Phototransformation of carboxin in water. Toxicity of the pesticide and its sulfoxide to aquatic organisms

Article abstract of DOI:10.1021/jf049737o

Sunlight exposure of aqueous suspensions of carboxin (1) causes its phototransformation to sulfoxide 2 and minor components. Similar effects are observed in the presence of humic acid or nitrate or at different pH values. Photoproducts 2-9 were isolated by Chromatographic techniques and/or identified by spectroscopic means. Carboxin 1 and its main photoproduct sulfoxide 2 were tested to evaluate acute toxicity to primary consumers typical of the aquatic environment: the rotifer Brachionus calyciflorus and two crustaceans, Daphnia magna and Thamnocephalus platyurus. Chronic tests comprised a producer, the alga Pseudokirchneriella subcapitata, and a consumer, the crustacean Ceriodaphnia dubia.

Full text of DOI:10.1021/jf049737o

6228 J. Agric. Food Chem. 2004, 52, 6228−6232
Phototransformation of Carboxin in Water. Toxicity of the
Pesticide and Its Sulfoxide to Aquatic Organisms
MARINA DELLAGRECA,† MARIA ROSARIA IESCE,*,† FLAVIO CERMOLA,†
‡
MARIA RUBINO,† AND MARINA ISIDORI
Dipartimento di Chimica Organica e Biochimica, Universita` Federico II, Complesso Universitario
Monte S. Angelo, Via Cinthia 4, I-80126 Napoli, Italy, and Dipartimento di Scienze della Vita,
Seconda Universita` di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
Sunlight exposure of aqueous suspensions of carboxin (1) causes its phototransformation to sulfoxide
2 and minor components. Similar effects are observed in the presence of humic acid or nitrate or at
different pH values. Photoproducts 2-9 were isolated by chromatographic techniques and/or identified
by spectroscopic means. Carboxin 1 and its main photoproduct sulfoxide 2 were tested to evaluate
acute toxicity to primary consumers typical of the aquatic environment: the rotifer Brachionus
calyciflorus and two crustaceans, Daphnia magna and Thamnocephalus platyurus. Chronic tests
comprised a producer, the alga Pseudokirchneriella subcapitata, and a consumer, the crustacean
Ceriodaphnia dubia.
KEYWORDS: Carboxin; solar light; nitrate; humic acid; toxicity; Brachionus calyciflorus; Daphnia magna;
Thamnocephalus platyurus; Pseudokirchneriella subcapitata; Ceriodaphnia dubia
INTRODUCTION
shown to sensitize oxygenation and other reactions involving
electronic energy transfer (10), whereas nitrate promotes es-
sentially OH radical formation (11).
Carboxin, continuously discharged in the environment, mainly
on the soil, can reach surface waters due to phenomena of runoff.
Therefore, the present study evaluates the ecotoxicological
effects of the pesticide and its main photoproducts on nontarget
aquatic organisms.
Pesticides are largely used to ensure crop yields and high
quality, and the variety and quantities of these substances present
in surface waters have dramatically increased. Most pesticides
resistant to chemical and/or photochemical degradation under
typical environmental conditions are detected in food and water
(1). In recent years, many studies have reported the possible
adverse effects that the presence of these pesticides may have
for human health and the equilibrium of ecosystems (2-5).
However, pesticides may be subjected to physical, chemical,
and biological transformations in the environment. Therefore,
the presence and possible effects of the transformation products
should be investigated, too. Here we report the photolysis of
the pesticide carboxin (1) in water by sunlight exposure.
Carboxin is a systemic fungicide used to control plant diseases
caused by Basidiomycetes (6). Recent studies have described
the photochemical behavior of the pesticide when irradiated with
UV light (filter Pyrex) in organic solvent (7) or in the presence
of humic substances and soil (8) and with a halogen lamp in
the presence of sensitizers (9). In this study we examine the
irradiation in water with solar light as close to natural conditions
as possible. The effect of pH or commercially available humic
acid or nitrate on the rate of degradation is also discussed.
Humate and nitrate, which are likely to be encountered in aquatic
environments, are reported to be capable of inducing the
photodegradation of organic compounds. The former have been
MATERIALS AND METHODS
Chemicals. Carboxin, analytical standard grade (99%), was supplied
by Labservice Analytika S.r.l.; all other chemicals were obtained from
Aldrich.
General Experimental Procedures. Nuclear magnetic resonance
(NMR) spectra were recorded at 500 MHz for 1H and at 125 MHz for
13C on a Varian 500 Fourier transform NMR spectrometer. Electronic
impact mass spectra (EI-MS) were obtained with an HP 6890
spectrometer equipped with an MS 5973 N detector. UV-vis spectra
were recorded in methanol on a Perkin-Elmer Lambda 7 spectropho-
tometer. IR spectra were recorded on a Jasco FT/IR-430 instrument.
The HPLC apparatus consisted of an Agilent 1100 HPLC system
equipped with a refractive index detector. The column was a Phenom-
enex Hydro RP-18, 4 µm, 250 × 4.5 mm. Analytical TLC was made
on Kieselgel 60 F254 or RP-18 F254 plates with 0.2 mm layer thickness
(Merck). Preparative TLC was performed on Kieselgel 60 F254 plates
with 0.5 or 1 mm film thickness (Merck). Flash column chromatography
(FCC) was conducted on Kieselgel 60, 230-400 mesh (Merck), at
medium pressure.
Phototransformation of Carboxin by Natural Solar Light. A
suspension of carboxin (20 mg) in 300 mL of deionized water was
exposed to natural sunlight in Pyrex flask, under aerobic conditions, at
Naples (Italy) in October 2003 (temperature range of 20-35 °C).
* Author to whom correspondence should be addressed (telephone 39-
081-674334; fax 39-081-674393; e-mail iesce@unina.it).
† Universita` Federico II.
‡ Seconda Universita` di Napoli.
10.1021/jf049737o CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/09/2004

Phototransformation and Toxicity of Carboxin in Water
J. Agric. Food Chem., Vol. 52, No. 20, 2004 6229
5 (in ∼2:1 molar ratio) in the organic layer, whereas the aqueous extract,
after evaporation, gave the oxanilic acid 7.
Table 1. Product Distribution by Sunlight Irradiation of Carboxin in
Water after 4 Days
Preparation of Disulfide 5. A solution of 1-mercaptoethanol (30
mg) in deionized water (300 mL) was saturated with oxygen and
exposed to solar light for 4 days. Then, the solution was extracted with
ethyl acetate, and the organic layer, analyzed by 1H NMR, showed the
presence of a product whose signals matched exactly those of compound
5.
Toxicity Testing. Acute toxicity tests were performed on primary
consumers typical of the aquatic environment: the rotifer Brachionus
calyciflorus and two crustaceans, Daphnia magna and Thamnocephalus
platyurus. Chronic tests (sublethal endpoints) comprised a producer,
the alga Pseudokirchneriella subcapitata, and a consumer, the crusta-
cean Ceriodaphnia dubia.
product distributionb (%)
reaction
conditiona
1
2
3
4
5
6
8
9
pH 7
pH 10
pH 2
humic acid, 10%
KNO3, 5%
55
76
47
63
27
23
20
38
24
45
5
3
7
3
5
14
1
2
1
-
2
10
18
<1
<1
5
5
<1
<1
2
<1
<1
a Suspension of carboxin (20 mg) in 300 mL of water after saturating with oxygen.
b Percentages have been deduced by 1H NMR of the mixture extracted with ethyl
acetate.
Carboxin and its photoderivative were previously dissolved in
dimethyl sulfoxide (Carlo Erba, Milan, Italy), and, in the final test
solutions, DMSO concentration was kept constant at 0.01% (v/v). A
solvent-only control was included in each experiment to detect the
possible effect of the vehicle. For all of the bioassays dissolved oxygen
and pH were measured in each sample both at the start and at the end
of testing. At the same time as acute and chronic toxicity tests, reference
assays were performed with potassium dichromate (Aldrich Chemical,
St. Louis, MO) for all of the organisms except C. dubia, for which
pentahydrate copper sulfate (Aldrich) was used.
Acute Bioassays. Juveniles (age, 0-2 h) of the rotifer B. calyciflorus
were hatched from cysts provided by MicroBioTest, Nazareth, Belgium,
after 16-18 h of incubation under a light source of 3000-4000 lx at
25 °C in synthetic reconstituted medium (moderately hard medium
EPA-600/4-85-013) and then exposed to the test sample (16). Five test
dilutions were prepared in a 50% dilution series for each sample with
six replicates of five animals (0.3 mL of test solution, slightly different
from the ASTM procedure applied). Test duration was 24 h, temperature
was 25 °C, in the dark. The test parameter considered was mortality,
and the concentration found to kill 50% of the rotifers in 24 h was
indicated as the LC50 (16).
The bioassay on the anostracan crustacean T. platyurus was
conducted using second- and third-instar fairy shrimp larvae hatched
from cysts provided by MicroBioTest, after 20-22 h of incubation at
25 °C in synthetic reconstituted freshwater (the same moderately hard
EPA medium as rotifers) under continuous illumination (light source
of 3000-4000 lx). Tests were performed in 24-well plates, 10
crustaceans per well (1.0 mL of test solution), 3 replicates per
concentration, 5 concentrations, and a negative control. Test duration
was 24 h, temperature was 25 °C, in the dark. The test parameter
considered was mortality, and the concentration found to kill 50% of
the crustaceans in 24 h was indicated as the LC50.
The test on D. magna Straus, during 24 h of exposure, was performed
on young organisms obtained from our laboratory cultures, <24 h old,
at 20 °C in the dark, following International Standard Organization
procedure 6341 for acute toxicity tests (17). The synthetic reconstituted
freshwater, aerated before use, was the ISO hard medium (hardness of
250 mg/L expressed as CaCO3). Tests were performed with five
daphnids per vessel (10 mL of test solution), four replicates for each
of the five concentrations. The test endpoint was the inhibition of
mobility, and the concentration found to immobilize 50% of the
crustaceans in 24 h was indicated as the EC50.
Similar experiments were carried out by adjusting the pH of suspension
to 2 with 10-3 M HCl and to 10 with 10-4 M KOH, in the presence of
KNO3 (10 mg/L), with humic acid (5 mg/L). Each experiment was
performed in duplicate, with one set of dark controls. After 4 days of
sunlight exposure, each reaction mixture was extracted with ethyl
acetate, yielding 10-15 mg of crude mixture. The organic layer was
1
analyzed by H NMR, and product distribution was reported for each
experiment in Table 1. The mixture was chromatographed by reverse
phase C-18 HPLC [H2O/CH3OH/CH3CN (5:3:2)] using a refractive
index detector to give unreacted carboxin (30-55%) and the photo-
products (altogether 20-35%). The aqueous layer was analyzed by 1H
NMR and showed the presence of only one product, which was
identified as oxanilic acid (7). All of the products were fully character-
ized by spectral means. Compounds 2, 3, 8, and 9 were identified by
comparing the spectroscopic data with those reported in the literature
(9, 12, 13). Compound 5 was identified by comparison of spectral data
with those of a pure sample suitably prepared. The unreported spectral
data for products 3 (12), 4 (14), 5 (15), 6 (7), and 7 (8) are given below.
Ketoamide 3: 13C NMR (CDCl3) δ 25.3 (Me), 33.8 (CH2S), 71.8
(CH2O), 92.1 (C-2), 119.8 (C-2′), 125.5 (C-4′), 129.2 (C-3′), 136.1
(C-1′), 156.2 (CON), 189.7 (CO); MS, m/z 251 [M]+, 103.
1
Acetate 4: IR (CHCl3) 2852, 1717 cm-1; H NMR (CDCl3) δ 2.08
(s, 3 H, -Me), 2.93 (t, J ) 6.3 Hz, 2H, CH2S), 4.33 (t, J ) 6.3 Hz,
2H, CH2O); 13C NMR (CDCl3) δ 23.7 (Me), 38.7 (CH2S), 68.1 (CH2O),
172.6 (CO2); MS, m/z 87 [M - SH]+, 60, 43.
1
Disulfide 5: H NMR (CDCl3) δ 2.88 (t, J ) 6.3 Hz, 4H, 2CH2S),
2.98 (br s, 2H, 2OH), 3.89 (t, J ) 6.3 Hz, 4H, 2CH2O); 13C NMR
(CDCl3) δ 41.2 (CH2S), 60.3 (CH2O); MS, m/z 154 [M]+, 92, 79, 64,
45.
3-[(2-Hydroxyethyl)thio]-4-methylquinolin-2(1H)-one (6): mp 153-
154 °C (CH2Cl2/hexane); IR (CHCl3) 3389, 1641 cm-1; NMR (CDCl3)
δ 2.92 (s, 3H, Me), 3.04 (t, J ) 5.2 Hz, 2H, CH2S), 3.70 (t, J ) 5.2
Hz, 2H, CH2O), 4.35 (br s, 1H, OH), 7.30 (t, J ) 7.5 Hz, 1H, H-6),
7.45 (d, J ) 7.5 Hz, 1H, H-8), 7.58 (t, J ) 7.5 Hz, 1H, H-7), 7.78 (d,
J ) 7.5 Hz, 1H, H-5), 11.75 (br s, 1H, NH); 13C NMR (CDCl3) δ 18.2
(Me), 38.8 (CH2S), 60.2 (CH2O), 116.7 (C-8), 120.6 (C-10), 123.2 (C-
6), 124.4 (C-3), 125.5 (C-5), 131.5 (C-7), 137.2 (C-9), 155.7 (C-4),
161.1 (C-2); MS, m/z 235 [M]+, 204, 143, 77, 43.
Oxanilic Acid (7): IR (KBr, wafer) 3473, 1697 cm-1 1H NMR
;
(DMSO) δ 7.03 (t, J ) 7.3 Hz, 1H), 7.27 (t, J ) 7.3 Hz, 2H), 7.75 (d,
J ) 7.3 Hz, 2H), 10.18 (br s, 1 H); 13C NMR (DMSO) δ 119.8 (C-2′),
125.5 (C-4′), 129.2 (C-3′), 136.1 (C-1′), 163.3 (CON), 165.5 (COOH);
MS, m/z 167 [M]+, 148 [M - OH]+.
Chronic Bioassays. The algal growth inhibition test was run in 72
h according to International Standard Organization procedure 8692 (18).
The algal inoculum was taken from an exponentially growing preculture
and added to 25 mL of test solution to obtain an initial cell density of
the order of 104 cells/mL. Each compound was tested in five
concentrations, three replicates, and a negative control. Flasks were
placed in a growth chamber at 25 °C under continuous illumination
(8000 lx). The cell density was measured at 0 time and every 24 h for
3 days by an electronic particle dual threshold counter (Coulter Counter
Z2, 100 µm capillary, Instrumentation Laboratory, Miami, FL), and
from these data the algal growth inhibition in percentage was calculated
by integrating the mean values from t0 to t72 h (area under the curve).
The test on C. dubia was based on a population growth inhibition
in 7 days and performed on young organisms, <24 h old, obtained by
acyclical parthenogenesis of individual adult females for at least three
Enol 9: 13C NMR (CDCl3) δ 21.3 (Me), 39.1 (CH2S), 59.7 (CH2O),
93.1 (C-3), 120.3 (C-2′), 124.5 (C-4′), 129.0 (C-3′), 137.3 (C-1′), 171.1
(C-2), 183.1 (C-7); MS, m/z 253 [M]+, 193, 135.
Sunlight Irradiation of Sulfoxide 2 in Water for 1 Month. Twenty
milligrams of this compound was dissolved in deionized water and
exposed to solar light for 1 month (between October 4 and November
1
3, 2003). After extraction with ethyl acetate, H NMR analysis of the
organic layer showed the presence of only the starting sulfoxide 2.
Preparation of Products 4, 5, and 7. Compound 8 (25 mg),
synthesized as reported in the literature (9), was dispersed in deionized
water (250 mL) and exposed to natural sunlight for 4 days. Then, the
mixture was extracted with ethyl acetate, giving acetate 4 and compound

6230 J. Agric. Food Chem., Vol. 52, No. 20, 2004
DellaGreca et al.
and processed by a regression analysis technique to obtain the respective
IC50 value (the test concentration corresponding to 50% reduction in
growth relative to the control).
RESULTS AND DISCUSSION
After 4 days of exposure to sunlight, the water suspension
of carboxin 1 (2.8 × 10-4 M) was extracted with ethyl acetate
and the organic layer was analyzed by 1H NMR. No appreciable
variations were observed using more dilute solutions (∼10-5
M).
The separation of this mixture (12 mg) by chromatographic
methods allowed us to isolate in addition to unreacted carboxin
(50%) five compounds, which were identified as sulfoxide 2,
ketoamide 3, acetate 4, disulfide 5, and quinolinone 6 (Figure
1) by spectroscopic means (1H NMR, 13C NMR, MS, IR) and/
or by comparison of some spectroscopic data with those
previously reported (8, 12, 13). A similar procedure was carried
out by sunlight exposing aqueous suspensions of carboxin at
pH 2 and 14, in the presence of humic acid (10%) or with nitrate
(5%). The conversion percentage and the composition of each
mixture was evaluated by the 1H NMR spectrum of the organic
layer and are reported in Table 1.
In acidic conditions the ester 8 and the enol 9 were identified
(Figure 1). Under all of the conditions used, evaporation of
the aqueous layer furnished a compound that was spectroscopi-
cally identified as oxanilic acid 7 (Figure 1). A control
experiment showed that this compound is obtained together with
acetate 4 and disulfide 5 when a suspension of ester 8 is exposed
to sunlight in water. Disulfide 5 is the oxidation product of
mercaptoethanol formed by hydrolysis of acetate 4. In all of
the experiments dark controls for the same length of time
showed no change of carboxin.
Figure 1. Carboxin and its photoproducts.
generations. The first females were born from the hatching of ephippia
provided by MicroBioTests. Organisms were exposed individually in
beakers with 20 mL of test solution for 7 days to seven concentrations
(2-fold dilutions) in synthetic reconstituted aerated hard ISO medium
(total hardness of 250 mg/L as CaCO3). Ten replicates were incubated
at 25 °C with a 16:8 h light/dark cycle (500 lx) (19). Daphnids were
fed at each renewal with 100 µL of a suspension of the alga P.
subcapitata (4 × 108 cells/mL), food fish (5 g/L), and yeast (5 g/L).
Organisms were monitored for survival, and released neonates were
counted each day prior to renewals and then discharged. The parameter
considered was the population growth inhibition, so that from the
comparison between the number of offspring born from live or dead
mothers at the end of the test in the sample batch and the control it
was possible to calculate the concentration that gave rise to 50%
population growth inhibition, indicated as the EC50 (19).
Data Analysis. All results, except the algal test, were analyzed using
the program Toxcalc (20). For acute toxicity tests, the LC50 and EC50
with 95% confidence intervals were calculated by concentration/
response regression using probit or the trimmed Spearman-Karber
method, as appropriate. For the test with C. dubia, the value of EC50
with 95% confidence intervals was calculated using the maximum
likelihood-logit method. Inhibition (percentage) values of algae were
reported against log-transformed data of concentrations (in micromolar)
As reported in Table 1, carboxin is readily photodecomposed
by natural sunlight, giving mainly sulfoxide 2 and acetate 4.
Ketoamide 3 is also found, whereas disulfide 5, quinolinone 6,
and ester 8 are obtained in very small amounts. Environmental
effects such as pH variation or the presence of humate appear
Figure 2. Mechanism of the photodegradation of carboxin.

Phototransformation and Toxicity of Carboxin in Water
J. Agric. Food Chem., Vol. 52, No. 20, 2004 6231
Table 2. Toxicity Tests of Carboxin and Its Sulfoxide 2 toward Aquatic Organisms
L(E)C50 in mg/L for acute toxicity testsa
L(E)C50 in mg/L for acute toxicity testsa
compound
B. calyciflorus
5.0 (2.60 7.32)
4.10 (2.72 6.19)
T. platyurus
D. magna
C. dubia
P. subcapitata
2.41 (2.09 2.77)
NE (30 ppm)
carboxin 1
sulfoxide 2
−
−
61.00 (55.25−67.35)
78.17)
22.59 (19.04−26.80)
0.64 (0.52−0.73)
0.79)
−
56.57 (40.94
−
NEb (80 ppm)
0.66 (0.31
−
a Ninety-five percent confidence limits in parentheses. b No effect.
to have little influence on the photodegradation rate, whereas a
significant increase is observed in the presence of the nitrate as
expected on the basis of its property of inducing photo-oxidation
(11).
various plant species and animals, too (21, 22). The metabolite
has non-fungitoxic activity and, as shown by our investigation,
exhibits similar or even lower acute toxicity toward aquatic
organisms.
Formation of the products can be explained on the basis of
photo-oxidative transformations as the main light-induced
pathways. According to previous studies (7), excited states of
the pesticide, formed directly by the absorption of the solar
radiation [carboxin exhibits an absorption band with a maximum
at 292 nm (log ꢀ of 3.2)], can react with ground-state oxygen,
which adds to sulfur or to the double bond leading to sulfoxide
2 or to the radicals 10 or 11 (Figure 2). The latter afford ester
8 via the unstable dioxetane 12, whereas intermolecular reactions
should be involved in the formation of ketoamide 3 via the
intermediate 13. These products could be formed via other
oxygenating species such as singlet oxygen generated by energy
transfer by the excited 1 (7). Control experiments showed that
the reaction is largely inhibited by DABCO (10-3 M) as a
quencher of singlet oxygen or energy transfer reactions (20%
of photodegradation after 4 days).
As proven by control experiments, hydrolysis of ester 8 leads
to acetate 4 and acid 7. Further decomposition of compound 4
gives disulfide 5. Quinolinone 6 is a photoisomerization product
and is formed also in the absence of oxygen (7). Enol 9 is
formed by both acid- and light-induced addition of H2O to
carboxin. Indeed, this compound is not found under neutral and
basic conditions, and carboxin was quantitatively recovered
under acid conditions in the dark after 4 days.
ACKNOWLEDGMENT
We thank Centro Interdipartimentale Chimico-fisiche for the
NMR facilities. NMR experiments were performed on a 500
MHz spectrometer of Consortium INCA (Project P0 L488/92,
Cluster 11A).
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bioactive at low concentrations mainly for the primary consumer
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Received for review February 17, 2004. Revised manuscript received
July 15, 2004. Accepted July 21, 2004.
JF049737O