ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 39, 10—20 (1998)ENVIRONMENTAL RESEARCH, SECTION B

ARTICLE NO. ES971603

Short-Term Toxicity of Lindane, Hexachlorobenzene, and Copper Sulfateto Tubificid Sludgeworms (Oligochaeta) in Artificial Media

Michael Meller,*,1 P. Egeler,* J. Rombke,* H. Schallnass,* R. Nagel,- and B. Streit‡* ECT Oekotoxikologie GmbH, Bo( ttgerstrasse 2-14, D-65439 Flo( rsheim/M, Germany; -Technische Universita( t Dresden, Institut fu( r Hydrobiologie,

D-01062 Dresden, Germany; and ‡ J. W. Goethe-Universita( t Frankfurt, Abt, O$ kologie und Evolution, D-60054Frankfurt/M, Germany

Received April 23, 1997

have been standardized on a national level (EPA, 1994;

1To whom correspondence should be addressed.

The toxicity of lindane, hexachlorobenzene, and copper sulfateto Tubifex tubifex and Limnodrilus hoffmeisteri was determinedusing an easily applicable and standardizable 72-h short-termtest system. It was designed for the quick assessment of sublethaland lethal effects of sediment-associated chemicals on the worms.An artificial sediment based on the Artificial Soil according toOECD Guideline No. 207 was used as test medium. The dataconfirm the common view that oligochaetes are highly tolerant oflethal effects. However, sublethal effects were detected at con-siderably lower concentrations than found for lethal effects. TheEC50 values for autotomy (172 mg/kg dry wt sediment) andsediment avoidance (217 mg/kg) for T. tubifex exposed to lin-dane-contaminated sediment were, for example, more than fivetimes lower than the LC50 value (''1000 mg/kg). The no-ob-served-effect concentration for reworking activity (8 mg/kg) wasmore than 125 times lower than the LC50 . Tubificids thus turnedout to represent useful test organisms for the assessment of theecotoxicological hazard potential of chemicals in the sedimentcompartment, because the sublethal effects not only affect theindividual, but can influence the population levels and, conse-quently, the composition of the benthic community. ( 1998 Aca-

demic Press

Key Words: artificial sediment; tubificids; autotomy; sedi-ment avoidance; reworking activity; lindane; hexachlorobenzene;copper sulfate.

INTRODUCTION

Over the last few years, many representatives of fresh-water benthos fauna were examined for their suitability astest organisms for the testing of chemicals, as well as for theecotoxicological assessment of sediment contamination.Most of the tested species were insects (e.g., Lydy et al., 1990;Fleming et al., 1994), oligochaetes (e.g., Ammon, 1985;Reynoldson et al., 1991; Phipps et al., 1993), and amphipods(e.g., Becker et al., 1995). To date, only a few test methods

100147-6513/98 $25.00Copyright ( 1998 by Academic PressAll rights of reproduction in any form reserved.

ASTM, 1995; BBA, 1995), and none have been acceptedinternationally, e.g., as an OECD guideline. Important cri-teria for choosing the test species include (1) the ecologicalrelevance of the organism with respect to its local and globaldistribution and its functional role within the ecosystem;(2) its tolerance to a wide range of abiotic sediment charac-teristics; (3) easy handling and culturing (Hill et al., 1993). Itis well known that tubificids fulfill these requirements (e.g.,Wachs, 1967; Hill et al., 1993). Another major selectioncriterion is the sensitivity of the test species. Tubificids dohave a reputation for being very tolerant of chemical stress(Chapman and Brinkhurst, 1984), but it has been found thatthis is not the case regarding sublethal effects (Keilty et al.,1988a, b; Reynoldson et al., 1991). Furthermore, as sedi-ment-ingesting endobenthic animals, they are subject toexposure to sediment-bound substances by all potentialroutes (overlying water, interstitial water, and ingestion ofsediment). Taking into consideration all of these character-istics, tubificids are regarded as suitable tools for ecotoxico-logical research.

Depending on the source of natural sediment, the pres-ence of micropollutants as well as indigenous organisms caninfluence toxicity tests (Reynoldson et al., 1994; Suedel andRodgers, 1994). In addition, comparison of results fromvarious laboratories is further complicated by the widevariety of abiotic sediment characteristics. It is thereforerecommended that an artificial test medium be used tostandardize whole-sediment toxicity tests.

The purpose of this study was to develop a test method toassess not only the lethal effects but more notably thesublethal effects caused by chemicals. The acute toxicity testwith ¹ubifex tubifex described by Ammon (1985) was usedas the basis for this work. Particular attention was paid toquick and simple performance of the test as well as the use ofan artificial sediment. For a better evaluation of the vari-ations between different species, two tubificid species (¹.tubifex, ¸imnodrilus hoffmeisteri) were used in all of theexperiments carried out in this study.

METHODS chlorocyclohexane) represents an insecticide of global distri-

SHORT-TERM TOXICITY TO TUBIFICIDS 11

Culture Conditions

Two common tubificid species were selected for labor-atory culture: ¹. tubifex (Muller) and ¸. hoffmeisteri(Claparede). The animals have been continuously kept inthe laboratory of ECT Oekotoxikologie GmbH (Florsheim,Germany) since March 1994. They were originally suppliedby FEE Fischfutter Etzbach (Mechernich-Bergheim, Ger-many). According to the supplier, the animals originatedfrom the River Mass, including its tributaries in Belgium.The worms were kept in semistatic single-species culturesusing an artificial sediment and reconstituted water accord-ing to OECD Guideline No. 203 (1983) as the overlyingmedium. Prior to setting up the cultures, mature animalswere identified according to Wachs (1967) and Brinkhurst(1971). The identification also was confirmed by P. Rod-riguez, Universidad del Pais Vasco, Bilbao. The cultureprocedure is described in detail by Egeler et al. (1997b).

Artificial Sediment

An artificial sediment based on the Artificial Soil accord-ing to OECD Guideline No. 207 (OECD, 1984) was used asculture and test medium. Artificial Soil was slightly modi-fied for use as sediment for tubificids by Egeler et al. (1995,1997a). The substrate was composed (percentages refer todry weight) of 2% sphagnum peat; 22% kaolinite clay(kaolinite content '30%); 76% quartz sand (grain size:more than 50% of the particles in the range 0.05—0.2 mm);approximately 0.05%. CaCO

3(pulverized, chemically pure).

The air-dried peat was shredded in a chaff-cutter (grain size41 mm). A suspension of the required amount of peatpowder in demineralized water (11.5]dry weight of peat)was prepared using a high-performance homogenizing de-vice. The pH of this suspension was adjusted to 5.5$0.5with CaCO

3. To establish a stable microorganism compon-

ent, the suspension was gently stirred for 48 h at roomtemperature. After this conditioning period the pH was6.0$0.5. To obtain a homogenous sediment with a watercontent of approximately 46% of the dry weight of thesediment, the suspension was mixed with the other constitu-ents and demineralized water. The pH of the completeartificial sediment studied be 6.0$0.5. More details anda complete description of the characteristics of this artificialsediment are published elsewhere (Egeler et al., 1997b).

Test Substances

The main criteria for selecting test substances were a cer-tain tendency to associate with sediments and ubiquitousoccurrence in freshwater sediments. Therefore, two organicchemicals, one with a moderate and the second with a highlipophilicity, and a metal were chosen. Lindane (c-hexa-

bution in aquatic systems with a moderate low P08

of 3.63(Rippen, 1991). It was supplied by Sigma Chemical Com-pany (St. Louis, MO; 99% c-hexachlorocyclohexane).The second organic chemical, hexachlorobenzene (HCB)('99% GC, Fluka Chemika, Buchs, Germany) is a widelydistributed hydrophobic pollutant that was used as a fungi-cide, and is generated as a by-product of chlorinated hydro-carbons. It has a logP

08of 5.72 (Rippen, 1991). Copper

sulfate (CuSO4 )

5H2O, p.a. quality, Roth, Karlsruhe, Ger-

many) was chosen as the metal compound, as it is fairlywater soluble [230.5 g/liter, 25°C (Royal Society of Chem-istry, 1994)]. Nevertheless, copper is known to be highlyparticle associated in aquatic systems (e.g., van de Plassche,1994).

Test System

The test system was designed as a 72-h static whole-sediment system using artificial sediment and reconstitutedwater according to OECD Guideline 203 (OECD, 1983) asthe overlying medium. The ratio of sediment :overlyingwater was 1 : 4, following Hooftman et al. (1993).

Spiking Procedure and Test Vessel Setup

Test substances were introduced into the system by spik-ing a bulk sediment for each test concentration, from whichdifferent replicates were subsampled before the overlyingwater was added. Lindane was dissolved in n-hexane andsubsequently diluted with n-hexane to prepare an applica-tion solution for each required concentration. The quartzsand fraction of the sediment was moistened with a definedvolume of this application solution in a glass vessel. Afterthe solvent had evaporated, the coated quartz sand wasthoroughly mixed with the other sediment constituents.Application of HCB followed the same spiking procedure,where cyclohexane replaced n-hexane as a solvent. Applica-tion of copper sulfate was performed using demineralizedwater as a solvent. The quartz sand and kaolinite fractionsof each bulk sediment were mixed with the peat suspension.Then, the aqueous application solutions were added to thisslurry. Concentrations of application solutions and addedvolumes were calculated to obtain a water content of 46%of dry weight of the sediment.

To disperse the test substances homogeneously within thesediment, the spiked bulk sediments were gently stirred for1 h at room temperature using a magnetic stirring device.Subsequently the sediment was immediately subsampled tothe test vessels (100-ml glass tubes, height 14.5 cm,H 3.4 cm), and the overlying water was added using anantiturbation device to minimize turbation of sediment par-ticles. Each test vessel contained a 2-cm layer of spikedartificial sediment and 62.5 ml of reconstituted water. Before

the test organisms were added, the test vessels were incu- required, since the test conditions were identical to culture

12 MELLER ET AL.

bated for a 24-h equilibration period in a climatic chamberunder the described test conditions (Table 1).

Range-finding tests with test substance concentrationsbetween 0.01 and 1000 mg/kg (dry weight of the artificialsediment) spaced by a constant factor of 10 according to theOECD Guideline No. 207 (1984) were performed to deter-mine the concentrations for the definitive tests. Range-find-ing tests with lindane indicated a wide range betweensublethal and lethal effects. Since the aim of the study was tocompare all described endpoints within one experiment, itwas necessary to use a constant factor of 5 for the geometri-cal concentration series in the definitive test with lindane. Inthe cases of lindane and HCB the test design includeda solvent control; this was prepared by adding only n-hexane and cyclohexane, respectively.

Exposure Period

Test organisms were sampled from the cultures by sievingthe sediment through a 1-mm mesh which retained the adultworms. Using the negative thermotaxis of the tubificidsaccording to the method of Wachs (1965), the animals wereremoved from the sieves into a vessel containing recon-stituted water. Only undamaged, actively creeping adults(with fully developed clitellum) of uniform size (5$2 cm)were selected as test organisms (Ammon, 1985). The wormswere transferred randomly into the test vessels using a softsteel forceps or a pipette. An acclimation period was not

TABLTest Conditions for Conducting Short-Term Whole-Sediment To

Parameter

Test system Static short-term whole-sediment testTemperature 20$2°CPhotoperiod 16L : 8D; 4100 lxTest vessel 100-ml glass tubes, height 14.5 cm, H 3

(ratio 1 : 4)Sediment Artificial sediment based on Artificial SoOverlying water Reconstituted water according to OECDApplication The test substance is applied into the sedTest design/concentrations Range-finding test: (0.01), 0.1, 1, 10, 100

a solvent control using 1 replicate perusing 4 replicates per treatment

Test organisms Adults with fully developed clitellum of uLoading 10 animals per test vesselSediment quality pH and redox potential at the beginningOverlying water quality pH, redox potential, hardness, conductivEquilibration period 24 h referring to test conditionsExposure period 72 h, no feeding, no aerationEndpoints Reworking activity, sediment avoidance,Validity Sediment avoidance, autotomy, mortalit

should not differ from controlEvaluation Usual statistical treatment to calculate E

conditions.The test vessels were then incubated under the described

test conditions, in a climatic chamber (Table 1). After 72 hthe animals were removed from the sediment. The sedimentwas suspended with the overlying water by shaking the testvessels. The test organisms could be easily sieved from thissediment suspension using a 1-mm mesh. According to therecommendations of the SETAC Workshop on SedimentToxicity Assessment (Hill et al., 1993), the sediment—watersystem was characterized at the beginning and the end ofexposure period (see Table 1).

To assess sublethal effects on the behavior of the tubi-ficids, the vessels were checked visually after approximately0.5—1 h and again after 24 and 48 h. At the end of the test,sublethal and lethal effects were recorded. Morphologicalchanges of the worms were determined using a binocularmicroscope.

Endpoints and Data Analysis

Table 2 gives an overview of endpoints and their classi-fications. The effect rates in precentages (mortality,autotomy, sediment avoidance) for each concentration (i)were calculated according to the following equations:

Mortality ini"

+ dead animals ini

+ animals exposed ini

]100.

E 1xicity Test Using Tubifex tubifex and Limnodrilus hoffmeisteri

Conditions

.4 cm containing a 2-cm layer sediment and 8 cm overlying water

il according to OECD Guideline No. 207 (Egeler et al., 1997b)Guideline No. 203iment, 1000 mg/kg (sediment dry weight) including a control and, if necessary,treatment; definitive test: concentrations based on results of range finding

niform size (5$2 cm), undamaged and actively creeping

and end of testity, ammonia, and dissolved oxygen at the beginning and end of test

autotomy, mortalityy (10% in the control; reworking activity in the lowest concentration

C50

and LC50

, e.g., probit analysis

TABLE 2Description of Endpoints for Short-Term Whole-Sediment Toxicity Test Using Tubifex tubifex and Limnodrilus hoffmeisteri

Endpoint Description

Reworking activity The animals leave traces while digging through the sediment (so-called galleries). To estimate the reworking activity, a qualitativecomparison on replicate level was performed. The reworking activity of all animals in one test vessel was defined as reducedwhen the visible number of galleries was distinctly lower than in the control vessels (Fig. 1).

Sediment avoidance According to Keilty et al. (1988a) a worm was considered unburrowed if more than an estimated 75% of its body was visible onthe sediment surface.

Autotomy Autotomy starts with a local constriction of the circular muscles, which can be seen macroscopically. The segments behind theconstriction are completely autotomized (Kaster, 1979). Autotomy was defined as all animals showing either constrictionand/or loss of segments.

Mortality Animals were recorded as dead when they did not respond to a gentle mechanical stimulus to the front end.

Autotomy ini" dose response after probit transformation, an arcsinus

SHORT-TERM TOXICITY TO TUBIFICIDS 13

+ dead animals ini#+ animals showing Autotomy in

i+ animals exposed in

i

]100.

Sediment avoidance ini"

+ animals showing Sediment avoidance ini

+ animals surviving ini

]100.

The LC50

and the EC50

and their corresponding 95%confidence intervals were determined using probit analysis(Finney, 1971). To perform probit analysis, concentrationswith 0% effect were set as 1 of n (n"number of exposed orsurviving animals) and concentrations exhibiting 100% ef-fect as (n!1) of n. If fewer than three data points between0 and 100% effect were available, or if there was no linear

FIG. 1. Schematic figure of a test vessel showing reduced reworking actvessel was defined as reduced when the visible number of galleries was dist

transformation of the data was performed. The LC50

orEC

50was then calculated by nonlinear interpolation be-

tween the two concentrations that bracketed theLC

50/EC

50value. In this case the 95% confidence interval

was determined using an independent binomial test (Peltierand Weber, 1985). Since the n of the endpoints mortality,autotomy, and sediment avoidance was small, a no-ob-served-effect concentration (NOEC) for each endpoint wasdefined as the highest concentration exhibiting an effect(10%, and lowest-observed-effect concentration (LOEC),as the lowest concentration with an effect 510%.

The animals leave traces while digging through the sedi-ment (so-called galleries). The quantity of galleries dependson the activity of the worms in the sediment. To estimatethis ‘‘reworking activity,’’ a qualitative comparison on repli-cate level was performed. The reworking activity of allanimals in one test vessel was defined as reduced when thevisible number of galleries was distinctly lower than in thecontrol vessels (Fig. 1).

ivity and a control vessel. The reworking activity of all animals of one testinctly lower than in the control vessels.

The maximum effect of the endpoint ‘‘reworking activity’’ LOEC ‘‘mortality’’; therefore, it was possible to calculate

14 MELLER ET AL.

was met when all replicates of a specific concentration levelindicated reduced reworking activity (see Table 2, Fig. 1).NOEC ‘‘reworking activity’’ was defined as the highestconcentration with no replicate demonstrating reduced re-working activity, and LOEC reworking activity, as the nexthigher concentration.

RESULTS

In both range-finding and definitive tests, sediment-bound HCB did not cause any sublethal or lethal effects onthe two tubificid species up to 1000 mg/kg. Table 3 gives anoverview of EC

50and LOEC/NOEC values obtained from

the experiments with lindane and copper sulfate. Lindaneinfluences the reworking activity of ¹. tubifex and ¸. hof-fmeisteri at sediment concentrations of 40 and 8 mg/kg,respectively. From a threshold value of 200 mg/kg onward,lethal effects as well as morphological changes (autotomy)and sediment avoidance were observed (see Fig. 2). Since themortality rates, even at the highest concentration, were farbelow 50%, no LC

50values were calculated (Table 3).

In the experiments with copper sulfate-spiked artificialsediment, ¸. hoffmeisteri appeared to be more sensitive tothe toxicant than ¹. tubifex (Fig. 3). As demonstrated withlindane, copper sulfate caused sublethal effects in both spe-cies, although the range between sublethal and lethal effectswas a lot smaller (Table 3). Since the number of deadanimals was only recorded once, at the end of the test, therate of sediment avoidance could only be calculated for 72 h.The test design did not allow an estimation of lethal effectsduring the exposure period. Copper sulfate caused sedimentavoidance in ¹. tubifex at several concentrations below the

TABLOverview of Sublethal and Lethal Effects of Lindane or Copper

T. tubifex and L

Lindane

Endpoints E(L)C50

95% Clb LOEC

¹. tubifexReworking activity — — 40Sediment avoidance 217 156—309 200Autotomy 172 40—1000 200Mortality '1000 — 200

¸. hoffmeisteriReworking activity — — 8Sediment avoidance 224 164—314 200Autotomy 200 c 200Mortality '1000 — 200

aAll data refer to nominal concentrations in mg/kg sediment dry weight.b95% confidence interval.cCI not determined, because EC

50value is just an approximation.

rates of sediment avoidance for these concentrations duringthe exposure period. Figure 4 indicates that most animalsinitially burrowed into the spiked sediment and thensome of them returned to the sediment surface during thefollowing 24 h.

In all presented experiments, lethal as well as sublethaleffects demonstrated a dose—response relationship. Only inone case was this relationship not found: the rate ofautotomy of ¸. hoffmeisteri as a response to lindane did notincrease at concentrations higher than the threshold value(Fig. 2). The presented EC

50value (Table 3) therefore rep-

resents the threshold concentration.

DISCUSSION

There was no obvious difference between the two tubificidspecies with respect to the toxicity of sediment-bound lin-dane. In accordance with Wiederholm et al. (1987) ¸. hof-fmeisteri seems to be slightly more sensitive to copper sulfatethan ¹. tubifex. HCB caused no adverse effects in the whole-sediment test (this study), even though it is known thattubificids quickly and strongly accumulate HCB (Oliver,1987; Egeler et al., 1997a). Referring to Nebeker et al. (1989),HCB is also not toxic to different benthic invertebrates inwater-only tests at concentrations in the range of its watersolubility. These authors presumed that the cause of the lackof acute effects on aquatic invertebrates is that HCB, basedon its high lipophilicity, is localized in lipids within theorganism and may not be available.

Although the sensitivity of tubificids to chemical stress isa matter of controversy, they fit most criteria for test speciesselection, e.g., ecological relevance, tolerance of a wide range

E 3Sulfate Associated with Artificial Sediment on the Tubificids

. hoffmeisteria

Copper sulfate

NOEC E(L)C50

95% Clb LOEC NOEC

8 — — 125 62.540 547 250—1000 250 12540 601 500—1000 500 25040 '1000 — 1000 500

1.6 — — 125 62.540 392 250—500 500 25040 349 294—403 125 62.540 516 458—581 500 250

FIG. 2. Dose—response relationship of lindane. Plots on the left-hand side show toxicity of lindane to ¹. tubifex and plots on the right to¸. hoffmeisteri. Error bars represent the SD of the four replicates in definitive tests.

SHORT-TERM TOXICITY TO TUBIFICIDS 15

FIG. 3. Dose—response relationship of copper sulfate. Plots on the left-hand side show toxicity of copper sulfate to ¹. tubifex and plots on the right to¸. hoffmeisteri. Error bars represent the SD of the four replicates in definitive tests.

16 MELLER ET AL.

FIG. 4. Time-dependent sediment avoidance of ¹. tubifex in copper sulfate-spiked artificial sediment.

No-Observed-Effect Concentration (NOEC) Values Obtainedfrom 72-h Whole-Sediment Testsa

Test organism NOECb Reference

Copper sulfate¹. tubifex 62.5 This study¸. hoffmeisteri 62.5 This studyC. riparius (first-instar larvae) 125.0 Meller et al. (1997)

Lindane¹. tubifex 8.00 This study¸. hoffmeisteri 1.60 This studyC. riparius (first-instar larvae) 0.08 Meller et al. (1997)

aTests with larvae of Chironomus riparius were conducted using the sameartificial sediment and the same spiking procedure used in this study(Meller et al., 1997). Presented values represent the NOEC ‘‘reworkingactivity’’ (tubificids) and the NOEC ‘‘body length’’ (chironomids).

bRefers to nominal concentration in mg/kg sediment dry weight.

of sediment characteristics, simple handling and culturing. the action of lindane as an insecticide, the 20 or 100 times

SHORT-TERM TOXICITY TO TUBIFICIDS 17

Therefore, the following discussion focuses on four areas:(1) the sensitivity of sublethal effects; (2) the power andinterpretation of the different endpoints; (3) the reproduci-bility of the results and (4) the definition of validity criteriafor a standardized performance of the test. The suitability ofartificial sediment for ecotoxicological tests, as well as theadvantages and disadvantages of breeding and keeping thetwo tubificid species, is discussed in detail by Egeler et al.(1997b).

Sensitivity

Oligochaetes are well known for being highly tolerant oflethal effects induced by chemicals (e.g., Wiederholm et al.,1987). Distinct physiological mechanisms are discussed asa possible explanation. The chloragog tissue, typicallyfound in oligochaetes, seems to have a key function withrespect to the availability of toxicants within the organisms(e.g., Hagens and Westheide, 1987; Klerks and Bar-tholomew, 1991; Fischer and Molnar, 1992). Nevertheless,the detection of sublethal effects reveals that tubificids areaffected by chemicals far below lethal threshold levels (e.g.,Keilty et al., 1988a, b; Reynoldson et al., 1991). Larvae ofChironomus riparius (Meigen) are well known as sensitivetest organisms (e.g., Hill et al., 1993). A comparison oftoxicity data from this study and results of experiments withcopper sulfate-spiked artificial sediment using first-instarlarvae of C. riparius (Meller et al., 1997) gives a first impres-sion of the tubificids’ high sensitivity (Table 4). Because of

TABLE 4

respectively lower NOEC for chironomids in lindane-spikedsediment is not unexpected (Table 4).

Endpoints

Based on its body size and resistance to mechanical stress¹. tubifex is easier to handle than ¸. hoffmeisteri. ¸. hoffmeis-teri is more inclined to injury, usually accompanied bybruising. To avoid autotomy from injury (Kaster, 1979) ¸.hoffmeisteri must be handled with extreme care. Autotomy

as a morphological response to chemical exposure isalso known for other oligochaetes (e.g., Roberts and

Reproducibility

18 MELLER ET AL.

Dorough, 1984; Rombke and Knacker, 1989). However,until now, this reaction has not been assessed as a quant-itative endpoint in toxicity tests. The data presentedprove that autotomy for both species of tubificids is avalid endpoint.

From the investigations of McMurtry (1984), it is knownthat tubificids have the ability to avoid stress induced bychemicals associated with the sediment. Keilty et al. (1988a)observed in experiments with endrin-spiked sediment that¸. hoffmeisteri and Stylodrilus heringianus initially burrowedinto the sediment and later returned to the sediment surface.Such avoidance behavior is neither restricted to endrin norconfined to both species of aquatic oligochaetes examinedby Keilty and co-workers (1988a). It was possible to provethis behavior also with ¹. tubifex in the present experimentsusing copper sulfate (see Fig. 4). Based on the presumptionthat autotomy leads to death of the single individualafter prolonged exposure, mortality and autotomy directlyaffect the population density of tubificids. Also, thecomposition of species within the benthic community can beindirectly changed below lethal threshold levels, e.g.,as a result of sediment avoidance, via migration anda greater threat of being eaten (Ahlf, 1995). Additionallythere is the risk of increased transmission of chemicalswithin the food chain if the animals accumulate the substan-ces and are more easily preyed on because they are on thesediment surface.

Keilty et al. (1988b) noticed a change in the reworkingbehavior of tubificids during longterm studies, usinga labor-intensive 137Cs marker layer burial technique, atconcentrations three to five magnitudes lower than theLC

50(96 h). However, the experiments with lindane- or

copper sulfate-contaminated sediment described here dem-onstrate that the reworking activity of the examined tub-ificids was already reduced within 72 h. Using this method,no complicated or costly equipment was necessary to assesschanges in reworking activity. For further investigations itshould be easy to establish a method to quantify reworkingactivity (e.g., using digital video analysis). Then a NOECcould be determined by a usual statistical treatment (e.g.,ANOVA).

Keilty and co-workers (1988b) also observed that de-creased reworking activity was accompanied by a reductionof worm biomass. They therefore presumed this effect toreflect decreased feeding rates. Lotufo and Fleeger (1996)recently demonstrated in experiments with pyrene- andphenanthrene-contaminated sediments that reproduction of¸. hoffmeisteri was reduced at those concentrations at whichthey had observed a decrease in the egestion rate. As a re-sult, a reduction in reworking activity affects the populationdynamics of tubificids and should therefore not be ignoredin the risk assessment of chemicals.

The reproducibility of the results is sufficiently ensured.All of the definitive tests performed met the expectations ofthe range-finding tests. This assumption was also confirmedin a second experiment using copper sulfate, where only themortality rate was recorded (data not provided). In thatexperiment, the LC

50for ¸. hoffmeisteri was almost exactly

reproduced, whereas the LC50

for ¹. tubifex with 627 mg/kgwas approximately two times lower than the results de-scribed here. These variations, which remain within therange of biological variability, could also be clarified bypossible diverse ages of the test organisms. A synchronizedculture would be essential to perform toxicity tests on ani-mals of the same age and physiological state. However, atthe time of the investigations, such a culture had not beenestablished.

Validity Criteria

In addition to common criteria of toxicity tests, thefollowing validity criteria for the standardized performanceof whole-sediment tests using tubificids are required:(1) With reference to the endpoint autotomy, the testorganisms should not exhibit any signs of injury at thebeginning of the test. (2) Rates of sediment avoidance,autotomy, and mortality must be less than 10% in thecontrol. (3) To determine a NOEC, the reworking activityat the lowest concentration should not differ from that ofthe control.

CONCLUSIONS

Oligochaetes are commonly known to be highly tolerantof chemically induced lethal effects (e.g., Wiederholm et al.,1987) and the results presented here confirm this for the twotubificid species ¹. tubifex and ¸. hoffmeisteri. However,based on the tubificids’ wide spectrum of potential re-sponses to chemical stress, the sublethal endpoints are farmore suitable for assessment of the ecotoxicological hazardpotential of chemicals and qualify tubificids as a useful toolin sediment ecotoxicology, especially because these sub-lethal effects not only affect the individual, but can influencepopulation levels and, consequently, the composition ofbenthic community in the medium term.

This test system ensures quick and simple handling.It allows the evaluation of sublethal effects, which arefar more sensitive than lethal effects. In addition, it hasalready been successfully used in distinguishing the effectsof various storage conditions on the toxicity of naturalsediment spiked with lindane (Hanne, 1997) and artifi-cial sediment spiked with copper sulfate (Walther,1997).

ACKNOWLEDGMENTS Vera( nderungen in der Ultrastruktur von Chloragog- und Darmzellen in

SHORT-TERM TOXICITY TO TUBIFICIDS 19

This study was sponsored by the Federal Environmental Agency (Berlin,Germany), R&D Project 106 03 106. Thanks to Dr. Pilar Rodriguez,Universidad del Pais Vasco, Bilbao, who confirmed the identity of theworms, and to Susan and Denis Squires and Rachel Gallagher, Universityof Cardiff, who improved the English. A special thanks to all the friends ofMichael Meller for a great farewell.

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