Nem2565.dvi

Nematology, 2011, Vol. 13(8), 901-909 A microcosm experiment on the effects of permethrin
on a free-living nematode assemblage
OUFAHJA , Badreddine SELLAMI, Mohamed DELLALI, Patricia AÏSSA, Coastal Ecology and Ecotoxicology Unit, Laboratory of Environment Biomonitoring, Faculty of Sciences of Bizerte, University of Carthage, 7021 Zarzouna, Tunisia Received: 20 January 2011; revised: 5 April 2011 Accepted for publication: 10 April 2011; available online: 3 June 2011 Summary – Microcosms were used to assess the impact of permethrin on the abundance and diversity of free-living marine nematodes.
The nematodes were exposed to three permethrin concentrations (50, 100 and 150 μg l−1), and the effects were examined after 25
days. The abundances of nematodes at all permethrin concentrations significantly exceeded those in the controls. Multivariate analyses
demonstrated that responses of nematode species to permethrin treatments were varied: Pselionema sp., Prochromadorella neapolitana
and Spirinia gerlachi were eliminated at the low dose and seemed to be species intolerant to permethrin; Trichotheristus mirabilis
and Xyala striata, which increased with increasing contamination levels, seemed to be ‘opportunistic’ and/or ‘resistant’ species.
Results showed significant differences between univariate measures of diversity of control nematodes and those from permethrin-
contaminated microcosms, where all decreased significantly with increasing abundance of the most tolerant species to permethrin. The
use of microcosms has allowed the effects of permethrin on nematodes to be assessed individually, which is not possible in the field.
Keywords – community composition, insecticide, marine nematodes, permethrin contamination, Prochromadorella neapolitana,
Pselionema sp., Spirinia gerlachi, Trichotheristus mirabilis, Xyala striata.
Many kinds of pesticides and their metabolites can phipod, Corophium volutator, with a 28-day LC50 rang- potentially enter water bodies by several routes and ing from 55 to 82 ng g−1. Research with the freshwa- be partitioned to bottom sediments even if they are ter amphipod, Hyalella azteca, indicated similar results appropriately used for crop protection in accordance with with growth inhibition at levels as low as 44-73 ng g−1 good agricultural practice (Gilliom, 2001). Spray drift, (Amweg et al., 2005; Holmes et al., 2008). Results from surface runoff, and field drainage are relevant routes Stueckle et al. (2009) suggested that permethrin disturbed of exposure, and contamination through groundwater limb regeneration and moulting in the mud fiddler crab, discharge can occur (Martin et al., 2003). Permethrin is Uca pugnax. Singh and Srivastava (1999) found a signifi- an insecticide in the pyrethroid family. It is a non-polar cant reduction in the activity of lactate dehydrogenase and chemical with low water solubility and a high affinity cytochrome oxidase and an enhancement in succinate de- for sediment, making it generally immobile in aqueous hydrogenase activity in the tissues of the freshwater fish, systems (Sharom & Solomon, 1981). Published field Channa striatus, exposed to permethrin. This species sur- studies indicate that permethrin concentrations ranged vives the dry season by burrowing in the mud at the bot- from 0.94 ng g−1 (San Francisco, CA, USA) (Woudneh & Oros, 2006) to 335 ng g−1 (Yorkshire, UK) (Bonwick The first step towards preventing the environmental risks of permethrin is knowledge of small bioindicators The occurrence of permethrin is of concern because at the base of the marine food web. This is the case of the pyrethroids are known to be highly toxic to aquatic organ- meiofauna (all metazoans between 40 μm and 1 mm ac- isms, especially those that are sediment-dwelling (Hill, cording to Vitiello and Dinet, 1979). The understanding 1989). In a sediment toxicity study, Bat and Raffaelli and awareness of the impact of permethrin on these or- (1996) found that permethrin is toxic to the mud am- ganisms encouraged the investigation of their major rep- ∗ Corresponding author, e-mail: fehmiboufahja@yahoo.fr Koninklijke Brill NV, Leiden, 2011 Also available online - www.brill.nl/nemy resentative component in terms of abundance (ca 23 mil- composed of coarse fraction (99.6 ± 0.37%) with sand lion m−2; Warwick & Price, 1979) and diversity (ca 4000 porosity of 0.26 ± 0.03. Sediments were homogenised in known species; Platt, 1977) of free-living nematodes. The the laboratory by gentle hand stirring with a large spatula small size and short generation times of these worms and the ease with which they can be maintained in laboratoryconditions greatly encourages their use in biomonitoring experimental studies (Guo et al., 2001; Schratzberger etal., 2002; Mahmoudi et al., 2007; Beyrem et al., 2010).
The microcosm containers were acrylic boxes with Although meiobenthic nematodes have been shown to be internal dimensions 29 cm long × 19 cm wide × 17 cm sensitive to many classes of pollutants, including diesel, high, as described and utilised successfully by Suderman metals and lubricants, no published work has examined and Thistle (2003). Each was gently filled with 2 kg of the effect of permethrin on these organisms. Moreover, homogenised sediment topped up with 4 l of seawater there are almost no studies on the effects of pesticides on from the native site. The experiments were conducted in nematodes except for entomopathogenic species (Rovesti a controlled environment (20◦C; 12 h light/dark cycle) & Deseo, 1990; Shannag et al., 1994; Radova, 2010).
and treatments consisted of three levels of permethrin: The current work aimed to assess changes in abun- 50 μg l−1 (termed Per (L)), 100 μg l−1 (Per (M)) and dance, diversity descriptors and species composition of a 150 μg l−1 (Per (H)) and an untreated control (C).
nematode assemblage from Bizerte Bay in Tunisia when Since the water solubility of technical grade permethrin exposed to crescent doses of permethrin.
is 200 μg l−1 at 20◦C (Bringolf et al., 2007), thehighest permethrin test dose in this study was limitedto 150 μg l−1. Four replicates of each microcosm were Materials and methods
randomly assigned to the control and each permethrintreatment. Each microcosm was aerated via an air stone diffuser. After 25 days, the experiments were terminatedand the sediments were fixed in 4% formalin. During this Natural meiobenthic communities were collected on 15 period, stability of salinity, temperature, dissolved oxygen August 2009 from the subtidal zone of El Kebir beach and pH was measured daily with a thermo-salinity meter (width 4 km, max. height 20 m) in Bizerte Bay, Tunisia.
(LF 196; WTW, Weilheim, Germany), an oxymeter (OXI According to Ben Garali et al. (2008), this ecosystem 330/SET, WTW) and a pH meter (pH 330/SET-1, WTW), is a dissipative beach principally driven by the physical forces of waves and tides (N-S and NW-SE directions).
There is practically no exposure to human activities from the bordering cities (Zarzouna & Menzel Jemil) due tothe presence at the south of Bizerte lagoon, which has Four plexiglass hand-cores with a section area of been classically used since 1950 as an open-dumping 10 cm2 down to the base of each microcosm container type solid waste disposal area. Maritime pine is planted were sampled at the end of the experiment. We consid- to stabilise movement of east coastal sand. This area is ered that a single core cannot provide an accurate rep- not open as a classical agricultural zone and potential resentation of the nematode community inside a micro- pollutants like fertiliser and pesticides are not applied. The cosm. Meiobenthic taxa were extracted from the sedi- extreme left part of El Kebir beach was chosen to keep the ment by resuspension-decantation methodology (Wieser, sampling location (37◦13 16.05 N, 9◦56 04.58 E) away 1960). After passing through a 1 mm sieve, and reten- from tourism-induced disturbances (presence of people tion on a 40 μm sieve according to Vitiello and Dinet on the beach and swimming in the surf zone). Hand- (1979), they were stained with Rose-Bengal (0.2 g l−1).
cores of 10 cm2 were used to a depth of 15 cm to All nematodes within each core were counted under a transfer sediment into a bucket. On the sampling day, stereo-dissecting microscope. Thereafter, every four cores the depth was 1.30 m, the water salinity was 38 PSU, from each microcosm were pooled, yielding one sample the dissolved oxygen concentration was 6.84 mg l−1, the per microcosm. One hundred individuals per sample were water turbidity was 3.7 NTU and the water pH was 8.22.
randomly separated under a stereo-dissecting microscope The sediment had a mean grain size of 0.32 ± 0.05 mm, (Kotta & Boucher, 2001), transferred to glycerol through organic matter content of 0.6 ± 0.10% and was totally a series of ethanol-glycerol solutions and finally mounted Effects of permethrin on free-living nematodes in glycerin on slides (Somerfield et al., 1994). Nema- todes were identified to the species level using the picto- rial keys of Platt and Warwick (1983, 1988), Warwick etal. (1998), and descriptions downloaded from the web site http://nemys.ugent.be/ developed by nematologists from Margalef’s richness (d = (S − 1)/ ln N) and Pielou’s = H / log S) were evaluated for each microcosm using the PRIMER software package (Clarke, from species 1 to species S, p of the ith species in a sample with N individuals andS total species. All data were first tested for normality(Kolmogorov-Smirnov test) and homogeneity of variance (Bartlett test). The one-way ANOVA was used to test for overall differences between abundance and indices of diversity and the Tukey HSD multiple comparisons test was used in pairwise comparisons of treatments and control. In all the above statistical significance testing, a significant difference was assumed when P < 0.05.
The community structure data were also presented in the form of k-dominance curves, where k refers to a set of species used to describe dominance (Lambshead et al., 1983). The graphs consist of a logarithmic x-axis with the rank of species by abundance, and a y-axis along which are plotted the cumulative percent abundances by species.
Multivariate data analysis was by non-parametric multi- dimensional scaling (MDS) ordination with the Bray- Curtis similarity measure performed on square-root- transformed species abundance data to determine whether the nematode assemblages responded to the permethrin contamination by changes in the abundance of species.
SIMPER (similarity percentages) was used to determine the contribution of individual species toward dissimilarity The summary of the abundance and indices of diver- sity for nematode assemblages from each microcosm (Ta- ble 1) illustrates clear treatment effects according to the permethrin concentrations. Results from multiple com- parisons tests (Tukey’s HSD test; Table 1) show that all descriptors of nematodes change significantly (P < 0.05) at all levels of permethrin contamination. The abun-dances of nematodes at all permethrin concentrations sig- MDS results (Fig. 2) indicate that the samples are ar- nificantly exceeded those in the controls. By contrast, ranged in a graded series according to the permethrin con- the Shannon-Wiener index (H ), Margalef’s richness (d), centrations. The replicates of all treatments are placed to Pielou’s evenness (J ) and the number of species (S) de- the left-hand side of the controls and the microcosms Per creased significantly with the increase of the level of per- (L) and Per (M) are arranged in sequence along a line with the most contaminated microcosm Per (H) at its left-hand end. These results indicate the clear effect of perme- thrin contamination on nematode assemblages. Addition-ally, the ANOSIM results showed a significant impact of The k-dominance curves (Fig. 1) graphically illustrate permethrin contamination on nematode assemblages. All a distinct picture of increasing dominance and decreasing permethrin-contaminated microcosms were significantly diversity with increased permethrin contamination. All different from controls (R statistic = 1, significance permethrin microcosms were less diverse and had greater level = 2.9%). SIMPER results reveal the most impor- tant average dissimilarities between Per (M) and Per (H)nematode assemblages and the control (Table 2).
A total of 20 nematode species were recorded in all the microcosms (Table 3). All microcosms including thecontrols were dominated by Trichotheristus mirabilis.
Further in the control microcosm Lauratonema hospitumand Latronema orcinum were the two next most frequentspecies besides T. mirabilis.
Significant differences between control and treated mi- crocosms mainly resulted from changes in the abun-dances of the most dominant species (Table 3). Elimi-nation of Pselionema sp., Prochromadorella neapolitanaand Spirinia gerlachi and an increasing number of T. Fig. 1. k-dominance curves for uncontaminated control micro-
mirabilis were responsible for the significant difference cosm (C) and permethrin-contaminated microcosms Microcosm between C and Per (L). An increasing abundance of T. treatments consisted of three levels of permethrin: 50 μg l−1 mirabilis and Paramesonchium angelae and the elimina- (Per (L)), 100 μg l−1 (Per (M)) and 150 μg l−1 (Per (H)). tion of Daptonema hirsutum and Marylynnia belbula were Fig. 2. Non-metric MDS ordination of square-root-transformed nematode species abundance data from uncontaminated control
microcosm (C) and permethrin-contaminated microcosms. Microcosm treatments consisted of three levels of permethrin: 50 μg l−1
(Per (L)), 100 μg l−1 (Per (M)) and 150 μg l−1 (Per (H)).
Effects of permethrin on free-living nematodes Table 2. Average dissimilarity (%) between microcosms.
treatment. The largest abundances in the high dose treat-ments belong to the Xyalids which constitute one of the families tolerant to strong chemical pollution with Come- somatidae and Linhomoeidae. By contrast, seven taxa (Odontophora villoti, Oncholaimellus calvadosicus, Vis- cosia cobbi, Chaetonema sp., Pselionema sp., P. neapoli- Microcosm treatments consisted of three levels of permethrin, tana and S. gerlachi) appear to be very sensitive as they 50 μg l−1 (Per (L)), 100 μg l−1 (Per (M)) and 150 μg l−1 are present in the control treatments but are eliminated at (Per (H)), and an untreated control (C).
It was not possible, based on this study, to define objec- responsible for the significant difference between C and tively the indicator species. To name a species as indicator Per (M). Increasing levels of permethrin contamination some particular methods of data analysis and a specific de- led to the elimination of Bathylaimus sp. and Sabatieria sign/test of the response of the potential indicator species celtica. The elimination of these species and the increase are needed (Gray & Pearson, 1982; Pearson et al., 1983; in the number of T. mirabilis and Xyala striata caused the Goodsell et al., 2009). These points are outside the scope significant difference between the control and the micro- of this study. Even so, two different types of species can be defined based on our community approach. First, thereare the species that increase in dominance and even ab- Discussion
solute abundance in the insecticide treatments. They arethe positive indicative species. Second, there are the neg-ative indicative species that disappear first because they Until now the effects of permethrin on nematodes were are most sensitive. Their absence may also be interpreted totally unknown and only Shannag et al. (1994) had de- as an indicator for the presence of the contaminant. It ap- termined the LC50 of a few entomopathogenic nematode pears that the remaining species, interestingly, belong to species from the genera Steinernema and Heterorhabdi- neither group. They are moderately tolerant to the lower tis. Therefore, experimental studies were required, espe-cially those to establish the relationship as a function of concentrations of permethrin, but disappear at the high dose. The first objective of this research was to establish concentration. Such species can be considered as the best a cause-and-effect relationship between permethrin con- indicative species. This is essentially the case of S. celtica, tamination levels and diversity descriptors of meiobenthic Calomicrolaimus parahonestus and Bathylaimus sp.
nematodes. The evidence presented here supports such a Genera such as Sabatieria and, to a lesser extent, Dap- cause-and-effect relationship. The univariate descriptors tonema are often considered as very tolerant to various of diversity in the contaminated microcosms were sig- kinds of toxicants like metals and hydrocarbons (Somer- nificantly reduced in comparison with controls (Tukey’s field et al., 1994; Beyrem & Aïssa, 2000; Boufahja et al., HSD test, P < 0.05). A similar result was clearly estab- 2011). Then, the elimination of S. celtica and D. hirsu- lished by using the k-dominance curves. The multivari- tum when exposed to the high dose of permethrin ap- ate species-dependent MDS (Fig. 2) was sensitive in dis- pears as somewhat counterintuitive. However, we think criminating the communities (stress = 0.01), suggesting that a given taxon is going to exhibit different responses significant differences between the control and all treated to different toxins or different threshold levels of toler- microcosms, and this was confirmed by ANOSIM anal- ance according to its biology and the mode of action of ysis. This indicates that the response of the studied ne- the toxin. Specifically with nematodes, biology and autoe- matode assemblage was dependent on the level of perme- cology are too poorly known for most of species includ- thrin contamination. In the MDS plot, all treatments were ing the most tolerant (Sabatieria, Daptonema, etc.). The separated from each other indicating a gradual change in mode of action of permethrin and the feeding behaviour of community composition with increased permethrin con- these species may partly explain such results. Pyrethroids act as neurotoxins and target the central nervous systems It was clear from Table 3 that T. mirabilis was dom- of insects (Narahashi et al., 1998). Permethrin primar- inant across all treatments and has a high tolerance for ily disturbs the axonic nerve impulse conduction caus- permethrin, as do X. striata and L. orcinum which also ing rapid muscle paralysis. In the case of non-selective showed increased abundance at the highest concentration deposit-feeding nematodes, including species belonging ± ± ± ± ± 0 0 0 0 0 0 0.0 0.0 0 0.0 0 0 0 ± 0 ± ± ± ± ± ± 0.0 0.0 0.0 0.0 ± 0 0 0.0 0 ± ± 0.0 ± 0.0 ± ± ± ± ± ± 0 0 0 0 ± ± ± 0 ± ± ± 0 ± 0 ∗ .0 .08 .01 .08 .04 .06 .0 .0 .0 .0 .06 .05 .07 .0 .04 .04 .36 .0 .05 .0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Effects of permethrin on free-living nematodes to Sabatieria and Daptonema, permethrin can be particu- X. striata, L. orcinum and P. angelae, and secondly larly effective by ingestion of contaminated detritus. It is Desmodora longiseta and L. hospitum can be classified known that the pharynx of nematodes is a pumping organ as permethrin-tolerant species. The increase of the ne- that serves the uptake and transport of food (Hoschitz et matofauna abundance with increasing level of permethrin al., 2001). There are four cell types in the pharynx: muscle contamination may best be explained by the proliferation cells, neurons, structural and glandular cells (Albertson & of these tolerant species. Differential tolerance to perme- Thomson, 1976). We supposed that the nervous system thrin may result in decreased competition and a subse- controls the pharyngeal pumping by modulation of depo- quent competitive release of more ‘opportunistic’ and/or larisation and repolarisation frequency in the muscle cells (Avery & Horvitz, 1989). Thus, it appears that permethrinmay eventually disturb the functioning of the nervous sys-tem and, hence, the pharyngeal pumping.
Acknowledgements
Based on our study, there is an increase in absolute abundance, at least in the tolerant species. There is also We thank Prof. Guy Boucher (National Museum of a clear decrease in diversity. By taking into account the Natural History, France) and Prof. Pierre Vitiello (Ocean- absence of any macrofauna and the presence of rare cope- ography Centre of Marseilles, France) for species iden- pods in the meiofaunal community in the control and tification. Thanks go also to Prof. Raymond Marshall treated microcosms (data unpubl.), we have rejected the (Universita Komenskeho, Bratislava, Slovakia) and Prof.
hypothesis that some biota which either prey on nema- William D. Hummon (Ohio University, Athens, OH, todes or compete with them could have been negatively USA) for the revisions they made to the English of this pa- affected by the permethrin, thus leading to increased ne- per. The detailed comments and suggestions of Prof. Pe- matode abundances. As there are fewer species present at ter Herman, Prof. Tom Moens, Prof. Denis Fichet, Prof.
the higher doses, there is less competition among the few David Karlen, Prof. Maickel Armenteros and Prof. Emil remaining species for space, food or other resources, al- Olafsson helped substantially in improving the quality of lowing for higher abundances of the most tolerant taxa, the revised manuscript. Funding for this research was pro- and also possible release from predation assuming that vided by the Tunisian Ministry of Scientific Research and some of the more sensitive taxa are predatory on other nematodes. We suggest that survivors fill the ecologicalniches vacated by the eliminated species.
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