The Science of the Total Environment, 132 (1993) 339-348 339 Elsevier Science Publishers B.V., Amsterdam

The fate of triazine pesticides in River Po water

Anna Brambilla a, Bruno Rindone a, Stefano Polesello b, Silvana Galassi c and Raffaella Balestrini c

aDipartimento di Chimica Organica e Industriale, Universita" di Milano, Via Venezian 21, 20133 Milan, Italy

bDipartimento di Chimica Inorganica e Metallorganica, Universita" di Milano, Via Venezian 21, 20133 Milan, Italy

Clstituto di Ricerca sulle Acque del Consiglio Nazionale delle Ricerche, Milan, Italy

ABSTRACT

A group of triazine herbicides has been monitored in river Po water over a period of three years. The N-deethylated metabolite of atrazine was also found. Laboratory tests of biodcgradation of triazine performed with River Po water as cultural medium did not show any significant triazine degradation, leading to the conclusion that triazine metabolites derive from de.~-adation in soil. The chemical reactivity of triazines toward oxidants was also tested in laboratory with lead(IV) acetate, cerium(IV) ammonium nitrate and ozone. A chemical degradation pathway for triazines is suggested.

Key ~vords: triazine pesticides; River Po; biodegradation; possible degradation pathway

INTRODUCTION

In a previous paper water samples from River Po were withdrawn from May 1988 to September 1989. Organic phosphates and pesticides were deter- mined by HRC~ using selective detectors. HRGCoMS was used for the analysis of organic micropollutants deriving from occasional pollution [l]. Herbicides and products therefrom resulted to be the most relevant pollutants from the ecotoxicological point of view [2,3].

The presence of a metabolite of atrazine (ATR) 1, N-deethylatrazine (DEA) 2, in some samples suggested a possible degradation pathway for ATR in River Po water. Experiments aimed to the quantification of triazine herbicides and their metabolites in River Po water are reported in this study. Moreover, attempts of microbiological and oxidative degradation of some triazines were performed.

0048-9697/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved

340 A. BRAMBILLA ET AL

EXPERIMENTAL

Sampling and filtration

Samples for herbicide analyses (10 liters) were taken from the River Po at Pontelagoscuro, about 100 km before the mouth, transported in glass vessels and extracted within 72 h, after filtration with Domnick Hunter filter, loaded with a teflon cartridge. The cartridge was previously activated with methanol and washed with 10 liters of MilliQ water. The filtration flow was fixed under argon pressure at 500 ml/min. Samples for biodegradation tests were col- lected at S. Nazzaro, halfway through the River Po course in October and in December 1990.

Concentration of the samples

The filtered sample of River Po water was then passed through a glass column (14 x 0.7 cm, i.d.) filled with XAD-2 resin (10 cm) at a flow rate of 30 ml/min. The resin was previously washed with ether and methanol. Elu- tion of organic compounds was performed with 30 ml of acetone. The eluates were lyophilised using a Edwards 4K Modulyo freeze dryer.

HRGC analyses

One to two microlitres of concentrated extracts were injected into a gas chromatograph C. Erba, Mega series 5160, with an on-column system, heated by a PTV, from 100 to 240°C. The analyses were carried out with a fused silica capillary column (25 m): PS 255, film thickness 1.9-2.0 ttm and OV 1701, film thickness 0.1-0.15 ~tm, respectively. The carrier gas was helium at 2 and 1 ml/min, respectively. An NPD 40 selective detector was used in the following conditions: 270°C, hydrogen flow, 30 ml/min; air flow, 300 ml/min; make up (helium), 30 ml/min.

HRGC-MS analyses

Concentrated extracts were injected into a Dani 3800 gaschromatograph equipped with a 25-m SE 54 capillary column in the split-splitless injection mode. The oven was kept at 60°C for 2 min, then heated to 250°C with a gra- dient of 10°C/min. This last temperature was kept for further 10 min. This instrument was connected with a VG 7070 EQ mass spectrometer operating in Electron Impact (EI) at 70 eV.

THE FATE OF TRIAZINE PESTICIDES IN RIVER PO WATER 341

Biodegradation assays

Unfiltered water samples (2 1) were added to 5 g/l of ATR, SIM or TBA and left under stirring at 20°C. Subsamples were taken at different times and preconcentrated on a C-I 8 disposable column for analytical determinations. In a second set of experiments, water samples (2 liters) were added to dif- ferent amounts of ATR. The analyses reported above were then performed.

Chemical degradation assays

With lead(IV) acetate A 10-2-M solution of substrate in methylene chloride was added to a 1.5:1

molar excess of lead(IV) acetate and left at room temperature for 48 h under nitrogen. In the case of TBA a starting concentration of 2 x 10 -3 M was used. The resulting suspension was filtered over Celite, washed with saturated sodium hydrogen carbonate solution and then dried over anhydrous sodium sulfate. After evaporation of the solvent under reduced pressure the residue was analysed by HRGC-NPD and HRGC-MS.

With cerium(IV) ammonium nitrate A 4.5 x 10 -3 M solution of substrate in acetonitrile was added to a 1.5:1

molar excess of cerium(IV) ammonium nitrate and left at room temperature until the reagent had disappeared (starch-iodine paper). The resulting suspension was filtered and the solvent was evaporated under reduced pressure. The residue was extracted with three 50-ml portions of ethyl acetate, which was filtered and evaporated under reduced pressure. This residue was analysed by HRGC-NPD and HRGC-MS.

With ozone Two-hundred milliliters of a 1.15 x 10-3-M solution of substrate water

pH 1 for sulphuric acid was treated with a 20-1iters/h stream of oxygen sub- j~:cted to a 2.2-A electric discharge. In these conditions ozone production was 1.0 g/h. Ozonation time was 1 h. The solution was then adjusted to pH 10 and extracted with three 150-ml portions of ethyl acetate and the organic ex- tracts were dried over anhydrous sodium sulfate. After evaporation of the solvent under reduced pressure. The residue was analysed by HRGC-NPD and HRGC-MS.

RESULTS AND DISCUSSION

A family of triazine pesticides ~nd their degradation products was

342 A. BRAMBILLA El" AL

monitored over a period of 3 years in River Po water. After a preconcentra- tion step, the eluates were analysed by HRGC with NPD selective detector.

Figure 1 shows the analytical results concerning (1) atrazine (ATR), and (2) N-deethylatriazine (DEA) determination, while Fig. 2 reports the concen- tration of (3) simazine (SIM) and (4) t-butylazine (TBA). The presence of DEA indicates that in the degradation process of triazine herbicides N- deethylation of atrazine is prevalent.

A control experiment performed to test herbicide recoveries showed that the efficiency in the recovery of ATR, SIM and TBA was 95%. DEA and DIP were recovered with 65% and 20% efficiency, respectively. Due to the poor recovery efficiency and the occurrence of interferring peaks in the chromatograms, DIP could not be quantitatively determined in many River Po water samples. In those samples in which DIP was detectable, its concen- trations were comparable to those of DEA.

Further experiments were devoted to test the possible microbiological orion of this N-deethylation reaction. Samples of ATR, SIM and TBA were dissolved in River Po water collected in a period in which triazines are not present and left for several days in dark bottles. HRGC analyses using the NPD detector showed that triazine concentrations in the cultural medium did not change with time (Table 1). Moreover, no significant metabolite for- mation was ob,3erved. Also over a longer period of time (14 and 32 d~ys) ATR (five different concentrations) was recovered unchanged (Table 2). Hence, no biological degradation of these triazines is to be expected in River Po water.

Atrazlne and DEA in the River Po

ngll I " A t r a z i n " ~ OEA 1

688 :300 m,

200

100

18 30 23 31 38 1~ 16 13 10 01 26 0S 33 22 27 29 09 36 30 30 24 11 May Sep Nov Jan Feb qsr May Jun Jul Aug Sep Dec Jan Feb Mar May Jun Mar Apt Apr May Jun

1988 1989 l tmn 1991

Fig. 1. Concentrations of atrazine (ATR) and N-deethylatrazine (DEA) in River Po water.

THE FATE OF TRIAZINE PESTICIDES IN RIVER PO WATER 343

Simazine and Terbutylazine in lhe River Po

] k~ Simazine , Terbutylazin. [ ng/I 3S0

300

250

200

150

100

SO

0 18 20 23 31 20 11 16 13 18 01 20 05 23 27 27 29 09 20 20 30 24 11

May Sep Nov Jan Feb Apt May Jun Jul Aug Sep Dec Jan Feb Mar May Jun Mar Apt Apt g,~y Jun 1988 1989 1990 1991

Fig. 2. Concentrations of simazine (SIM) and terbutylazine (TBA) in River Po water.

Oxidative N-dealkylation reactivity was tested reacting ATR, SIM and TBA with three chemical oxidants, e.g. lead(IV) acetate, Pb(OCOCH3)4 (LTA) in methylene chloride; cerium(IV) ammonium nitrate, Ce(NH4)2 (NO3)e (CAN) in acetonitrile; ozone, 03 in water. LTA and CAN allowed to obtain data in the absence of oxygen and hydrolytic phenomena. Ozone operated in aqueous medium and in the presence of oxygen. Different chemical phenomena could be present in ozone oxidation and the recovery of reaction products from the aqueous solution could be a major problem.

TABLE 1

Attempted microbiological degradation of triazines in River Po water. Initial concentration, 5 ~g/l. Samples were concentrated with a C-18 column which was eluted with methanol. Analysis by HRGC-NPD

Time (h) Atrazine Simazine Terbutylazine

24 3.7 3.1 4.8 48 4.3 3.4 4. I

120 3.5 4.1 4.3 216 4.3 3.3 5.4 312 3.7 2.6 3.5 384 3.1 2.5 4.2

344 A. BRAMBILLA ET AL,

TABLE 2

Attempted microbiological degradation of atrazine in River Po water after 0, 14 and 32 days. Samples were concentrated with a C-18 column which was eluted with methanol. Analysis by HRGC-NPD, concentrations in/~g/l

0 14 32

lO 9.03 9.4 I 0.93 1.25 0.I 0.076 0.16 0.01 0.0082 0.01 0.001 0.0013 0.001

Conditions of low conversion were used with LTA in order to observe primary reaction products. After two days at room temperature the reactant in excess was decomposed with sodium hydrogen carbonate and the reaction mixtures were analysed both in HRGC-MS and in HRGC-NPD. Both DEA and DIP were detected in the reaction of ATR and the ratio DEA/DIP was 4.11 (Table 3). Hence, the N-deethylation of atrazine resulted faster than the Nodeisopropylation. Also SIM underwent the Nodeethylation reaction to give DIP. Only traces of N-deethylterbutylazine (DET) (6) were observed with TBA.

In CAN oxidation, after two days at room temperature the reactant has been consumed and with ATR the ratio DEA/DIP was I 1.5 (Table 3). Again, the Nodeethylation of ATR resulted faster than the Nodeisopropylation. Ozone oxidation of ATR gave after one hour at room temperature the DEA/DIP ratio of 13.2 (Table 3). Control experiments of recovery of DEA and DIP in these conditions allowed to correct *.his ratio to 4.06. Again, the N-deethylation of atrazine resulted faster than the N-deisopropylation. In the reaction of ATR and TBA, also traces of the amides 7 and $ were detected.

Triazine herbicides in water are an important problem. Tile-drain water in Canada [4], agricultural watersheds in Quebec [5], lakes and rain in Switzerland [6], rivers in France [7] contain these compounds. Also their presence in soils is relevant [8,9].

Possible degradation pathways in water were suggested to be the sensitized photooxidatioL~ [10], the photocatalytic process [l l] and the ozonation pro- cess [12]. In these processes the Nodeethylation of ATR was shown to be the most important primary degradation pathway.

DEA is also the major urinary metabolite of atra:Jne [13] and is probably formed via an oxidative pathway catalysed by cytochrome P-450 as already

THE FATE OF TRIAZINE PESTICIDES IN RIVER PO WATER 345

TABLE 3

The chemical oxidation of triazines

SIM ATR TBA

N-deethylation % LTA

TRIAZ 97 93 99 DIP DEA 3 6 DET tr

CAN TRIAZ 33 30 13 DIP 67 DEA 64 DET 86

Ozone TRIAZ 65 79 86 DIP 35 DEA 20 DET 14

N-dealkylation % LTA

DIP tr DET

CAN DIP 6 DET

Ozone DIP l DET

shown for aromatic tertiary and secondary amines [14]. However, DIP is formed too, even if to a lesser extent.

These findings are in line with the fact that the oxidative N-dealkylation of small linear alkyl groups is faster than that of branched alkyl groups both with chemical oxidants [15,16] and with enzymatic systems [17]. We suggest that also in the environment these triazine herbicides are degraded via the mechanism shown for ATR in Scheme 1. Here, one-electron transfer reagents such as LTA and CAN form the cation radical 9 which is an acid- base equilibrium with the two carbon radicals 10 and 11.

The chemoselective N-deethylation reaction is the result of the competi- tion between the rates of removal of the protons to nitrogen by an external

346 A. BRAMmLL ̂~r AL

CI

' JJ-..

N NHR

Fig. 3. Triazine substrates and reaction products. (1) ATR, R - Et, R ' - / - P r ; (2) DEA, R = H, R ' = i-Pr; (3) SIM, R = Et, R ' = ET; (4) TBA, R = Et, R ' = t-Bu; (5) DIP, R = Et, R ' = H; (6) DET, R = H, R ' = t-Bu; (7) R = COCH3, R ' =/-Pr; (8) R = COCH 3, R ' = t-Bu.

base. This was the acetate ion with LTA, the nitrate ion with CAN. In the oxidation of N,N-dialkylanilines the removal of the proton is easier with small linear alkyl groups than with branched groups [18]. Hence, formation of the radical 10 is preferred to 11. These carbon radicals to nitrogen are very oxidizable [19] and their further oxidation gives the cations 12 and 13. Both undergo solvolysis and DEA and DIP are formed with a ratio which depends on the ratio between the radicals 10 and 11.

A confirmation of these observations derive from the finding that the ratio DEA/DIP was higher with CAN than with LTA indicating a higher selectiv- ity of the nitrate ion over the acetate ion in the removal of the proton. The higher pKB of acetate results in higher reactivity and lower selectivity.

A different situation occurs in the ozonation reaction at low pH. Here, ozone itself abstracts one hydrogen atom [20] and forms the radicals 10 and 11. The chemoselectivity observed is in line with other selective N- dealkylation reactions performed with dioxygen and hydroxyl radical [21] and is probably the result of steric hindrance in the attack of ozone [22]. The formation of the radical 10 during the ozonation is suggested also from the detection of the amides 7-8. In fact, they derive from the insertion of dioxy- gen at the radical center in 10 to give the hydroperoxyl radical 14 which decomposes to an acetamide.

Steric factors seem to play a role in the reactivity. An equilibrium forma- tion of a metal-substrate complex has been shown to occur both in LTA [23] and in CAN [24] oxidations. Hence, the electron abstraction to give the cat- ion radical 2 is an innersphere process. This equilibrium is expected to be strongly affected by the steric bulk of the triazine. In ozone oxidations at pH 1, the position of the protonatio, n should play a role.

In conclusion, these experiments suggest that N-dealkylation of triazine

THE FATE OF TRIAZINE PESTICIDES IN RIVER PO WATER 347

C! N "~'N e" CI

H3C "kFCH (1) N N ~ (9)

Cl O2 y ~ H÷ O.o N 'N cl o

H3C "~ NI-I "~ N i~ NH

H3C ")¢"<CH3 H3C "J~ CH3 04)

(10) (11)

(7) e - ~ ~ e

C! Cl N "~N £

N * ' N H H .~ .~ + J~. ,I H3C"~NH N Nil H_

DEA (2) ~ H3C ''~'NI'I'~N/'~ NH H3C CH3 ,,]KH (13) (12)H3 C CII3 /

DIP (5)

Scheme 1. Mechanistic scheme for the Pb-, Ce- and ozone-mediated oxidation of herbicide triazines.

depends on the structures of the Noaikyl group. Deethylation appears to be the faster dealkylation process. Therefore, although DIP should to be the main metabolite occurring in River Po, being the only one common to all the triazine herbicides, it was detected at very low concentrations probably because of its low recovery and high degradability. This is important for practical purposes because the structures with linear alkyl groups will be the ones which degrade first and will have a minor persistence in the environ- ment. Moreover, the rv~tio DEA/ATR and DIP/ATR will help to know the time application of ATR in field [25].

348 ^. BRAMBILLA ET AL,

ACKNOWLEDGEMENTS

We thank our student E. Andreoni for technical support and Mr A. Cremonesi for the nmss spectra. This work was supported by a grant of the Consiglio Naziona!e delle Ricerche.

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