1085

Environmental Toxicology and Chemistry, Vol. 20, No. 5, pp. 1085–1091, 2001q 2001 SETAC

Printed in the USA0730-7268/01 $9.00 1 .00

EFFECTS OF DI(2-ETHYLHEXYL) PHTHALATE AND DIBUTYL PHTHALATE ON THECOLLEMBOLAN FOLSOMIA FIMETARIA

JOHN JENSEN,*† JUDITH VAN LANGEVELDE,‡ GUNNAR PRITZL,§ and PAUL HENNING KROGH††National Environmental Research Institute, Department of Terrestrial Ecology, Soil Fauna and Ecotoxicology Unit, Vejlsøvej 25,

P.O. Box 314, DK-8600 Silkeborg, Denmark‡Department of Nematology, Wageningen Agricultural University, Binnenhaven 10 NL-6709 Pd Wageningen, The Netherlands

§National Environmental Research Institute, Department of Environmental Chemistry, Frederiksborgvej 399, P.O. Box 358,DK-4000 Roskilde, Denmark

(Received 7 April 2000; Accepted 28 September 2000)

Abstract—Lethal and sublethal effects of di(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) on adult individuals ofthe collembolan Folsomia fimetaria were investigated in the laboratory by the use of small microcosms. Effects of DEHP and DBPwere also tested on newly hatched collembolans in a multidish system. The endpoints were juvenile mortality, growth, anddevelopment. When exposed to DEHP, adults and juveniles were unaffected at all test concentrations, that is, up to 5,000 mg/kg.However, DBP caused increased adult mortality at 250 mg/kg and juvenile mortality at 25 mg/kg. For DBP, adult reproduction wasa more sensitive endpoint than was survival, with an EC10 and EC50 of 14 and 68 mg/kg, respectively. Juvenile molting frequencyseems to be a sensitive parameter, because number of cuticles produced by young springtails was reduced at 1 mg/kg. Toxicitywas reduced when soil spiked with DBP was stored at 208C for a period of up to 28 d before adding the animals. Reduction intoxicity of DBP may be due a combination of degradation, evaporation, and adsorption of DBP to soil material. This was confirmedby chemical analyses, which showed a rapid initial disappearance followed by a much slower disappearance. Our results lead tothe overall conclusion that significant adverse effects of phthalates on collembolans are not likely to occur as a result of normalsewage sludge application.

Keywords—Soil fauna Phthalates Plasticizers Ecotoxicology Sewage sludge

INTRODUCTION

Globally, the majority of consumed phthalates are used asplasticizers in production of polyvinyl chloride. Important non-plasticizer uses of phthalates include paint, lacquer, printingink, adhesives, fillers, and dielectric fluid in capacitors. Onlyan insignificant amount of phthalates is formed naturally. Di(2-ethylhexyl) phthalate (DEHP) is one of the most widely usedindustrial chemicals. Annual DEHP production is approxi-mately 1.3 million tons, which is about one half of the totalconsumption of phthalates. In Denmark, annual phthalate con-sumption is approximately 10,000 tons [1].

Because of their ubiquitous use, phthalates can be foundalmost everywhere in the environment. Phthalates are gener-ally weakly associated to plastic and may slowly leak or evap-orate to the environment. Chemical inertness and low anaer-obic degradation rates may result in high concentrations ofDEHP and other phthalates in sewage sludge [2,3]. In Denmarkand many other countries, sewage sludge is commonly usedas fertilizer by farmers. To ensure that the amendment of ag-ricultural soils with sewage sludge is conducted in an envi-ronmentally sound manner, a Danish ordinance includes a cut-off value for DEHP of 100 mg/kg dry weight in organic wasteused for agricultural purposes. This limit will be lowered to50 mg/kg dry weight by the year 2000. No cutoff value existsfor dibutyl phthalate (DBP), the second most common phthal-ate in Danish sludge. The average DBP concentration is nor-mally lower than 5 mg/kg dry weight, but may reach morethan 25 mg/kg dry weight [3]. Phthalates in sludge may po-

* To whom correspondence may be addressed(john.jensen@dmu.dk).

tentially affect soil organisms or they may be taken up in edibleparts of plants, leading to undesired exposures of humans orlivestock. However, little information is available thus far forjudging the actual risk.

Di(2-ethylhexyl) phthalate has low water solubility andhigh octanol–water partition coefficient (KOW), thus soil ab-sorption is high, whereas DBP with an intermediate log KOW

has a moderate mobility. Even large DEHP concentrationshave been shown to be relatively easily degraded by micro-organisms under aerobic conditions. Maag and Løkke [4]found half-lives of DEHP between 33 and 85 d in soil initiallycontaining 21,900 mg/kg. However, DEHP degradation is veryslow or even nonexistent under anaerobic conditions [5].Shanker et al. [6] observed large differences in aerobic andanaerobic DEHP biodegradation. In flooded soil, only 30% ofthe incubated phthalate was degraded in 30 d, whereas 90%was degraded in aerobic soil. No information was found onhalf-lives of DBP in soil.

Phthalates are considered to be moderately to highly toxictoward aquatic organisms [7]. The European Union directiveon risk assessment of new and existing substances includes afirst step of hazard identification primarily used for marketingand labeling of hazardous substances. According to this pro-cedure, classification of DEHP and DBP as, respectively, toxicand very toxic for the aquatic environment has been suggested[8]. In general, phthalates with alkyl chain length shorter thanfive carbon atoms are considered most toxic because of theirhigher water solubility. A few phthalates may disrupt the en-docrine system [9,10]. Only very limited information is avail-able for assessing risk of phthalates in the terrestrial environ-ment. To improve the scientific basis for assessing the risk to

1086 Environ. Toxicol. Chem. 20, 2001 J. Jensen et al.

soil fauna, this paper presents studies of the effects of twocommonly used phthalates, DEHP and DBP, on adults andjuveniles of the springtail Folsomia fimetaria. Folsomia fi-metaria is widely distributed in temperate regions, where, incontrast to the more often used springtail Folsomia candida,it is common in the upper layers of agricultural soils andgrassland. The objective of this paper is to evaluate the acuteand sublethal effects of DBP and DEHP on F. fimetaria inthe laboratory and to discuss the risk of sludge-born phthalatesto terrestrial ecosystems.

MATERIALS AND METHODS

The present experiments were conducted with laboratorycultures of the euedaphic, nonpigmented, eyeless collembolanF. fimetaria, which reproduces sexually. The laboratory cul-ture of F. fimetaria was established from field-collected ani-mals and was maintained in petri dishes on a substrate ofmoistened plaster of paris and charcoal. The collembolans werefed 15 mg of dried baker’s yeast weekly and every two tothree months, adult animals were transferred to new petri dish-es with fresh substrate and yeast. Before the experiment asynchronized culture was produced by collecting eggs (approx.7 d old) from cultures stimulated for egg-laying by new sub-strates. Eggs were allowed to hatch over a 3-d period. Animalshatched during this time were subsequently used for the fol-lowing experiments at the age of 23 to 26 d (for more detailssee the work of Wiles and Krogh [11]).

The test soil was a topsoil (20 cm) from a sandy soil col-lected at the agricultural field station Askov, Jutland, in Den-mark. This soil has a composition of approximately 4% clay,4% silt, 25% fine sand, and 65% coarse sand. The organiccarbon content is less than 1.5%. The soil was dried at 808Cto eliminate undesired soil fauna. Before use, the soil wassieved through a 2-mm mesh. The chemicals used (DBP, DEHP,and acetone) were obtained from Merck Schuchardt, Darm-stadt, Germany.

Adult survival and reproduction

Animals were exposed to phthalates in microcosms con-taining 30 g of moist soil (27 g of dry soil and 3 ml of de-mineralized water). Phthalates were dissolved in acetone and3 ml of acetone–phthalate mixture was mixed into dry soil theday before starting the experiments. During the night, the sol-vent evaporated and 3 ml of demineralized water was mixedinto the soil just before the start of the experiments. The sameamount of acetone–water was added to the control. Experi-ments were conducted at constant temperature (208C), with a12:12 h light:dark regime. To measure adult survival and re-production, 10 adult females and 10 adult males (aged 23–26d) were added to each replicate microcosm on day 0 and in-cubated for 21 d. In a synchronous culture of F. fimetaria, sexcan be discerned by size because adult females are much largerthan adult males. Animals were fed dried baker’s yeast (15mg dry weight) at day 0 and day 14. The soil was remoistenedafter 14 d and soil pH was measured at the end of the exper-iment to exclude toxic effects of pH changes. Test concentra-tions used were DEHP at 0, 1,000, 2,000, 3,000, 4,000, and5,000 mg/kg dry weight and DBP at 0, 100, 250, 500, 750,and 1,000 mg/kg dry weight. Four replicated microcosms wereused per concentration.

At the end of the laboratory experiments, all animals wereextracted in a high-gradient extractor of the MacFayden typeand collected in a cooled (38C) collecting dish [11]. For each

microcosm, surviving adults and juveniles were counted au-tomatically using a digital image processing system [12]. Thedigital image processing system is an automated counting andmeasuring technique based on a video camera connected to aframe-grabber employing subsequent computerized treatmentof data. The digital image processing technique is fast, ac-curate, and highly reproducible. Using digital image process-ing data, number of viving animals, overall reproduction, andsize (measured as final body-surface area) were calculated.Using an ordination technique (principal component analysis)[13], separation of the collembolans into adult females, adultmales, and juveniles was possible.

Juvenile survival, growth, and molting

To elucidate whether the observed effect on reproductionwas due to a lower production of offspring, inability of eggsto hatch, or reduced survival of juveniles, newly hatched ju-veniles were exposed for one week to DBP and DEHP. Forthis experiment, multidishes of plastic (Nunclony, Nalge NuncInternational, Roskilde, Denmark) with 24 small circularchambers (diameter 5 1.6 cm; area 5 2.0 cm2) were used toobserve each animal individually as described by Folker-Han-sen et al. [14]. Each chamber was filled with 2.65 g of testsoil. The surface had to be compressed so that the animalswere unable to hide in the soil. A single 0- to 1-d-old juvenilespringtail was added to each chamber. To minimize water evap-oration, multidish chambers were sealed by rubber plugs im-mediately after adding the animals. Lost water was replacedweekly if necessary. The animals were fed granules of driedbaker’s yeast, which were renewed whenever necessary.

Test concentrations were DBP at 0, 1, 5, 10, and 25 mg/kg dry weight and DEHP at 0, 100, 250, 500, and 1,000 mg/kg dry weight. Twenty individuals were exposed to each con-centration. No replicates were made. Phthalates were dissolvedin acetone and mixed into the dry soil 1 d before to the startof the experiments. The next day, evaporated solvent was re-placed by demineralized water before starting the experiments.The same amount of acetone–water was added to the control.Experiments were run at constant temperature (208C), with a12:12 h light : dark regime.

Juveniles were counted and measured twice a week for sixweeks with the digital image processing system. Growth ofthe animals was determined manually at the screen by mea-suring the length from the posterior end of the abdomen tothe anterior end of the head. Sex was determined at the endof the experiment by analyzing slides of the animals in amicroscope as described by Folker-Hansen et al. [14]. Thegrowth pattern of both sexes fitted a more or less sigmoid-shaped curve. The growth rate coefficient b in the logisticmodel was used to test significance.

During the first three weeks, covering an entire F. fimetarialife cycle, exuviae of growing juveniles were recorded everysecond day and removed if present. An observation frequencyof 2 d is sufficient for F. fimetaria because this springtail doesnot eat its own exuviae [15].

To study whether exposure of animals on compressed soilin multidishes was comparable to exposure in loose microcosmsoil in the standard test, juvenile survival in a one-week acutetest was studied. Ten juveniles (0–1 d old) were added to eachof two replicated microcosms containing the same five con-centrations of phthalates as in the multidish experiment, thatis, DBP at 0 to 25 mg/kg and DEHP at 0 to 1,000 mg/kg.Animals were fed 2 mg of dried baker’s yeast.

Sublethal effects of phthalates on collembolans Environ. Toxicol. Chem. 20, 2001 1087

Fig. 1. Degradation of dibutyl phthalate (DBP) in soil. Fraction oftotal initial DBP concentrations in the 50 mg/kg (dry weight) assays(squares) and 500 mg/kg (dry weight) assays (circles), and modelestimates of total (solid line) and dissolved (broken line) DBP con-centrations over a 30-d period. The data points are almost identicaland the standard error of the mean (vertical bars) is very small. De-scription of the model is found in the Materials and Methods.

After one week, the microcosms were extracted with a high-gradient extractor of the MacFayden type and collected inbeakers with a plaster of paris–charcoal substrate. Survival inthe microcosm soil was compared with the one-week survivalof juveniles exposed to compressed contaminated soil in mul-tidishes.

Effects of aging on DBP toxicity

To study the time course of observed DBP toxicity and tolink DBP toxicity with its chemical fate in soil, soil spikedwith DBP was stored at 208C for 3, 7, 14, or 28 d beforeadding animals. After adding animals, tests were conductedas for the 21-d reproduction test described above. Four rep-licates of the test concentrations of DBP at 0, 50, 100, 200,300, and 500 mg/kg were made. To minimize water loss, mi-crocosms were closed. Because evaporation may contribute todisappearance of DBP (vapor pressure is 0.0097 Pa), micro-cosm lids were removed for 5 min every day during the storageperiod to ensure that DBP evaporation was not prohibited asa result of equilibrium between air and soil air.

Chemical analysis

Fate of DBP was studied during the above-mentioned stor-age period. Five subsamples of 25 g each were taken fromeach of the four replicate (total of 20) of the lowest and highesttest concentrations, that is, DBP at 50 mg/kg dry weight and500 mg/kg dry weight, at days 1, 3, 7, 14, and 28. Tests werenot conducted in parallel but rather in two groups. Therefore,samples from 7 d of storage were collected from both sets toevaluate possible differences. No significant differences werefound between the two sets of day 7 data. Samples were storedat 2208C before analysis. All glassware was solvent rinsedand heated overnight at 4508C before use. After thawing, sam-ples were homogenized in an agate mortar, and 10 g was trans-ferred to a 50-ml centrifuge tube sealed with glass stopper,and spiked with 500 ml of deuterated DBP (4D-DBP, CIL,Chattanooga, TN, USA) at 1 mg/ml. After 1 h equilibration,samples were extracted with 24 ml of dichloromethane–ace-tone mixture (9:1, v/v) by ultrasonication for 1 h and thenshaking at 350 rpm for 2 h. The suspension was centrifugedat 3,500 g for 10 min and the supernatant was decanted. Theextraction was repeated two times. Pooled supernatants werereduced under a gentle stream of nitrogen to 5 ml, and analyzedwithout further clean-up. All preparations were made in du-plicate.

The DBP analysis was carried out by a Varian Saturn 4Dgas chromatography–mass spectrometry system with a Varian3400 gas chromatograph equipped with a Varian 1078 SPIinjector (Varian, Walnut Creek, CA, USA), operated in on-column single-ion monitoring electron ionization mode. Thecolumn was a 30-m-high 3 0.25-mm–inner diameter HP-5mass spectrometer (Hewlett Packard, Avondale, PA, USA),with 0.25 mm 5% Ph-Me silicone, and a temperature programof 1 min at 408C, 408C/min to 1208C (2 min), and 108C/minto 2708C (6 min). Injection volume was 1 ml and the carriergas was helium 6.0 with a constant flow at 1 ml/min. Theionization energy was 70 eV, and the measured ions mass-to-charge ratios (m/z) were 149 and 153. Confirmation of identitywas made by comparison with the National Institute of Stan-dards and Technology (Gaithersburg, MD, USA) mass spectralibrary. Quantification was performed toward a five-point stan-dard curve, with concentrations of DBP at 0.1, 1.0, 10, 100,and 500 mg/ml in acetone (Merck, Darmstadt, Germany). Re-

coveries, measured by the deuteriated DBP spike, were be-tween 82 and 93%. The overall limit of detection (calculatedas S/N ratio at 3) was 1 mg/kg dry weight.

Mathematical model for DBP disappearance

Assuming a constant rate adsorption and desorption be-tween the soil matrix and soil liquid, the following equationdescribes the adsorbed concentration (S):

dS5 k 2 c 2 k ·S (1)1 21dt

where k1 (1/d) is the first-order adsorption coefficient, c is thedissolved concentration (mg/kg dry weight), k21 is the rate ofdesorption (1/d), and S is the adsorbed concentration (mg/kgdry weight). The dissolved concentration c can be describedassuming first-order removal from the batch of the dissolvedphase as

dc5 2(k 1 k ) ·c 1 k ·S (2)1 dgr 21dt

where kdgr is the first-order removal constant out of the system(1/d), which can be due to either biological or nonbiologicalprocesses.

Equations 1 and 2 are solved and the parameters k1, k21,and kdgr are calibrated to the measurements in Figure 1. Arather close relationship between model and measurements waspossible for k21 5 0, which indicates that the desorption pro-cess from the soil matrix to the dissolved liquid phase is un-important and can be neglected. By neglecting k21, Equation1 becomes independent of Equation 2 and by a simple solutionof these two equations, total measured (c 1 S) concentrationin the batch can be calculated as

S 1 c k k1 1 2(k 1k )t1 dgr5 1 1 2 ·e (3)1 2c k 1 k k 1 k0 1 dgr 1 dgr

where c0 is the initial concentration, where no substance isadsorbed to the soil matrix. Equation 3 predicts the ratio be-

1088 Environ. Toxicol. Chem. 20, 2001 J. Jensen et al.

Fig. 2. Survival of adult Folsomia fimetaria and number of juvenilesproduced per test container after three weeks of exposure to differentconcentrations of dibutyl phthalate (DBP) (mg/kg dry weight). Ver-tical bars indicate standard error of the mean.

Fig. 3. Growth of male and female Folsomia fimetaria after threeweeks of exposure to different concentrations of dibutyl phthalate(mg/kg dry weight). Vertical bars indicate standard error of the mean.

Table 1. Summary of the effects of dibutylphthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) on survival, reproduction, and growth of thespringtail Folsomia fimetaria. Effect concentrations (mg/kg dry wt) with 95% confidence interval in parentheses

EC/LC10

DBP DEHP

EC/LC50

DBP DEHP

Survival—adultsSurvival—juvenilesReproductionGrowthNumber of cuticles

33 (250–370)11.3a

14 (10–46).100.5 (0.36–6.2)

.5,000

.1,000

.5,000

.1,000

.1,000

305 (256–45)19.4a

68 (48–185).10.10

.5,000

.1,000

.5,000

.1,000

.1,000

a No replicates were available in this study; therefore, confidence intervals cannot be estimated.

tween the total measured concentration (c 1 S) and the initialconcentration (c0) to be the same for different initial concen-tration values at fixed time. Although not verified throughmeasurement of, for example, pore-water concentrations, theprediction of Equation 3, involving a first-order removal (pre-sumably biodegradation, volatilization, or both) combinedwith a first-order adsorption to active sites in the soil matrix,seems rather close to the experimental results (Fig. 1).

Statistical analyses

Both EC10 and EC50 were estimated using a boot-strappingmethod [16,17]. The LC10 and LC50 were estimated usingprobit analysis [17]. Determination of no-observed-effect con-centration and lowest-observed-effect concentration for repro-duction and mortality and cuticle numbers was accomplishedwith one-way analysis of variance [17] after checking for anal-ysis of variance requirements with SAS/LABt [18]. Differ-ences between treatments and control were analysed by Dun-nett’s t test.

RESULTS

Lethal and sublethal effects of DEHP and DBP

The DEHP did not seem to be toxic, because it had noeffect on survival, reproduction, growth, or molting of juvenileF. fimetaria, whereas survival and reproduction of springtailswere strongly affected by DBP (Fig. 2). Little reproductionoccurred at DBP concentrations above 100 mg/kg, and at 500mg/kg almost all adults died. In some of the tests mortality incontrol samples exceed 20%, which lessened the certainty ofthe test results.

Juvenile mortality was similar when exposed in multidishes

with a hard soil surface and in microcosms with loose soil(data not shown). At DBP at 1, 5, and 10 mg/kg, one weekof exposure had no adverse effects on juvenile survival, where-as DBP at 25 mg/kg killed all juveniles after 1 d of exposure(Table 1). If the springtails survived, that is, at concentrationslower than 25 mg/kg, DBP did not affect their growth in theentire exposure period of 50 d (Fig. 3). Because of smallersize, growth stabilized earlier for males than for females. Dur-ing the first three weeks of exposure, DBP significantly re-duced molting frequency, that is, the average number of cu-ticles produced per individual during the test period (Table 2).However, because molting frequency seems to be highly var-iable for this batch of springtails, that is between 3.47 and 5.3for control animals, to what degree DBP may affect moltingof collembolans remains unclear.

Effects of aging on DBP toxicity

Although not completely consistent and significant, theanalysis of the results shows that one to two weeks of storagebefore exposure in general leads to higher EC10 and EC50values for reproduction and survival of adult springtails (Table3 and Fig. 4).

Chemical analyses of DBP in soil revealed that some dis-appearance did occur during the 28 d of storage at 208C (Fig.1). Disappearance of DBP in soil seems to become asymptoticduring the experiment, thus approaching a constant value.Therefore, a first-order removal relationship is not valid todescribe the data because this would result in a more distinctconcentration decrease towards zero. A simple mathematicalmodel may instead describe the measured concentrations (seeMaterials and Methods). The model predicts a half-life forDBP of more than 30 d. However, the half-life of dissolved

Sublethal effects of phthalates on collembolans Environ. Toxicol. Chem. 20, 2001 1089

Table 2. Effects of dibutyl phthalate (DBP) and di(2-ethylhexyl)phthalate (DEHP) on sprintail development. Mean number of cuticlesper juvenile during three weeks of exposure in multidishes. The 95%confidence intervals are given in square brackets. Numbers followedby different superscript letters are significantly different (Dunnett’s t

test, p , 0.05)

Tests concentration(mg/kg dry wt)

No. of cuticles(95% CI)

DBP0 (n 5 15)1 (n 5 12)5 (n 5 17)10 (n 5 13)

5.3a [4.9–5.7]4.1b [3.5–4.7]4.3b [3.8–4.8]4.2b [3.7–4.7]

DEHP0 (n 5 17)100 (n 5 15)250 (n 5 14)500 (n 5 16)1,000 (n 5 14)

3.47a [3.0–4.0]3.53a [3.0–4.0]3.36a [2.8–3.9]3.19a [2.7–3.6]3.36a [2.9–3.8]

Fig. 4. Reproduction of Folsomia fimetaria in aging studies. Numberof juveniles per test container after three weeks of exposure to differentconcentrations of dibutyl phthalate (DBP) (mg/kg dry weight). TheDBP-spiked soil was preincubated 1, 3, 7, 14, or 28 d before animalswere added to the soil. Vertical bars indicate standard error of themean.

Table 3. The EC10 and EC50 values (mg/kg dry wt) for dibutylphthalate (DBP) on adult survival and reproductive output afterdifferent storage times of DBP-spiked soil. Effect concentrations arebased on initial concentrations (mg/kg dry wt) after spiking, notmeasured concentrations at test start. The 95% confidence intervals

are given in parentheses

Storage(d) EC10 EC50

Adult survival037

1428

33.7 (8.2–66.5)29.5 (4.9–61.3)266 (175–320)268 (193–318).500

277 (177–440)362 (254–605)438 (380–504)473 (414–564).500

Reproduction037

1428

50.0 (218–63)13.9 (6.0–161)30.4 (9.1–87.3)131 (49–255)61 (213–176)

84.2 (5.1–96)125 (28.4–171)107 (73–171)245 (133-335)583 (138–1,027)a

a Estimated by fitting to a linear dose–response relationship beyondthe highest test concentration (500 mg/kg). For comparison, theEC10 fitted by the same linear dose–response relationship is 117mg/kg.

DBP is only approximately 5 d, and one month after additiondissolved DBP approached zero (Fig. 1).

DISCUSSION

Relatively high variation was observed in the data sets.Nevertheless, the results clearly show that DEHP was not toxicto collembolans even at high concentrations, because neithersurvival nor reproduction was effected. On the other hand,DBP was toxic for adult collembolans at concentrations downto 250 mg/kg and juvenile springtails may have been affectedbelow 1 mg/kg. Phthalates are considered moderately to highlytoxic to aquatic organisms. In general, the phthalates with analkyl chain length shorter than five carbon atoms are consid-ered most toxic, because of their higher water solubility [7].Di(2-ethylhexyl) phthalate has low water solubility and highKOW, thus absorption to soil is high, whereas DBP with anintermediate log KOW has a moderate mobility [19]. The majorexposure route of springtails in soil is likely to be through the

water phase. However, for soil-ingesting animals such as earth-worms, Belfroid et al. [20,21] showed that for highly lipophiliccompounds such as hexachlorobenzene (log KOW 5 5.7) in-testinal uptake may be of importance. The same may be truefor DEHP (log KOW 5 5.2). For example, Wams [22] estimatedthat approximately 90% of DEHP is readily adsorbed by soilparticles. Analysis of our results also indicates that a certainfraction of DBP is unavailable for biological degradation ortoxic action after a period of time (Fig. 4). The measured totalconcentration of DBP in the 500-mg/kg assay was still 265mg/kg 28 d after addition. Although this concentration is sim-ilar to the LC50 value of 277 mg/kg in the assays with nostorage, no mortality could be detected in soil stored for 28 d(Table 3). Nevertheless, more conclusive research is neededbefore any definite conclusions can be made about changes inbioavailability of phthalates.

Reduced availability may also hampers degradation. TheDBP degradation rates may differ among soil types and mi-crobial activity because availability and half-lives of organicsubstances are depending on the organic matter content ofsoils. Our test soil was a sandy agricultural soil not especiallyhigh in organic matter (,1.5%) and lower availability ofphthalates could be expected in soil richer in organic matter.The observed relatively long half-life of DBP in this study isnot consistent with other findings in the literature. Half-livesof DBP shorter than one week are generally reported, as longas the starting concentrations are not extremely high or thetemperature very low [6,23]. The microbial activity in the soilused in this experiment likely is not comparable to naturalfield soil because of the 808C defaunation process.

Adult and juvenile springtails are active in and on the soilsurface and therefore are exposed via similar pathways. So thedifference in sensitivity between the two life stages of spring-tails are rather explained by the larger surface to volume ratioof juveniles, which will increase the relative exposure anduptake of phthalates. Furthermore, the cuticle of adults, be-cause of a thicker wax layer, is likely to be more resistant todamage of membranes and associated proteins by phthalatesand other toxicants [15]. Collembolans molt regularly through-out their life, for example, every 3 to 5 d at 208C, and theanimals probably are most vulnerable to toxicants such as DBPin the period just after molting, when their new cuticle is softand thin.

1090 Environ. Toxicol. Chem. 20, 2001 J. Jensen et al.

We did not find any significant difference between the tox-icity of DBP on collembolans when exposed through a loosesoil and a compacted soil. When the soil is compressed a thinlayer of water appears on the surface of the soil. Uptake ofchemicals through the ventral tubus, which F. fimetaria con-stantly is protruding onto the surface, is then comparable inthe two situations. Unpublished results from our laboratorywith the pesticide dimethoate confirm that the toxicity is almostequal in the two exposure situations.

Very little information concerning phthalate toxicity to oth-er soil-dwelling organisms is found in the literature. Neuhauseret al. [24] determined LC50 values for several organic com-pounds, including dimethyl phthalate, in the European Eco-nomic Community artificial soil test on earthworms. ObservedLC50 values for four earthworms species ranged from 1,064to 3,335 mg/kg. In a filter paper contact test with earthworms,Neuhauser et al. [25] investigated dimethyl phthalate toxicityalong with four other phthalates. Dimethyl phthalate (45) wasthe most toxic followed by diethyl phthalate (30), di-iso-octylphthalate (8), and DEHP (1), with the relative acute lethaltoxicity given in parentheses. Dragonflies nymphs feeding onDEHP-contaminated sediment (587–623 mg/kg) showed sig-nificantly lower predation efficiency compared to control andalso a significant higher body concentration of DEHP (14.7mg/kg vs 2.9 mg/kg fresh weight in the controls) [26]. Sev-enteen phthalates were found to be nontoxic to female houseflies (Musca domestica) when applied at high doses topicallyor by injection [27]. However, antagonistic and synergisticinteraction was observed between DEHP and DBP with severalorganophosphates. The same ranking of toxicity of phthalatesin soils has been found in phytotoxicity tests. Hulzebos et al.[28] studied the phytotoxicity of several phthalates with lettuce(Lactuca sativa) and found EC50 values (growth) of 387 mg/kg for DBP and more than 1,000 mg/kg for DEHP. Herringand Bering [29] did not observe any inhibition on the growthof spinach due to exposure to DEHP, whereas dimethyl phthal-ate had a marked negative effect on the growth of seedlings.

Because of chemical inertness and low anaerobic degra-dation rates, high concentrations of DEHP and other phthalatesmay be found in sewage sludge [2,3]. This may lead to anexposure in the terrestrial environment in cases where sludgeis used for soil amendment. Although release to the environ-ment is large and adsorption to soil is high, only relativelylow concentrations of DEHP have been found in terrestrialecosystems. Wams [22] reported without further specificationsa concentration of 1.5 mg/kg in a contaminated Dutch soil,and Persson et al. [30] found 0.5 mg/kg in soil collected inthe vicinity of a Finnish DEHP manufacturing plant. In a Swed-ish clay loam soil the DEHP concentration was below detectionlimit [2]. No monitoring data on DBP levels in terrestrial eco-systems could be found. If one assumes a homogenous mixingof sewage sludge containing the maximum load of DEHP andDBP found in Danish sludge (170,000 and 26,000 mg/kg dryweight), the theoretical soil concentration of the first 10 cmis below 1.0 mg/kg for DEHP and 0.1 mg/kg for DBP.

Both the predicted and the few reported measured soil con-centrations of DEHP are significantly lower than the level atwhich effects of phthalates have been observed in the labo-ratory. The difference between the lowest-observed-effect con-centration and the predicted soil concentrations (PEC) is atleast three orders of magnitude, that is, EC50 . 1,000 andPEC , 1.0. Collembola only constitute a minor fraction ofthe ecosystems in soils and species may vary greatly in sen-

sitivity. Nevertheless, safety factors of 100 to 1,000 are gen-erally considered sufficient for full protection of both structureand functions of ecosystems [31]. This makes the risk of eco-logical effects caused by DEHP as a result of normal sludgeapplication negligible. For DBP the margin of safety is ap-parently lower. Our results have shown that adverse effects ofDBP may occur at 25 mg/kg dry weight. An environmentalconcentration of DBP of 25 mg/kg is nevertheless unlikely tooccur as a result of typical sewage sludge amendment, becauseit is at least 250 times higher than the predicted environmentalconcentration shortly after normal sludge application. Fur-thermore, phthalate present in sludge could be anticipated tobe less available to soil organisms than the pure chemicalsused in the present study. This is currently being investigatedin our laboratory, because extrapolation from the sandy testto soils rich in organic matter should be made with caution.

The observed effect on molting is difficult to intrepret.Folsomia fimetaria in stock cultures reaches maturity afterfive molts, normally after 19 to 20 d (M. Holmstrup, personalcommunication). Therefore, effects on molting frequency willretard sexual development. However, on basis of the knowl-edge we have today, extrapolating from the observed prolon-gation of molting in the laboratory to long-term ecologicalconsequences in the field is difficult. This is further compli-cated by the high variation in molting frequency observed inthe control animals of this study.

The potential of especially DEHP and to a lesser extentDBP to adsorb to the soil matrix may pose a risk of accu-mulation in soil or biota in cases of repeated application ofhigh loads of contaminated sewage sludge. Persson et al. [30]found DEHP concentrations of 2.8 mg/kg in soil arthropodscollected in the vicinity of a DEHP-producing factory locatedin a rural area of Finland. The soil concentration in the samearea was 0.5 mg/kg. Uptake of DEHP in plants is generallylow. Schmitzer et al. [32] found less than 0.001% of the applied14C-DEHP in plants harvested two growing seasons after soiltreatment. Aranda et al. [33] observed bioconcentration factorsfrom 0.01 to 0.03 for DEHP in fescue, lettuce, carrots, andchillies. Kirchmann and Tengsved [2] found that DBP andDEHP uptake in barley grain amounted to approximately 0.1to 0.2% of the initial added amount. Shea et al. [34] foundDBP uptake in plants from soil to be insignificant. This wasconfirmed by Muller and Kordel [35], who concluded thataccumulation of airborne phthalates in plant cuticles may beof importance, whereas root uptake from soil is negligible.Løkke and Rasmussen [36] concluded that even airborneDEHP and DBP were unlikely to lead to any significant ac-cumulation in plants. The small plant uptake of DEHP andDBP from soil supports the view of low bioavailability of thesephthalates in soil. In any case, biomagnification of phthalatesin higher trophic levels of terrestrial ecosystems is not likely,because an efficient metabolism and excretion of phthalates isobserved in many higher organisms, for example, fish andhumans. Earthworms are also able to metabolise phthalates[37].

On the basis of the data from this study and informationabout fate and occurrence of phthalates in the terrestrial en-vironment the conclusion is reached that long-term adverseeffects of phthalates on collembolans are not likely to occuras a result of a normal application of sewage sludge. Neitherdoes the available information indicate a significant risk ofphthalates accumulating in biota or soils. Therefore, the currentcutoff values for phthalates in Danish sludge seem sufficient

Sublethal effects of phthalates on collembolans Environ. Toxicol. Chem. 20, 2001 1091

to protect the terrestrial environment from any long-term ad-verse effects.

Acknowledgement—This study was done during the practical term ofJ. van Langevelde at the Department of Terrestrial Ecology. This workwas funded by the Centre for Sustainable Land Use and Managementof Contaminants, Carbon and Nitrogen, and the Danish EnvironmentalProtection Agency.

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