Haloacetic acids, phytotoxic secondary air pollutants

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-halocarbonswhich are important solvents and propellants. Levds 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 coniferneedlesat concentrations up to 0.7/~mol/kg, MCA up to 0.2/amol/kg. At the Finnish sites, TCA-concentrationsand branch degenerationsymptoms of Scots pine are correlated. Monochloroacetate (MCA) has been determinedin needlesamples from SouthernGermanyin concentrations exceeding its phytotoxicity threshold towards photoautotrophic organisms. Data on atmospheric chloroacetate levels in Germanyare also given; ambient air levelsof chloroaceticacids range from about 2 pmol/m3 (TCA) to 390 pmol/m3 (MCA). TCA and dichloroacetic acid (DCA) arise from atmospheric oxidation of airborne C2-chlorocarbons,while the source of MCA is not yet known; several tentative pathways are suggested.

1

Introduction

Haloacetic acids are atmospheric oxidation products of halogenated C2-hydrocarbons [1,2]. Quantitatively most relevant 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 released into the atmosphere. Some of them have strongly increased in their atmospheric levels during the last two decades [5] because of long atmospheric residence times. Chlorofluorohydrocarbons, suggested as substitutes for long-lived chlorofluorocarbons, may also be oxidized to various haloacetic acids [7]. Halocarbon exposure at relatively high levels result in degradation of photosynthetic pigments [8] of conifers and deciduous 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 derivatives thereof are known as herbicides [14,15]: Trichloroacetate (TCA) has been employed as sodium salt or as ester or amide derivatives against perennial grasses. Monochloroacetate (MCA) is the phytotoxic principle in the herbicides Alachlor, Propachlor, Metazachlor, and Metolachlor.

4

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 contribute to the induction of phytotoxicological symptoms on conifers and other forest trees observed since a decade in remote areas [22,23]. The exquisite algal toxicity of monohaloacetic acids [24,25] and the potential increase in environmental burden of haloacetic acids when introducing C2-chlorofluorohydrocarbons [5] as substitutes for chlorofluorocarbons (CFC) urge detailed assessments of environmental fates and ecotoxicological properties of the former.

2 2.1

Experimental 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 isolated 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~ 5 km west of Herrenberg); Sch6nbuch (spruce, 460 m a.s.l., 48~ north of Tfibingen).

8~ 9~

8 km

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

Research Articles

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 index 0.6) on dry sandy heathland on level ground. During the growing season of 1992, average wind passages of 39 k m / d at Hietaper~i I and 78 k m / 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 arriving 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. Phytotoxicity was assessed as branch damage abundance according to JUKOLA-SULONENet al [27]. 2.3

Determination of Chloroacetic Acids in Conifer Needles

Twigs were cut with clean scissors into the respective annual 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 chloroacetic 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 chlorinated aliphatic acids were extracted with diethyl ether, methylated 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 carboxylate 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., center tubes 500 x 9 mm i.d.) coupled by ground joints [30].

ESPR-Environ. ScF & Pollut. Res. 1 (1) 1994

Haloacetic Acids

They were internally coated with 1 % sodium bicarbonate in glycerol in the following manner: two tubes were connected, 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 homogeneously wetted, they were placed vertically on filter paper 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 bellows gas counter (NB 3, Rombach, Kartsruhe). Another coated denuder was stored for the same period in closed condition to serve as blank. Calibration curves were established 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 aspiration of the same volume of purified air. After sampling, the glycerol coat of both halves of the denuder was rinsed with 6 mL each of potassium hydroxide solution (1 mmol/L) into a round bottom flask under continuous 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 mixing with a vibration mixer for 60 seconds. An aliquot of 0.6 mL of the ethereal layer was withdrawn, an equal volume of freshly prepared ethereal diazomethane or 1-(pentafluorophenyl)-diazoethane (800 mg/ml) solution was added, and the reaction mixture was kept at room temperature for 5 minutes or overnight, respectively. One microliter 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 suspension containing a wet cell mass of 200 mg/mL were added to 50 mL medium, to yield suspensions with an initial cell mass of 33 mg/mL. For assessment of relative phytotoxicity, the respective compounds were added in dimethyl sulphoxide solution (60 a L per 60 mL cell suspension) to yield final concentrations between 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 established, the growth inhibition was calculated, and EC~0- and ECs0-values were determined [32].

5

Haloacetic Acids 3

Research Articles

Results

3.1

Environmental Monitoring

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

(ng/g)

(pmol/kg

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 p m o l / k g fresh weight. When spruce and fir were monitored simultaneously (Mauzenberg, Zwieselberg), the former species exhibited higher levels than the latter. Maximum levels were reached in September 1990, mi-

(ng/g)

(prnol/kg

120

Mauzenberg, s p r u c e

110 100

II

needle age 88

90

I

I needle age 90

0.7

0.7

Mauzenberg, fir

0.6

II

needle a g e 88

I

I needle age 90

0.6

0.5

80

0.5 p- .

70 0.4

60

0.4

50 -

0.3

30

0.2 o.15

0.2

0.1

0.09

2O 10

-

0.3

0 Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr 1990

Jun Jul Aug Sep Oct Nov Dec Jan Feb MarApr

1991

1990

la

1991

Ib

(rig/g)

(wmol/kg

(ng/g)

(pmol/kg

120 1lO -

Bernstein, spruce

loo -

II

0.7

0.7

Altsteigerskopf, spruce I

9o 80

needle age 88

-

II

_

I--I needle age 90

0,5

I---I needle age 90

7O 6O 50 40130 ~

0.6

i ,

I

i

m ~

20

needle a g e 88

o.6 0.5

0.4

0.4

0.3

0.3

~ 0,2 0.165

0.2 ~

0.1

0.11

10 0

Ic

6

Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr 1990 1991

Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr 1990 1991 Id

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

Research Articles

Haloacetic Acids

Fig. 1 continued

(ng/g)

(pmol/kg)

(/.tmol/kg

(rig/g)

120 110 100 90

Zwieselberg, spruce II

needle age 88

0.7

II

0.6

{Z] needle age 90

80

0.7

Zwieselberg, fir needle age 88

0.6

r I needle age 90 0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.09

0.1 0.07

70 60 50 40 30

10 0

Jun Jul Aug Sep Oct Nov Dec Jan Feb MarApr 1f

1990

1991

le

120 110 100

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.4

0.30

0.3

0.2

0.2

9O 80 7O 60 5O 4O 30 2O

0.11

0.1

10 0 Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr 1990 1991 lg

Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apt 1990 1991 lh

Fig. 1: Levelsof 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 Mauzenberg 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.

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

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 p m o l / k g (--* Table 1 a) [33].

7

Haloacetic Acids

Research Articles

Table 1: Chloroaceticacids 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

a) June 1992 1992 1992 1992

MCA

DCA

TCA

100 210 210 80

-

40 70 40 20

b) 26.03.-29.03.92 31.03.-04. 04.92 04.04.-08. 04.92 08. 0 4 . - 1 3 . 04, 92 14.04.-16.04. 92

42 •

30

5.4 •

3

27. 0 4 . - 3 0 . 04. 92 126 •

40

43

• 23

390 •

1 1 . 0 7 . - 1 6 . 07. 92

160 •

160 60

67 92

• 54

31.07.-04.

190 •

75

66

• 39

Range

• • • • •

5.5 1.2 0.7 1.2 0.9

3.0 • 0.9

30.04.-06.06. 92 20.05.-26.05. 92 1 9 . 0 6 . - 2 6 . 06. 92 08.92

7.3 6.7 11.0 1.8 3.0

42 - 3 9 0

• 40

5 - 90

2.4 • 0.3 3.7 • 0.7 6.7 • 3.7

16.0 • 3.7 10.4 • 1.8 2 -

16

Ambient air levels of MCA, DCA, and TCA were determined at the southern edge of the Sch6nbuch Forest close to Tiibingen (--' Table 1 b). The chloroacetic acids were present in concentrations between 2 (TCA, minimum) and 390 p m o l / m 3 (MCA, maximum). The second monitoring campaign for TCA in conifer needles was started in August 1990. Samples were collected from three spruce trees in the Erzgebirge, a mountain range bordering on Northern Czechia where the extent of tree damage is similar to the Black Forest [34]. Again, TCA-levels in twoyear-old needles were higher than in young ones (--* Fig. 2 ac). 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 / l m o l / k g 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 minima 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, minima in August 1991 and January 1992. Altogether, the levels 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 clearcut 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 k m / 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 8

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 a n d / o r interception of TCA depend upon differences in canopy closure, local wind velocity, and probably on many other local microclimatic, topographical, and ecological features. At Hietaperii II, the concentration of TCA as putative phytotoxicant 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 correlation coefficient of 0.79 (p ___ 0.001). Table 2: Phytotoxicityof chloroacetates, non-ionic derivatives, and selected phytotoxicants; (a) ECs0 and (b) EC10 (toxicity threshold) [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 suspicatus [25] or Scenedesmus quadricauda [54]; (d) typical maximum concentrationsin pine needles [/amol/kgfresh weight], concentrations calculated on the basis of aqueous needle tissue compartment are shown in parentheses Compound Trichloroethanot Chloral Trichloroacetate Pentyl trichloroacetate

Trichloroacetyl amide Methyl trichloroacetate Tetrachlorooxirane Dichloroacetate Monochloroacetate Alachlor Monofluoroacetate

Pentachloro~henol 4,6-Dinitro-o-

a

b

3O00 3600 600 140

220 160 200 56

180

2

60

4

60

6

1200

20O

400 60

120

14

c 17 1000

0.07

d

0.8 (1.6)

0.2 (0.4)

0.7 2

0.7

0.11 [62] 0.041601 (0.08) 22

0.21631 (0.4)

cresol

Phenol Potassium cyanide

Atrazine

3.2

8o 0.46 0.14

Phytotoxicity of Chloroacetic Acids

The acute phytotoxicity of the chloroacetates and some lipophilic derivatives of TCA has been determined as growth inhibition of bean cell suspension cultures (--' Table 2, columns 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 ESPR-Environ. Sci. & Pollut. Res. 1 (1) 1994

Research Articles

Haloacetic Acids (rig/g)

2a

(/Jmol/kg 1

120 0.7

Fichtelberg, spruce 1 needle age 88 I--q needle age 90

110 100 90

0.6 0.5

80 70

0.4

60 50

0.3

4O

~

20 10 0 120

2b

100

Fichtelberg, spruce 2 needle age 88

9O

[E3 needle age 90

110

0.1

0.7 0.6

0.5

80 i

7oi

0.2 0.16

i

0.4

60 50

0.3

30

0.2

20

0.15 0.1

10 0 120 2 c

0.7

Fichtelberg, spruce 3

110 1O0

needle age 88

90

r---i needle age 90

0.6

0.5

80 70

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 tetrachlorooxirane exhibit highest toxicity in this series. The data are compared to algal toxicity threshold concentrations published for several compounds ( ~ Table 2c) and the con-

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

centrations of some relevant herbicides found in conifer needles ( ~ Table 2 d). The concentrations are calculated relative 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).

9

Haloacetic Acids

4 4.1

Research Articles

Discussion TCA-Deposition and

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).

Elimination

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 TCAuptake 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-trichloroethane 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 kilometer 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-trichloroethane) days [38, 39] (-o Table 3), the amount degraded by atmospheric oxidation (Ca) can be calculated according to Cd

=

Ca

9

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

C2Cl4 CClaCH3

Atmosph. First-order Ambient Daily Daily lifetime constant air levels oxidation TCA-formation r [d] [d4 ] [mol [mmol [mmol km-3] km-3d-1] km-3d-1] 70 2200

1.43 X 1 0 .2 4.5 x 104

1.8

10.0

25

4.5

18

4

This is of similar magnitude as calculated for the foliar uptake 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 vegetation. It is noteworthy that ambient air levels of TCA (2

10

(1) CH 2 __ CH 2

*cl~

CICH2_ C H 2 0 . 2

+-o. H2 ~

_

ClCH2_(~H2 -*NO NO;

*-O'2H O2

4. NO

9

CICH 2 + H20 - HCI

CCI 2OH

-o2 =

C I C H - - CCI2OH

_ HCl~ CICH 2 -

"OH

-oa

COCI

ClCH 2 - CHCl ~oa =

O l C H a _ C H C l O . 2 9 NOa NO,. +Oa -0.2~ +H20 HCl-

4.O'2 H

CICH2 - - COOH

(3) C l C H 2 - - C I ~ C l ~

ClCH2

(4) ClCH ---- CH 2

--

OICH 2 -

COCl

COOH

"OH

+'OH

= ClCH - - CH2

/o\

*Oz -O.2H

,.,

ClHC--CH *Oa

CliO

/ O" + 0"2 H C l C H a - - C O O H

(2) CICH - - COl 2 § OH=

-

ClCH 2 -

(~O -o2= ClCH2__ C,oOO-

ClCH 2 -

-NO~- C I C H 2 - - C~O

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).

Chlorocarbon

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

2

-__ ClCH2

"NO

4.0.2 H

-NO2

-Oa

+ "OH

CliO

_ H20

C I C H 2 - - COOH

Schemes 1 - 4: Tentative pathways suggested as sources of airborne monochloroacetic acid (MCA): Chlorine radical-initiated oxidation of ethene (1); hydroxyl radical-dependent oxidation 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 molecules/cm3; ethylene is an abundant and ubiquitous VOCcomponent 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 production in Western Europe [41] was about 150 000 t, so the global annual production may be in the range of half a million 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 viESPR-Environ. Sci. & Pollut. Res. 1 (1) 1994

Research Articles

nylchloride; it has also been used as scavenger in leaded petrol but is presently being phased out. (4) Vinylchloride emissions during polyvinyl chloride production are probably very small but formation via microbial anaerobic metabolism of the solvents tri- and tetrachloroethene may contribute as source of unknown emission strength [42]. Whether hydrolysis of airborne monochloroacetyl herbicides [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 condensation of volatile ubiquitous pollutants in colder climate zones [44]. It has been suggested that kraft pulp chlorination 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 ambient air (air concentrations, - Table I), the chloroacetic acids are probably present in free vapor form. This is supported by the fact that cryogenic sampling, adsorption cartridge sampling, and denuder sampling all yield similar results (to be published); the latter method is regarded as selective for gas phase pollutants. Correspondingly, the main mechanism of deposition may be diffusive equilibrium uptake. 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 correspondingly smaller at lower wind velocity, as apparent for Hietaper~i I and II. Wind velocity may also influence deposition 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 HAAGENSMIT [48], and the potential importance of airborne halocarbons to forest decline symptoms has been discussed [49]. A causal link between pollutant exposure level and toxic response may be assumed when both are quantitatively correlated. 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 regression line suggests a toxicity threshold (EC10) of T C A for Scots pine of about 15 ng/g (0.09/lmol/kg) under the particular growth conditions at Hietaper~i. In another recent study [50], TCA-levels have also been found to be correlated to altered reflectance spectroscopic properties of conifer

E S P R - E n v i r o n . Sci. & Pollut. Res. 1 (1) 1994

Haloacetic Acids

needles, but also lead and cadmium. The latter underlines the well known fact that a dose/response correlation is a necessary but insufficient requirement for proving the potential 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 physicochemical 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 entrapped 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 culture growth by single initial doses of MCA, TCA, and several lipophilic derivatives thereof are listed in Table 2, columns 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), depending upon which derivative is considered. In the open environment, 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, haloacetic 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 potent algal toxicants known so far. The high levels of MCA in the atmosphere (-o Table 1 b), and the fact that its concentration in pine needles exceeds the toxicity threshold for photoautotrophic organisms (green algae, Scenedesmus suspicatus; blue algae Microcystis aeruginosa, see below) suggest the high relevance of MCA as environmental phytotoxicant. 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

11

Haloacetic Acids

Research Articles

(ng/g)

(rig/g)

(#mol/kg

(/.zmol/kg '

120

Hietaper~, spruce 1 needle age 89

110

0.7

100

0.7

Hietaper&, pine 3 needle age 89

0.6

0.6

90 80 70 60 50

0.5

0.5

0.4

0.4

0.3

0.3

40 0.2

30

II lllnlnnl

20 10 0

I

~

i I JlJaJL,JJ33

0.06

Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug 1991 1992

0.1 L07

Aug Sop Oct Nov Dec Jan Feb Mar Apr May Jun Jui Aug 1991 1992 3b

3a

(ng/g) 120

(pmol/kg) Hietat nee(

110

(ng/g) 0.7

I O0

(/amol/kg) Hietaper~, pine 6 needle age 89

~ss 124 ~

0.7

0.6

0.6

80

0.5

0.55 0.5

70;

0.44 0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

90

60

40 30 20 10 0 Aug Sep Oct Nov IDecJan Feb Mar AprMay Jun Jul Aug 1991 1992

Aug Sep Oct Nov Dec Jan Feb Mar Apt MayJun Jul Aug 1991 1992 3d

3c

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 d a m a g e 1991/92 60 50 40 30 20 10 0

10

20 0.1

12

30 0.'2

40

(ng/g) 50 013 (prnot/kg)

TCA

Fig. 4: Correlation of trichloroacetate levels in needles and branch damage abundance of 20 trees located at Hietaper~ I and II in Northem Finland.

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

Research Articles

are obviously quite insensitive to it [53], simple photoautotrophic 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, photoautotrophic organisms are more sensitive to haloacetic acids than heterotrophic ones [25,54].

Haloacetic Acids

Acknowledgements Financial support of the Federal Ministry of Research and Technology and of the Ministry of the Environment of the State of BadenWfirttemberg is gratefully acknowledged. The help of M. LINDNERin collecting the needle samples at the Fichtelberg site is appreciated.

6

4.4

Putative Biochemical Mechanisms of Chloroacetate Toxicity

The molecular targets of T C A in phytotoxic doses to terrestrial plants have been suggested as the enzymes involved in coenzyme A synthesis [55]; this m a y be followed by disturbed development of epicuticular waxes [56] and weakened defense against fungal infections [57]. M C A may exert its toxicity in a similar manner as fluoroacetate, 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 output may be one of the biochemical lesions underlying some forest decline symptoms is supported by the fact that phosphoenol pyruvate carboxylase (PEPC) is consistently induced 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. chloroand nittophenols, are potent uncouplers of mitochondrial oxidative phosphorylation. Pentachlorophenol has been found in conifer needles throughout Europe in concentrations 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 phenols act in a synergistic manner on the same organelle at different levels of mitochondrial ATP-production deserves further scrutiny.

5

Conclusions

Phytotoxic chloroacetic acids have been found in various environmental compartments in low-polluted regions. M C A is present in conifer needles in concentrations which surpass the toxicity threshold to photoautotrophic algae about threefold ( ~ 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 primary producers. T C A is of medium phytotoxicity but may be regarded as indicator compound for the distribution and deposition of secondary air pollutants arising from atmospheric oxidation of ubiquitous anthropogenic C2-chlorocarbons and of other volatile organic air pollutants of similar physicochemical properties and atmospheric reactivity.

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

References

[1] K. SCHOTT;H.J. SCHUMACHER:Die photochemische Chlorierung und die durch Chlor sensibilisierte photochemische Oxydation von Tetrachlor/ithylen. Z. Physikal. Chemie 49, 107-125 (1941) [2] B.W. GAY;P.L. HANST;J.J. BuraLtNh R.C. NOONAN:Atmospheric Oxidation of Chlorinated Ethylenes. Environ. Sci. Technol. 10, 58 - 67 (1976) [3] E.C. TUAZON;R. ATKINSON;S.M. ASCHMAN;M.A. GOODMAN; A.M. WINER:Atmospheric Reactions of Chloroethenes with the OH Radical. Intern. J. Chem. Kinetics 20, 241-265 (1988) [4] International Trade Commission USA. Chem. Eng. News 70 (15) 17 (1992) [5] P. FABIAN:Halogenated Hydrocarbons in the Atmosphere. In: O. HUTZINGER(Ed.), The Handbook of Environmental Chemistry, Vol 4A, Springer Berlin, 1986, pp. 23 - 51 [6] P. MIDGLrY:The Production and Release to Atmosphere of Industrial Halocarbons. Ber. Bunsenges. Phys. Chem. 96, 293- 296 (1992) [7] E.O. EDNEY;B.W. GAY;D.J. DRiscou.: Chlorine Initiated Oxidation Studies of Hydrochlorofluorocarbons: Results for HCFC-123 and HCFC-1416. J. Atmos. Chem. 12, 105-120 (1991) [8] H. FRANK;W. FRANK:Chlorophyll-Bleachingby Atmospheric Pollutants. Naturwissenschaften 72, 139- 141 (1985) [9] R. DEBUS;P. SCHRODER:Wirkungen yon Halon 1211 (Bromchlordifluormethan) auf Kresse. VDI-Bericbte 745, VDI-Vertag, Dfisseldorf 1989, pp. 563- 572 [10] R.A. PETERS"Lethal Synthesis and Carbon-Fluorine Compounds. In: BiochemicalLesions and Lethal Synthesis, Pergamon Press, Oxford, 1963, pp. 88- 130 [11] M.P. QUICK:Sodium Monochloroacetate Poisoning of Cattle and Sheep. Vet. Rec. 113, 155-156 (1983) [12] K.C. BARRONS;R.W. HUMMER:Basic Herbicidal Studies with Derivatives of TCA. Agricult. Chem. 6, 48 - 121 (1951) [13] H. ZOTVL:Untersuchungen fiber die Wirkung yon Trichloracetat, und anderen Halogenacetaten auf pflanzliches Gewebe. Z. Naturforschg. 8 b, 317-323 (1953) [14] C. FoY: The Chlorinated Aliphatic Acids. In: P.C. KEARNEYand D.D. KAUFMANN,Degradation of Herbicides, Marcel Dekker, New York, 1969, pp. 207- 254 [15] G. MATOLCSY;M. NADASY;V. ANDRISKA-Pesticide Chemistry. Elsevier, Amsterdam, 1988, pp. 577- 580 [16] H. FRANK; J. VITAL; W. FR~qI(: Oxidation of Airborne C2-Chlorocarbons to Trichloroacetic and Dichloroacetic Acid. Fresenius Z. Anal. Chem. 333, 713 (1989) [17] G.R. FuCHS; K. B/ICHMANN:Ionen-chromatographische Bestimmung yon Chloracetat und Dichloracetat in Niederschlagswasserproben. Fresenius Z. Anal. Chem. 327, 205-212 (1987) [18] I. RENNER; R. SCHLEYER;D. MI3HLHAUSEN:Gef/ihrdung der Grundwasserqualit~t durch anthropogene organische Luftverunreinigungen. VDI-Berichte Nr. 837, 705- 727 (1990) [19] A. A~THO;K. GROB;P. GIGER:Trichloressigs~iure in Oberfl/ichen-, Grund-, und Trinkw~issern. Mitteilung Gebiete Lebensm. Hyg. 82, 487-491 (1991) [20] H. FRANK:Airborne Chlorocarbons, Photooxidants, and Forest Decline. Ambio 20, 13-18 (1991) [21] H. FRANK;H. SCHOLL;S. SUTINEN;Y. NOROKORPI:Trichloroacetic Acid in Conifer Needles in Finland. Ann. Bot. Fennici 29, 263 - 267 (1992) [22] H. FRANK:Photoaktivierung luftgetragener Chlorkohlenwasserstoffe. Nachr. Chem. Tech. Lab. 34, 15- 20 (1986)

13

Haloacetic Acids

[23] R. OPEN; K.S. WERK;J. MEYER;E.D. SCHULZE.In: Forest Decline and Air Pollution, Springer Berlin, 1989, pp. 24 ff [24] G. BR~GMANN;R. KOHN:Comparison of the Toxicity Thresholds of Water Pollutants to Bacteria, Algae, and Protozoa in the Cell Multiplication Inhibition Test. Water Res. 14, 231 -241 (1980) [25] R. KOHN;M. PATTARD:Results of the Harmful Effects of Water Pollutants to Green Algae (Scenedesmus suspicatus) in the Cell Multiplication Inhibition Test. Water Res. 24, 3 1 - 3 8 (1990) [26] Y. ILVESSM.O:Mets/inarvioiminen (W. SODERSTROMOY, Poroo, Helsinki, 1965), p. 231 [27] E.L. JUrOLA-SOLONEN;K. MIKKOLA;M. Sa/_EM~: The Vitality of Conifers in Finland, 1986- 88. In: P. IOit/vPI,P. ANTTILLA,K. KE~TTKMIES, Acidification in Finland, Springer Berlin, 1990, pp. 523 - 560 [28] H. FRANK;A. VINCON;J. REISS;H. SCHOLL:Trichloroacetic Acid in the Foliage of Forest Trees. J. High Resol. Chromatogr. 13, 733 - 736 (1990) [29] U. HOrM~qN; S. HOLZER;C.O. MEESE:Pentafluorophenyldiazoalkanes as Novel Derivatization Reagents for the Determination of Sensitive Carboxylic Acids by Gas Chromatography-NegativeIon Mass Spectrometry. J. Chromatogr. 508, 349- 356 (1990) [30] D.J. EATOUGH;C.L. BENNER;J.M. BAYONA;G. RICHARDS;J.D. LAMa; M.L. LEE; E.A. LEWIS;L.D. HANSEN:Chemical Composition of Environmental Tobacco Smoke 1. Gas-PhaseAcids and Bases. Environ. Sci. Technol. 2 3 , 6 7 9 - 6 8 7 (1989) [31] T. MURASHIGE;F. SKOOG:A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. Physiol. Plant. 15, 473 - 497 (1962) [32] ,Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus suspicatus and Selenastrurn capricornutum" International Standard ISO/DIS 8692, Geneva, Switzerland (1989) [33] H. SCHOLL:Ph.D. Thesis, University of Tiibingen, 1993 [34] Daten zur Umwelt 1990/91, Umweltbundesamt, Schmidt, Berlin, 1992, pp. 164-179 [35] P.N.P. CHOW:Adsorption and Dissipation of TCA by Wheat and Oats. Weed Sci. 1 8 , 4 9 2 - 4 9 6 (1970) [36] H. KRAMER;H. GUSSONE;R. SCHOBER:Waldwachstumslehre. Paul Parey, Hamburg, 1988, p. 23 [37] W. FRANK;H.J.C. NEVES;H. FRANK:Levels of Airborne Halocarbons at Urban and Mountain Forest Sites in Germany and at the Atlantic Coast. Chemosphere 23, 6 0 9 - 626 (1991) [38] B.J. FINLAYSON-PITTS;J.N. PITTS:Atmospheric Chemistry. Wiley & Sons, New York, 1986, p. 1026 [39] R. PRINN;D. CUNNOLD;R. RASMUSSEN;P. SIMMONDS;F. ALYEA; A. CRAWFORD;P. FRASER;R. ROSEN:Atmospheric Trends in Methylchloroform and the Global Average for the Hydroxyl Radical. Science 238, 945 - 950 (1987) [40] H.B. SINGH;J.F. KASTING:Chlorine-Hydrocarbon Photochemistry in the Marine Troposphere and Lower Stratosphere. Atmos. Chem. 7, 261-285 (1988) [41] P. HERBERT;P. CARBONNIER;L. RIVOLTA;M. SERVmS;F. van MENSCH; I. CAMPBELL:The Occurrence of Chlorinated Solvents in the Environment. Chem. Ind. 1986, 861- 869 [42] T.M. VOGEL;P.L. McCARTY:Biotransformation of Tetrachloroethyleneto Trichloroethylene, Dichloroethylene,Vinylchloride,and Carbon Dioxide under Methanogenic Conditions. Appl. Environ. Microbiol. 49, 1080-1983 (1985) [43] M. TPEVlSAN;C. MONTEPIANI;L. RAGOZZA;C. BARTOLETTI;E. IOANILLI;A.A. DEL RE: Pesticides in Rainfall and Air in Italy. Environ. Pollution 80, 3 1 - 39 (1993) [44] F. WANIA;D. MACKAY:Global Fractionation and Cold Condensation of Low Volatility Organochlorine Compounds in Polar Regions. Ambio 22, 1 0 - 1 8 (1993)

14

Research Articles

[45] S. Jt/UTI; A. HIRVONEN; J. TARHANEN;J.M. HOLOPAINEN; J. ROUSKANEN:Trichloroacetic Acid in Pine Needles in the Vicinity of a Pulp Mill. Chemosphere 26, 1859 - 1868 (1993) [46] Beilstein, Handbuch der Organischen Chemie, Volume 2, Springer Berlin; (1920) pp. 194 if; E1 (1928) pp. 87 if; E2 (1942) pp. 187 ft. [47] T. ASHENDEN;T. MANSFIELD:Influence of Wind Speed on the Sensitivity of Ryegrass to SO2. J. Experimental Botany 28,729 - 735 (1977) [48] A.J. H~GEN-SMIT: Chemistry and Physiology of Los Angeles Smog. Industr. Eng. Chem. 44, 1342-1348 (1954) [49] H. Fa~qK: Waldsch~idendurch Photooxidantien? Nachr. Chem. Tech. Lab. 32, 298-305 (1984) [50] O. M~IN~,~DERkP.P~TERO: Application of Singular Value Decomposition to High Resolution Spectrometry for Forest Damage Detection. Contribution to the Conference on Environmetrics, 17-21 Aug. 1992 [51] F. Z~a~EL:Atmospheric Degradation of Volatile Halocarbons. Toxicol. Environ. Chem. 1993, in press [52] M.B. CHENOWETH:Monofluoroacetic Acid and Related Compounds. Pharmacol. Rev. 1 , 3 8 3 - 424 (1949) [53] J.S.C. MaR~s: Monofluoroacetic Acid. The Toxic Principle of ,Gifblaar" Dichapethalum Cymosum. J. Vet. Sci. Animal Ind. 20, 6 7 - 73 (1944) [54] G. BRINGMANN;R. KOHN:Grenzwerte der Schadwirkung wassergefiihrdender Stoffe gegen Blaualgen (Microcystis aeruginosa) und Grfinalgen (Scenedesmus quadricauda) im ZeUvermehrungshemmtest. Vom Wasser 50, 4 5 - 60 (1978) [55] J.L. HILTON; J.S. ARD; L.L. JANSEN; W.A. GENTNER: The Pantothenate-Synthesizing Enzyme, a Metabolic Site in the Herbicidal Action of Chlorinated Aliphatic Acids. Weeds 7, 381 - 396 (1959) [56] B.E. JUNIPER:The Effect of Pre-Emergent Treatment of Peas with Trichloroacetic Acid on the Submicroscopic Structure of the Leaf Surface. New Phytologist 58, 1 - 5 (1959) [57] R. SCHELL;U. KRISTEN:Trichloressigs/iurebegtinstigt Pilzinfektionen yon Fichtennadeln. In W. MICHAELISand J. BAUCH(Eds.): Luftverunreinigungen und Waldschfiden am Standort ,Postturm", Forstamt Farchau/Ratzeburg. GKSS-ResearchCenter Geesthacht, 1992, pp. 353- 363 [58] S. TIETZ;S. SCHNEIDER;J. GILL;A. WILD:PEPC-Aktivit~it,Biochemischer Indikator fiir den Sch~idigungsgrad bei Fichten. UWSF- Z. Umweltchem. C)kotox. 3,206 - 209 (1991) [59] E.D. SCHULZE;O.L. La_~GE;R. OREN: Forest Decline and Air Pollution, Springer Berlin 1989, p. 193- 207 [60] G. EILIKSSON;S. JENSEN; H. KYLIN; W. STRACHAN:The Pine Needle as a Monitor of AtmosphericPollution. Nature 341, 42 - 4 4 (1989) [61] A. REISCHL;M. REtSSINGER;O. HUTZINGER:Organic MicropolImams and Plants. In: E.D. SCHULZE,O.L. LANGE;R. OREN: Forest Decline and Air Pollution, Springer Berlin 1989, p. 193 - 207 [62] H. GEYER;I. SCHEUNERT;F. KORTE:The Effect of Organic Environmental Chemicalson the Growth of the AlgaeScenedesmus Subspicatus: A Contribution to Environmental Biology. Chemosphere 14, 1355 - 1369 (1985) [63] M. Hn,n~L; A. REIsc~IL;K.-W. SCHR~tM;F. TV,A ~ R ; M. REtSSINGER;O. HUTZ1NGER:Concentration Levels of Nitrated Phenols in Conifer Needles. Chemosphere 18, 2433- 2439 (1989)

Received April 15, 1993 Accepted June 28, 1993

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