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16 Eucoleus aerophilus (syn. Capillaria aerophila) and Other Trichinelloid Nematodes in Dogs from Liguria (Northwest Italy)
F. Macchioni et al.
The genus Capillaria has been revised several times. It is currently divided into
Eucoleus spp. for parasites found in the airways, Aonchotheca spp. for those living
in the gastrointestinal tract, and Pearsonema spp. for parasites of the urinary tract
(Moravec 1982; Burgess et al. 2008).
Eucoleus aerophilus (syn. Capillaria aerophila) lives in the trachea, bronchi,
and bronchioles of domestic and wild carnivores. Infection with this parasite can
cause verminous tracheo-bronchitis. It is occasionally reported in humans
(Laloševic et al. 2008). It is present in Europe, and North and South America
(Conboy 2009). In Italy E. aerophilus has been reported in wild animals (Rossi
et al. 1983; Balestrieri et al. 2006; Magi et al. 2009) and in dogs and cats (Traversa
et al. 2009; Di Cesare 2010). The adult females lay eggs after mating. The eggs
reach the pharynx, are swallowed, and then eliminated through the feces. The eggs
have an elliptical shape and asymmetrical polar plugs and show a reticular surface
with anastomosed ridges. They are 60–83 lm long and 26–40 lm wide (Campbell
1991). Outside of the host, they mature in 30–45 days. Infecting larvae can reach
the definitive host directly or via an earthworm, the intermediate host. Infesting
larvae migrate in 7–10 days from the enteric wall of the host toward the site of
choice (Euzeby 1961).
E. boehmi (syn. C. boehmi) lives in the nasal passages and sinuses of wild and
domestic canids. This species has been reported in America and Europe (Conboy
2009). The eggs measure 54–60 9 30–35 lm. They exhibit a characteristic space
between the embryo and the shell and have a smoother surface with small wells
(Campbell 1991; Campbell and Little 1991). E. boehmi was identified for the first
time in Germany in the silver fox (Supperer 1953).
A. putorii (syn. C. putorii) inhabits the stomach and small intestine of domestic
and wild carnivores. It has been reported in North America and Europe (Campbell
1991). In Italy it has been reported only in foxes (Iori et al. 1990; Di Cerbo et al.
2008; Guardone et al. 2010). The eggs measure 53–70 9 20–30 lm and have a
reticular surface organized in a longitudinal pattern (Campbell 1991).
P. plica (syn. C. plica) is localized in the urinary bladder of dogs, cats, and wild
carnivores. It has been reported in North America and Europe (Campbell 1991;
Whitehead 2009; Senior et al. 1980). In Italy it has been reported in foxes and in a
dog (Iori et al. 1990; Callegari et al. 2010). The eggs measure 58–74 9 23–31 lm,
and have a rough surface (Campbell 1991).
T. vulpis is a cosmopolitan parasite of dogs and wild canids and has
occasionally been reported in humans (Dunn et al. 2002; Traversa 2011). It
inhabits the large intestine, especially the cecum. The eggs, which are similar in
size to those of the genus Capillaria, measure 70–84 9 30–40 lm. They have a
typical symmetrical barrel shape, are yellowish-brown in color, and have a very
smooth surface (Campbell 1991).
The aim of this study was to confirm the presence of Capillaria spp. and
T. vulpis in dogs in an area where these parasites are known to infect wild animals,
especially foxes (Guardone et al. 2010).
Eucoleus aerophilus (syn. Capillaria aerophila)
16.2 Materials and Methods
Between January 2010 and March 2011, we examined fecal samples from 270
dogs (163 from hunting dogs and 107 from dogs in kennels) in the province of
Imperia (Liguria, Northern Italy). Fresh fecal samples were examined using
flotation in zinc sulphate (specific gravity = 1.350), combined with centrifugation.
Using an optical microscope at 1009 and 4009 magnification, trichurid eggs were
identified according to a morphological and morphometric analysis following in
the reported descriptions (Campbell 1991).
Out of a total of 270 samples of fresh dog feces, 48 were positive for Trichuridae
eggs. Fifteen dogs were positive for Capillaria spp., and 28 were positive for
T. vulpis. Five dogs were infested with two species. The overall prevalence of
infection with Capillaria spp. was 7.4 % (95 % confidence interval [CI],
4.3–10.5 %). The prevalence of infection with T. vulpis was 12.2 % (95 % CI,
Among the 20 dogs infected with Capillaria spp., 12 were positive for eggs of
E. aerophilus (prevalence, 4.4 %; 95 % CI, 2.0–6.8 %). In nine cases, we found
eggs belonging to the genus Capillaria that had different morphological
characteristics (one dog had a double infestation of E. aerophilus and Capillaria
spp.). In six dogs (2.2 %), the eggs had characteristics similar to eggs of
E. boehmi. Most of them were partially embryonated and a few contained a living
embryo. In three dogs (1.1 %), the eggs had characteristics similar to those of
A. putorii. Samples collected during this study are currently being examined with
biomolecular tools (Traversa et al., Deplazes et al., unpublished data) to confirm
With the exception of three dogs, which had respiratory symptoms with cough
and nasal discharge, most of the dogs infected with Capillaria spp. were
The prevalence of Capillaria spp. was 8.6 % in the hunting dogs and 5.6 % in
the kennel dogs. This difference was statistically significant. The prevalence of
T. vulpis in the hunting dogs was 2.4 and was 27.1 % in the kennel dogs.
These differences were highly significant. However, dogs from a heavily infested
kennel were present in the kennel dog samples.
Some of the 48 dogs were coinfested by Toxocara canis, Toxascaris leonina,
Ancylostomatidae, Angiostrongylus vasorum, Crenosoma vulpis, or Dipilydium
F. Macchioni et al.
In Italy, the prevalence of infestation by E. aerophilus is likely underestimated.
Reports are more common in wild animals, in which the coprological diagnosis is
confirmed by finding adult worms in the trachea or lungs during necropsy
examinations. In the same geographic area of this study, we detected E. aerophilus
in the airways and A. putorii and T. vulpis in the intestines of red foxes.
The sharing of habitat between wild and domestic animals can lead to an increased
risk of infestation for domestic species. The prevalence of E. aerophilus in dogs
(4.4 %) found in this study does not differ significantly from that reported by
Traversa et al. (2009) and Di Cesare et al. (2010). The prevalence of T. vulpis
(12.2 %) found in dogs in the Liguria region contributes to the data reported by
other authors in other Italian regions (Poglayen et al. 2000; Perrucci et al. 2001;
Rinaldi et al. 2006; Zanzani et al. 2010).
In conclusion, the various capillarid eggs display very similar morphological
and morphometric characteristics. Therefore, new biomolecular methods should be
developed to aid in the identification of these parasites.
Balestrieri A, Remonti L, Ferrari N, Ferrari A, Lo Valvo T, Robetto S, Orusa R (2006) Sarcoptic
mange in wild carnivores and its co-occurence with intestinal helminthes in the Western
Italian Alps. Eur J Wild Res 52:196–201
Burgess H, Ruotsalo K, Peregrine AS, Hanselman B, Abrams-Ogg A (2008) Eucoleus aerophilus
respiratory infection in a dog with Addison’s disease. Can Vet J 49:389–392
Callegari D, Kramer L, Cantoni AM, Di Lecce R, Dodi PL, Grandi G (2010) Canine
bladderworm (Capillaria plica) infection associated with glomerular amyloidosis. Vet
Campbell BG (1991) Trichuris and other Trichinelloid nematodes of dogs and cats in the United
States. Compend Contin Educ Vet 13(5):769–778
Campbell BG, Little MD (1991) Identification of the eggs of a nematode (Eucoleus boehmi) from
the nasal mucosa of North American dogs. J Am Vet Med Assoc 198:1520–1523
Conboy G (2009) Helminth parasites of the canine and feline respiratory tract. Vet Clin North
Am Small Anim Pract 39:1109–1126
Di Cerbo AR, Manfredi MT, Bregoli M, Ferro Milone N, Cova M (2008) Wild carnivores as
source of zoonotic helminths in north-eastern Italy. Helmintologia 45(1):13–19
Di Cesare A, Meloni S, Milillo P, Castagna G, Otranto D, Paoletti B, Bartolini R, Avolio S,
Traversa D (2010) Feline and canine cardio-pulmonary nematodes in central and southern
Italy. Parassitologia 52(1–2):307
Dunn J, Columbus S, Aldeen W, Davis M, Carroll K (2002) Trichuris vulpis recovered from a
patient with chronic diarrhea and five dogs. J Clin Microbiol 40(7):2703–2704
Euzeby J (1961) Trichuroidea In: Les maladies vermineuses des animaux domestique, Tome I,
Maladies dues aux nemathelminthes. Vigot Frères Editeurs, Paris, France, p. 54
Guardone L, Macchioni F, Prati MC, Mignone W, Torracca B, Magi M (2010) Common parasites
of dogs (Canis familiaris) and foxes (Vulpes vulpes) in an area of Maritime Alps (northwestern Italy). LXIV Convegno Nazionale S.I.S.Vet Asti, Italy, 8–10 Sep 2010
Eucoleus aerophilus (syn. Capillaria aerophila)
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Laloševic D, Laloševic V, Klem I, Stanojev-Jovanovic0 D, Pozio E (2008) Pulmonary
capillariasis miming bronchial carcinoma. Am J Trop Med Hyg 78(1):14–16
Magi M, Macchioni F, Dell’Omodarme M, Prati MC, Calderini P, Gabrielli S, Iori Cancrini G
(2009) Endoparasites of Vulpes vulpes in central Italy. J Wildl Dis 45(3):881–886
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Capillaridae. Folia Parasitol 29:119–132
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Poglayen G, Giannetto S, Macrì B, Garippa G, Scala A, Cambosu C, Giangaspero A, Paoletti B,
Montauti AE, Traldi G, Habluetzel A (2000) Canine zoonoses by environmental faecalization.
Rinaldi L, Biggeri A, Carbone S, Musella V, Catelan D, Veneziano V, Cringoli G (2006) Canine
faecal contamination and parasitic risk in the city of Naples (southern Italy). BMC Vet Res
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volpi presente nel parco regionale La Mandria. Parassitologia 25:340–343
Senior DF, Solomon GB, Goldschmidt MH, Joyce T, Bovee KC (1980) Capillaria plica infection
in dogs. J Am Vet Med Assoc 176:901–905
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des Fuchses Z Parasitenk 16:51–55
Traversa D (2011) Are we paying too much attention to cardio-pulmonary nematodes and
neglecting old-fashioned worms like Trichuris vulpis? Parasit Vectors 4:32
Traversa D, Di Cesare A, Milillo P, Iorio R, Otranto D (2009) Infection by Eucoleus aerophilus
in dogs and cats: is another extra-intestinal parasitic nematode of pets emerging in Italy? Res
Vet Sci 87:270–272
Whitehead M (2009) Urinary capillariosis in a cat in the UK. Vet Rec 165:757
Zanzani S, Bonassi L, Maffi S, Mandredi MT (2010) Prevalence of intestinal parasites in dogs and
cats from two provinces of Lombardy region and perception of related risks by the pet owners.
Helminths in Sheep on Farms
of the Basilicata Region of Southern Italy
A. Bosco, L. Rinaldi, V. Musella, D. Pintus, M. Santaniello,
M. E. Morgoglione, G. Zacometti and G. Cringoli
Abstract The aim of this study was to obtain up-to-date information on the
presence and distribution of helminths in grazing sheep from the Basilicata region
of southern Italy. A cross-sectional coprological survey was conducted on 98
sheep farms and the FLOTAC dual technique was used to detect and count parasitic elements. The most frequent nematodes found were gastrointestinal (GI)
strongyles (91.8 %), in particular Haemonchus (76.9 %), Trichostrongylus
(91.8 %), Teladorsagia (88.8 %), Cooperia (77.6 %), and Oesophagostomum
(72.5 %). They were followed by lungworms (50.0 %), including Muellerius
(37.8 %), Neostrongylus (13.3 %), Protostrongylus (4.4 %), Cystocaulus (2 %),
and Dictyocaulus (2 %). We also found Trichuris (39.8 %), Nematodirus
(24.5 %), Strongyloides (4.1 %), and Skrjabinema (2.0 %). Trematoda were also
found with the following prevalence: Calicophoron daubneyi (10.2 %), Dicrocoelium dendriticum (61.2 %), and Fasciola hepatica (1.0 %). Moniezia was
found on 35.7 % of farms. Interestingly, the spatial analysis showed clusters for C.
daubneyi and D. dendriticum.
Keywords Helminths Sheep GIS FLOTAC Epidemiology Southern Italy
A. Bosco (&) Á L. Rinaldi Á D. Pintus Á M. Santaniello Á M. E. Morgoglione Á G. Cringoli
Department of Pathology and Animal Health,
University of Naples Federico II, Cremopar, Italy
Department of Clinical and Experimental Medicine,
University of Catanzaro Magna Graecia, Catanzaro, Italy
Provincial Association of Farmers, Potenza, Basilicata, Italy
C. Boiti et al. (eds.), Trends in Veterinary Sciences,
DOI: 10.1007/978-3-642-36488-4_17, Ó Springer-Verlag Berlin Heidelberg 2013
A. Bosco et al.
Sheep farming in the Basilicata region of southern Italy (378,966 head; ISTAT,
2010) is an important part of the region’s rural economy. Information on the
distribution of helminths on sheep farms in this region, and on the health aspects
related to these parasitic infections, are scant and out of date. Therefore, the aim of
this study was to update the data on the presence and distribution of helminth
infections on sheep farms in the Basilicata region. A cross-sectional coprological
survey was conducted using geographical information systems (GIS) for territorial
sampling and for the representation of results with parasitological maps. In
addition, a spatial analysis was conducted to detect possible clusters of positive
17.2 Materials and Methods
A GIS of the Basilicata region was constructed using administrative boundaries at
the provincial and municipal levels as data layers. The region was divided into
ninety-eight 10 9 10 km quadrants. The centroid of each quadrant was identified
and the farm closest to each centroid was sampled. A total of 98 sheep farms were
sampled, one for each quadrant. On each farm, 20 sheep (15 adults and 5 juveniles) were subjected to parasitological investigation, for a total of 1,960 subjects.
In the laboratory, individual samples were grouped into four composites (each
consisting of equal parts of five individual samples), one for juveniles and three
for adult animals.
Copromicroscopic analyses were performed using the FLOTAC dual technique
(Cringoli et al. 2010) with an analytic sensitivity of two eggs/larvae per gram
(EPG/LPG) of feces. Two flotation solutions were used, the first consisting of
sodium chloride [specific gravity (SG) = 1.200] to detect and count eggs of the
following genera/groups of nematoda: gastrointestinal (GI) strongyles, Nematodirus, Strongyloides, and Trichuris. A second solution consisting of zinc sulfate
(SG = 1.350) was used to detect and count eggs/larvae of Skrjabinema, Fasciola,
Calicophoron, Dicrocoelium, Moniezia, and lungworms. Coprocultures were
performed for each farm to identify the genera of GI strongyles (van Wyk et al.
2004). The GIS software ArcGIS 9.3 GIS (ESRI, Redlands, CA, USA) was used
for spatial analysis to detect cluster distribution of each parasite.
The results showed the presence of GI strongyles on 91.8 % (90/98) of farms,
including Haemonchus 76.9 % (78/98), Trichostrongylus 91.8 % (90/98), Teladorsagia 88.8 % (87/98), Cooperia 77.6 % (76/98), and Oesophagostomum
Helminths in Sheep on Farms of the Basilicata Region of Southern Italy
Fig. 17.1 Distribution of C. daubneyi and D. dendriticum in the Basilicata region. Parasitological maps with proportional circles and clusters detected by spatial analysis
72.5 % (71/98). The following genera of nematoda were also found: Trichuris
39.8 % (39/98), Nematodirus 24.5 % (24/98), Strongyloides 4.1 % (4/98), and
Skrjabinema 2 % (2/98). The most frequent lungworms were Muellerius, found on
37.8 % (37/98) of farms, followed by Neostrongylus 13.3 % (13/98), Protostrongylus 4.4 % (4/98), Cystocaulus 2 % (2/98), and Dictyocaulus 2 % (2/98).
Cestoda of the genus Moniezia were found on 35.7 % (35/98) of farms. Among
trematoda, D. dendriticum was found on 61.2 % (60/98) of the farms, followed by
C. daubneyi on 10.2 % (10/98), and F. hepatica on 1 % of the farms (1/98). The
two parasitological maps with proportional circles shown in Fig. 17.1 show the
distribution and intensities of D. dendriticum and C. daubneyi. Spatial analyses
showed a cluster distribution pattern for both D. dendriticum (P = 0.05) and C.
daubneyi (P = 0.01).
This survey showed a noteworthy distribution of helminths on sheep farms of the
Basilicata region. The most prevalent helminths were GI strongyles, detected on
most of the farms (91.8 %), and comprised the genera Haemonchus (76.9 %),
Trichostrongylus (91.8 %), Teladorsagia (88.8 %), Cooperia (77.6 %), and
Oesophagostomum (72.5 %). Lungworms were also widely distributed (50 %) and
Muellerius was most commonly detected genus (37.8 %). These findings are
consistent with similar studies performed in other Apenninic areas of southern
Italy (Cringoli et al. 2002; Biggeri et al. 2007). The prevalence values for Trichuris
(39.8 %), Nematodirus (24.5 %), Strongyloides (4.1 %), and Skrjabinema (2 %)
are also consistent with those recently reported on sheep farms in other Italian
regions (Bosco et al. 2011). D. dendriticum was the most prevalent liver fluke
A. Bosco et al.
(61.2 %), whereas F. hepatica was reported on only 1 % of the farms. Paramphistomes (C. daubneyi) were reported on 10.2 % of farms.
Spatial analysis detected an interesting cluster distribution of D. dendriticum
and C. daubneyi in the study area, likely due to soil and environmental characteristics on the biology of the intermediate hosts. Further studies are needed to
clarify this finding.
In conclusion, considering that the health status of livestock is fundamental for
the productive and reproductive performances of these animals, the economic
impact of this parasitological situation is likely very significant, although not
quantified in this study (Cringoli et al. 2004, 2007, 2008). Local knowledge about
the presence and distribution of parasitic infections in sheep is a basic requirement
to better plan and implement appropriate control protocols and targeted strategies.
Acknowledgments Part of this research was funded by the European Union Seventh Framework
Programme FP7 –KBBE-2011-5 under grant agreement n° 288975. The authors would like to
express sincere appreciation to Giovanna Cappelli, Ida Guariglia, Davide Ianniello, and Mario
Parrilla for the laboratory analyses.
Biggeri A, Catelan D, Dreassi E, Rinaldi L, Musella V, Veneziano V, Cringoli G (2007)
Multivariate spatially-structured variability of ovine helminth infections. Geospat Health
Bosco A, Rinaldi L, Musella V, Pintus D, Morgoglione ME, Santaniello M, Prestera G, Zacometti
I (2011) Mappe parassitologiche degli elminti nei bovini semibradi in Basilicata. J Italian
Assoc Buiatrics 6:43–47
Cringoli G, Rinaldi L, Veneziano V, Capelli G, Malone JB (2002) A cross-sectional coprological
survey of liver flukes in cattle and sheep from an area of the southern Italian Apennines. Vet
Cringoli G, Taddei R, Rinaldi L, Veneziano V, Musella V, Cascone C, Sibilio G, Malone JB
(2004) Use of remote sensing and geographical information systems to identify environmental
features that influence the distribution of paramphistomosis in sheep from the southern Italian
Apennines. Vet Pararasitol 122(1):15–26
Cringoli G, Veneziano V, Rinaldi L, Sauve C, Rubino R, Fedele V, Cabaret J (2007) Resistance
of trichostrongyles to benzimidazoles in Italy: a first report in a goat farm with multiple and
repeated introductions. Parasitol Res 101:577–581
Cringoli G, Veneziano V, Jackson F, Vercruysse J, Greer AW, Fedele V, Mezzino L, Rinaldi L
(2008) Effects of strategic anthelmintic treatments on the milk production of dairy sheep
naturally infected by gastrointestinal strongyles. Vet Parasitol 156:340–345
Cringoli G, Rinaldi L, Maurelli MP, Utzinger J (2010) FLOTAC: new multivalent technique for
qualitative and quantitative copromicroscopic diagnosis of parasites in animals and humans.
Nat Prot 5:503–515
van Wyk JA, Cabaret J, Michael LM (2004) Morphological identification of nematode larvae of
small ruminants and cattle simplified. Vet Parasitol 119:277–306
Pharmacology and Clinical Science
Effects of Veterinary Drugs on Swimming
Activity in Two Freshwater Organisms
M. Dalla Bona, V. Di Leva and M. De Liguoro
Abstract Alterations in swimming activity may influence ecologically relevant
performance, such as predation avoidance, prey capture, growth, stress resistance,
mating, and longevity. The evaluation of swimming activity supports toxicological
investigation with endpoints other than traditional LC50s values and may aid in
investigating the environmental relevance of low-level exposures and determining
‘‘no observable effect concentration’’ (NOEC) and ‘‘lowest observable effect
concentration’’ (LOEC). In this chapter, some veterinary antibacterial compounds
that may contaminate the aquatic environment due to their use in livestock and/or
mass aquaculture treatments are evaluated for their effects on swimming activity
of Daphnia magna (primary consumer) and Poecilia reticulata (secondary consumer). Results show that the chosen endpoint may call to the attention of ecotoxicology some compounds that are otherwise negligible, based on lethality tests.
Keywords Swimming activity
Á Daphnia magna Á Poecilia reticulata Á Toxicity
Behavioral changes represent integrated responses of the whole organism. These
altered responses may be associated with reduced fitness and survival, resulting in
adverse consequences at the population level (Bridges 1997).
Until recently, behavioral endpoints have been slow to be integrated into
aquatic toxicology, not only because of the poor understanding of their consequences on ecologically relevant activities, such as predation avoidance, prey
M. Dalla Bona (&) Á V. Di Leva Á M. De Liguoro
Department of Biomedicine and Food Safety, University of Padua,
Viale dell’Università 16 35020 Legnaro, PD, Italy
C. Boiti et al. (eds.), Trends in Veterinary Sciences,
DOI: 10.1007/978-3-642-36488-4_18, Ó Springer-Verlag Berlin Heidelberg 2013
M. Dalla Bona et al.
capture, growth, and reproduction, but also because it is difficult to obtain quantifiable and reproducible data (Kane et al. 2005).
Recent improvements in computer and video automation have made significant
progress in the ease, utility, and affordability of obtaining, interpreting, and
applying behavioral endpoints in a variety of applications, including aquatic
toxicity tests, possible. Currently, after exposing an aquatic organism to a substance, it is possible to acquire a film by means of video tracking software and
analyze it to accurately evaluate the swimming activity through graphical and
statistical elaboration of data.
Acute toxicity tests, while demanding little time and labor, are generally less
sensitive than chronic toxicity tests. Their sensitivities can be improved by evaluating sublethal effects such as the behavioral consequences. Generally, the acute
toxicity (lethality) of antibacterial toward nontarget organisms such as crustaceans
and fishes occurs only at concentrations higher than 100 mg/L; substances with
EC50 values higher than this threshold are considered safe for the aquatic environment (Regulation 1272/2008/EEC).
In this chapter, some methodologies are described that can be used for swimming activity evaluation of fishes and crustaceans after exposure to antibacterial. It
is shown that significant effects of these drugs on swimming activity may be
detected at concentrations that are unable to cause any lethality of the test
18.2 Materials and Methods
In accordance with the fish acute toxicity test (OECD 203 1992), the following
compounds were tested at a concentration of 100 mg/L (limit test) on Poecilia
reticulata: enrofloxacin (EFX), its metabolite ciprofloxacin (CFX), trimethoprim
(TMP), sulphamethazine (SMZ), sulphaguanidine (SGD), and sulphaquinoxaline
(SQO). As no lethality was recorded after 96 h, the test was extended to 14 days
(OECD 204 1984). At the end of this prolonged toxicity test and before sacrificing
the animals with an overdose of MS-222, each group of seven fish was allocated in
a small, round tank (20 cm in diameter) under standard conditions of light and
temperature and filmed from above for 12 min using a digital video camera (JVC
EVERIO GZ-MS100E); the middle 2 min of each video sequence was analyzed on
a PC AcerAspire M3201, using Swistrack 4.0 (Lochmatter et al. 2008). The spatial
coordinates (in two dimensions) of the movements were then exported to Excel
(MicrosoftÒ) software to perform graphical and statistical elaborations.
Based on the results of this test, the following compounds were selected to be
tested on Daphnia magna: EFX, TMP, SGD, and SQO. Ten daphnids (6 days of
age) were allocated in Roux flasks (75 cm2) at 20 ± 1 °C and with a 16 h photoperiod (200 lux), and exposed for 24 h to a single compound dissolved in ADaM
medium (Klüttgen et al. 1994) at 100 mg/L concentration. At the end of the test,
each daphnid was frontally filmed for 5 min (see above), and the middle 2 min of