al Sa
ge
imazapyr, and the surfactant
l
lim
Biolo´
ed fo
The herbicide arsenal 250NA, its technical-grade active ingredient imazapyr, and the surfactant nonylphenol ethoxylate (NP)
between active ingredientsand their formulationswith
insecticides, and other formulated products (Hawrelak
et al., 1999; Junk et al., 1974; Kim et al., 2002). Many
how these components that cause toxicological concern,
2.1. Test substances employed
ARTICLE IN PRESSstudies have shown that NP has been implicated in the
disruption of endocrine functions in wildlife (Gaido
The following test substances were used, Imazapyr
(technical herbicide), chemical family of imidazolinone,
molecular formula C
13
H
15
N
3
O
3
, M
w
261.3, CAS No.
081334-34-1, 99.7% purity, Batch 70304. Arsenal 250
NA, Batch 0011-98-7776, formulation of imazapyr with
*Corresponding author. Fax: +55-61-273-4942.
E-mail address: grisolia@unb.br (C.K. Grisolia).
0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ecrespect to toxicity and genotoxicity. Surfactants and
other so-called ‘‘inert’’ components generally increase
the toxicity of these formulations (Mann and Bidwell,
1999; Wagner et al., 2003). Oakesand Pollak (2000)
demonstrated that inert components such as surfactants
contributed approximately 50% of the overall toxicity
of the complete pesticide formulation.
Commercial imazapyr productscontain nonylphenol
ethoxylate, which givesrise to concern for human health
and the environment. Nonylphenol (NP) iswidely used
as a component of detergents, paints, herbicides,
when added to the active ingredients, modify their
toxicological properties. Genotoxic and toxicological
studies were therefore carried out to evaluate the
technical-grade active ingredient imazapyr, itsformula-
tion arsenal 250NA, and nonylphenol ethoxylate. These
test systems were chosen for their genetic endowment, in
addition to their easy availability.
2. Materials and methodsThe results showed that arsenal, imazapyr, and NP did not cause chromosome aberration in Allium cepa nor increase the frequency
of micronuclei in mice. However, toxicological evaluations showed that NP was the most toxic compound to mice, A. cepa,
Drosophila melanogaster, and Biomphalaria tenagophila. In this evaluation, it was observed that the adverse effects were produced by
the surfactant additive of the pesticide formulation.
r 2004 Elsevier Inc. All rights reserved.
Keywords: Arsenal; Imazapyr; Nonylphenol; Toxicity; Genotoxicity
1. Introduction
Many studies with pesticides are showing differences
et al., 1997; Soto et al., 1991). The objective of thisstudy
wasto evaluate the potential adverse effectsof inert
components of pesticide formulations to understandwere evaluated through genotoxicity and toxicity studies in different organisms. A comparative study of these three compounds was
carried out to assess how the addition of surfactant components may pose the highest toxicological risk to pesticide formulations.Ecotoxicology and Environment
A comparative toxicologic and
arsenal, its active ingredient
nonylpheno
Cesar Koppe Grisolia,* Marina Ro
Departamento de Gene´tica e Morfologia, Instituto de Cieˆncias
Received 6 November 2002; received in revis
Abstractoenv.2004.01.014ethoxylate
Bilich, and Lia Menezes Formigli
gicas, Universidade de Brası´lia, 70910-900 Brası´lia, DF, Brazil
rm 11 July 2003; accepted 28 January 2004fety 59 (2004) 123–126
notoxic study of the herbicide

interval and sacrificed 24 h after the second injection.
ARTICLE IN PRESS
d Envcomponents25 g/L imazapyr, 186 g/L of ammonium
hydroxide, 18 g/L nonylphenol ethoxylate, and water.
Nonylphenol ethoxylate (RENEX 95%, nonylphenol
with nine ethoxylate units), Batch 970919-202, CAS No.
25154-52-3. All three chemicalswere obtained from the
BASF Company of Brazil.
2.2. Toxicity test
Acute toxicity in mice was tested as follows. The
median lethal dose (LD50%) after an intraperitoneal
single injection for 7 days was determined. Arsenal,
imazapyr, and nonylphenol were administered at six
dose levelsfor each group of five malesand five females
of 12-week-old Swiss albino mice from the Central
Animal Facility of the University of Brası´ lia. The
LD50% wascalculated using the Trimmed Spearman–
Karber method (Hamilton et al., 1977).
Acute toxicity in Drosophila melanogaster wastested as
follows. Groups of 10 Drosophila were randomly isolated
from the stock of the Genetics Laboratory of the
University of Brası´ lia for each treatment group. Treat-
mentswere carried out through diet. Arsenal, imazapyr,
and NP were diluted at six multiple-dose levels in 5mL of
Drosophila Instant Medium (Carolina Biological Supply
Company). Adultswere introduced into the vials, where
they were fed for 5 days. The LD50 was also calculated
using the Trimmed Spearman–Karber method.
Lethal concentration (LC50) in the snail Biomphalaria
tenagophila wasmeasured asfollows. Thistest iscompar-
able to the LD50, but iscarried out with aquatic
organisms by whole-body exposure. Wild type B. tenago-
phila from Southern Brazil have been bred and kept in the
Malacology Laboratory of the University of Brası´ lia for
more than 8 years. The animals, 8–12mm in diameter,
were 4–6 monthsold. They were kept isolated in 125-mL
vials. Snails were exposed to eight different concentrations
of each test-compound for 72 h for the determination of
LC50, using the Trimmed Spearman–Karber method.
2.3. Mouse micronucleus test
Swiss mice, from the Central Animal Facility of the
University of Brası´ lia, were acclimatized to laboratory
conditionsfor 1 week prior to the study. Malesand
females(10–12 weeksold), weighing 3072 g, were fed
Purina mouse chow and filtered water ad libitum. The
negative control received distilled water. Cyclopho-
sphamide (Enduxan) was injected at 30mg/kg, as a
positive control. Arsenal was tested at 65.5, 131.6, and
196.7mg/kg body weight, which means25%, 50%, and
75% of LD50 (262.33mg/kg), respectively. Imazapyr
wastested at 374.5, 749.0, and 1123.5mg/kg body
weight, representing 25%, 50%, and 75% of LD50
(1498mg/kg), respectively. Nonylphenol was tested
C.K. Grisolia et al. / Ecotoxicology an124only at the maximum tolerated dose of 57.27mg/kg,The treatment protocol wascarried out according to the
half-life of these pesticides in rodents. Test substances
were administered intraperitoneally. The bone marrow
preparationsfor micronucleusanalysiswere made
according to Schmid (1975). The slides were fixed with
methanol and stained with Giemsa. Two thousand cells
per animal were counted and classified as polychromatic
erythrocytes(PCE) and normochromatic erythrocytes
(NCE). The PCEs/NCEs relationship was determined
by the first 1000 PCEs or NCEs counted.
2.4. Allium cepa test
This assay was carried out according to the test
protocol proposed by Rank and Nielsen (1993) to screen
the genotoxicity of complex mixtures. Commercial
onion bulbswere obtained from organic growers,
without any treatment with growth inhibitors. For each
test solution 10 onions were set up. Genotoxicity tests
were then carried out with different concentrationsof
each pesticide. Growth inhibition tests were carried out
for each pesticide prior to the genotoxicity test to find
the toxicity level of the test chemical. Root tips were
exposed to four concentrations of arsenal and imazapyr
and three concentrationsof NP for 48 h. These were
continuously shaken during the period of treatment in a
horizontal shaker. Filtered and dechlorinated tap water
(pH 7.0) of good quality wasused asa negative control
and for dilution of pesticides. Methylmethanesulpfonate
(MMS) at 10mg/L was used as positive control
mutagen. At the end of exposure, five or six root tips
from each bulb were prepared for the microscopic slides.
Ten bulbs were used per treatment and 100 metaphase–
telophase cells were analyzed, giving 1000 cells per
treatment. The root tipswere fixed and macerated in
a solution of 45% acetic acid (9 parts) and 1N HCl
(1 part) at 50


C for 5min, followed by squashing them
in a 2% orcein stain in 45% acetic acid. Slides were kept
in a freezer and examined within 2 weeks. One hundred
cells per onion in metaphase or anaphase were examined
and classified in the following categories: bridges,
fragments, and lagging chromosomes. All slides were
coded and examined blind. The mitotic index was
determined by counting all stages of mitotic cell out of
1000 cells. Statistical analysis was performed using the
Mann–Whitney U-test, a ¼ 5%.
3. Results
Asshown in Table 1, there was no statisticallypreviously determined in our laboratory. They were
housed at random in groups of 10 and were dosed twice
with pesticides at a volume of 0.5ml within a 24-h
ironmental Safety 59 (2004) 123–126significant difference in the means of the frequencies of