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Water Air Soil Pollut (2012) 223:5917–5930 DOI 10.1007/s11270-012-1328-9

Acute Toxicity of Seven Selected Pesticides (Alachlor, Atrazine, Dieldrin, Diuron, Pirimiphos-Methyl, Chlorpyrifos, Diazinon) to the Marine Fish (Turbot, Psetta maxima) Lazhar Mhadhbi & Ricardo Beiras

Received: 4 April 2012 / Accepted: 10 September 2012 / Published online: 28 September 2012 # Springer Science+Business Media B.V. 2012

Abstract The present study evaluated the short-term toxicity of seven selected pesticides: four insecticides (chlorpyrifos, dieldrin, diazinon and pirimiphosmethyl) and three herbicides (diuron, alachlor and atrazine). With this aim, a standard toxicity test with the highly sensitive early life stages (ELS) of a marine fish was used. The turbot, Psetta maxima, is abundant in shallow estuarine and costal habitats and is currently the most commonly cultivated fish species in Galicia, NW Spain. According to the turbot ELS test results, chlorpyrifos was the most toxic pesticide tested for both embryos and larvae and was followed in order of decreasing toxicity by dieldrin, pirimiphos-methyl, diazinon, alachlor, atrazine and diuron. Larvae were more sensitive than embryos to the seven pesticides. The median lethal concentrations of the selected pesticides during a 48- and a 96-h exposure for turbot embryos and larvae were, respectively (in micrograms per litre): chlorpyrifos, 116.6 and 94.65; dieldrin, 146 and 97; pirimiphos-methyl, 560 and 452; diazinon, 1,837 and 1,230; alachlor, 2,177 and 2,233; diuron, 10,076 and L. Mhadhbi : R. Beiras Estación de Ciencias Mariñas de Toralla (ECIMAT), 36331 Vigo, Galicia, Spain L. Mhadhbi (*) Unité d’Hydrobiologie, Laboratoire de Biosurveillance de l’Environnement, Faculté des Sciences de Bizerte, Université de Carthage, Zarzouna 7021, Tunisia e-mail: [email protected]

7,826; and atrazine, 11,873 and 9,957. According to their acute toxicity, the insecticides were more toxic than the herbicides. Furthermore, all insecticides and herbicides appear to be teratogenic to turbot ELS. Keywords Turbot . Early life stage . Acute toxicity . Sublethal effects . Pesticides

1 Introduction The adverse effects of toxicants become significant when they affect economically important organisms or organisms consumed by economically important animals or human beings, producing stress conditions either in the form of physiological and biochemical damage to the vital organs or even in the form of death of living organisms of the terrestrial and aquatic environment (Kumar et al. 2011). Widely used all over the world for pest control in agriculture and fish farming, pesticides ultimately find their way into aquatic ecosystems, thus posing risk to economically important non-target species. The toxic effects of pesticides may range from alterations in a single cell to changes in whole organisms or even populations (Giari et al. 2008). Among the many forms of chemical pesticides, organophosphates (chlorpyrifos, dieldrin, pirimiphosmethyl, diazinon) and herbicides (alachlor, atrazine, and diuron) are considered to be the most hazardous environmental pollutants since they are very persistent,

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non-biodegradable and biaccumulative (Kumar et al. 2011; Barbieri 2009). Thus, contamination by pesticides is a serious water pollution issue, which may cause an environmental imbalance and an increase in poisoning of fish and other aquatic species (Barbieri 2009; Aguiar et al. 2004). In recent years, selected insecticides/herbicides have been intensively and widely used in the Galician area (AEPLA 2012), and all selected pesticides, except diazinon and pirimiphos-methyl, are on the list of priority substances (According to Annex II of the Directive 2008/105/EC). Fish are suitable bio-indicators of aquatic environmental pollution since they are exposed to the chemicals resulting from agricultural production either directly or indirectly of their ecosystem (Lakra and Nagpure 2009). The turbot provides a good biological model for toxicological studies (Mhadhbi et al. 2010) thanks to several of its characteristics, namely its high growth rates, great resistance to diseases, simple handling practices, easy reproduction in captivity at a prolific rate and good tolerance to a wide range of environmental conditions. Previous studies evaluated the acute toxicity of pesticides to adult fish species; however, the early life stages of fish are generally regarded as the life history stages which are most sensitive to toxic agents (Hutchington et al. 1998). During early ontogenesis, critical development of the tissues and organs takes place, a process which can easily be disrupted by unfavourable environmental conditions including exposure to toxic compounds (Foekema et al. 2008; Kammann et al. 2009). As to the best of our knowledge few studies of the consequences of embryonic and larval fish exposure to pesticides have been performed, their lethal and sublethal effects are not yet completely understood. Therefore, our objective was to test the toxicity of the most abundant insecticides/herbicide used in Galicia on the early life stages (ELS) of turbot. For this purpose, toxicant effects were examined through diverse endpoints, such as hatching success, embryo–larval morphology malformations and larval survival (Mhadhbi et al. 2010, 2012).

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NW Spain). The eggs were transported to the laboratory in plastic bags inside portable ice boxes and maintained in aquaria with running natural seawater (salinity, 34‰). Eyed eggs were acclimated to laboratory conditions for 24 h at 14±1 °C (hatchery–rearing temperature) before the experimental exposures to the toxicants. 2.2 Experimental Solutions and Exposures Technical grade atrazine (97.5 % purity), alachlor (99.2 % purity), chlorpyrifos (99.9 % purity), diazinon (98 % purity), dieldrin (98 % purity), diuron (98 % purity) and pirimiphos-methyl (99.5 % purity) were obtained from Sigma Chemical, Co. (St. Louis, MO). Selected physicochemical properties of these pesticides are listed in Table 1. The stock solutions of each pesticide were made in 100 % dimethyl sulfoxide (DMSO, Sigma-Aldrich, Steinheim). Pesticide grade DMSO was used as a carrier (0.1 %, as this was found to be non-toxic in the preliminary test) in all tests. DMSO was added to the control groups equal to the amount of the carrier solvent used for the toxicity tests. Six concentrations in a 2× geometric scale, plus one solvent control and one control with no toxicants added, were tested using four replicates for each condition. The experimental concentrations were chosen on the basis of range-finding trials and data from the literature. The concentrations tested for each compound were always below their water saturation levels. Incubations were made in 1,000-mL glass beakers to avoid losses of the tested compounds from the solutions. All glassware was acid-washed (HNO3 10 vol%) and rinsed with acetone and distilled water before the experiments. The physicochemical conditions of the experiments were 34.20±0.15 ppt salinity, 7.32±0.70 mg/L O2 and 8.29±0.11 pH. The experimental design followed the recommendations from the OECD guidelines (OECD 1998) and the EU Commission Directive 92/69/ EEC, with the modifications indicated below. The modifications included a higher number of eggs per experiment and the number of reported non-lethal endpoints. All of the experiments were performed using a semistatic test with water renewal every 48 h.

2 Materials and Methods 2.3 Fish Embryo Exposure and Toxicity Assay 2.1 Biological Material Turbot (Psetta maxima) eggs were obtained from a single stock of adults (Insuiña S.L., Mougás, Galicia,

Immediately after their arrival at the laboratory, within 72 h post-fertilization, the floating fertilized eggs were collected and the non-fertilized eggs at the bottom

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Table 1 Physical and chemical properties of the pesticides tested for acute toxicity to the seawater flatfish turbot (P. maxima) Common name

Chemical name

Class

Water solubility (mg/L)

Log Kow

Adsorption coefficient (log Koc)

Alachlor

2-Chloro-N-[2,6-diethyphenyl]-N-[methoxymethyl) acetamide]

Herbicide

140

2.63

2.08–2.28

Atrazine

2-Chloro-4-ethylamino-6-isopropylamino-s-triazine

Herbicide

28

2.75

122

Chlorpyrifos

O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate

Insecticide

1.18

4.7–4.9

3.83

Diazinon

O,O-diethyl-O-(6-methyl-2-{1-methylethyl}-4-pirimidinyl phosphorothioate [1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a, 5,6,7, 8,8a-octahydro-exo-1,4-endo-5,8-dimethanonaphthalene] C9H10Cl2N2O, 3-(3,4-diclorofenil)-1,1-dimetilurea

Insecticide

60

3.81

3.12

Insecticide

0.2

4.55

3.87

Herbicide

36.4

2.68

418–560

Dieldrin Diuron

2.13 Pirimiphos-methyl

O-[2-(diethylamino)-6-methylpyrimidin-4-yl] O,O-dimethyl phosphorothioate

Insecticide

4

4.2

2.14

Log Kow 0log octanol/water partition coefficient. Water solubility is given in milligrams per litre

discarded. The eggs were examined under a dissecting microscope, and those embryos exhibiting normal development that had reached the blastula stage were selected for subsequent experiments. Briefly, 50 normal fertilized eggs were randomly selected and carefully distributed into exposure glass beakers containing 500 mL filtered seawater and spiked with the test solutions. The treatments were incubated per quadruplicate in an isothermal room (18±1 °C), in the dark. The control beakers were similarly set up. Neither food nor aeration was provided during the bioassays. The eggs were transferred into each beaker from the lowest to the highest concentration to minimize the risk of cross-contamination. The tests were run for 6 days. The effects of the toxicants on the turbot embryos and larvae were observed daily throughout the 6-day exposure period (from 0 to 2 days embryonic exposure and from 2 to 6 days larval exposure). The number of dead eggs/ embryos was recorded 48 h after incubation. Hatching was defined as the rupture of the egg membrane, and partially as well as fully hatched larvae were counted as hatched. The survival and the malformation of larvae were observed and recorded every day after hatching. Mortality was identified by coagulation of the embryos, missing heartbeat, failure to develop somites and a non-detached tail. The recorded sublethal endpoints included embryo malformations—yolk sac alterations, no rupture of the egg membrane, pericardial oedema and skeletal deformities—and hatching success.

The observations were made using a thick slide with a concave chamber, which was filled with clean seawater. Each larva was carefully placed in the chamber and observed under a binocular (magnification, 1.5×1.6) using MultiScan (Nikon SMZ1500) computer image analysis. 2.4 Statistical Analyses The dose–response relationships were described using the modified Weibull model. Fitting procedures and parametric estimations were performed by minimization of the sum of quadratic differences between the experimental and the model-predicted values using the nonlinear least-squares (quasi-Newton) method provided by the ‘Solver’ macro of the Microsoft Excel spreadsheet. Parametric estimates were confirmed in the nonlinear section of the Statistica 8.0 pack, which was also used to calculate the parametric confidence intervals and model consistency (Student’s t and Fisher’s F tests, respectively, in both cases with α00.05). The maximum no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were established through ANOVA and Dunnett’s post hoc test using the SPSS application, version 19.0. Non-parametric tests, Kruskal–Wallis and the Mann–Whitney U, were used when the data did not meet the requirements of normality and homoscedasticity. Differences were considered as significant when ppirimiphos-methyl> diazinon>alachlor>atrazine>diuron, which is consistent with previous studies on invertebrates and amphibians (Sparling and Fellers 2007). 4.2 Teratogenic Effects A number of studies focussed on the sublethal effects of pesticide exposure on olfaction, behaviour, kidney histology and tissue growth (Gagnon and Rawson 2009). However, few studies address the teratogenic effects caused by pesticides for marine fish ELS, and there are only a few previously published studies for

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Table 4 Comparison of LC50 for the toxicity of selected pesticides tested to fish species Chemical

Fish

Life stage

Study time

LC50

Source

Alachlor

Bluegill (L. macrochirus)

Adult Adult Adult

96 h 96 h 96 h

2,500 μg/L 500 μg/L 3,730 μg/L

Juvenile Embryo–larvae Adult Adult Juveniles Embryo–larvae Adult Adult Embryo Post-larvae Juveniles Adult

96 h 96 h

PED (2000) PED (2000) Chaturvedi and Agrawal (1991) Peebua et al. (2008) Present study Nwani et al. (2010) Neškovic et al. (1993) Broderius et al. (1995) Present study Belden and Lydy (2006) Jarvinen et al. (1983) Phillips et al. (2002) Phillips et al. (2003) Kikuchi et al. (1996) Humphrey and Klumpp (2003) Eder et al. (2009) Rao et al. (2005) USEPA (1986) USEPA (1986) De Silva and Samayawardhena (2002) Present study Banaee et al. (2011) USEPA (1999) USEPA (1999) Girón-Pérez et al. (2007) USEPA (1999) Sharma (1990) Aydın and Köprücü (2005) Present study Peakall (1996) Peakall (1996) Adema and Vink (1981) Adema and Vink (1981) Present study Nebeker and Schuytema (1998) 27,100 μg/L

India catfish (H. fossilis)

Atrazine

Chlorpyrifos

Nile tilapia (O. niloticus) Turbot (P. maxima) Breathing fish (C. punctatus) Carp (C. carpio) Rainbow fish (M. fluviatilis) Turbot (P. maxima) Fathead minnows (P. promelas) Bluegill (L. macrochirus) Walleye (S. vitreum) Rainbow trout (O. mykiss) Rainbow fish (M. splendida splendida) Chinook salmon (O. tshawytscha)

Diazinon

Dieldrin

Diuron

Juveniles

Lake trout (S. namaycush) Goldfish (C. auratus) Guppy (P. reticulata)

Adult Adult Juveniles

Turbot (P. maxima) Rainbow trout (O. mykiss) Bluegill (L. macrochirus) Fathead minnows (P. promelas) Nile tilapia (O. niloticus) Cutthroat trout (O. clarki) Sheepshead minnow (C. variegatus) Carp (C. carpio) Turbot (P. maxima) Bluegill (L. macrochirus) Rainbow trout (O. mykiss) Common goby (G. microps) Plaice (P. platessa) Turbot (P. maxima) Fathead minnows (P. promelas)

Embryo–larvae Adult Adult Adult Adult Adult Adult Embryo–larvae Embryo–larvae Adult Adult Adult Adult Embryo–larvae Embryo–larvae

96 96 96 96 96 48 48 96 96

h h h h h h h h h

380 μg/L 1,838 μg/L 42,300 μg/L 18,800 μg/L 5,600–10,400 μg/L 9,957 μg/L 200 μg/L 3.6 μg/L 225 μg/L 29 μg/L 45 μg/L 396 μg/L

96 96 96 96 96

h h h h h

7.1–81 μg/L 297 μg/L 98 μg/L 806 μg/L 7.1 μg/L

96 96 96 96 96 96 96 96 96 24 24

h h h h h h h h h h h

94.65 μg/L 1,170 μg/L 460 μg/L 7,800 μg/L 7,830 μg/L 2,150 μg/L 1,400 μg/L 1,500 μg/L 1,230 μg/L 0.0055 mg/L 0.0019 mg/L 0.0035 mg/L 0.0017 mg/L 97 μg/L 11,700 μg/L

96 h 7 day

Juvenile

Turbot (P. maxima) Pirimiphos-methyl

10 day

Nebeker and Schuytema (1998) Striped bass (M. saxatilis) Embryo–larvae

Larvae 96 h

96 h 7,826

500 μg/L Present study

Hughes (1973)

Rainbow trout (O. mykiss) Australian blue eye (P. signifier) Guppy (P. reticulata) Turbot (P. maxima)

Adult Adult Adult Embryo–larvae

96 96 96 96

354 μg/L 91 μg/L 19 μg/L 452 μg/L

Mensink (2008) Brown et al. (1998) USEPA (1999) Present study

h h h h

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other fish species in the literature. Thus, the aim was also to determine the morphological abnormalities caused by selected pesticides on turbot early life stage, namely hatching success, yolk sac alteration, pericardial oedema and skeletal deformities. Under our experimental conditions, both insecticides and herbicides were capable of affecting the survival and development of embryos and larvae, although mainly at high concentrations. The results also show that pesticides above the threshold concentrations of 25 μg/L for chlorpyrifos and dieldrin, 400 μg/L for pirimiphos-methyl, 800 μg/L for diazinon, 1,250 μg/L for alachlor and 2,500 μg/L for atrazine and diuron, respectively, reduced hatching success and caused many deformities in turbot. One-way ANOVA using ranks showed that the treatment had a statistically significant effect (p65 μg/L; Birge et al. 1983), zebrafish (concentration, >1,300 μg/L; Görge and Nagel 1990) and bluegill (concentration, >46 μg/L at 90 days; Macek et al. 2003). Birge et al.

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(1983) suggested that the exposure of fish embryos to atrazine may induce abnormalities. Specific types of abnormalities associated with atrazine exposure were not reported, although the report notes that defects of the head and the vertebral column, dwarfed bodies, and absent or reduced eyes and fins were reportedly the most common symptoms across studies and species. The relatively high embryo mortality and reduced survival rate reported here and in other studies (Brown et al. 1998; Birge et al. 1983) could be due to the fact that the properties and high lipophilicity of pesticides (Table 1) allow them to partially overcome the chorion barrier that protects the egg. As in previous studies, larvae exposed to pesticides showed a consistent and highly repeatable set of morphological deformations (Gagnon and Rawson 2009; Humphrey and Klumpp 2003; De Silva and Samayawardhena 2002). These aberrations included the development of an abnormal dorsal curvature of the trunk and tail observed at an exposure concentration of 25 μg/L of chlorpyrifos in our study compared to 155 μg/L in Jarvinen et al. (1983). Also, the guppy was less sensitive to chlorpyrifos than the turbot, where De Silva and Samayawardhena (2002) observed evident high percentages of malformations, such as tail abnormalities, ventral swelling and haemorrhaging in guppy at 2 μg/L. In addition, Karen et al. (1998) demonstrated that exposure to 10 μg/L chlorpyrifos can cause stress at failure of the caudal vertebrae in Fundulus heteroclitus. Danio rerio larvae exposed to 250 μg/L chlorpyrifos showed a significant increase in the percentage of individuals with morphological deformations (i.e. heart oedema and spine deformation; Kienle et al. 2009). Our results document a higher sensitivity of turbot embryo–larvae to diazinon when compared to the early life stages of other fish species such as D. rerio, for which it was observed in Modra et al. (2011) that severe oedema, abnormal gut development and simple axial abnormality were the most frequent malformations at the concentration of 12 mg diazinon per litre. In developing turbot, a prominent result of diazinon was oedema, with the greatest effects observed when exposure was initiated in the late embryonic and larval period. Our observations are in agreement with Hamm and Hinton (2000). Embryos were less sensitive to pesticides than larvae, possibly because the chorion protected the embryos. For some toxicants, the egg chorion acts as a barrier that protects the embryo (Hallare et al. 2006).

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These findings are similar to those of previous studies which found that newly hatched larvae were more sensitive than eggs when post-fertilized eggs and larval Melanotaenia fluviatilis were exposed to esfenvalerate and rainbow fish to chlorpyrifos (Humphrey and Klumpp 2003; Barry et al. 1995). This is consistent with the morphological abnormalities reported for catfish exposed to atrazine (Birge et al. 1983) and pink snapper (Pagrus auratus) exposed to diuron (Gagnon and Rawson 2009) during embryogenesis. The reduced larval survival observed in our study also agrees with the findings of Humphrey and Klumpp (2003) and Kienle et al. (2009) who observed reduced juvenile survival in killifish and rainbow fish exposed to pesticides. For adult fish, the literature indicates a lack of effects (Humphrey and Klumpp 2003; Belden and Lydy 2006; Eder et al. 2009). Our results therefore support the conclusion that the marine fish P. maxima seems to be more sensitive than the classical toxicological model freshwater zebrafish. There is some evidence that saltwater fish are more susceptible to pesticide exposure than freshwater fish. In addition, it is well known that the early life stages are much more sensitive than juveniles or adults. Therefore, the use of adult freshwater fish in aquatic risk assessment does not cover the potential harmful effects on marine fish ELS.

5 Conclusions Our study demonstrates that the sensitivity of P. maxima to various pesticides makes the turbot suitable as an indicator test species and useful for studying the effects of pesticides on marine fish and that it can be viewed as a representative species for costal aquatic ecosystems. Chlorpyrifos and dieldrin were extremely hazardous, pirimiphos-methyl was highly hazardous, diazinon and alachlor were moderately hazardous, and atrazine and diuron were less hazardous to P. maxima. The larvae of P. maxima were more sensitive to pesticides than the embryos. Pesticides have been proven to be teratogenic to the embryo–larval stages at concentrations above 25 μg/L for chlorpyrifos and dieldrin, 40 μg/L for pirimiphosmethyl, 400 μg/L for diazinon, 1,250 μg/Lfor alachlor and 2,500 μg/L for atrazine and diuron, leading to malformations in embryo development, failure to hatch and consequent egg coagulation at 48 h. At higher

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concentrations, pesticides caused a significant increase in embryo mortality. Surviving organisms suffered a significant decrease in hatching success, malformations, pericardial oedema and skeletal deformation. Acknowledgments The authors gratefully acknowledge the cooperation of all the workers and personnel at the ECIMAT and Laboratory of Marine Ecology, especially those workers who willingly participated in the hatchery, Damián Costas and Arantxa Martínez. The authors would like to thank David Chavarrias (Insuiña S.L., Mougás, Galicia, Spain). This study was financially supported by MAE-PCI (Ministry of Foreign Affairs, Spain), Ministry of Higher Education, Scientific Research and Technology in Tunisia and the Spanish Ministry of Science and Innovation (MCINN) through the research project Environmental Quality Criteria for Marine Ecosystems (ENVICRISYS).

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