L-1 of nominal

459

glyphosate was determined as non-lethal. This concentration can also be regarded as

460

ecotoxicologically important. As cited before, concentrations around 0.4 mg L-1 of glyphosate

461

have been detected in waters from Europe and US. (Horth, 2012 and Battaglin et al., 2014).

462

In water samples from Argentina and Brazil, steams were detected at higher concentrations of

463

glyphosate: 0.7 and 1.48 mg L-1, respectively (Peruzzo et al 2008 and Tzaskos et al., 2012).

464

Although the concentration used in this work (0.5 mg L-1 of glyphosate) is close to higher

465

concentrations of glyphosate found in the environment, it still maintains environmental

466

relevance.

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Biochemical parameters are effective in demonstrating sublethal physiological effects

468

of pesticides and elucidating their mode of action. Specifically, oxidative parameters are

469

often investigated in the livers of animals, as the liver is the key organ responsible for

470

xenobiotic detoxification and is particularly susceptible to oxidative damage. J. multidentata

471

exposed to the herbicides RO and RT showed a clear imbalance in the oxidative condition

19

ACCEPTED MANUSCRIPT that resulted in a significant increase in LPO when compared to the non-exposed fish (Fig. 5).

473

Livers of fish exposed to RT indicated a significant increase in ROS at 96 h, with a reduction

474

in GSH content at the same time; however, antioxidant capacity and GST activity were

475

maintained throughout the experiment (Fig. 1, 3 and 4, respectively). Even without an

476

increase, the antioxidant activity appeared to be sufficient to neutralize excess ROS detected

477

at 96 h and to avoid an increase in LPO from 24 to 96 h (Fig. 5). LPO levels at 96 h in liver

478

of RT fish remained higher than in controls. Despite an increase in antioxidant capacity in

479

liver tissue of fish exposed to RO formulation at 96 h, this remained insufficient to prevent a

480

significant increase in LPO from 24 to 96 h of experiment (Fig. 2 and 5, respectively). The

481

observed increase in ACAP of fish exposed to RO, as well as the maintenance of ACAP in

482

the RT exposed group may be associated with levels of antioxidants other than GSH, as GSH

483

concentrations decreased in the liver of those fish (24 to 96 h of exposure; Fig. 3). Amado et

484

al. (2011) found that GSH levels and antioxidant capacity are inversely correlated in the gills

485

of the zebrafish Danio rerio. The authors suggested that the lower values of GSH might

486

reflect a lower capacity of GSH to neutralize peroxyl radicals. On the other hand, the

487

decreased in GSH levels in liver of fish exposed to RO and RT at 96 h can also be a

488

consequence of GSH consumption in both direct reactions with oxidizing or conjugation

489

reactions mediated by the GST. The tripeptide GSH is an important agent of the antioxidant

490

defense system, as it participates in the detoxification of chemicals and elimination of lipid

491

peroxidation products (Deneke and Fanburg, 1989).

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Concerning the GST enzyme (Fig. 4), there was a tendency toward increased the

493

activity with the time of exposure in fish exposed to RO and RT, however this parameter was

494

only significant in the liver of control animals, which may result in a decrease of LPO from

495

24 to 96 h. GST is a multifunctional enzyme, which not only catalyzes xenobiotic binding

496

with GSH for elimination, but also reacts directly with the peroxidized lipids, neutralizing

20

ACCEPTED MANUSCRIPT them and preventing oxidative membrane damage. GST is frequently used as biomarker for

498

ROS production (Modesto and Martinez, 2010a, Glusczak et al., 2011 and Murussi et al.,

499

2016). Toni et al. (2013) reported that reductions in GST activity might be associated with

500

activity of the enzyme Cytocromo P450 (CYP). CYP produces different metabolites that may

501

compete with GST substrates for active sites in the enzyme. Thus, the presence of oxidants

502

may lead to the activation or inhibition of this enzyme. Generally, decreases in GST activity

503

negatively affect the detoxification process and oxidative damage may occur. An example is

504

the increase in peroxidized lipids (LPO). In the present study, GST activity was not sufficient

505

to protect J. multidentata liver tissue against the occurrence of LPO. Nevertheless, results

506

regarding oxidative parameters suggest that both RO and RT formulations are able to induce

507

oxidative stress; however, RO seems to be more harmful due to the higher levels of LPO that

508

indicate oxidative damage.

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There are several authors that corroborate glyphosate formulations as inducing oxidative stress and promoting damage to unsaturated fatty acids such as the case in LPO

511

(Modesto and Martinez, 2010a, Glusczak et al., 2011 and Murussi et al., 2016). Formation of

512

lipid peroxides is the general response of fish species exposed to glyphosate formulations,

513

and seems to be stimulated by the surfactants present in the herbicides (Cooper and Hausman,

514

2007). An increase in LPO in the liver of fish after 96 h of exposure to glyphosate

515

formulations has been observed by various authors, including: Murussini et al. (2016) in

516

Rhamdia quelen exposed to 2.5 and 5 mg L-1 of different glyphosate formulations (Orium®,

517

Roundup Original® and Biocarb®); Sinhorin et al. (2014) when Psuedoplatystoma sp were

518

exposed to 7.5 mg L-1 of glyphosate as Roundup Original®; and by Glusczak et al. (2011),

519

when Leporinus otusidens was exposed to Roundup®. Modesto and Martinez (2010a) showed

520

a significant increase in LPO in the liver at an earlier stage (6 h of exposure) in Prochilodus

521

lineatus exposed to 1 and 5 mg L-1 of Roundup Transorb®. However, Harayashiki et al.

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ACCEPTED MANUSCRIPT (2013) did not observe an increase in LPO levels in the liver of Poecilia vivipara exposed for

523

96 h to 0.70 mg L-1 glyphosate in Roundup®. In the present study, LPO was observed in liver

524

tissues of J. multidentata exposed to RO and RT formulations at 0.5 mg L-1 of glyphosate

525

over 24 and 96 h. This concentration is lower than those reported above and suggest that J.

526

multidentata is quite sensitive to glyphosate formulations.

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Hepatosomatic index (HI) reflects the involvement of the liver in xenobiotic

528

detoxification. The significant effect of RO on HI can be related to high levels of LPO and

529

other oxidative damages. Histological alterations have been shown in livers of different

530

species of fishes exposed to glyphosate formulations. Ayoola et al. (2008) detected

531

vacuolization in the liver of Oreochromis niloticus exposed to glyphosate. In a similar study,

532

Murussini et al. (2015), observed alterations ranging from vacuolization to focal necrosis in

533

the livers of R. quelen, hypothesizing that an increase in the detoxifying functions of the liver

534

may have occurred and led to the inability to regenerate new hepatocytes in response to this

535

exposure. According to Jiraungkoorskul et al. (2003), vacuolization in the liver may be an

536

imbalance between the rate of synthesis of lipid substances in the parenchymal cells and the

537

rate of release in systemic circulation. Lipid changes in the hepatocytes may indicate that fish

538

are concentrating lipophilic substances (such as surfactant POEA) in the hepatocytes,

539

resulting in a reduced availability of xenobiotics (Sarkar et al., 2005 and Ramírez-Duarte et

540

al., 2008). With respect to J. multidentada, Hued et al. (2012) found significantly higher

541

values of HI in fish exposed to Roundup in comparison with the control fish, and increased

542

HI in a concentration and time dependent manner of exposure. According to the authors, the

543

severity and extension of the hepatic injuries were greater in the highest concentration of

544

Roundup (35 mg L-1 of Roundup Max Granular®) under acute exposure in J. multidentada.

545

Moreover, they did not observe differences between male and female HI, and subsequently

546

compiled data from males and females together, similar to the present study.

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ACCEPTED MANUSCRIPT 547

Although glyphosate is not considered a classical AChE inhibitor, various studies report a reduction in AChE activity in the brain and muscle tissue of fish following exposure

549

to pure glyphosate, as well as its commercial formulations (Glusczak et al., 2006, 2007,

550

Modesto and Martinez, 2010 a, b, Salbego et al., 2010 and Gholami-seyedkolaei et al., 2013).

551

In an in vitro experiment, Sandrini et al. (2013) showed that glyphosate inhibits AChE

552

activity in brain and muscle tissues of two species of fish: D. rerio and J. multidentata.

553

Values of IC50 were about 1 g L-1 of pure glyphosate and these tests were conducted exposing

554

the tissue extracts to the herbicide and not the whole animal. The authors also observed a

555

major inhibition of AChE activity present in the membrane fraction of both brain and muscle

556

tissues. In the present study, RT exposure resulted in a more pronounced effect on AChE

557

activity, significantly reducing AChE activity over time (24 to 96 h in the mussel) or in

558

relation to the respective control (at 24 and 96 h in the brain and muscle). Giesy et al. (2000),

559

suggests that AChE inhibition following exposure of fish to commercial herbicides is more

560

likely to be related to the surfactant used in the formulated products than to glyphosate itself.

561

Do not only restricted to fish, a study from Southern Brazil has also shown AChE inhibition

562

by 30 % in small-scale agricultural workers that do not use appropriate equipment (Nerilo et

563

al., 2014).

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AChE is a member of the cholinesterase enzyme family and is responsible for the

565

breakdown of the neurotransmitter acetylcholine in cholinergic synapses. This enzyme is

566

typically located on the extracellular side of the plasmatic membrane and controls the ion

567

currents in excitable membranes, playing a critical role in nerve conduction (Sinha et al.,

568

2010). AChE may also be found in a monomeric soluble form on the cytoplasmatic side.

569

AChE may be classified within both globular and asymmetric structure types. The main

570

globular forms consist of monomers (G1), dimers (G2) or tetramers (G4). The G1 unit is

571

connected to the cytosol, while the G4 is connected to the cell membrane. The G4 type is the

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ACCEPTED MANUSCRIPT 572

most commonly observed in the nervous and muscular system (Xie et al., 2007). Thus,

573

inhibition of AChE can cause neurological and behavioral disturbances. In this sense,

574

instabilities in the swimming patterns or court behavior of fish may occur as a result of

575

exposure to neurotoxic pesticides (Bretaud et al., 2002). The results described above indicate that RT represents the most dangerous

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formulation, due to it promoting the highest mortality, levels of LPO and inhibition of AChE

578

activity of J. multidentata. However, the severity that RO affects the liver may suggest that

579

oxidative stress is the main mechanism of RO toxicity while inhibition of AChE is the

580

primary mechanism of the RT formulation. Results also suggest that AChE inhibition is more

581

aggressive to fish lives than oxidative imbalance, considering that fish acutely exposed to RT

582

experienced approximately 60 % mortality at 5 mg L-1 of nominal glyphosate while no fish

583

died when they were exposed to RO at the same concentration of glyphosate.

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Motility and sperm concentration were affected by exposure to herbicides in J. multidentata. Motility of spermatozoa (Fig. 7a) represents the most sensitive parameter as it

586

was inhibited by glyphosate in the three experimental formulations (RO, RT and RWG) at 24

587

and 96 h of exposure. Lopes et al. (2014) reported a similar effect from exposing zebrafish to

588

5 mg L-1 of pure glyphosate for 24 h. However, these parameters returned to regular

589

conditions after 96 h of experiment. Sperm condition and motility are directly related to the

590

ability of an organism to reproduce (Cosson et al., 1999), as non-motile sperm do not find the

591

oocyte for fertilization (Rurangwa et al., 2004). Regarding sperm concentration (Fig. 7b), the

592

results observed in J. multidentata showed that this parameter is affected by all formulations

593

at 24 h, however sperm concentration in fish exposed to RT recovered at 96 h. Lopes et al.

594

(2014) observed a time-dependent decrease in the percentage of sperm cells in zebrafish

595

exposed to 5 and 10 mg L-1 of pure glyphosate. Also, Harayashiki et al. (2013), observed a

596

significant reduction in the percentage of spermatozoa in P. vivipara exposed to RO at 0.13

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ACCEPTED MANUSCRIPT and 0.70 mg L-1 of glyphosate for 96 h, as well as a decrease in mitochondrial function. In a

598

study with the same important issue, Hued et al. (2012) found that Roundup decreases sexual

599

courtship in males from J. multidentata. Male fish have a copulatory organ called the

600

gonopodiun, which is introduced in the female genital pore following a successful courtship

601

(Bisazza et al., 2000). Taking all together, the effects found in J. multidentata indicate that

602

Roundup is detrimental to their reproduction.

603

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Overall, results discussed above show an effect of the three Roundup formulations RO, RT and RWG on the fish J. multidentata. Even with the same active ingredient,

605

differences between formulations are directly related to their inclusion of components other

606

than glyphosate, which are described in their respective labels as “inert compounds”.

607

However, it remains unknown if and how components within the Roundup formulations other

608

than glyphosate may be significantly contributing to its toxicity. Are these “inert

609

compounds”, including the surfactants, more toxic to the fish than pure glyphosate? Do they

610

facilitate glyphosate penetrating into different tissues promoting acute or chronic toxicity?

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Results presented here also support the potential use of J. multidentata as a species for regional bio-monitors (Ferreira et al., 2012 and Pinto et al., 2015). The livebearing lives

613

associated with irrigated rice crops of Rio Grande do Sul (Southern Brazil) that receives tones

614

of pesticides every year, thus this fish is under Roundup formulations exposure in the

615

environment. Further, parameters such as LPO, AChE and sperm motility can be suggested as

616

biomarkers to respond to aquatic contamination with Roundup.

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Finally, it is important to keep in mind that although 0.5 mg L-1 of glyphosate (the

618

main concentration tested in this work) is an environmentally relevant concentration, it is

619

close to the maximum limit of what is found in most of the environments (Peruzzo et al.,

620

2008, Horth, 2012 and Battaglin et al., 2014). Moreover, as we have tested only this

621

concentration, we cannot determine exactly how the biomarkers would respond if the fish

25

ACCEPTED MANUSCRIPT 622

were exposed to a range of glyphosate concentrations, including some of greater

623

environmental relevance. These are limitations that may be considered in the use of our data

624

in environmental risk assessment.

625

Acknowledgement

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The authors of the present study thank the Brazilian CNPq – Conselho Nascional de Desenvolvimento Ciêntífico e Tecnológico (Process 449695/2014-0) for financial support.

630

Jessica Albañil Sanchez is supported by the Brazilian CAPES - Coordenação de

631

Aperfeiçoamento de Pessoal de Ensino Superior. R. D. Klein (Process 159061/2014-8), A. S.

632

Varela Junior (Process 307195/2015-7), C. D. Corcini (Process 306356/2014-7), E. G. Primel

633

(Process DT 310517/2012-5) are researches fellow from the Brazilian CNPq. Authors thank

634

Andrew Taylor for revision of English and grammar.

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Abdelghani, A., Tchounwou, P., Anderson, A., Sujono, H., Heyer, L., Monkiedje, A., 1997.

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Toxicity evaluation of single and chemical mixtures of roundup, garlon-3A, 2,4-D, and

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microchirus), and crawfish (Procambarus spp). Environ. Toxic. Water. 12, 237-243.

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Amado, L., Garcia, L., Ramos, P., Freitas, R., Zafalon, B., Ferreira, R., Yunes, J., Monserrat,

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J., 2009. A method to measure total antioxidant capacity against peroxyl radicals in aquatic

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organisms: application to evaluate microcystins toxicity. Sci. Total. Environ. 407, 2115 -

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2123. https://doi.org/10.1016/j.scitotenv.2008.11.038.

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Amado, L., Longaray, M., Baptista, P., Yunes, J., Monserrat, J., 2011. Influence of a toxic

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in common carp Cyprinus carpio (Teleostei: Cyprinidae). Arch. Environ. Contam. Toxicol.

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60, 319–326. https://doi.org/10.1007/s00244-010-9594-2.

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ANVISA. Agência Nacional de Vigilância Sanitária, 2010. Programa de Análise de Resíduo

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Figure Captions

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Figure 1. Reactive oxygen species (ROS) in the livers of Jenynsia multidentata kept in

944

control conditions or exposed for 24 and 96 h to the formulations Roundup Original® (RO)

945

and Roundup Transorb® (RT) at a concentration of 0.5 mg L-1 of glyphosate. Data are

946

expressed as mean ± standard error. Lowercase letters represent significant differences (p

947

<0.05) between experimental times. Capital letters represent significant differences (p <0.05)

948

between treatments each sample time. (ANOVA – two way).

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Figure 2. Total antioxidant capacity against peroxyl radicals (ACAP) in the livers of Jenynsia

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multidentata kept in control conditions or exposed for 24 and 96 h to the formulations

952

Roundup Original® (RO) and Roundup Transorb® (RT) at a concentration of 0.5 mg L-1 of

953

glyphosate. Data are expressed as mean ± standard error. Lowercase letters represent

954

significant differences (p <0.05) between experimental times. Capital letters represent

955

significant differences (p <0.05) between treatments each sample time. (ANOVA – two way).

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Figure 3. Glutathione (GSH) concentration in the livers of Jenynsia multidentata kept in

958

control conditions or exposed for 24 and 96 h to the formulations Roundup Original® (RO)

959

and Roundup Transorb® (RT) at a concentration of 0.5 mg L-1 of glyphosate. Data are

960

expressed as mean ± standard error. Lowercase letters represent significant differences (p

961

<0.05) between experimental times. Capital letters represent significant differences (p <0.05)

962

between treatments each sample time. (ANOVA – two way).

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963 964

Figure 4. Glutathione-S-transferase (GST) activity in the livers of Jenynsia multidentata kept

965

in control conditions or exposed for 24 and 96 h to the formulations Roundup Original®

39

ACCEPTED MANUSCRIPT 966

(RO) and Roundup Transorb® (RT) at a concentration of 0.5 mg L-1 of glyphosate. Data are

967

expressed as mean ± standard error. Lowercase letters represent significant differences (p

968

<0.05) between experimental times. Capital letters represent significant differences (p <0.05)

969

between treatments each sample time. (ANOVA – two way).

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Figure 5. Lipid peroxization (LPO) in the livers of Jenynsia multidentata kept in control

972

conditions or exposed for 24 and 96 h to the formulations Roundup Original® (RO) and

973

Roundup Transorb® (RT) at a concentration of 0.5 mg L-1 of glyphosate. Data are expressed

974

as mean ± standard error. Lowercase letters represent significant differences (p <0.05)

975

between experimental times. Capital letters represent significant differences (p <0.05)

976

between treatments each sample time. (ANOVA – two way).

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Figure 6. Acetylcholinesterase (AChE) activity in the brain and muscle of J. multidentata

979

kept in control conditions and exposed for 24 and 96h to Roundup Original® (RO) and

980

Roundup Transorb® (RT) at a concentration of 0.5 mg L-1 of glyphosate. (A) brain soluble

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fraction; (B) brain membrane fraction; (C) muscle soluble fraction; (D) muscle membrane

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fraction. Data are expressed as mean ± standard error. Lowercase letters represent significant

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differences (p <0.05) for the same treatment in different experimental times. Capital letters

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represent significant differences (p <0.05) between treatments each sample time. (ANOVA –

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Figure 7. Sperm quality of J. multidentata males kept in control conditions and exposed for

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24 and 96 h to Roundup Original® (RO), Roundup Transorb® (RT) and Roundup WG® at a

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concentration of 0.5 mg L-1 of glyphosate. (A) Motility; (B) Sperm concentration; (C)

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Integrity of the membrane; (D) DNA integrity; (E) Mitochondrial Functionality. Data are

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expressed as mean ± standard error. Lowercase letters represent significant differences (p

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ACCEPTED MANUSCRIPT

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Universidade Federal do Rio Grande – FURG Instituto de Ciência Biológicas Laboratório de Toxicologia o Av. Itália, km8 s/n – Carreiros – 96201-900, Rio Grande RS – Brazil Phone: +55(53) 3293-5162 Fax: +55(53) 3233-6848

MS ref Numer: CHEM44018

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Revised Highlights

Effects of Roundup herbicides were studied in the fish Jenynsia multidentata.

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Oxidative stress and AChE activity inhibition are responses to exposure to Roundup. Sperm quality of J. multidentata was also disturbed by Roundup at 0.5 mg L-1. Toxicity varied between formulations due to components other than glyphosate.

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J. multidentata is a sensitive fish species and an effective regional bio-monitor.

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Camila De Martinez Gaspar Martins Universidade Federal do Rio Grande - FURG Instituto de Ciências Biológicas Av. Itália km 8 – Campus Carreiros 96.203-900 – Rio Grande – RS – Brazil Phone: + 55 53 393-5162 FAX: + 55 53 3233-6848 E-mail:[email protected]