Studies of mobility of di-iso-butyl phthalate (DiBP), di-N-butyl phthalate (DBP), and di-(2-ethyl hexyl) phthalate (DEHP) by plant foliage treatment in a closed terrestrial simulation chamber

Chemosphere, Vol.lO, No.ll/12, pp Printed in Great Britain

STUDIES

OF M O B I L I T Y

PHTHALATE FOLIAGE

(DBP), AND TREATMENT

Hans Laboratory versity

1223 - 1235, 1981

OF D I - I S O - B U T Y L DI-(2-ETHYL IN A C L O S E D

Lekke

and

of E n v i r o n m e n t a l

of D e n m a r k ,

Building

OO45-6535/81/121223-13502X)O/O ~ 1 9 8 1 Pergamon Press Ltd.

PHTHALATE

(DiBP),

HEXYL) PHTHALATE TERRESTRIAL

Finn

(DEHP)

SIMULATION

DI-N-BUTYL BY

CHAMBER.

Bro-Rasmussen

S c i e n c e s and E c o l o g y , T e c h n i c a l 224,

PLANT

DK-28OO

Lyngby,

Uni-

Denmark.

ABSTRACT A climate chamber was constructed for model studies of mobility and effects of chemical substances

within simplified terrestrial systems. The chamber functions and its performance

were tested by foliar applications of phthalates on higher plants. A low elimination rate from foliage was observed for DEHP. More than 95% of DiBP and DBP were eliminated within 15 -2 of DBP caused chlorosis on the leaves of Sinapis alba L.

days. 1.5 ~E cm

INTRODUCTION Phthalic acid esters are today recognised as widespread contaminants in the biosphere. The occurrence and biological activity have been reviewed by Peakall (1975), and the toxicity and metabolism have been considered by Daniel (1978). Thomas et al. (1978) have reviewed the biological effects of DEHP. Most data have been reported on the occurrence, mobility, and fate of phthalates in aquatic systems, but very few articles deals with the deposition and fate in terrestrial systems, especially soil and higher plants. The occurrence of phthalates in marine atmosphere was measured at the concentration level I n E m "3 (Giam et al. 1977, 1980, Cautreels

et al., 1977). Typical concentrations in continental air of industrial regions were founa in the range 10-1OO n E m -3 (Cautreels and Van CauwenberEhe , 1976, 1978, Bove et al., 1978). It was found that DEHP and DBP were partly present in the gas phase, partly adsorbed on particles in the atmosphere (Giam et al., 1980, Cautreels and Van CauenberEhe, 1978). Thus, it is likely that an entering of phthalates to the terrestrial environment can take place from the atmosphere by solid as well as by wet deposition. The uptake and fate of I~C-DEHP in a model ecosystem was studied by Metcalf et al. (1973) by application of the carbonyl-labeled compound to Sorghum plants in the terrestrial end of the system. However, no data were reported on the fate in plants or soil. In the present study a mixture of DiBP, DBP, and DEHP was applied to the foliage of 3inapis albaL. (white

1223

1224

mustard), Lapsana communis L. (nipplewort},and achillea millefoliumL.(milfoil).The aim was to study the possible phytotoxic effects and the transport by evaporation from the leaf surfaces simulating a situation of final deposition on the plants. During many routine analyses of organic pollutants, the interference of phthalates from reagents, laboratory equipment, and buildinE materials is a wellknown problem (Dudman and Whittle, 1976, Giam et al., 1975, 1976, SinE,,aster and Crosby, 1976, Andersen and Lam, 1979). In order to reduce the leaching of phthalates from plastic materials a special closed-circuit climate chamber was constructed from inert materials, such as glass, stainless steel, and PTFE.

MATERIALS AND METHODS

Climate chamber The climate chamber flow principle is shown in Fig. I. The system was tightly closed and the necessary carbon dioxide was supplied through a pressure regulator and a calibrated restrictor. The air flow was driven by a variable centrifugal air pump, and the flow was measured by a standard orifice plate (20 ~m bore, DIN 1952) placed in a straight glass tube (1,3 m, i. d. 50 mm) and connected to a manometer. The carbon dioxide concentration level was checked by absorption in a flow-regulated by-pass (20 ml min -I) with two serie-connected washing glasses with standard amounts of sodium hydroxide solution. The absorption liquid was daily checked by titration with standard hydrochloric acid, and the mean concentration was calculated from the flow rate, the absorption time and the amount of acid required.

1225

Fig.

1.

DIAGRAM OF THE CLOSED CLIMATE

1

IL~

14 | I

13

CHAMBER

SYSTEM

4

12 -

-

9

-

-

11 I. Chamber

8. Air washer and cooler

2. Light source

9. Cooling bath

3. Air filter

10. Water circulation pump

4. Orifice plate

11. Water sampling system

5. C02-inlet

12. Heating element

6. Air circulatlon pump

13. Psycrometer and temperature controller

7. C02-trap

14. Manometer

1226

Fig.

2.

\

,

Phthalate-treated plants Plants treated with solvents only Untreated plants

I

1227

The circulating air was cooled to the dew point temperature and wetted in an air washer specially constructed from stainless steel and insulated with Rockwool. The air washer water was in direct contact with the climate chamber air, and the water was continuously pumped through a closed cotter tublng system placed in a cooling bath (500 watt) to maintain a constant temperature. Evaporated water from test plants was condensed in the air washer. Surplus water from thewasher was drained via a

pressure compensated overflow system into a flask for

volume recording, and for chemical analysis in order to check the efficiency of the air adsorption filter. Water drops were trapped before the outlet of the air washer, and the air was reheated to the temperature desired in a glass tube (I m, i. d. 50 mm) surrounded by a heating tape element (1.5 m, 150 watt) insulated with Rockwool. The relative humidity was measured at the entrance to the chamber by an aspiration psycrometer, and continuously recorded. For sensors were used two-ter~,inal integrated circuit temperature transducers (Analog Devices, Massachusetts, U.S.A., type AC 2626-L4). A constant water supply was maintained to the wet sensor. The temperature of the system was measured in six

positions and was continuously recorded.

For the chamber was used standard glassware (Duran Glass, Jena'er Glaswerk Schott & Gen., D-6500 Mainz, West Germany). The chamber (Schott ABS 450/200) was provided with a plane glass lid (5 mm) clamped on a PTFE packing ring. In the chamber a liner of stainless steel (i. d. 413 mm, height 500 mm) was placed, see Fig. 2. The liner was carried by a flange and had a perforated bottom. The inner wall was high mirror finished to allow reflection of light and to minimize a possible adsorption of test compounds on the wall. A vertical glass plate divided the chamber in two identical halves. The air flow went through the perforated bottom in the liner to the lid and via the space between the liner and the glass wall to the broad outlet tube (200 mm). The air flux was not constant throughout the chamber area giving higher values near the outlet side of the chamber. However, the total flow was the same in both chamber halves. In the outlet tube from the chamber a filter holder of stainless steel was placed in a coupling with PTFE gaskets. The air filter material was placed in a

uniform horizontal layer

between small-meshed clothes of polyam/de. Activated carbon was used as filter material (1OO g, Pittsburgh Chemical Co., 2-3 m ) .

Depending on the pressure drop in the air filter a slight

over-pressure was observed in the chamber (15-65 ,~ water height}. Light was provided to the climate chamber by three circular concentric fluorescent lamps (Philips 'TL, colour 33, 40 ÷ 32 + 22 watt) placed 2 cm above the chamber glass lld in a ventilated reflector. The light was measured with a solarimeter (Kipp & Zonen, type CM 5). The irradlance was 35 watt m -2, and the illuminance 11 klux at the soll surface.

Test plants The cultivation of unifor~ test plants were conducted by a modification of a procedure described by Thonke (1975) for herbicide screenlng tests. The plants were grown in test tubes of brown glass (1OO mm, i. d. 18 mm) with an opening in the bottom (i. d. 13 mm) and a collar

1228

on the top allowing them to hang in 20 -,, holes. The tubes were packed with 3.0-3.5 g granules of Rockwool (made by passing fibers through a 8 mm network). Seeds of test plants were soaked in HoaEland no. 3 nutrient solution (Hewitt, 1966) for 5 hours. Two seeds were sown in each tube at 5 "" depth. The tubes were wetted with nutrient solution (5 ml) and placed under climatical conditions corresponding to the experiments. After the emergence of all plants the smallest plant in each tube was removed. The tubes were placed in 20 mm holes in an aluminium lid, and the tubes were dipped 2 cm into a nutrient solution bath (Hoagland no. 3). The plants were allowed to grow for 3-4 weeks (Slnepls elba) or 6 weeks (Lapsana communis, Achillea mll-

lefollum) before they

were taken for experiments. The nutrient solution was changed every 3-4

days. Gas chromatographic analyses Di-iso-butyl phthalic acid ester (~99,5%, BASF AG, D-67OO LudwiEshaven, West Germany), din-butyl phthalic acid ester (>99,5~, Fluke AG, CH-9470 Buchs, Switzerland), and di-(2-ethyl hexyl) phthalic acid ester (>99%, Scandiflex A/S, DK-2860 Seborg, Denmark) were used as test compounds and as well as analytical standards without further purification. All reagents were checked for traces of phthalates and redistilled or washed if necessary. Plants were extracted with dichloromethane by homoEenization in a WarinE blendor stainless steel cup (I00 ml) provided with sealinE and lid of PTFE. After concentration to I-2 ml the extracts were cleaned up by passinE alumina column (3 g per 10 E of sample wet weight, 10% of water, and loaded with I% of silver nitrate). 10 ml of dichloromethane was used as solvent. The eluate volume

was adjusted to 10 ml. A Perkin Elmer Sigma 3 gas chromatograph was used equipped

with flame ionization detector. As column material was used 5% OV-I01 on Chromosorb WHP packed in a glass column (1.8, i. d. 3 mm). The column temperature was progj-ammed from 200 to 240°C with a rate of 3°C mln -I. 2 ~i of the solutions were injected and peak heiEhts were compared with those of standard solutions with known concentrations. The limits of determination for the analyses of plant material were calculated from blank values of untreated plants in each analytical series. Contaminating amounts of phthalates from reaEents and equipment appeared in the followinE ranges: 0.3-4 ~E DiBP sample "I, 0.4-5 ~ E D B P sample -I, and 8-37 ~ E D E H P sample -I. Water samples were shaken with dichloromethane, and activated carbon from air filters were Soxhlet extracted for 12 hours with dichloromethane. The extracts were concentrated for Eas chromatographic analyses. Blank values by analyses of activated carbon were 9 ~E kg -I DiBP, 3 ~ E k g - ; DBP, and 3 ~ E k E "I DEHP. Recoveries of the analytical procedures were regularly checked by spiking untreated plant material, activated carbon, or distilled water with test compounds at the concentration levels of interest. The following recovery ranges were found: DiBP, 77-98%, DBP 73-98~, and DEHP 79-I05~. All results were corrected for blank values. However, no results were corrected for losses caused by the analytical procedures as this was considered of minor importance compared with the scattering caused by biological and experimental variations.

1229

Experimental procedure Spraying solutions were prepared from acetone solutions (2 ml) of a mixture of three phthalic acid esters. A solution of Tween 20 (polyoxy ethylene sorbitane monolaurate) in distilled water (18 ml) was added dropwise by vigourous mixing. Unlfon~ plants were selected and treated with 1OO or 250 ~i of the spraying solution by use of a photo laboratory retouching sprayer (Steffens and Wieneke,

197~). The originally applied dose on each plant was estl-

mated by random sampling of eight treated plants which were deep-frozen for two weeks and analyzed by the described gas chromatographic procedure. Inevitably, some spraying solution was lost in the space around leaf cover. Spraying efflclences were estimated to be 65-75% for Si-

napis alba and Lapsana communis, and 40-50% for Achillea millefollum. The experiments included plants treated with solvent only, i.e. acetone ( ~ )

+ water (80%) + Tween 20 (0.;%), at the

same rate as phthalate treated plants. A new air filter with activated carbon as filter material was installed, and 32 plants were placed in the climate chamber. Of those were 16 plants treated with phthalates, 8 plants were untreated, and_8 plants were treated with solvent only. The phthalate treated plants were placed within the same chamber-half, and shielded from untreated and solvent-treated plants in the other half, as shown by Fig. 2. The plant cultivating tubes were hanging in alumlnium llds and dipped into nutrient solutions in one lltre Jars of brown glass covered with alumlnium foll to exclude light. Each Jar contained 4 plants. Sampling was performed at days I, 3, 7, and 15 after the start of the experiment. Any air flux variation in the chamber was compensated for by random sampling of one plant from each Jar at each sampling time. The sampling of plants left open holes in the alumlnlum lld allowing sufficient air contact with the nutrient solution surface. This solutlon was changed only once during the experiments, namely on day 7. At each sampling time the carbon air filter material was sampled and replaced with a new batch. Leaf areas were determined by photocopying the leaves of untreated plants, cutting, and comparing the paper weight with that of standard paper areas. Fresh weights of leaves, stems, and the heights of stems of each sampled plant were recorded. All green parts were taken for chemical analysis. The amount of water evaporated was recorded, and water samples from the air washer were taken for analysis. During the experiments the climatic conditions were kept at constant levels. The temperature was 16.7-17.2°C, the relative humidity was 67-76%, and the mean wind speed O.13-O.15 m sek -I. Plants received 11 klux at the soll surface with a 16-h daily photoperiod. Carbon dioxide was supplled in daytime only, and was kept between 300 and 480 ppm. By the end of experiments, the inner walls of the chamber and the stainless steel liner were carefully washed with acetone to detect traces of test compounds adsorbed.

1230

RESULTS AND DISCUSSION Table I shows the mass balance for an experiment involving 16 plants of Sinapis alba, at 5-7 leaves stage, treated with 100 ~I of a mixture of DiBP, DBP, and DEHP at a rate of 2.5 t~ -2 cm of each compound on the leaf surfaces. After 15 days some loss was observed of DBP (24%),

Table I.

Mass balance study (15 day experiment) for a mixture of three phthalic acid esters in a closed simulation system with Sinapis alba plants. DiBP mg

Plants

b)

Carbon air filter b) c) Chamber walls d) Air washer Amounts appllcated

at

Difference (loss)

a)

DBP

DEHP

~

mg

%

mg

0.45

19

0.79

31

2.47

1.68

71

1.10

43

n.d.

-

89

0.006

0.3

0.002

0.1

0.15

5

0.06

3

0.04

2

0.05

2

2.35

I00

2.54

100

2.78

I00

-0.15

-7

-0.61

-24

-0.11

-4

value calculated on basis of analysis of eight treated plants randomly sampled at start of experiment

b)

amounts removed from the system by sampling of carbon air filters or plants at days I, 3, 7, and 15

c) d)

acetone washings at end of experiment sampling after 3 days. Only insignificant amounts were found after 15 days.

n.d. = not detectable.

The experimental blanks are substracted from the reported values.

but the calculated losses of DiBP (7%), and of DEHP (4%} were not significant. Only small amounts of DEHP evaporated compared with DiBP (71%7 and DBP (43%}. Analysis of air washer water showed that only insignificant amounts of test compounds passed the carbon air filter, and an acetone washing of the chamber walls after the experiment was ended showed that only small amounts of test compounds were adsorbed on the walls. Due to variations in size and development of test plants a statistical comparison was not possible of the growth rate of treated and untreated items. However, on the third day chlorosis developed in the new leaves (no. 6-8) of IO out of 12 treated plants. After the first appearance no further chlorotic

1231

leaves were developed. The experiment was duplicated with the same mixture of phthalates in a sprayln8 solution in which Tween 20 was replaced with Sun Superior Spray Oil 11-E (highly refined petroleum distillate, Sun Oil Company, Belgium) as emulsifying agent. Each plant was treated with 250 ~I -2 of the spraying solution at a rate of 2.5 ~ cm of each compound on the surface of the leaves. In this experiment the elimination patterns of DiBP, DBP, and DEHP was the same as in the former experiment.

11 out of 12 treated plants developed chlorosls in the new leaves on

the third day. Further experiments were performed by treatment of plants with pure phthalates without any addition of acetone or emulsifying agents. It was observed that chlorosis was caused by DBP only. Due to the very low water solubility of the phthalates the application was performed

by transferring drops of the pure liquid substances. By this procedure no quantita-

tive evaluation was possible. No adverse visible effects were observed by treatment with pure DiBP or with DEHP.

Table 2.

Residues from a mixture of three phthalates on the leaves of Sinapis alba during a 15 day experiment. Time

Mean leaf area

days

cm 2 plant -I

Leaf area concentration, ~

cm

-2

DiBP

DBP

DEHP

0

65

2.3

2.~

2.7

1

77

1.1

1.6

2.1

3

88

0.2

0.5

1.9

7

118

0.1

0.2

1.2

15

223

0.02

0.03

0.8

In Table 2 the concentration~s o£ DIBP, DBP, and DEHP are shown on the leaves of Sinapis

alba in the same 15 day experiment as shown by the mass balance in Table I. Tween 20 was used as emulsifying agent. The leaf surface concentration of DiBP and DBP declined rapidly. The decline of DEHP concentration was caused by growth dilution only as compared with a constant compound level ( ~ plant -I) during the experiment. The evaporization rate (ng cm -2 h -I ) and air mean concentration increments per plant (ng m -3 plant -I) in the same experiment are shown in Table 3. In the present plant experiments the pressure of DBP from 16 treated plants was I x 10-5 Pa over the foliage in the simulation chamber in the first day of the experiments. This corresponded to a value 200 times lower than saturation pressure. Theoretically the rate of loss of a substance from a given surface is independent of the thickness of the substance layer. Under constant climatic conditions, especially ventilation, the evaporation will proceed at a constant rate until the remaining amount of substance no longer covers the surface. As shown by Table 3, there was a rapid decrease of the evaporation

1232

Table 3.

Evaporation of a mixture of three phthalates from the leaves of Sinapis alba during a 15 days experiment. Time interval

Evaporlzation rate a)from

Air mean concentration a)

leaves, ng cm -2 h -I

increments, ng m'3plant -I

DiBP

DBP

DEHP

DiBP

340

33

17

n.d.< 0.8

2- 3

840

14

7

4- 7

15OO

3

3

8-15

3200

I

0.5

days

O- I

Air volume b) m3

DBP

DEHP

151

76

n.d.< 2

n.d.< 0.6

72

50

n.d.< 2

n.d.< 0.6

21

18

n.d.< 2

n.d.< 0.5

10

5

n.d.< 3

a) Vaporization and air mean concentration increments were determined on basis of substance amounts adsorbed in carbon air filter. b)

Calculated by use of treated chamber-half air flow volume only.

n.d. = not detectable.

rate during the first part of the experiment. The initial fast rate might be explained as an evaporation from a continuous film deposit, until the film gradually retreats into smaller, dropllke areas. Such retreat is the obvious result of dissappearance of the thinnest parts of the surface film layer and, as well, of the growth of the leaf area. In the last part of the experiment, Table 3 indicates a more slowly release to the atmosphere of DiBP and DBP, which could be interpreted as a result of a dominance of adsorptive forces on the leaf surface. A certain contribution to the evaporation in this latter phase, could come from a delayed release of phthalates after an earlier penetration into the outer epidermal wall of the leaf surface. Such process will mostly be controlled by the relatively low diffusion coefficients within the leaf tissues. The effect of the individual phthalate DBP emulsified with Tween 20 was studied by treatment of Sinapis alba plants at the four leaves stage. The doses were 1.5, 0.25, and 0.05 Hg -2 cm respectively of the whole leaf area. In the same experiment were included plants of Lapsans communis, and of Achilles millefolium which were treated with a mixture of DiBP, DBP and DEHP. The residues found of DBP at different sampling times are shown in Fig. 3. At the high dosage level of DBP (1.5 ~g cm -2) chlorosis was observed after three.days in the new leaves of Sinapis alba. This effect level was in agreement with the results from all experiments with this plant. However, no visible effects appeared at the lower dosages or on the Lapsana communis nor Achillea millefolium plants. The evaporation of DE}~ from the latter plants was negligible, but the dissappearance rate of DiBP and DBP seemed to be slower from Achillea millefollum than from

the other plants.

1233

Fig.

3.

Residues

of DBP

application

in

of D B P

three

different

as a f u n c t i o n

plants

after

of t i m e .

pg DBP Plant-I

,2°l

0

Sinapis alba,

•

Sinapis alba, 0.25

1.5 I/g cm

I/g cm

Lapsana communis D Achillea millefolium

20

[]

01

""---[3 3

7

£3

15 Time ( d a y s )

-2 -2

1234

In this study it was not possible to observe any influence on the evaporization rate from the simultaneous presence of three test compounds, acetone, and emulsifying agent. The use of acetone and emulsifying agent was necessary for the solubillzation of the hydrophobio phthalate compounds. In the

spraying solutions the concentration of Tween 20 was 40% of that

of single phthalate test compound~However, the acetone and the emulsifying agent might have played a role in the uptake and biodistribution of phthalates in the plants. CONCLUSION The climate chamber which has been constructed is found to function satisfactorily permitring studies of mobility and effects of phthalates in higher plants. A considerable elimination of contamination was achieved by avoiding plastizer-contalning materials in the chamber construction and by exoludlng excess of contamination from laboratory equipment and building materials. The system might as well be used for the examination of organic pollutants other than phthalates. The present experiments showed that application of DBP on the leaves of Sinapis alba caused chlorosls at 1.5 DE cm-2. The damages developed in the new leaves and they appeared at the third

day after application. In parallel experiments at the same concentration level, there

were no observed, adverse effects on the leaves of Achillea millefollum and of Lapsana communis. A single high dose of phthalates as involved in the present experiments is not resempling natural conditions in which continuous low level exposure most likely are to occur. However, the study indicates the need for further investigations on the uptake and cellular effects of DBP in higher plants. The experiments have further shown a high stability of DEHP on the leaves of higher plants after deposition.

ACKNOWLEDGEMENTS The authors wish

to thank Mr. P r e e n J~rgensen for construction of the electronic equip-

ment in the climate chamber and for valuable technical assistance. The Danish Council for Scientific and Industrial Research is thanked for financial support,

1235

REFERENCES Andersen, K. S., Lain, J. (1979) J. Chromatogr.

169, 101-106.

Bore, d. L., Dalven, P., Kukreja, V. P. (1978) Intern. J. Environ. Anal. Chem. 5, 189-194. Cautreels, W., Van Cauwenberghe, K. (1976) Atmospheric Environment I0, 447-457. Cautreels, W., Van Cauwenberg~e, K., Guzman, L. A. (1977) Sci. Total Environment 8, 79-88. Cautreels, W., Van Cauwenberg~e, K. (1978)Atmospheric

Environment 12, 1133-1141.

Danlel, J. W. (1978) clin. Toxicol. 13, 257-268. Dudman, W. F., Whittle, C. P. (1976) Carbohydr. Res. 46, 267-272. Giam, C. S., Chan, H. S., Neff, G.S. (1975) Anal. Chem. 47, 2225-2229. Glare, C. S., Chan, H. S. (1976) Nat. Bur. Stand. (U.S.), Spec. Publ. 422, vol. 2, 701-708. Glare, C. S., Atlas, E., Chan, H., Neff, G. (1977) Rev. Int. Oceanogr. Ned. 47, 79-84. Giam, C. S., Atlas, E., Chan, H. S., Neff, G. S. (1980) Atmospheric Environment 14, 65-69. Hewitt, E. J. (1966) Sand and Water Culture Methods used in the Study of Plant Nutrition. Tech. C o ~ .

Commonwealth Bur. Hort. Plant. Crops no. 22. Rev. 2 ed. East Malling,

Maidstone, Kent. C.A.B.

Farnham Buchs.

Metcalf, R. L., Booth, G. M., Schuth, C. K., Hansen, D. J., Lu, P.-Y. (1973) Environ. Health Perspect. 4, 27-34.

Peakall, D. B. (1975) Res. Rev. 54, 1-41. Singmaster III, J. A., Crosby, D. G. (1976} Bull. Environ. Contamino roxicol. 16, 291-300. Steffens, W., Wleneke, J. (1974) Landwirtsch. Forsch. 27, 38-45. Thonke, K. E. (1975) Proceedings European Weed Res. Soc. SNmp.; Status, BioloEy and Control of Grassweeds in Europe, 2-3 dec. 1975, Paris. Thomas, J. A., Darby, T. D., Wallin, R. F., Garvin, P. J., Martls, L. (1978) Toxicol. AppI. Pharmacol. 45, 1-27.

(Received in ~ e

Netherlands IO S e p t e ~ r

1981}