Parasitology of juvenile mullet (Mugil liza) and effect of formaldehyde on parasites and host

Aquaculture 354–355 (2012) 111–116

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Parasitology of juvenile mullet (Mugil liza) and effect of formaldehyde on parasites and host Eduardo Pahor-Filho a, Kleber Campos Miranda-Filho b,⁎, Joaber Pereira Júnior c a b c

Aquaculture Center, University of São Paulo UNESP - CAUNESP, Jaboticabal - SP, Brazil Laboratory of Aquaculture, Federal University of Minas Gerais, Belo Horizonte - MG, Brazil Laboratory of Parasitology of Aquatic Organisms, Federal University of Rio Grande - FURG, Rio Grande - RS, Brazil

a r t i c l e

i n f o

Article history: Received 24 March 2012 Accepted 28 March 2012 Available online 5 April 2012 Keywords: Mullet Parasitic control Toxicity test Formaldehyde Monogenoidea Endoparasites

a b s t r a c t Few studies have been performed with parasites of marine and estuarine fish in southern Brazil. In the present study, unpublished results show the ways of parasitism of juvenile mullet by parasites. The toxicity of formaldehyde and the effectiveness of this chemotherapy in controlling parasites in reared juvenile mullet Mugil liza were also studied. Juvenile mullets (1 ± 0.26 g; 4.1 ± 0.4 cm) were exposed to different concentrations of 37% formaldehyde: control group and five formaldehyde concentrations which were tested: T1 (13.5), T2 (21.6), T3 (40.5), T4 (81) and T5 (135) mg L − 1 with 8 fish per repetition in triplicate. To verify the drug effectiveness in parasitic control, juvenile mullets were exposed to 1 h prophylactic bath of 37% formaldehyde with a control group and five formaldehyde concentrations: T1 (67.5), T2 (135), T3 (270), T4 (405) and T5 (540) mg L− 1, 8 fish per repetition in triplicate. Ligophorus cf. uruguayensis (Monogenoidea: Ancyrocephalidae) and Solostamenides cf. platyorchis (Monogenoidea: Microcotylidae) were identified in the gills. Digenea and Nematoda were observed in the intestines. This is the first occurrence of S. cf. platyorchis in Brazil. During the toxicity test, the LC50-96 h was estimated at 20.77 mg L− 1 of formaldehyde. During the 1 h formaldehyde prophylactic bath, all parasites were eliminated in formaldehyde concentrations between 135 and 540 mg L− 1. High survival rate was observed in all treatments. Values of prevalence and intensity of infestation observed in this study showed the potential damage caused by Monogenoidea to mullet. Formaldehyde baths with 135 mg L− 1 are recommended to control Monogenoidea in mullet and the safe limits for formaldehyde use were presented. Besides, the endoparasites were tolerant to formaldehyde exposure. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Mullets represent an important fishery resource on several continents and are successfully used in coastal aquaculture (FAO, 2010; Khemis et al., 2006; Leber and Arce, 1996; Miranda-Filho et al., 2010). In western South Atlantic, the mullet is abundant and has a great consumer market (Reis and D'incao, 2000; Silva and Araújo, 2000). There is much information about the use of mullets in aquaculture (Godinho et al., 1993; Miranda-Filho et al., 1995; Okamoto et al., 2006; Poersch et al., 2007; Sampaio et al., 2001, 2002). Recent studies (Fraga et al., 2007; Heras et al., 2009; Menezes et al., 2010) conclude that the nominal species Mugil platanus is a junior synonym of Mugil liza. Considering that the M. liza distribution in the extreme north on the east coast of the Atlantic is in Bermuda (Fishbase, 2011), and that

⁎ Corresponding author at: Universidade Federal de Minas Gerais, Escola de Veterinária, Departamento de Zootecnia, Laboratório de Aquacultura - LAQUA, Av. Antônio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brasil. Tel.: +55 31 3409 2217; fax: +55 31 3409 2190. E-mail addresses: [email protected], [email protected] (K.C. Miranda-Filho). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.03.032

the nominal species M. platanus distribution in the extreme south is Puerto Madryn in Argentina (42°45′S, 64°50′O) (Cousseau et al., 2005), we may say that M. liza extends from Bermuda to Puerto Madryn. Parasitic infestations can compromise the immunity of its host (Secombes and Chappell, 1996) and fish quality for consumption (Manera et al., 2003). Studies have reported the occurrence of parasites in M. liza (Failla-Siquier and Ostrowski de Núñez, 2009; Knoff and Boeger, 1994; Knoff and Serra-Freire, 1993; Marcotegui and Martorelli, 2009; Oliveira et al., 2007). The index of severity of parasitic infection per infestation and its associated diseases suggests the need for studies to minimize or eradicate these parasitoses, considering its pathogenic potential effects on hosts (Hutson et al., 2007; Mansell et al., 2005; Ranzani-Paiva and Silva-Souza, 2004). Fish parasitic control can be achieved with chemotherapeutic administration (Hirazawa et al., 2000, 2001; Kim and Choi, 1998; Tubbs and Tingle, 2006). Among these drugs, formaldehyde has been effective against ectoparasites (Fajer-Ávila et al., 2007; Katharios et al., 2006; Rowland et al., 2006) and its use is approved in aquaculture (Costello et al., 2001). Formaldehyde is indicated for Protozoa and Monogenoidea control (Daniel, 2009). In addition for fish safety, there is also a concern

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group and five treatments: T1 = 67.5 mg L − 1, T2 = 135 mg L − 1, T3 = 270 mg L − 1, T4 = 540 mg L − 1 and T5 = 1080 mg L − 1 of formaldehyde with 8 fish per treatment, without replicates.

with the Maximum Residue Limits (MRL), defined by the legislation (Council of the European Communities, 1981a, 1981b, 1987; Daniel, 2009). However, the information available about this chemotherapy is controversial and unclear. There are no known concentrations of formaldehyde which can be used effectively and without risk to different species of fish. There are no studies demonstrating the formaldehyde effectiveness for the parasitic control and the chemotherapy toxicity for marine or estuarine species. In order to establish an experimental model for the parasitosis control in M. liza, parasites of juvenile mullet were investigated. The acute toxicity of formaldehyde estimated by median lethal concentration (LC50) and the effectiveness of chemotherapy in controlling parasites in juvenile mullet raised in captivity were also determined.

2.4.2. Experiment 2 — definitive test of formaldehyde acute toxicity (LC50) Taking into account the results of preliminary acute toxicity test, 144 juveniles were exposed to different concentrations of formaldehyde for 96 h, a control and T1 = 13.5 mg L − 1, T2 = 21.6 mg L − 1, T3 = 40.5 mg L − 1, T4 = 81 mg L − 1 and T5 = 135 mg L − 1 of formaldehyde with 8 fish in each of the three replicates. In order to estimate the median lethal concentration (LC50) and 95% confidence intervals, the software Trimmed Spearman Karber method was used (Hamilton et al., 1977).

2. Material and methods

2.5. Effectiveness of formaldehyde to juvenile mullet parasitic control

2.1. Animals used

2.5.1. Experiment 3 — lethal concentration — formaldehyde bath for one hour Thirty juveniles were exposed to formaldehyde bath for one hour in five treatments: T1 = 540, T2 = 1080, T3 = 1620, T4 = 2160 and T5 = 2700 mg L − 1 of formaldehyde and a control group with 5 fish per treatment, with no replicates. The tolerance time (until the moment of death) of mullet exposed to each formaldehyde concentration was monitored.

Juvenile mullet (1.0 ± 0.26 g; 4.1 ± 0.4 cm) was captured using 3 m × 1.5 m × 5 mm net in an estuary that flows into the Cassino Beach (32°11′55″S, 52°11′14″W) in Southern Brazil, in August 2009. Fish were kept in a 100 L tank with water from the estuary, with a daily renewal of 80%, constant aeration, and ambient temperature (21 °C) and photoperiod (12 h). 2.2. Experimental design Fish were acclimated to laboratory conditions for at least ten days before being exposed to the baths of formaldehyde. Commercial diet INVE® with 28% crude protein was used to feed the fish during the period of acclimatization. The water was maintained at 21 °C, salinity 5‰, and photoperiod 12 h, besides constant aeration. Under these conditions four experiments were performed in a germinating chamber (TE-401), where mullet juveniles were exposed to formaldehyde (37%) (MERK®) in 1 L beakers during acute toxicity tests. Control group was kept in water without addition of formaldehyde and animals were fasted during the lethal experiments. 2.3. Ways of parasitism of juvenile mullet 2.3.1. Necropsies To establish the initial state of the parasites diversity and their parasitological index, 40 juvenile mullets were necropsied with a sample minimum size to establish fish parasitological index defined by Marques and Cabral (2007). We examined the gills, esophagus, stomach, intestines, spleen, liver, heart, muscles and the abdominal cavity. The hosts were euthanized by section of the medullae to the head. The option of not using anesthetic was taken to avoid changing the parasitological index (Eiras et al., 2006). The collection, fixation and preparation for identification of parasites were performed according to Amato et al. (1991) protocol. Monogenoidea were preserved in formalin 5% and transferred to ethanol 70% after 24 h. Some specimens were stained with Masson trichrome or Semichon carmine, dehydrated in a series of increasing ethanol concentrations between 70 and 100%, cleared in Faia creosote and mounted in Canada balsam for identification. Other specimens were mounted in Grey–Wess' medium to study the eclerotized structures of haptor and male and female copulatory apparatuses. The endoparasites were preserved in AFA for 24 h, transferred to ethanol 70%, stained with Semichon carmine, clarified in Faia creosote and mounted in Canada balsam.

2.5.2. Experiment 4 — prophylactic bath with formaldehyde to control parasites After the preliminary test, 144 juveniles were exposed to formaldehyde bath for one hour. Treatments were a control group and five formaldehyde concentrations: T1= 67.5, T2= 135, T3= 270, T4= 405 and T5 = 540 mg L − 1 of formaldehyde, 8 fish per replicate in triplicate. After one hour of exposure to formaldehyde, fish were transported to other beakers with water prepared in the same physical and chemical conditions, but without formaldehyde for survival analysis. After 24 h, five fish from each treatment were euthanized for parasitological analyses. 2.6. Physical–chemical parameters of water During the acute toxicity test (Experiment 2), a daily monitoring of physical–chemical parameters of water, such as, temperature (°C), pH, salinity (‰) and dissolved oxygen (mg L − 1) were measured with a pH Benchtop Meter (Hanna HI 2212, Romania), refractometer (Atago — Japan) and oxymeter (YSI 55/12 FT, USA), respectively. The level of total ammonia was measured with an ammonia electrode (Orion 9512 BN, USA). 2.7. Statistical analysis The form of parasitism was evaluated according to Bush et al. (1997). Upon resuming, we calculate the prevalence index (% P), intensity of infection per infestation (II), mean intensity of infection per infestation (IMI) and mean abundance (AX). A comparison of parasitological index was performed with the appropriate protocol of statistical analysis established by Reiczigel and Rózsa (2005) using quantitative parasitology. In Experiment 2, the statistics of the water physical and chemical parameters and fish survival in the different treatments were performed analyzing significant difference between the means using one-way ANOVA (α = 0.05) with Statistica 6.0. 3. Results

2.4. Toxicity of formaldehyde for juvenile mullet

3.1. Ways of parasitism of juvenile mullet

2.4.1. Experiment 1 — preliminary test of acute toxicity Forty eight juveniles were exposed to formaldehyde for 96 h to determine the lethal range. The test was composed of a control

Through studies conducted to identify the diversity and parasitological index in the juvenile mullet, Ligophorus cf. uruguayensis Euzet & Suriano, 1977 (Ancyrocephalidae) and Solostamenides cf. platyorchis

E. Pahor-Filho et al. / Aquaculture 354–355 (2012) 111–116 Table 1 Parasitological index of juvenile mullet (n = 40). P — prevalence (%); II — intensity of infection/infestation; IMI — average intensity of infection/infestation; AX — average abundance. Parasites

Ligophorus cf. uruguayensis Solostamenides cf. platyorchis Digenea Nematoda

Parasitological index P

II

IMI

AX

100 10 92.5 6.7

2–110 1–3 1–40 1

23.4 1.5 2.67 1

23.4 0.15 2.47 0.07

Jianyin and Tingbao, 2001 (Microcotylidae) both Monogenoidea were found. Unidentified Digenea and Nematoda were observed among endoparasites. Table 1 shows the parasitological index in juvenile mullet at the time of capture and not treated with formaldehyde. 3.2. Toxicity of formaldehyde for juvenile mullet 3.2.1. Experiment 1 — preliminary test of acute toxicity In the preliminary test carried out to determine the definitive lethal concentrations of formaldehyde, total survival was observed in the control group. In T1 there was a 50% survival at 96 h and in T2 to T5 there was no survival after 24 h. 3.2.2. Experiment 2 — definitive formaldehyde acute toxicity test After a 96 h exposure to acute concentrations of formaldehyde, the LC50 for juvenile mullet was 20.77 mg L − 1 (Table 2). During the experimental period, control group maintained 100% survival. Treatments T1 to T4 showed 95.8, 91.6, 75.0 and 29.1% survival in 96 h, respectively. T5 showed 100% mortality within 48 h. The means of water quality parameters (temperature, salinity, pH, dissolved oxygen and total ammonia) were obtained for each treatment and no significant differences were observed (Table 3). 3.3. Effectiveness of formaldehyde to mullet parasitic control 3.3.1. Experiment 3 — lethal concentration — formaldehyde bath for one hour The survival of juvenile mullet was inversely proportional to formaldehyde concentrations. The median time of survival can be observed in Table 4. The LC50-1 h was estimated at 317.73 mg L − 1 formaldehyde, with a confidence interval between 273 and 368.87 mg L − 1. The LC100-1 h was set at 400 mg L − 1 formaldehyde. 3.3.2. Experiment 4 — formaldehyde prophylactic bath to parasitosis control After 1 h exposure, 100% survival was observed in the control group and also in T1 to T4. In T5, 87.5% survival of organisms tested was observed. In control group, L. cf. uruguayensis (Ancyrocephalidae) and S. cf. platyorchis (Microcotylidae) were identified. In T2 to T5, 100% of parasites were eliminated (Table 5). The formaldehyde concentration of 135 mg L − 1 was effective in Monogenoidea control. The parasitological index of juvenile mullet is shown in Table 6.

Table 2 Values of LC50 for juvenile mullet exposed to formaldehyde. LC50 = median lethal concentration (mg L− 1) and CI = confidence interval (mg L− 1). Time

LC50

CI

24 h 48 h 72 h 96 h

38.02 33.41 25.92 20.77

(33.47–43.18) (29.63–37.67) (21.75–30.88) (17.19–25.10)

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Table 3 Water quality parameters. T = treatments, Temp = temperature (°C), S = salinity, DO = dissolved oxygen (mg L− 1) and N-NH4+ + NH3 = total ammonia (mg L− 1). T1 = 13.5 mg L− 1, T2 = 21.6 mg L− 1, T3 = 40.5 mg L− 1, T4 = 81 mg L− 1 and T5 = 135 mg L− 1 formaldehyde. T

Temp

S

pH

DO

N-NH4+ + NH3

C T1 T2 T3 T4 T5

22.6 ± 0.19 22.5 ± 0.22 22.8 ± 0.20 22.5 ± 0.04 22.5 ± 0.11 22.5 ± 0.04

5 5 5 5 5 5

7.52 ± 0.10 7.50 ± 0.07 7.61 ± 0.04 7.52 ± 0.03 7.56 ± 0.10 7.47 ± 0.09

6.22 ± 0.45 5.68 ± 0.43 5.50 ± 0.56 5.49 ± 0.60 5.30 ± 0.38 5.11 ± 0.54

0.38 ± 0.04 0.41 ± 0.03 0.45 ± 0.05 0.46 ± 0.04 0.36 ± 0.04 0.48 ± 0.03

4. Discussion 4.1. Parasitology and method to use the hosts by parasites Records of Ligophorus species are specifically related to Mugil, suggesting particularity for this host group (Dmitrieva et al., 2007). Among Ligophorus species, only L. cf. uruguayensis was reported parasitizing juvenile mullet in this study. Marcotegui and Martorelli (2009) identified only Ligophorus saladensis in juvenile of the same host on the coast of Argentina. These authors argue that a diversity of parasites information was observed in this life stage of mullet. The fact of the same host being parasitized by different Ligophorus species at different latitudes may suggest that environmental differences determine this variation. Severe levels of prevalence and intensity of Ligophorus spp. infestation parasitizing the gills of Mugil species have also been reported (Failla-Siquier and Ostrowski de Núñez, 2009; Sarabeev and Balbuena, 2004; Sarabeev et al., 2005). Corroborating these results, severe index of prevalence (100%) and intensity of infestation (2–110) of L. cf. uruguayensis in the gills of M. liza juveniles were found. These results may be related to low efficiency of the immune system in juvenile stage of mullet. Tort et al. (2003) suggest that fish immunocompetence is observed in advanced stages of life, in which the contact with parasites and the number of immunocompetent cells are greater. Furthermore, severe index may be related to the specificity of Ligophorus spp. by Mugil species, or by direct life cycle of these parasites, encouraged by favorable environmental conditions such as low salinity, water temperature and availability of specific host in the estuary. Microcotylidae are common parasites of mullets (Fletcher and Whittington, 1998; Jianyin and Tingbao, 2001; Kohn and Cohen, 1998). Solostamenides sp. was detected in Mugil sp. in South America (Mendoza-Garfias and Pérez-Ponce de Leon, 1998) and this was the first record of Solostamenides species in Brazilian fish. The specimens are close to Solostamenides platyorchis Jianyin and Tingbao, 2001. In this study, the prevalence was 10% and infestation intensity was 1–3 by S. cf. platyorchis parasitizing the mullet gills. These values are relatively discrete in comparison to parasitological index observed for L. cf. uruguayensis. However, Microcotylidae are oviparous and have high proliferation capacity in intensive fish cultures, possibly Table 4 Average time of survival (min) of juvenile mullet in relation to the concentration of formaldehyde (mg L− 1). C = control, T1 = 540 mg L− 1, T2 = 1080 mg L− 1, T3 = 1620 mg L− 1, T4 = 2160 mg L− 1 and T5 = 2700 mg L− 1 formaldehyde. Treatments

Average time of survival

C T1 T2 T3 T4 T5

>60 >60 32 22 15 10

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Table 5 Parasitological index of Monogenoidea infestation in juvenile mullet: T = treatments, T1 = 67.5 mg L− 1, T2 = 135 mg L− 1, T3 = 270 mg L− 1, T4 = 405 mg L− 1 and T5 = 540 mg L− 1 formaldehyde, C = control, (n = 24 fish each). P (%) = prevalence; II = intensity of infestation; IMI = average intensity of infestation and AX = average abundance. Monogenoidea

T

Ligophorus cf. uruguayensis Solostamenides cf. platyorchis L. cf. uruguayensis S. cf. platyorchis L. cf. uruguayensis S. cf. platyorchis L. cf. uruguayensis S. cf. platyorchis L. cf. uruguayensis S. cf. platyorchis L. cf. uruguayensis S. cf. platyorchis

T1 T2 T3 T4 T5 C

Parasitological index P

II

IMI

AX

13.3 – – – – – – – – – 100 13.3

3–152 – – – – – – 1 – – 1–121 1–3

77.5 – – – – – – – – – 34 2

10.33 0 0 0 0 0 0 0 0 0 34 0.26

causing mortality in the hosts, as observed in Pagrus pagrus (Katharios et al., 2006). Values of prevalence and intensity of infestation of Monogenoidea observed in this study show the potential damage that these parasites may represent to fish cultures. Previous records of Digenea were presented by Oliveira et al. (2007), Merella and Garippa (2001) and Carnevia and Speranza (2003). Birds are often definitive hosts of Digenea, and newlyhatched larvae (in miracideo life stage) infect snails. These mollusks are the primary intermediate host and can be eaten by fish, which are considered secondary intermediate host (Poulin and Cribb, 2002). In this study many species of herons (Ardea cocoi Linnaeus, 1766; Bubulcus ibis Linnaeus, 1758; Casmerodius albus Linnaeus, 1758; Egretta thula Molina, 1782) and cormorants (Phalacrocorax brasilianus Gmelin, 1789) were observed and these birds are common in the estuary in which fish were collected (Votto et al., 2006). In juvenile mullet, severe prevalence index (92.5%) and intensity of infection (10–40) by Digenea were observed in the intestines. On the other hand, we found discrete index of prevalence (6.7%) and intensity of infection by nematode. The incidence of these parasites may be associated with the presence of birds in the estuary.

Table 6 Parasitological index of endoparasite infection in juvenile mullet. T = treatments, T1 = 67.5 mg L− 1, T2 = 135 mg L− 1, T3 = 270 mg L− 1, T4 = 405 mg L− 1 and T5 = 540 mg L− 1 formaldehyde infection, C1 = Digenea control and C2 = Tetraphyllidea control (n = 24 fish each). P (%) = prevalence; II = intensity of infection; IMI = average intensity of infection and AX = average abundance. Endoparasites

Digenea Tetraphyllidea Nematoda Digenea Tetraphyllidea Nematoda Digenea Tetraphyllidea Nematoda Digenea Tetraphyllidea Nematoda Digenea Tetraphyllidea Nematoda Digenea Tetraphyllidea Nematoda

T

T1

T2

T3

T4

T5

C

Parasitological index P

II

IMI

AX

40 40 – 40 33.33 – 40 66.66 – 33.33 60 – 46.66 66.66 6.7 80 80 –

1–10 1–102 – 1–12 1–8 – 2–70 1–107 – 1–32 1–270 – 2–40 1–172 1 1–67 1–99 –

4 30.83 – 4.83 3.8 – 20.16 30.1 – 9.4 50.88 – 9.71 52.7 1 22.58 25 –

1.6 12.33 0 1.93 1.26 0 8.06 20.06 0 3.13 30.53 0 4.53 35.13 0.07 18.06 20 0

4.2. Toxicity of formaldehyde for juvenile mullet 4.2.1. Experiment 1 — preliminary acute toxicity test The range of formaldehyde lethality for juvenile mullet was observed at concentrations between 0 and 67.5 mg L− 1. Thus, the definitive acute toxicity test was delineated according to those data. 4.2.2. Experiment 2 — definitive test of formaldehyde acute toxicity There was a great variation in fish responses comparing the acute toxicity data (estimated as LC50) in fish exposed to formaldehyde. Cruz and Pitogo (1989) studying milk fish (Chanos chanos) juveniles with average weight of 6 g, estimated the LC50-24 h in 322 mg L − 1, nearly ten times more tolerant than M. liza (LC50-24 h =38.02 mg L− 1). The results of acute toxicity of formaldehyde for mullets showed total mortality when animals were subjected to concentration >135 mg L− 1 at 24 h. A comparison for three days of acute exposure showed that juvenile blowfish Sphoeroides annulatus are more tolerant (LC50-72 h =79 mg L− 1) (Fajer-ávila et al., 2003) to formaldehyde than juvenile mullet (LC5072 h = 25.92 mg L− 1). The LC50-96 h estimated for juvenile mullet was 20.77 mg L− 1, showing more tolerance to formaldehyde toxicity in comparison to striped bass Morone saxatilis (LC50-96 h =10.84 mg L− 1) (Reardon and Harrell, 1990) and betray Hoplias lacerdae (LC5096 h = 7.47 mg L− 1) (Cruz et al., 2005). However, the LC50-96 h estimated for the Atlantic salmon Salmo salar (LC50-96 h = 69 mg L− 1) (Bills et al., 1977), and for the milk fish C. chanos (LC50-96 h =232 mg L− 1) (Cruz and Pitogo, 1989) showed that these species are more tolerant to formaldehyde than M. liza. During the experimental period, the increase of concentrations of formaldehyde did not interfere in the physical and chemical parameters of water, which remained within the limits required for aquatic species, not interfering, in juvenile mullet survival. The ammonia values were expressed as total ammonia N-NH4+ + NH3, because values of gas ammonia were insignificant. 4.3. Effectiveness of formaldehyde to mullet parasitic control 4.3.1. Experiment 3 — lethal concentration — formaldehyde bath for one hour Juveniles of mullet were more tolerant when subjected to formaldehyde at concentrations up to 540 mg L − 1 for 1 h. According to Francis-Floyd (1996), the concentration of formaldehyde used for parasitosis control (baths for 1 h) in aquaculture, should not exceed 250 mg L− 1. Studying the stress of “tambaqui” Colossoma macropomum caused by formaldehyde, it was reported that formaldehyde can be used in concentrations of 50, 100 and 150 mg L − 1 in baths of 30, 60 and 120 min, respectively, or at concentrations of 200 and 250 mg L − 1 in baths of 30 min, without compromising the fish homeostasis (Araújo et al., 2004). According to the results of preliminary test, the definitive test with formaldehyde baths for 1 h was performed up to 540 mg L − 1. The LC50-1 h estimated to juvenile mullet was 317.33 mg L − 1 of formaldehyde, while in the concentration of 1080 mg L − 1 total mortality was observed, confirming the results of Francis-Floyd (1996). The LC50-1 h demonstrated that this species is more sensitive to formaldehyde than Nile tilapia Oreochromis niloticus (LC50-2 h = 429.6 mg L − 1) (Macniven and Little, 2001) and blowfish (LC50-1 h = 972 mg L− 1) (Fajer-ávila et al., 2003). 4.3.2. Experiment 4 — formaldehyde prophylactic bath to parasitosis control Formaldehyde at high concentrations, causes lesions in the gills, decreasing the efficiency of gas exchange and osmoregulation, resulting in the death of fish (Noga, 2010). The mortality of juvenile mullet observed in T5 appears to be related to the histological lesions in the gills, causing death by hypoxia, confirming what was previously described by Noga (2010).

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Recent studies describe Ligophorus species (Abdallah et al., 2009; Failla-Siquier and Ostrowski de Núñez, 2009; Marcotegui and Martorelli, 2009) and Solostamenides (Jianyin and Tingbao, 2001) parasites of Mugil, but these studies did not examine the effectiveness of anthelmintics in parasite control. Formaldehyde is used for Monogenoidea control in aquaculture, but the effective concentration has been discussed by many authors, revealing conflicting results. The severe parasitological index of L. cf. uruguayensis and S. cf. platyorchis infestation found in this study suggests the negative potential risk that these parasites may represent in fish farming. In the physical and chemical conditions in which the experiment was done, in T1, formaldehyde was effective to control 100% of S. cf. platyorchis in 1 h baths, nevertheless, this concentration was not effective in eliminating the L. cf. uruguayensis. In T2, formaldehyde was effective to Monogenoidea control. These results suggest a possible tolerance of L. cf. uruguayensis to formaldehyde at concentrations 67.5 mg L− 1. These results corroborate Rowland et al. (2006), showing that 40 mg L− 1 of formaldehyde was effective in Lepidotrema bidyana and Gyrodactylus sp. (Monogenoidea) control in Bidyanus bidyanus (Terapontidae), but in concentrations of 20 and 25 mg L− 1, fish continued to be infested with Monogenoidea. On the other hand, Fajer-Ávila et al. (2007) observed that formaldehyde baths of 51 mg L − 1 for 1 h were not fully effective to control Haliotrema sp. and Euryhaliotrema sp. (Monogenoidea) in Lutjanus guttatus, eliminating 72% of parasites. In T3 to T5, formaldehyde was totally effective in Monogenoidea control, but the possibility that these treatments cause injury and/or mortality of the hosts is high. Corroborating these results, other authors have shown that formaldehyde concentrations above 150 mg L − 1 are also effective in Monogenoidea control for other fish species. Katharios et al. (2006) found that 200 mg L − 1 of formaldehyde for 1 h, eradicated 100% of Microcotyle sp. in P. pagrus. Sharp et al. (2004) studying the control of Monogenoidea in Seriola lalandi lalandi (Carangidae), observed that 400 mg L − 1 of formaldehyde was effective in eliminating 99% of Benedenia seriolae and Zeuxapta seriolae (Monogenoidea). However, these studies did not have a histological analysis of treated fish to give proof of injuries caused by formaldehyde. Francis-Floyd (1996) reported that formaldehyde was not effective in endoparasite control. The results of the present study corroborate this latter author, because the formaldehyde did not control the infection by Digenea with the concentrations tested; Nematoda and Tetraphyllidea were also found in the intestines of all mullets. It is possible that formaldehyde is not absorbed by the body of hosts during fish exposition. In other studies, high prevalence and intensities of infection have been found in Mugil spp. by Nematoda (Lymbery et al., 2002) and by Digenea causing histological changes in hosts (Oliveira et al., 2007). Tetraphyllidea infection was also reported in the intestines, but this infection was not reported in the present study with mullets. This group of parasites is strictly marine, indicating that the infestation is occurring in seawater. The severe parasitological index of infection by Digenea and Tetraphyllidea in juvenile mullet found in this study, reveals the need for studies aiming the reduction or total eradication of endoparasites using anthelmintics such as mebendazole (Stoskopf, 1988), levamisole and praziquantel (Fujimoto et al., 2006). Until now, there are no studies demonstrating the efficacy of these drugs to endoparasites of Mugil spp. In conclusion, this study showed that formaldehyde was effective in controlling S. cf. platyorchis and L. cf. uruguayensis at concentrations of 67.5 mg L − 1 and 135 mg L − 1, respectively. Survival can corroborate these claims, since there were no mortalities in mullets exposed to these concentrations. However, none of the concentrations tested were effective in controlling endoparasites. The different responses of host exposed to formaldehyde prophylactic bath, in similar studies indicate that the results do not extend to other species already studied.

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Acknowledgments We would like to manifest our gratitude to Dr. Luis Alberto Romano of Federal University of Rio Grande and to Dra. Ana Luísa Schifino Valente of the Federal University of Pelotas for their suggestions.

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