Haloacetic Acids Research Articles

Research Articles

Haloacetic Acids, Phytotoxic Secondary Air Pollutants

Hartmut Frank 1, Harald Scholl t, Dirk Renschen 1, Benolt Rether z, Abdelkrim Laouedj 2, Yrj6 Norokorpi 3

1 Institute for Toxicology, University of Tiibingen, Wilhelmstra~e 56, D-72074 Tfibingen, Germany 2 Laboratoire de Biologie V~g&ale Appliqu&, 3, rue de l'Argonne, Universit6 Louis Pasteur, F-67000 Strasbourg, France 3 Finnish Forest Research Institute, Rovaniemi Research Station, P. O. Box 16, FIN-96301 Rovaniemi, Finland

Abstract

Haloacetic acids are atmospheric oxidation products of airborne C2-halocarbons which are important solvents and propellants. Le- vds of trichloroacetate (TCA) in conifer needles from mountain ranges in Germany (Black Forest, Erzgebirge) and from two sites in Finland are compared; TCA is present in conifer needles at con- centrations up to 0.7/~mol/kg, MCA up to 0.2/amol/kg. At the Finnish sites, TCA-concentrations and branch degeneration symp- toms of Scots pine are correlated. Monochloroacetate (MCA) has been determined in needle samples from Southern Germany in con- centrations exceeding its phytotoxicity threshold towards photo- autotrophic organisms. Data on atmospheric chloroacetate levels in Germany are also given; ambient air levels of chloroacetic acids range from about 2 pmol/m 3 (TCA) to 390 pmol/m 3 (MCA). TCA and dichloroacetic acid (DCA) arise from atmospheric oxi- dation of airborne C2-chlorocarbons, while the source of MCA is not yet known; several tentative pathways are suggested.

Growth inhibition of plant cell suspension cultures show that lipophilic ester and amide derivatives of chloroacetic acids are more potent phytotoxicants than the free acetates. Chloroacetic acids are detectable in relatively low-polluted regions in the major environmental compartments, i.e. the pedosphere [16], hydrosphere [ 1 7 - 1 9 ] , and biosphere [20,21]; this raises the question whether they could contri- bute to the induction of phytotoxicological symptoms on co- nifers and other forest trees observed since a decade in re- mote areas [22,23]. The exquisite algal toxicity of monoha- loacetic acids [24,25] and the potential increase in environ- mental burden of haloacetic acids when introducing C2-chlorofluorohydrocarbons [5] as substitutes for chloro- fluorocarbons (CFC) urge detailed assessments of environ- mental fates and ecotoxicological properties of the former.

1 In t roduc t ion

Haloacetic acids are atmospheric oxidation products of ha- logenated C2-hydrocarbons [1,2]. Quantitatively most rele- vant are the technically important C2-chlorocarbons 1,1,1-trichloroethane, trichloroethene and tetrachloroethene [3,4]. The annual global production of the C/-halocarbons is presently over two million tons [5,6], most of which is re- leased into the atmosphere. Some of them have strongly in- creased in their atmospheric levels during the last two de- cades [5] because of long atmospheric residence times. Chloro- fluorohydrocarbons, suggested as substitutes for long-lived chlorofluorocarbons, may also be oxidized to various halo- acetic acids [7]. Halocarbon exposure at relatively high levels result in de- gradation of photosynthetic pigments [8] of conifers and de- ciduous trees and affect xenobiotic metabolism of plants [9]. More potent in toxicity [10,11] and phytotoxicity [12,13] are their atmospheric oxidation products, in particular the haloacetic acids. Various chlorinated aliphatic acids and de- rivatives thereof are known as herbicides [14,15]: Trichlo- roacetate (TCA) has been employed as sodium salt or as ester or amide derivatives against perennial grasses. Monochlo- roacetate (MCA) is the phytotoxic principle in the herbi- cides Alachlor, Propachlor, Metazachlor, and Metolachlor.

2 Exper imenta l

2.1 Sampling Sites

Samples were collected from European silver fir (Abies alba MILLER), Norway spruce (Picea abies (L.) K~ST), and Scots pine (Pinus sylvestris L.) by removing twigs from branches 2 m above the ground from the southwest quadrant of iso- lated trees or those located at southern to western fringes of forest stands.

In the Northern Black Forest, samples were collected from trees at the following locations:

Mauzenberg (fir, spruce), elevation 720 m above sea level (a.s.1.) (48~ 8~ 6 km east of Gaggenau); Bernstein (spruce, 620 m a.s.l., 48~ 8~ 5 km east of Gaggenau); Altsteigerskopf (spruce, 950 m a.s.l., 48~ 8~ Zwieselberg (fir, spruce, 830 m a.s.l., 48~ 8~

Two sites were at lower elevation east of the Black Forest range:

Herrenplatte (spruce, 560 m a.s.l., 48~ 8~ 5 km west of Herrenberg); Sch6nbuch (spruce, 460 m a.s.l., 48~ 9~ 8 km north of Tfibingen).

4 ESPR-Environ. Sci. & Pollut. Res. 1 (1) 4-14 (1994) �9 ecomed publishers, D-86899 Landsberg, Germany

Research Articles Haloacetic Acids

In the Erzgebirge, needle samples were taken from spruce trees located at the southwestern slope of the Fichtelberg peak (1214 m a.s.l., 50~ 12~ between 1140 and 1200 m.

In Northern Finland, samples were collected from Scots pine and Norway spruce at two sites (160 m a.s.l., 66~ 26~ i.e. Hietaper~i I (spruce 1, pine 3) and II (pine 5, pine 6), both at a distance of 1.2 km from each other [21]: Hietaperd/is a mixed Scots pine and Norway spruce stand (canopy density index 0.8; 1.0 = full closure, 0 = bare [26]) on moist heathland on a gentle north-easterly slope. Hietaperd llis an open Scots pine stand (canopy density in- dex 0.6) on dry sandy heathland on level ground. During the growing season of 1992, average wind passages of 39 km/d at Hietaper~i I and 78 km/d at Hietaperfi II were measured with a cup anemometer placed 2 m above ground. All needle samples were sent to Tiibingen by air mail arri- ving within two to three days.

2.2 Degree of Defoliation

For correlation of TCA-levels and needle loss, 20 Scots pine trees at Hietaperfi II were sampled in early June 1992. Phy- totoxicity was assessed as branch damage abundance accord- ing to JUKOLA-SULONEN et al [27].

2.3 Determination of Chloroacetic Acids in Conifer Needles

Twigs were cut with clean scissors into the respective an- nual sections. They were placed in clean screw-cap vials (7 x 2.5 cm) with aluminium-lined butyl rubber septa; the vials had been kept at 120 ~ C overnight before sampling.

For gas chromatographic analysis, the needles (about 3 g each) were cut from the respective twig sections and rinsed with distilled water to remove superficially adsorbed chlo- roacetic acids. Two aliquots (1.5 g) were homogenized by hand in a porcelain mortar with liquid nitrogen (100 mL). 2,2-Dichloropropionic acid (340 ng) was added as internal standard. The homogenized tissue was extracted with distilled water (6.0 mL), and an aliquot of the aqueous phase (4.0 mL) was acidified to pH 0 with 200/~L sulphuric acid. The chlori- nated aliphatic acids were extracted with diethyl ether, me- thylated with diazomethane, and analyzed by capillary gas chromatography and electron capture detection [28]. The results were verified by negative-ion chemical-ionization mass spectrometry (NCI-MS) and selected-ion monitoring (SIM) of the ions m/z 35 and m/z 37 (CI-). Calibrations were done by spiking a needle homogenate with increasing amounts of the chloroacetic acids dissolved in water. For determination of MCA, the same general procedure was followed, but detection and quantification were based upon derivatization to the corresponding 1-(pentafluorophenyl)- ethylesters [29] and NCI-MS-SIM of the respective carb- oxylate anions.

2.4 Determination of Chloroacetic Acids in Air

Denuders employed for air sampling consisted of 4 glass tubes (Duran, front and tail end tubes 500 x 3 mm i.d., cen- ter tubes 500 x 9 mm i.d.) coupled by ground joints [30].

They were internally coated with 1 % sodium bicarbonate in glycerol in the following manner: two tubes were connec- ted, positioned at a tilt of 15 degrees, and a few milliliters of the glycerol solution were applied to the upper opening under axial rotation of 30 rpm. When both tubes were ho- mogeneously wetted, they were placed vertically on filter pa- per for 4 hours to remove excess glycerol solution. For air-sampling, all four tubes were assembled, positioned horizontally, and unfiltered air (20 m 3) was aspirated at a flow of 5 L/min by means of a membrane pump (WISA 203, Sauer, Wuppertal). Volume flow was determined with a bel- lows gas counter (NB 3, Rombach, Kartsruhe). Another coated denuder was stored for the same period in closed con- dition to serve as blank. Calibration curves were estab- lished by placing appropriate amounts of the chloroacetic acids in a short tube attached to the front end of a denuder assembly and determining its transfer to the tubes after aspi- ration of the same volume of purified air. After sampling, the glycerol coat of both halves of the de- nuder was rinsed with 6 mL each of potassium hydroxide solution (1 mmol/L) into a round bottom flask under con- tinuous axial rotation (30 rpm) in tilted position (10~ 2,2-Dichloropropionic acid in aqueous solution (200/aL, 0.4/~g/mL) was added as internal standard. Concentrated sulphuric acid (350mg, 15drops), sodium chloride (500 mg), and freshly distilled diethyl ether (1.0 mL) were added, and the chloroacetic acids were extracted under mix- ing with a vibration mixer for 60 seconds. An aliquot of 0.6 mL of the ethereal layer was withdrawn, an equal vo- lume of freshly prepared ethereal diazomethane or 1-(pentafluorophenyl)-diazoethane (800 mg/ml) solution was added, and the reaction mixture was kept at room tem- perature for 5 minutes or overnight, respectively. One mi- croliter of the solution was injected for gas chromatographic analysis, as described previously [28].

2.5 Cell Culture Experiments

Bean cell suspension cultures (Phaseolus vulgaris L.) were started from callus cultures obtained from the hypocotyl of germinating seeds. Cells were transferred every ten days to fresh medium [31] supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid. Ten milliliters of a stock sus- pension containing a wet cell mass of 200 mg/mL were ad- ded to 50 mL medium, to yield suspensions with an initial cell mass of 33 mg/mL. For assessment of relative phytotoxicity, the respective com- pounds were added in dimethyl sulphoxide solution (60 aL per 60 mL cell suspension) to yield final concentrations bet- ween 10 z and 10 -7 mol/L. The incubations were performed in triplicate at each concentration level; the control cultures were incubated with 60/tL neat dimethyl sulphoxide. The cell suspensions were maintained for 15 days at 25 ~ C in a thermostated shaker (120 rev/min) at a day/night cycle of 12/12 h. Every third day, aliquots of 5 mL were withdrawn with sterile pipets and filtered through a Nylon filter (mesh size 25/~m). The cells on the filter were blotted dry and the fresh weights were determined. Growth curves were estab- lished, the growth inhibition was calculated, and EC~0- and ECs0-values were determined [32].

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Haloacetic Acids Research Articles

3 Results

3.1 Environmental Monitoring

The first monitoring campaign of TCA-levels in conifer nee- dies was performed from June 1990 to April 1991 on trees

from four sites in the Black Forest ( - ' Fig. I a-f) and two sites about 40 km to the east (-~ Fig. 1 g-h). The levels ranged from 0.02 to 0.55 pmol /kg fresh weight. When spruce and fir were monitored simultaneously (Mauzenberg, Zwiesel- berg), the former species exhibited higher levels than the lat- ter. Maximum levels were reached in September 1990, mi-

120

110

100

90

80

70

60

(ng/g) (pmol/kg

Mauzenberg, spruce

I I needle age 88

I I needle age 90

0.7

0.6

0.5

0.4

(ng/g) (prnol/kg

p- .

Mauzenberg, fir

I I needle age 88

I I needle age 90

0.7

0.6

0.5

0.4

50 - 0.3 - 0.3

30 0.2 0.2 o.15

2O 0.1 0.09

10

0 Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Jun Jul Aug Sep Oct Nov Dec Jan Feb MarApr

1990 1991 1990 1991

l a I b

(r ig/g) (wmol/kg (ng/g) (pmol /kg 120

0.7 0.7 1 lO - Bernste in, spruce A l ts te igerskopf , spruce

I

loo - I I needle age 88 0.6 - I I needle age 88 o.6

9o - I---I needle age 90 - I - - I needle age 90 80 0,5 _ 0.5

7O 0.4 0.4

6O

50 0.3 0.3

401- i I i m ~ 0,2 0.2 30 ~ , ~ 0.165

20 ~ 0.11 0.1 10

0 Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr

1990 1991 1990 1991

I c I d

6 ESPR-Environ. ScL& Pollut. Res. 1 (1) 1994

Research Articles Haloacetic Acids

Fig. 1 continued

(ng/g) 120

110

100

90

80

70

60

50

40

30

Zwieselberg, spruce

I I needle age 88

{Z] needle age 90

(pmol/kg)

0.7

0.6

0.5

0.4

0.3

0.2

(rig/g) (/.tmol/kg

Zwieselberg, fir

I I needle age 88

r I needle age 90

0.09 10

0 Jun Jul Aug Sep Oct Nov Dec Jan Feb MarApr

1 f 1990 1991 l e

120 0.7

110

100 0.6

9O

80 0.5

7O 0.4

60

5O 0.30

4O

30 0.2

2O 0.1

10

0 Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apt

1990 1991 1990 1991 l g l h

0.7

0.6

0.5

0.4

0.3

0.2

0.1 0.07

0.7

0.6

0.5

0.4

0.3

0.2

0.11

Fig. 1: Levels of trichloroacetate in conifer needles in Southwest Germany. The average TCA-concentrations in needles of the age group 1988 over the whole monitoring period are shown at the right ordinate of each panel (.~)

nima occurred in June 1990 and February 1991. At Mau- zenberg and Bernstein, low concentrations were also found in December 1990; both sites are at a distance of 1 km from each other. Average levels in two-year-old needles over the whole monitoring period in the Black Forest ranged from 0.07 (Zwieselberg, fir) to 0.165 ~mol/kg (Bernstein, spruce). Older needles exhibited higher levels than younger ones.

A similar temporal pattern was observed at the two sites east of the Black Forest (Sch6nbuch, Herrenplatte), again with minima in early summer 1990 and mid-winter 1991 (~ Fig. 1 g-h). The highest level of 0.55/~mol/kg and the highest average of the campaign (0.30/~mol/kg) occurred at the Sch6nbuch site. Spruce needles collected in June 1992 contained MCA up to 0.2 pmol /kg (--* Table 1 a) [33].

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Haloacetic Acids Research Articles

Table 1: Chloroacetic acids in (a) spruce needles [nmol/kg or pmol/kg] and (b) in ambient air [pmol/m 3 or pmol/g] at the site .Sch6nbuch"

Sampling period MCA DCA TCA

a) June 1992 100 - 40

1992 210 - 70 1992 210 - 40 1992 80 - 20

b) 2 6 . 0 3 . - 2 9 . 0 3 . 9 2 7.3 • 5.5 3 1 . 0 3 . - 0 4 . 0 4 . 9 2 6.7 • 1.2 0 4 . 0 4 . - 0 8 . 0 4 . 9 2 11.0 • 0.7 08. 0 4 . - 1 3 . 04, 92 42 • 30 5.4 • 3 1.8 • 1.2 1 4 . 0 4 . - 1 6 . 0 4 . 92 3.0 • 0.9 27. 04. -30. 04. 92 3.0 • 0.9 3 0 . 0 4 . - 0 6 . 0 6 . 92 126 • 40 43 • 23 2.4 • 0.3 2 0 . 0 5 . - 2 6 . 0 5 . 92 3.7 • 0.7 1 9 . 0 6 . - 2 6 . 06. 92 390 • 160 67 • 40 6.7 • 3.7 11.07.-16. 07. 92 160 • 60 92 • 54 16.0 • 3.7 3 1 . 0 7 . - 0 4 . 0 8 . 9 2 190 • 75 66 • 39 10.4 • 1.8

Range 42 - 390 5 - 90 2 - 16

Ambient air levels of MCA, DCA, and TCA were deter- mined at the southern edge of the Sch6nbuch Forest close to Tiibingen (--' Table 1 b). The chloroacetic acids were pre- sent in concentrations between 2 (TCA, minimum) and 390 pmol /m 3 (MCA, maximum).

The second monitoring campaign for TCA in conifer nee- dles was started in August 1990. Samples were collected from three spruce trees in the Erzgebirge, a mountain range bor- dering on Northern Czechia where the extent of tree damage is similar to the Black Forest [34]. Again, TCA-levels in two- year-old needles were higher than in young ones (--* Fig. 2 a- c). For spruce 1, the levels were close to 0.2 pmol/kg from August 1990 to June 1991, with maxima in December 1990, February, May and July 1991, and minima of 0 .1 / lmol /kg in March and June 1991. From August 1991 to February 1992, the levels remained at about 0.1/amol/kg. For spruce 2, the levels fluctuated more strongly but maxima and mi- nima coincided with those of spruce 1. In autumn 1991 TCA declined to reach a minimum of 0.05 gtmol/kg in February 1992. In spruce 3, the levels were between 0.05 and 0.15/~mol/kg throughout the monitoring period. Maxima were recorded in December 1990, April and June 1991, mi- nima in August 1991 and January 1992. Altogether, the le- vels in the Erzgebirge showed less fluctuation than in the Black Forest, while average concentrations were similar (0.11 to 0.16 pmol/kg). The seasonal variations were less clear- cut than in the Black Forest, but minimum levels occurred in both regions in late winter.

In Northern Finland, large differences were found between individual trees (-o Fig. 3 a-d, S. 9) during the campaign from August 1991 to August 1992. In the forest stand with dense canopy and an average wind passage of 39 km/d in 1992 (Hietaper~i h spruce 1, pine 3), TCA was present at relatively low levels (averages 0.06 - 0.07/~mol/kg) whereas highest levels were determined (averages 0.44, 0.55/amol / kg) at the forest stand with open canopy and an average wind

passage of 78 km/d (Hietaper~i Ih pine 5, pine 6). Similar to the situation in Germany, the levels fluctuated seasonally. Pine 5 reached a maximum of 0.6/~mol/kg in October 1991, pine 6 almost 0.8 pmol /kg in July and August 1992. Minima were observed for pine 5 in March 1992 (0.28/amol/kg), and for pine 6 in May 1992 (0.25 pmol/kg). The differences of almost one order of magnitude at short distance suggest that deposition and/or interception of TCA depend upon differences in canopy closure, local wind velo- city, and probably on many other local microclimatic, to- pographical, and ecological features. At Hietaperii II, the concentration of TCA as putative phy- totoxicant was correlated to branch damage abundance [27] as quantifiable measure of phytotoxicity for a total number of 20 Scots pine trees ( ~ Fig. 4, S. 9), resulting in a corre- lation coefficient of 0.79 (p ___ 0.001).

Table 2: Phytotoxicity of chloroacetates, non-ionic derivatives, and se- lected phytotoxicants; (a) ECs0 and (b) EC10 (toxicity thre- shold) [pmol/L] of cellular growth of bean cell suspension, growth period 2 weeks; (c) toxicity threshold EC10 [/amol/L] in the multiplication inhibition test with Scenedesmus suspi- catus [25] or Scenedesmus quadricauda [54]; (d) typical ma- ximum concentrations in pine needles [/amol/kg fresh weight], concentrations calculated on the basis of aqueous needle tis- sue compartment are shown in parentheses

Compound

Trichloroethanot Chloral Trichloroacetate Pentyl trichloro- acetate Trichloroacetyl amide Methyl trichloro- acetate Tetrachloro- oxirane Dichloroacetate Monochloro- acetate Alachlor Monofluoro- acetate

Pentachloro- ~henol 4,6-Dinitro-o- cresol Phenol Potassium cyanide Atrazine

a

3O00 3600

600 140

180

60

60

1200 20O

120

2

b

220 160 200

56

2

4

6

400 60

14

0.7

c d

17 1000

0.07

0.7

0.11 [62]

22

8o 0.46

0.14

0.8 (1.6)

0.2 (0.4)

0.041601 (0.08)

0.21631 (0.4)

3.2 Phytotoxicity of Chloroacetic Acids

The acute phytotoxicity of the chloroacetates and some li- pophilic derivatives of TCA has been determined as growth inhibition of bean cell suspension cultures (--' Table 2, co- lumns a, b). TCA has moderate acute phytotoxicity; growth is inhibited by 50 % when TCA is added to the medium in a single dose to yield an initial concentration of 1400/~mol/L. Trichloroethanol and chloral, also previously

8 ESPR-Environ. Sci. & Pollut. Res. 1 (1) 1994

Research Articles Haloacetic Acids

2 a

2b

2 c

120

110

100

90

80

70

60

50

(rig/g)

Fichtelberg, spruce 1 needle age 88

I--q needle age 90

4O

20

10

0 120

110

100

9O

80 i i

7oi 60

50

(/Jmol/kg 1

0.7

0.6

0.5

0.4

0.3

0.2 0.16

~ 0.1

Fichtelberg, spruce 2 needle age 88

[E3 needle age 90

0.7

0.6

0.5

0.4

0.3

30 0.2

20 0.15 0.1

10

0 120

110

1 O0

90

80

70

Fichtelberg, spruce 3

needle age 88

r---i needle age 90

0.7

0.6

0.5

0.4 60

50 0.3

40

30 0.2

2010 ~ 0.11

0 Aug Sep Oct Nov Dec Jan Feb Mar Apt Maydun Jul Aug Sep Oct Nov Dec Jan Feb

1990 1991 1992

Fig. 2: Levels of trichloroacetate in three spruces located within a distance of 1 km on the southwestern slope of the summit of the Fichtelberg peak in the Erzgebirge. The average TCA-concentrations in needles of the age group 1988 over the whole monitoring period are shown at the right ordinate of each panel (2)

employed as herbicides, are still weaker. DCA is of similar potency as TCA. All other TCA-derivatives and MCA are more strongly phytotoxic; methyl trichloroacetate and te- trachlorooxirane exhibit highest toxicity in this series. The data are compared to algal toxicity threshold concentrations published for several compounds (~ Table 2c) and the con-

centrations of some relevant herbicides found in conifer nee- dles (~ Table 2 d). The concentrations are calculated rela- tive to the flesh needle weight, so the actual cytosolic levels of the hydrophilic haloacetic acids may be about double the concentrations per fresh weight (given in parentheses in Table 2, column d).

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Haloacetic Acids Research Articles

4 Discussion

4.1 TCA-Deposition and E l i m i n a t i o n

In the Black Forest, typical TCA-concentrations in conifer needles are about 0.15/amol / kg (150/lmol / ton). TCA-levels in the needles represent steady state concentrations (%) of uptake and metabolism/mobilization. In experiments with spruce [20], the first-order rate constant of TCA-elimination by metabolism/mobilization (lql) at 22 ~ C was 7 x 10 .2 d 4, a similar value as for wheat (Triticum aestivum L.) [35]. Thus, the uptake rate (Vup) can be calculated from css as:

Yap Lumol kg 4 d 4] = q, Lumol kg -1] x kr [d4]. An average TCA-level (0.15/amol/kg) in needles reflects an average uptake rate of 11 nmol kg 4 d 4. The needle fresh weight of a spruce stand in Southern Germany is in the range of 25 t /ha [36]. Correspondingly, the daily foliar TCA- uptake per hectare is about 0.27 mmol (45 mg), or 27 mmol (4.5 g) per square kilometer.

4.2 Atmospheric Formation of Chlorinated Acetic Acids

TCA probably arises from atmospheric oxidation of the C2-chlorocarbons tetrachloroethene and 1,1,1-trichloro- ethane within the planetary boundary layer, i.e. up to an altitude of about 2 kin. The mean ambient air levels (Ca) of both chlorocarbons in rural atmosphere in Central Germany (-~ Table 3, fourth column) are about 2 and 10 mol/km 3 (nmol/m3), respectively [37]. The amounts of both solvents contained within the boundary layer above a square kilo- meter of forest are about 3.5 mol tetrachloroethene and 20 mol 1,1,1-trichloroethane. With atmospheric lifetimes (r) of about 70 (tetrachloroethene) and 2200 (1,1,1-trichloro- ethane) days [38, 39] (-o Table 3), the amount degraded by atmospheric oxidation (Ca) can be calculated according to

C d = C a �9 e "t/r, as 25 mmol/km 3 tetrachloroethene and 4.5 mmol/km 3 1,1,1-trichloroethane per day (-0 Table 3, fifth column). If yields of conversion to TCA of 70 % for tetrachloroethene [3] and of 90 % for 1,1,1-trichloroethane are assumed [39], TCA-formation within the boundary layer may be expected at a rate of 44 mmol d -t km 2 (7 g d 4 krn2).

Table 3: Atmospheric life times, ambient air levels, daily atmospheric oxidation, and TCA-formation from the two major atmo- spheric C2-chlorocarbons

Chloro- Atmosph. First-order Ambient Daily Daily carbon lifetime constant air levels oxidation TCA-for-

mation r [d] [d 4 ] [mol [mmol [mmol

km-3] km-3d-1] km-3d-1]

C2Cl 4 70 1.43 X 1 0 .2 1 . 8 2 5 1 8

CClaCH 3 2200 4.5 x 104 10.0 4.5 4

This is of similar magnitude as calculated for the foliar up- take per square kilometer of forest (see above) suggesting that most of the trichloroacetate found in needle tissue arises from atmospheric oxidation and subsequent deposition upon ve- getation. It is noteworthy that ambient air levels of TCA (2

to 20 pmol/m3; ~ Table 1 b) are of similar magnitude as the amount daily formed (1 pmol /m a = 1 mmol/km3; -* Table 3, last column).

As to the origin of atmospheric MCA, various possible pre- cursors and oxidation pathways are presented in schemes 1 - 4; the suggestions are tentative, and not all of the reac- tions are mechanistically proven.

CH 2 __ CH 2 * c l ~ C lCH2_(~H2 (1)

CICH2_ C H 2 0 . 2 *NO * O 2 - N O ; -O'2H ClCH 2 - C l i O

+-o. ,oOO- _ H2 ~ ClCH 2 - (~O -o2= ClCH2__ C

4. NO / O" + 0"2 H C lCH a - - COOH -NO~- CICH2-- C~O -o2 =

�9 4. O'2 H § OH C I C H - - CCI2OH -oa (2) CICH - - COl 2 =

CICH 2 - CCI 2OH _ HCl~ CICH 2 - COCI + H20

- CICH2 - - COOH - HCI

"OH (3) C lCH2- -CI~Cl ~ ClCH 2 - CHCl ~oa =

O l C H a _ C H C l O . 2 �9 NO,. +Oa NOa -0.2~ OICH 2 - COCl

+H20 - HCl- ClCH2 - - COOH

+'OH (4) ClCH ---- CH 2 = ClCH

/ o \ ,.,

C l H C - - C H 2 -__

* O a "NO 4.0.2 H

-NO2 -Oa

"OH *Oz - - CH2 -O.2H

+ "OH ClCH2 Cl iO _ H20

C I C H 2 - - COOH

Schemes 1 - 4: Tentative pathways suggested as sources of airborne mo- nochloroacetic acid (MCA):

Chlorine radical-initiated oxidation of ethene (1); hydroxyl radical-dependent oxi- dation of trichloroethene (2); hydroxyl radical-dependent oxidation of 1,2-dichlnroethane (3); hydroxyl radical-dependent oxidation of vinyl chloride (4)

(1) Chlorine radicals have been suggested to occur in the free atmosphere [40] at concentrations of about 103 mole- cules/cm3; ethylene is an abundant and ubiquitous VOC- component arising from petrol production and use, and from plant sources. (2) Trichloroethylene is an important industrial solvent; its current annual production is uncertain; in 1985 the produc- tion in Western Europe [41] was about 150 000 t, so the global annual production may be in the range of half a mil- lion tons. (3) 1,2-Dichloroethene probably represents a minor source of MCA although its global annual production exceeds 15 million tons, mostly for non-emissive production of vi-

10 ESPR-Environ. Sci. & Pollut. Res. 1 (1) 1994

Research Articles Haloacetic Acids

nylchloride; it has also been used as scavenger in leaded pet- rol but is presently being phased out. (4) Vinylchloride emissions during polyvinyl chloride pro- duction are probably very small but formation via micro- bial anaerobic metabolism of the solvents tri- and tetrachlo- roethene may contribute as source of unknown emission strength [42]. Whether hydrolysis of airborne monochloroacetyl herbi- cides [43] is a source of MCA remains to be elucidated. In any case, the question as to the origin of atmospheric MCA awaits further investigation.

TCA-levels in Finland tend to be higher than in Germany. Since atmospheric levels of the most abundant C2-chlorocarbons may be expected to be lower in Finland, the relatively high burden of haloacetic acids is surprising; this may be another example of the global cryogenic con- densation of volatile ubiquitous pollutants in colder climate zones [44]. It has been suggested that kraft pulp chlorina- tion in paper and pulp industry may be a source of TCA [21], but the results of a corresponding TCA-monitoring study do not support this speculation [45]. With dissociation constants of 1 . 5 5 x 1 0 3 (MCA), 5.0 x 10 "2 (DCA), and 0.13 (TCA), fractions between 0.15 % (MCA) and 20 ppm (TCA) are protonated at pH 5.6 (CO2-saturated aerosol droplets). Since the vapor pressures at 20 ~ C (calculated by extrapolation from published data [46]) of about 30 Pa (= 10 mmol/m 3) (MCA), 25 Pa (= 8 mmol/m 3) (DCA), and 0.1 Pa (= 30/amol/m 3) (TCA) are far beyond the maximum partial pressure in am- bient air (air concentrations, - Table I), the chloroacetic acids are probably present in free vapor form. This is sup- ported by the fact that cryogenic sampling, adsorption car- tridge sampling, and denuder sampling all yield similar re- sults (to be published); the latter method is regarded as se- lective for gas phase pollutants. Correspondingly, the main mechanism of deposition may be diffusive equilibrium up- take. At an average wind velocity of 1 m/s (86 km/day), about 0.5/lmol TCA per day pass through a canopy cross section of one square meter; the amount deposited is corres- pondingly smaller at lower wind velocity, as apparent for Hietaper~i I and II. Wind velocity may also influence depo- sition onto foliage by affecting the boundary layer diffusion resistance [47].

4.3 Phytotoxicity of Chloroacetic Acids and Derivatives

The relevance of secondary air pollutants to plant damage has been shown already several decades ago by HAAGEN- SMIT [48], and the potential importance of airborne halo- carbons to forest decline symptoms has been discussed [49].

A causal link between pollutant exposure level and toxic re- sponse may be assumed when both are quantitatively corre- lated. At Hietaper/i I and II, TCA-levels in August 1992 and branch damage abundance, a component of defoliation, show a high degree of correlation (-, Fig. 4). The regres- sion line suggests a toxicity threshold (EC10) of TCA for Scots pine of about 15 ng/g (0.09/lmol/kg) under the par- ticular growth conditions at Hietaper~i. In another recent study [50], TCA-levels have also been found to be correla- ted to altered reflectance spectroscopic properties of conifer

needles, but also lead and cadmium. The latter underlines the well known fact that a dose/response correlation is a ne- cessary but insufficient requirement for proving the poten- tial causal involvement of a putative toxicant. Thus, TCA may just be an indicator compound for uptake of many other phytotoxic anthropogenic air pollutants.

The various haloacetic acids have largely different physico- chemical properties (see below). MCA is readily taken up by plant cells due to its relatively small dissociation constant, probably by active transport [33]. TCA, however, crosses lipoid membranes only slowly due to its large dissociation constant [20]. The fact that TCA is within the needle tissue suggests that lipophilic precursors are involved which may efficiently traverse cuticulae, cell walls, and cell membranes; upon entering the cytosol they may be hydrolyzed and en- trapped in the cell. The relevance of peroxytrichloroacetyl nitrate [51], trichloroacetyl chloride, tetrachlorooxirane, or trichloroacetamide as atmospheric transport forms remains to be elucidated.

Another important factor driving the uptake of medium and weakly acidic acids may be the pH-gradient across the cell boundary: while atmospheric aerosol has a pH of 5.6 (CO2-saturation) or less, the intracellular pH is about 7.4.

Lipophilicity is dearly important in respect to phytotoxicity. The results of experiments on the acute inhibition of cell cul- ture growth by single initial doses of MCA, TCA, and seve- ral lipophilic derivatives thereof are listed in Table 2, co- lumns a and b. Non-ionic derivatives of TCA exhibit higher phytotoxicity than the parent compounds. The same can be seen when comparing MCA and Alachlor. The acute toxicity thresholds (EC10's) of TCA and TCA-derivatives in this test system (-~ Table 2, column b) and the TCA-levels in conifer needles (~ Table 2, column d), differ by factors of 2.5 (trichloroacetylamide) to 250 (trichloroacetate), de- pending upon which derivative is considered. In the open en- vironment, airborne phytotoxicants act chronically; it is not uncommon that concentrations eliciting acute and chronic toxicity differ by such margins. About half of the wet weight of spruce needles is aqueous compartment; at pH 7.4, ha- loacetic acids are preferentially soluble in the cytosol. Thus, the actual intracellular concentrations may in fact be twice the concentration calculated on fresh weight basis.

In algal toxicity test systems, monohaloacetic acids are about thousand- to tenthousand-fold more potent than TCA [24,25] (~ Table 2, column c); MCA is one of the most po- tent algal toxicants known so far. The high levels of MCA in the atmosphere (-o Table 1 b), and the fact that its con- centration in pine needles exceeds the toxicity threshold for photoautotrophic organisms (green algae, Scenedesmus sus- picatus; blue algae Microcystis aeruginosa, see below) sug- gest the high relevance of MCA as environmental phytoto- xicant.

Typical for haloacetic acids are large differences in species susceptibility; the lethal doses of fluoroacetate for differently susceptible animals [52] cover a range between 0.05 (dog), 20 (monkey), and 500 mg/kg bodyweight (frog), i.e. vary by a factor of ten thousand. This seems to be true also for plants; while some higher plants synthesize fluoroacetate and

ESPR-Env i ron . Sci. & Pollut. Res. 1 (1) 1994 11

Haloacetic Acids Research Articles

120

110

100

90

80

70

60

50

40

30

20

10

0

3 a

(ng/g) (#mol/kg

Hietaper~, spruce 1 needle age 89

I Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug

1991 1992

0.7

0.6

0.5

0.4

0.3

(rig/g) (/.zmol/kg '

II lllnlnnl ~ 0.06

Hietaper&, pine 3 needle age 89

i I J lJaJL,JJ33 Aug Sop Oct Nov Dec Jan Feb Mar Apr May Jun Jui Aug

1991 1992 3b

0.7

0.6

0.5

0.4

0.3

0.2

0.1 L07

(ng/g) (pmol/kg) 120

110

I O0

90

80

70;

60

40

30

20

10

0

3 c

Hietat nee(

Aug Sep Oct Nov IDec Jan Feb Mar AprMay Jun Jul Aug 1991 1992

0.7

0.6

0.5

0.44 0.4

0.3

0.2

0.1

(ng/g) (/amol/kg)

Hietaper~, pine 6 ~ss 124 needle age 89 ~

Aug Sep Oct Nov Dec Jan Feb Mar Apt MayJun Jul Aug 1991 1992

3 d

0.7

0.6

0.55

0.5

0.4

0.3

0.2

0.1

Fig. 3: Levels of trichloroacetate in pine and spruce needles in Northern Finland in two adjacent stands with relatively dense (spruce 1, pine 3) and open canopy (pine 5 and 6)

% branch damage 1991/92 60

50

40

30

20

10

0 10

(ng/g) 20 30 40 50

0.1 0.'2 013 (prnot/kg)

TCA Fig. 4: Correlation of trichloroacetate levels in needles and branch da- mage abundance of 20 trees located at Hietaper~ I and II in Nor- them Finland.

12 ESPR-Environ. Sci. & Pollut. Res. 1 (1) 1994

Research Articles Haloacetic Acids

are obviously quite insensitive to it [53], simple photoauto- trophic organisms are extremely susceptible; multiplication of the blue algae Microcystis aeruginosa [54], for instance, is inhibited at a hundredfold lower concentration than the green algae Scenedesmus quadricauda. In general, photoau- totrophic organisms are more sensitive to haloacetic acids than heterotrophic ones [25,54].

4.4 Putative Biochemical Mechanisms of Chloroacetate Toxicity

The molecular targets of T C A in phytotoxic doses to terres- trial plants have been suggested as the enzymes involved in coenzyme A synthesis [55]; this may be followed by dis- turbed development of epicuticular waxes [56] and weakened defense against fungal infections [57]. MCA may exert its toxicity in a similar manner as fluoro- acetate, i.e. by attenuation of the mitochondrial citric acid cycle via formation of halocitrate and subsequent inhibition of aconitase [9]. The speculation that attenuated mitochondrial energy out- put may be one of the biochemical lesions underlying some forest decline symptoms is supported by the fact that phos- phoenol pyruvate carboxylase (PEPC) is consistently in- duced in declining conifers [58], perhaps an attempt of the affected plants to overcome the block in mitochondrial energy production by elevating the pro-substrate oxaloacetate.

Numerous other airborne herbicides [59-61] may contribute to forest tree pathology. Several phytotoxic compounds which have actually been detected in foliar tissue, i.e. chloro- and nittophenols, are potent uncouplers of mitochondrial oxidative phosphorylat ion. Pentachlorophenol has been found in conifer needles throughout Europe in concentra- tions of about a twentieth of its toxicity threshold to bean cell cultures and about a third of the toxicity threshold to green algae [62]; 4,6-dinitrocresol has been found [61] at about a hundredth of its toxicity threshold to green algae. The possibility that haloacetic acids and the uncoupling phe- nols act in a synergistic manner on the same organelle at dif- ferent levels of mitochondrial ATP-production deserves fur- ther scrutiny.

5 C o n c l u s i o n s

Phytotoxic chloroacetic acids have been found in various en- vironmental compartments in low-polluted regions. MCA is present in conifer needles in concentrations which surpass the toxicity threshold to photoautotrophic algae about three- fold ( ~ Table 2, columns c, d). Its occurrence in air and rain water may also be of relevance to aquatic ecosystems since algae are the most important pr imary producers. TCA is of medium phytotoxicity but may be regarded as indicator com- pound for the distribution and deposition of secondary air pollutants arising from atmospheric oxidation of ubiquitous anthropogenic C2-chlorocarbons and of other volatile orga- nic air pollutants of similar physicochemical properties and atmospheric reactivity.

Acknowledgements

Financial support of the Federal Ministry of Research and Technology and of the Ministry of the Environment of the State of Baden- Wfirttemberg is gratefully acknowledged. The help of M. LINDNER in collecting the needle samples at the Fichtelberg site is appreciated.

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Received April 15, 1993 Accepted June 28, 1993

14 ESPR-Environ. Sci. & Pollut. Res. 1 (1) 1994