Sublethal parameters in morphologically deformed Chironomus larvae: clues to understanding their...

Freshwater Biology (1998) 39, 179–191

APPLIED ISSUES

Sublethal parameters in morphologically deformedChironomus larvae: clues to understanding theirbioindicator value

L . J A N S S E N S D E B I S T H O V E N , * † P. N U Y T S , † B . G O D D E E R I S ‡ A N D F. O L L E V I E R ††Catholic University of Leuven, Laboratory of Ecology and Aquaculture, Naamsestraat 59, B-3000 Leuven, Belgium‡Royal Belgian Institute of Natural Sciences, Freshwater Biology, Vautierstraat 31, B-1040 Brussels, Belgium

*Author to whom correspondence should be sent.

S U M M A R Y

1. Parameters of condition between normal and deformed fourth instar larvae ofChironomus gr. thummi (Kieffer, 1911) were compared in four populations: one reference(PE) and two metal-polluted sites (NP and SCH) in the River Dommel, and one sitepolluted by domestic sewage and copper in the River Ijse (NEI).2. The site PE ranked lowest for metal body burdens, deformities, mortalities andemergence duration, while SCH and NEI ranked highest.3. Deformed and non-deformed larvae most often did not differ in length and weight(in situ end-points for growth); when differences occurred, deformed larvae tended to besmaller.4. The energy content and dry weights in one population (NEI) were lower in normallarvae than in the weakly deformed ones. The percentage of ash-free dry weight waslower in deformed larvae of the polluted Dommel sites, compared to the normal ones.5. The in vitro emergence rate (end-point for development of fourth instars) for thereference population PE, both in its own sediment and in artificial cellulose substrate,was better than for the other sites. In two populations (NEI, SCH) the development ofdeformed larvae in their own sediment was slower, with higher mortality, than for thenormal larvae. In one population (NP), normal and deformed larvae survived anddeveloped equally well. The emergence rates of the respective populations weresimilarly ranked when the larvae were raised in an artificial cellulose substrate.6. Elution peaks of alleged metal-binding proteins were lower in deformed larvae fromSCH and NEI, but higher in deformed larvae from NP, than in normal larvae.7. A different development rate and mortality of deformed larvae in non-adaptedpopulations and the possibility of metal adaptation, as in site NP, may modulate thefinal outcome of deformity frequencies, thus having an impact on the biomarker valueof deformities in benthic midge larvae.

Introduction

Brinkhurst, Hamilton & Herrington (1968) reportedmidge larvae (Chironomidae) with aberrant mouthparts from St Lawrence Great Lakes (Canada) thatwere polluted with heavy metals and toxic organics.

© 1998 Blackwell Science Ltd 179

This work and other circumstantial data (overview inJanssens de Bisthoven, 1995) indicate that deformitiesin benthic midge larvae are a reflection of sedimentpollution by heavy metals and toxic organic com-

180 L. Janssens de Bisthoven et al.

pounds and not of pollution by domestic sewage orhigh organic loads (Hamilton & Saether, 1971;Warwick, 1980a, b, c; Lenat, 1993). The study ofchironomid deformities in North America has focusedon the Great Lakes and their pollution burdens (Hare& Carter, 1976; Warwick et al., 1987; Warwick & Tisdale,1988; Dermott, 1991), with detailed descriptions of themorphological deformities (Warwick, 1985). In Europe,attention has focused on small lakes, canals and rivers,with emphasis on temporal patterns, sublethal charac-teristics of mentum deformities and their impact onpopulation dynamics (Frank, 1983; Wiederholm, 1984;Van de Guchte & Van Urk, 1989; Van Urk, Kerkum &Smit, 1992).

Krantzberg (1992) mentions growth and reproduc-tion impairments as consequences of biochemical dis-ruptions after exposure to contaminated sediments.Effects at the organism or the population level includeslower development (Wentsel, McIntosh & Atchison,1978a; Wentsel, McIntosh & McCafferty, 1978b), lowerreproductive capacity (Reynoldson, Thompson &Bamsey, 1991) and deformities (Warwick, 1985).Because the effects are often interrelated, knowledgeof one may bring insights into other effects at a higherlevel of organization, and vice versa.

The present study investigates the occurrence ofdeformities (gross anatomical aberrations at the indi-vidual level) in Chironomidae in relation to othersublethal effects at different levels of biologicalorganization. In addition the possible signal functionof anthropogenic contamination at the ecosystem levelis considered. Parameters related to performance(growth, development and energy content) and thepresence of metal-binding proteins are assessed. Toler-ance of aquatic organisms to heavy metals has oftenbeen explained by the induction of metallothioneins(MTs) in many aquatic organisms (Roesijadi, 1992). Ifthe presence of MTs is a measure of metal tolerance,their measurement in normal and deformed Chirono-mus larvae could provide clues about whetherdeformed individuals are, on average, more or lesstolerant to heavy metals than normal larvae. Further-more, measurements of bioaccumulation of selectedheavy metals may give clues about the possible toxicagents responsible for the stress (Maltby & Crane,1994). Populations were studied in Belgium and theNetherlands at sites on the River Dommel (Meusecatchment; heavily polluted with cadmium and zincfrom a point pollution source) and on the River

© 1998 Blackwell Science Ltd, Freshwater Biology, 39, 179–191

Ijse (Schelde catchment; polluted by domestic sewageand copper).

Materials and methods

Sampling and deformity screening

Fourth instar Chironomus gr. thummi (Kieffer, 1911)larvae were collected with a 500-µm handnet in theRiver Ijse (tributary of the River Dijle, Schelde drain-age) at Neerijse (coded ‘NEI’) (23 August 1993) andin the River Dommel (tributary of the River Meuse)at Peer (PE), Overpelt (NP) and Schaft (SCH) (16August 1993) for the study of emergence and mortality,metal-binding proteins, ash-free dry weight (AFDW),dry weights (DW), energy content, deformity screen-ing and metal measurements.

The sites NEI, PE and NP are located in Belgium,while the site SCH is located in the Netherlands. Therivers Ijse and Dommel are first- or second-orderlowland rivers. The site NEI is situated 3 km from theconfluence of the River Ijse with the River Dijle andis polluted by organic enrichment and householdeffluents. The site PE is situated in the upstream sectionof the River Dommel and is relatively unpolluted. Thesite NP is situated 300 m downstream of the confluenceof the River Eindergatloop with the River Dommel.The Eindergatloop discharges high loads of zinc andcadmium originating from industry into the RiverDommel (Janssens de Bisthoven, 1995; Postma, 1995).The site SCH is situated 15 km downstream of NP onthe River Dommel.

From each population, thirty reference larvae werefixed for later microscopical observation and screeningof deformities (mounting method, see Moller Pillot,1984).

From each site, about 500 fourth instar larvae werekept alive in their own aerated water in the laboratory.From this stock, living larvae were held one-by-oneunder the dissecting microscope at magnification 50 3

between two microscope glass slides and positionedso that the ventral side of the head was visible.Anomalies in the mentum were noted and the larvaewere assigned to a ‘normal’, a ‘weakly deformed’(split medial mentum tooth, unusual tooth shapes andnumbers) and a ‘strongly deformed’ larval group, with‘Kohn gap’, after a deformity described by Kohn &Frank (1980). The three larval groups were used inthe emergence bioassays. Any possible handling stresswas the same for all morphological groups.

Sublethal parameters in deformed Chironomus 181

Since the analyses of emergence, metal-binding pro-teins, energy content, DW and AFDW were eitherdestructive or occurred on live larvae which weredifficult to measure, another large batch of larvae froma previous study on the site NEI, sampled in June1992, was used for the comparison of lengths betweennormal and deformed larvae.

Metal concentrations

The total concentrations of the heavy metals cadmium,lead, copper and zinc were measured for each popula-tion in whole body tissues of fourth instar Chironomuslarvae, according to the method described in Janssensde Bisthoven, Timmermans & Ollevier (1992). Compar-isons of bioaccumulation between normal anddeformed larvae were reported in Janssens deBisthoven et al. (1992), Janssens de Bisthoven (1995)and Janssens de Bisthoven, Postma & Parren (1998).

Sizes

Body lengths were measured in normal and deformed(mentum, mandibles, pecten epipharyngis and anten-nae) fourth instar larvae. Length was measured onpreserved (70% ethanol), decapitated larvae (headshad previously been used for microscopy) as thedistance between the implantation of the anal setaeand the anterior side of the base of the anteriorparapods when the body was stretched between twomicroscopic slides (measured on millimetre paper,graded with a fine pen up to 0.1 mm). Dry weightswere measured on a precision balance (0.01 mg) after48 h drying of fresh material at 80 °C.

Ash-free dry weight and energy content

The AFDW was measured according to Lindegaard &Maehl (1993) on sediments and on the surplus larvaewhich were not used in the emergence tests: normaland weakly deformed larvae from all sites. Pelletswere dried overnight at 100 °C, weighed, dried for 1 hat 300 °C and then for 4 h at 550 °C and weighed. TheAFDW was expressed as a percentage of DW. Theenergy content was only measured in normal andweakly deformed larvae from NEI, because of a limitednumber of larvae. Seven pooled larvae in triplicatewere dried for 48 h at 80 °C and mixed with 0.5 g


benzoic acid into a compact pellet and combustedaccording to Thornton & Wilhm (1974).

Emergence and mortality

From the sites PE, NP, SCH and NEI, fifteen normal,fifteen weakly deformed and fifteen strongly deformedfourth instar Chironomus larvae were placed in duplic-ate plastic tanks (18 3 12 3 11.5 cm). The normal lar-vae were assigned to a set-up with cellulose as artificialsubstrate and a set-up with sediments from the respect-ive sampling sites. The deformed larvae were raisedin their own sediment. The densities were equivalentto 700 individuals m–2. The larvae in cellulose wereput in 1.1 L ‘artificial water’ (solution of distilled waterwith 25.2 mg Na1 L–1, 6.5 mg K1 L–1, 86.3 mg Ca21

L–1, 9.4 mg Mg21 L–1) and cellulose substrate made of200 Whatman filters [41 Cat. no. 1441 090 with lowash content (0.01%)] mixed with 3 g commercial troutfood (trouvit). To each tank, 65 g cellulose wet weightwas added. The other (normal and deformed) larvaewere put in their own river water and sediment (130 gdry weight sediment) without the addition of food.The sediment was dried for 3 days at room temper-ature prior to the experiment in order to kill all othermacroinvertebrates present. The tanks were kept in awater bath at a constant water temperature of 16 °Cand a photoperiod of 16 h light : dark period with30 min of twilight. The emerged imagos were counteddaily at 10.00 h.

Metal-binding proteins

Normal and strongly deformed larvae from NP, SCH,NEI and PE (only normal) were analysed for theoccurrence of metal-binding proteins. Larvae (fifteenpooled larvae per population) were washed in 0.1 mM

ethylenediaminetetraacetic acid (EDTA) solution andsubsequently in demineralized water. The larvae wereplaced in 1 mL Tris-HCl buffer pH 7.4 and homogen-ized at 0 °C under N2-gas in an ice bath. The homogen-ate was transferred to centrifuge vials. The volumesof the different vials were brought to the same levelwith Tris HCl buffer pH 7.4. The centrifuge vials wereput in a cooled rotor and centrifuged at 43 000 g for65 min. The supernatant (2.5 ml) was preserved at280 °C. Sephadex G-75 is able to separate globularproteins with a molecular weight between 3000 and80 000 Da. The Sephadex G-75 gel was first saturated


with Tris-HCl buffer for at least 3 h. Elution wasperformed with 10 mM Tris-HCl pH 8.6 and a velocityof 5 mL h–1. The absorbance of the eluent was meas-ured at 254 nm. Five mL fractions of the eluent werecollected in a fraction collector placed in a refrigeratorat 4 °C. The column was continuously cooled at 5 °C.Calibration occurred with protein fractions with amolecular weight ranging from 13 700 Da (ribonucleinA) to 67 000 Da (albumin).

Statistical analyses

The deformity frequencies were compared withexpected values in a Chi-squared test. The metalconcentrations were tested for stability of varianceand normality and compared between sites with one-way ANOVA and post-hoc Tukey test at 5% signific-ance. The same was applied to the larval DW, lengthsand arcsin√—

x-transformed frequencies of AFDW oflarvae and sediments, which were compared amongstand within sites, between normal and deformed larvae.Energy contents were corrected to DW and comparedwith the non-parametric Kruskal–Wallis and mediantests. Emergence was graphically expressed as follows(Fig. 1): at each day when an increase in number ofemerged adults in either of both replicates was noted,a datapoint was plotted as the mean 6 SD for the tworeplicate tanks of the cumulative total of emergedadults for that particular day. In order to test differ-ences between replicates, the non-parametricFriedman test (Siegel & Castellan, 1988) was appliedon k related samples (each score for each tank wasrelated in time). In order to test differences betweensites and between morphological groups, one-wayANOVA, followed by post-hoc Tukey tests was per-formed for five variables: (i) log10(x 1 1)-transformedday of first emergence; (ii) log10(x 1 1)-transformedday of last emergence; (iii) log10(x 1 1)-transformedday when half of the surviving population hademerged (integer value); (iv) arcsin√—

x-transformed fre-quency of the total number of emerged imagos (sur-vival); and (v) arcsin√—

x-transformed frequency of thenumber of emerged imagos at day 10. The first peaksin the protein elution profiles were compared bymeans of the non-parametric median test.

Results

Metal concentrations and deformities

All sites differed in metal concentrations (ANOVA,F3,50 5 12.29–19.5 (range), P , 0.00001; Table 1). The


larvae from the reference site PE had significantlylower concentrations of copper and zinc than thelarvae from SCH and NEI (Cu) and the larvae fromNP and NEI (Zn), respectively. Further, the NEI larvaehad higher copper concentrations than the larvae ofall other sites. The larvae from SCH contained higherconcentrations of lead and cadmium than the larvaeof the other sites (post hoc Tukey tests, P , 0.05).

The larvae from PE showed the lowest deformityfrequencies of all sites (Table 2). This population isconsidered to provide a relatively unpolluted refer-ence. The frequencies in mentum deformities increasedin the river Dommel from Peer to Schaft in a down-stream direction (Kohn gap in the mentum: significantat 10% level; abnormal number of teeth in the mentum:significant at 3% level). The other deformity frequen-cies were not different amongst the sites. There is acorrespondence between whole body concentrations oftrace metals in the larvae (Table 1) from the respectivepopulations and the trends seen in the deformityfrequencies (Table 2). The deformity frequencies in NPwere lower than the other polluted sites for most typesof deformities.

Sizes

Normal NEI larvae were larger than the deformedones at a significance level of P 5 0.058 (Table 3). MeanDW of normal and deformed larvae did not differwithin each site (Table 4). The larvae from PE weighedless than the larvae from the other Dommel sites NPand SCH.

Ash-free dry weight and energy content

No differences in percentage AFDW were foundbetween normal and deformed larvae in each site.There was an overall site effect in the percentageAFDW of the larvae and the sediments; the sites beingranked PE , NEI , SCH , NP (Table 4).

In the batch of larvae used for the assessment ofenergy content (NEI), normal larvae had significantlylower DW (0.68 6 0.70 mg) than deformed larvae(0.85 6 0.30 mg) (three groups of seven pooled larvaefor each morphological condition, ANOVA, F1,4 5

10.44, P 5 0.03). Therefore, the energy content of thenormal and deformed larval groups was corrected forDW (energy per g larva). The energy content correctedfor DW of the normal larvae (1858.50 6 63.84 J g–1)


Fig. 1 Cumulative emergence curves (n 5 two replicates offifteen larvae, mean 6 SD) for fourth instar Chironomus gr.thummi larvae. (a) Normal larvae in cellulose substrate (in thePeer (PE) condition, one additional sixteenth larva wasdetected a posteriori after count of emerged imagos).(b) Normal larvae in their own sediment. (c) Normal anddeformed larvae in cellulose and sediments of the River Ijse atNeerijse (NEI), (d) in the River Dommel at Schaft (SCH), and(e) at Overpelt (NP).

Table 1 Concentrations (mean 6 SD mg kg–1 DW) of total copper, lead, cadmium and zinc in fourth instar Chironomus gr. thummilarvae (3–5 larvae pooled) from the River Dommel and the River Ijse (n 5 14, except for SCH (Cu and Pb) and PE (Zn),where n 5 (13)

River Site Cu Pb Cd Zn

Dommel PE 27.30 6 11.48 6.23 6 5.41 0.71 6 1.06 171.90 6 60.79NP 44.60 6 16.88 27.52 6 20.81 6.76 6 6.49 681.63 6 368.40SCH 53.08 6 17.22 72.21 6 43.45 31.14 6 27.79 1337.30 6 1128.14

Ijse NEI 81.47 6 30.83 32.74 6 34.76 0.98 6 0.84 440.67 6 142.86



Table 2 Deformity frequencies (%) in larvae (n 5 30) ofChironomus gr. thummi collected in the River Dommel and theRiver Ijse. K 5 Kohn gap; S 5 weakly split medial mentumtooth; MI 5 strongly split medial mentum tooth; MNR 5

mentum with unusual number of teeth; MA 5 mandibledeformity (unusual number of teeth); UH 5 antennal segmentof unknown homology; PM 5 premandible deformity(splitting, aberrant shape). Statistical significance is indicatedby an asterisk

Deformity type

Site K S MI MNR MA UH PM

PE 0.0 27.6 3.4 7.1 0.0 7.1 2.5

NP 7.7 26.9 3.8 25.0 3.7 0.0 0.0SCH 18.5 39.1 4.3 44.0 5.5 10.0 0.0

NEI 14.8 22.2 10.7 42.8 6.90 5.5 0.0

χ2 6.07 1.57 0.56 9.20 1.98 2.79 3.00P 0.10* 0.67 0.90 0.03** 0.58 0.43 0.43

Table 3 Mean lengths (mm) of fourth instar larvae(n 5 number measured) from the River Ijse (NEI, collected on2 March 1992). N 5 normal; K 5 mentum Kohn gap;M 5 mentum other deformities; MD 5 deformed mandibles;ALL 5 all deformities pooled. Some larvae had multipledeformities

Morphology n x 6 SD (mm)

N 98 14.21 6 1.68a

K 16 13.38 6 1.67M 17 13.97 6 1.40MD 20 13.89 6 1.65ALL 53 13.76 6 1.57b

a,b difference: one-way ANOVA, F1,146 5 3.62; P 5 0.058

Table 4 Mean 6 SD of larval dryweights (DW) and of the percentages ofash-free dry weight (AFDW) in thesediments (n 5 3) and in normal (N)and weakly deformed (WD) fourth-instar larvae of Chironomus gr. thummifrom the River Ijse (NEI) and the RiverDommel (PE, NP, SCH). n 5 number ofreplicates of two pooled larvae

Morphological DW (mg) % AFDW % AFDWSite condition n larvae larvae sediment

PE N 5 0.38 6 0.20a 88.1 6 5.2 2.1 6 0.1a

NP N 15 0.95 6 0.35b 94.1 6 2.5a 6.2 6 0.5b,c

WD 5 0.62 6 0.20 88.6 6 2.8

SCH* N 15 0.65 6 0.34 91.2 6 4.9a 4.4 6 3.2WD 2 0.38 6 0.11 78.9 6 7.8

NEI N 15 0.69 6 0.31b 80.5 6 11.9b 3.6 6 0.8b,d

WD 14 0.65 6 0.31 77.2 6 12.6b

*No comparison between N and WD, due to low n for WD.One-way ANOVA on DW: F6,64 5 3.03; P 5 0.01; post-hoc Tukey differences betweena and b (P , 0.05).One-way ANOVA on arcsin√—

x-transformed AFDW frequencies: larvae, F6,64 5 10.28,P , 0.05; sediments, F3,8 5 7.9, P , 0.009; post-hoc Tukey differences between a and b,and c and d (P , 0.05).


was lower than for the deformed larvae(2176.62 6 415.80 J g–1) (Kruskal–Wallis test, H 5 3.86,P , 0.05; median test, χ2 5 6, P 5 0.01).

Emergence and mortality

All morphological conditions, including the stronglydeformed larvae, were able to pupate and developinto emerging adults. Differences between replicateswere found for the normal larvae of Peer in thesediment (Friedman test: χ2

1,19 5 15.00, P , 0.0001),the normal larvae of Overpelt (χ2

1,24 5 9.94,P , 0.0016), the weakly deformed larvae of all sites(χ2

1,19–26 5 19–22 (range), P , 0.002) and the stronglydeformed larvae of Schaft and Ijse (χ2

1,22–26 5 9–15.20,P , 0.0001) (Fig. 1).

There was an overall site effect on the day oflast-emerged imago, the total number of emergedindividuals (survival) and the number of emergedadults at day 10 (Table 5). The survival of the PE-population, reared in cellulose and in the sediment,and the NP-population of weakly deformed larvaewas higher than the deformed and cellulose-rearedNEI-populations. Also, the PE-population in cellulosesurvived better than the weakly deformed SCH-larvaeand the normal SCH-larvae in cellulose. The trend ofbetter development and survival in PE and NP isalready apparent in the emergence data at day 10(Table 5): these populations had a higher number ofemerged adults (normal and deformed) at day 10 andat the end of the experiment than the SCH and NEIpopulations (ANOVA, F3,24 5 10.38 (end), 21.19 (day


Table 5 Parameters of development and emergence for fourth instar Chironomus gr. thummi originating from four lowland riversites and raised in cellulose (only normal larvae) and in their own sediments (normal and deformed larvae) until day of lastemergence. Mean 6 SD for two replicate tanks (n 5 15 larvae) per site for each morphological condition. N 5 normal larvae;WD 5 weakly deformed larvae; SD 5 strongly deformed larvae; N, WD and SD refer to larvae raised in sediment; BL 5 normallarvae in cellulose. (1) day of first emergence; (2) day of last emergence; (3) halftime day of emergence; (4) total percentageemerged larvae; and (5) % emerged larvae at day 10

Parameter

Site Condition (1) (2) (3) (4) (5)

PE BL 4.5 6 0.7c 13.5 6 0.7c 9.0 6 0.0 100.0 6 0.0 70.0 6 14.1N 6.0 6 1.4a 16.5 6 3.5a 9.0 6 0.0 93.3 6 9.4 66.7 6 18.9

NP BL 4.0 6 0.0 11.5 6 0.7f 7.0 6 1.4 73.3 6 0.0 53.3 6 9.4N 4.0 6 0.0 23.5 6 0.7 10.0 6 1.4 80 6 28.3 40.0 6 9.4WD 4.0 6 0.0e 24.5 6 2.1f 9.5 6 0.7 93.3 6 9.4 56.7 6 14.1SD 5.5 6 2.1 24.0 6 2.8 12.0 6 1.4 76.7 6 14.1 23.3 6 4.7

SCH BL 5.0 6 1.4d 18.5 6 0.7 9.5 6 0.7 36.7 6 4.7 23.3 6 4.7N 5.0 6 1.4 21.0 6 2.8f 8.5 6 0.7 63.3 6 4.7 46.7 6 0.0WD 6.0 6 1.4d 23.5 6 0.7b,d 12.5 6 0.7 43.3 6 14.1 16.7 6 4.7SD 3.5 6 0.7 22.5 6 4.9 10.0 6 1.4 56.7 6 23.6 23.3 6 4.7

NEI BL 6.0 6 2.8b,d,f 12.0 6 2.8d 7.0 6 4.2 26.7 6 9.4 20.0 6 0.0N 4.0 6 0.0 16.0 6 9.9 6.5 6 3.5 56.7 6 23.6 36.7 6 4.7WD 11.5 6 10.6b,d,f 20.0 6 2.8b,d,e 12.5 6 12.0 23.3 6 4.7 10.0 6 14.1SD 8.0 6 0.0b,d,f 15.5 6 4.9b,d 10.0 6 0.0 26.7 6 0.0 16.7 6 4.7

One-way ANOVA on log10(x 1 1)-transformed variables (1), (2) and (3):(1) F13,14 5 5.97, P 5 0.001;(2) F13,14 5 6.24, P 5 0.0008; post-hoc Tukey differences at 5% significance indicated by subscripts ab, cd, etc.One-way ANOVA on arcsin√—

x-transformed variables (4) and (5): (4) F13,14 5 2.7, P 5 0.04. No post-hoc Tukey difference.

10), P , 0.05). Within the PE and NEI-populations themorphological conditions did not show differences forthe five parameters (P . 0.05). The normal NP-larvaereared in cellulose emerged in half the time taken bythe other NP-larvae reared in sediment (ANOVA,F3,4 5 40.29, P 5 0.001). The normal SCH-populationreared in sediment showed higher emergence at day10 and at the end of the experiment (higher survival)than the deformed larvae and the larvae reared incellulose (ANOVA, F3,4 5 16.17, P 5 0.01).

Metal-binding proteins

Typically, four peaks appeared in the protein elutionprofiles (Fig. 2). A median test on the percentages offirst peak proteins, corrected to total proteinabsorbance, did not reveal differences between sitesand morphological groups. Comparisons were thenperformed on absolute absorbances of the first peak,which lies in the 7000–10 000 Da range, typical for themolecular weight of MTs. The elution profile of thenormal larvae of PE could be considered as a controlagainst the other populations. It was characterized by


a high third peak, as compared to the other threepeaks. In NP, normal larvae exhibited the same elutionprofile as in PE, with the majority of the proteinssituated in the third peak. However, deformed larvaeshowed a strong first peak compared to the otherpeaks (d.f. 5 1, χ2 5 10.27, P , 0.001). The elutionprofiles of the deformed larvae of SCH and NEI weresimilar to the PE elution profile, while the profilesof the normal larvae showed a higher first peak(d.f. 5 2, χ2 5 10.92, P , 0.001). For the normal SCH-larvae, zinc was more concentrated in the heavierprotein fractions.

Discussion

Low deformity frequencies, whole body concentra-tions of heavy metals and emergence periods inChironomus larvae from PE justified the choice of thispopulation as an unpolluted reference. In naturalChironomus gr. thummi populations from Belgian low-land rivers, fourth instar larvae, classified as normaland deformed, often did not differ in size. Whendifferences occurred, deformed larvae generally


Fig. 2 Sephadex G-75 elution profile and zinc concentrations in the supernatant of fourth instar Chironomus gr. thummi larvae: (a)Normal larvae from the River Dommel at Peer (PE, reference site). (b) Normal and deformed larvae from the River Ijse at Neerijse(NEI). (c) Normal and deformed larvae from the River Dommel at Schaft (SCH), and (d) at Overpelt (NP). Peaks 1–4 are referredto in the text.

showed slightly lower values for AFDW and length.However, DW and energy contents for a batch oflarvae from NEI were higher in deformed larvae thanin normal ones. Changes in energy content in an animalare indicative of changes in the relative proportion ofhigh energy molecules, such as lipids or carbohydrates(Thornton & Wilhm, 1974). The deformed NEI-larvae,which were under stress, as attested by the copperbody burdens, the deformity frequencies and theemergence data, might have invested energy in lipidsas a storage for pollutants. AFDW, on the other hand,is a measure of biomass or carbon content (Lindegaard& Maehl, 1993) and was equal in normal and deformedindividuals within each site. The inter-population dif-ferences in larval AFDW were reflected in the inter-site differences of sediment-AFDW (Table 4), hencethe suggestion that larval size in terms of carboncontent may be closely related to the amount of carbon


as a food source in the sediments, as pointed out byRasmussen (1985).

Test end-points such as development, survival andgrowth (Pascoe, Kendall & Green, 1989; Reynoldson,1994) are often used to evaluate toxic stress in benthicaquatic invertebrates. Wentsel et al. (1978b) demon-strated a reduced and delayed emergence of adults ofChironomus when the larvae were exposed to a sedi-ment containing high concentrations of cadmium (alsoshown by Pascoe et al., 1989), zinc and chromium. VanUrk & Kerkum (1986) concluded that deformed larvaedeveloped more slowly than normal larvae in thesame environment and thus remained in the sedimentslonger, while normal larvae developed to emergingadults. However, Janssens de Bisthoven & Ollevier(1989) could not demonstrate experimentally a differ-ence in emergence rate between normal and deformedlarvae. In the present study, normal larvae either


showed a better survival and a higher developmentrate (SCH and NEI) than deformed larvae, or nodifference was noted (NP). This shows that differencesbetween normal and deformed larvae are more site-or population-specific than initially expected. TheSchaft and Ijse larvae showed a better survival innatural compared to artificial substrate. An interestinggradient was also noted for the weakly deformedlarvae, from fast development and high survival inOverpelt, to slower development and lower survivalin Schaft and Neerijse, respectively. For the stronglydeformed larvae, the same pattern was observed butwas less obvious, because the Schaft microcosmsshowed a high disparity within replicates, due tothe inherent variability in sediment composition. Theranking of the sites PE, SCH and NEI for emergenceand survival rates coincided with the site ranking fortrace metal accumulations and the deformity frequen-cies. However, site NP showed lower deformity fre-quencies than expected from the metal body burdensand a better survival and faster development rate.

The lower fitness of deformed individuals in SCHand NEI may be interpreted in various ways: deformedindividuals may be genetically less fit, they may behampered in their feeding behaviour because of theiraberrant mouthparts or they may have an impairedmetabolism because of a higher sensitivity to accumu-lated pollutants or a higher accumulation of pollutants.Combinations of these are possible. Under experi-mental conditions, however, deformed individualswere still able to develop to adults. If deformed larvaedevelop more slowly in the field, while normal onestend to emerge faster, it could generate a relativeincrease in deformity frequencies in the population.On the other hand, higher mortalities amongst thedeformed larvae could temper that trend, and this hasconsequences for biomonitoring. Deformity frequen-cies in the winter tend to be higher than in the summer(authors’ unpublished data), probably because of thedifferent population dynamics of normal anddeformed larvae.

Biochemical and physiological variables have beenproposed as potential sublethal indicators of contamin-ant stress. Frank (1981) did not find any difference inGADPH activity between Chironomus plumosus larvaefrom polluted and unpolluted waters, althoughdeformed individuals from the polluted site showedslightly lower values. In the same study, mentum-deformed C. plumosus and C. riparius larvae exhibited


lower values (only significant for C. plumosus) of lactatedehydrogenase activity, which is a zinc metallo-enzyme (Parisi & Valle, 1969). Aquatic invertebratescan develop tolerance to pollutants (Klerks & Weis,1987). Krantzberg & Stokes (1989) demonstrated thatchronic exposure to elevated metal levels of Chirono-mus larvae may result in the development of metaltolerance. When exposed to toxic stress, tolerant popu-lations showed better growth than non-tolerant ones.In aquatic insects, surplus metals may be stored ininert granules (Brown, 1982), or metal regulation mayconsist of binding surplus metal ions by specificproteins and releasing the metals when needed formetabolic use. These proteins, called metallothioneins(MTs), play an important role in the regulation ofessential metals, used as co-factors in biochemicalreactions and in the detoxification of non-essentialmetals (Roesijadi, 1992). Yamamura et al. (1983) identi-fied cadmium-binding proteins in cadmium-exposedlarvae of Chironomus yoshimatsui. Chironomus gr.thummi larvae in the River Dommel downstream ofOverpelt (NP) are metal-tolerant because of a historicalexposure of many generations (estimated to be at least200 generations) to cadmium and zinc pollution froman industrial site (Postma, 1995; Groenendijk, Postma& Admiraal, 1996). A higher concentration of a lightmolecular protein fraction (peak 1 in Fig. 2: ™ 7500 Da),with relatively higher zinc concentrations, was foundin the populations originating from cadmium- andzinc-contaminated sites (NP and SCH), compared tothe control population (PE). The molecular weight,the co-occurrence with a trace metal (Zn) and the lowvalues in the unpolluted reference site lend supportto the conjecture that this protein fraction is a metal-binding protein belonging to the protein family of theMTs (Petering & Fowler, 1986). Metal redistributionduring sample preparation is possible and co-occur-rence of zinc with the protein fractions is still no proofof a ligand relationship between them (Roesijadi,1992). However, the zinc concentration profiles overthe different protein fractions, including the heavierones, showed similarities to the findings of Bengtsson,Ek & Rundgren (1992) for Dendrobaena octaedra. Intwo of the three sites (SCH and NEI) normal larvaecontained relatively more (alleged) MT than thedeformed specimens. In contrast, the normal larvaefrom NP contained less MT than the deformed larvae.Since only the protein elution profile of one homogen-ate cytosol sample of larvae per population was


Fig. 3 Conceptual model representing diverging growth and development of normal and deformed chironomid larvae in a non-adapted population and hypothesis of positive feedback towards higher bioaccumulation in deformed larvae. Question marks areplaced where the differences between normal and deformed larvae are not directly observed but rather inferred, or where thedifferences are not clear. The length of the development stages is not to scale.

assessed, these differences should be interpreted withcaution. Nevertheless, it is interesting to note that,specifically in the NP population, where lowerdeformity frequencies and no differences in growth,emergence and mortality between normal anddeformed larvae could be found, the deformed larvaeshowed a higher MT peak than their normal counter-parts. It was also this site which contained the highestamount of organic matter in its sediments (Table 4).These results suggest that the deformed individualsin the NP population had less difficulty in investingextra energy in the synthesis of MTs than deformedindividuals from other populations. One of the reasonsfor this may be better food conditions, as attested toby the amount of organic matter in the sediments.However, adaptation of the population to a fairlyconstant and chronic input of cadmium and zincfrom an industrial plant in Overpelt should also beconsidered. Such adaptation of chironomid larvae tometal stress has been reported by Wentsel et al. (1978b),Krantzberg & Stokes (1989) and Gerhardt & Janssensde Bisthoven (1995).

Normal and deformed larvae co-occur within popu-lations. It is hypothesized here that this may be dueto different individual exposures to contaminants atthe micro-habitat scale, as suggested by Dickman,


Brindle & Benson (1992) and Janssens de Bisthovenet al. (1992), and to small genetic differences: indi-viduals that are less able to neutralize metal stressby, for example, synthesizing metal-binding proteins,would undergo sublethal effects with the eventualappearance of deformities. When selection pressureincreases (less food, enhanced competition, inadequatesubstrate), growth, development rate and survivalmay further diverge between the groups of larvae.The observed sublethal effects in deformed larvaeare summarized in Fig. 3. A positive feedback tobioaccumulation is hypothesized for the larvae withimpaired growth and/or development. The smallersize of deformed larvae may enhance the concentrationof bioaccumulated pollutants because of allometricsize changes (the ratio of surface to volume increases,Krantzberg & Stokes, 1989), although the essentialmetals, such as copper and zinc, are expected to remainunder homeostatic control within certain concentrationranges. Further, if deformed larvae mature over alonger time than normal larvae, they will be exposedto the contaminated sediments for longer, which couldresult in more bioaccumulation (Krantzberg & Stokes,1989). This positive feedback hypothesis could par-tially explain why, in some populations, deformedlarvae show more bioaccumulation of metals than


normal larvae (Janssens de Bisthoven et al., 1992;Janssens de Bisthoven et al., 1998). The same wasreported for PAHs (Dickman et al., 1992). The possi-bility of metal adaptation or of lower fitness andresulting higher mortality in deformed chironomidlarvae may have an effect on the final outcome ofdeformity frequencies. These will be governed by asubtle balance between early instar recruitment ofdeformed larvae to the population and differentialmortality and emergence in normal and deformedlarvae. That may be different in adapted populations.This study demonstrated that caution should be takenin interpreting deformities in midge larvae as bio-markers for sediment toxicity, as the deformity per-centages may depend on: (i) environmental pressures;and (ii) the history of a population with the possibilityof adaptation to heavy metals.

Acknowledgments

L.J.d.B. was recipient of an IWONL-scholarship(Instituut voor aanmoediging van WetenschappelijkOnderzoek in Nijverheid en Landbouw, no. 870053)during part of the study. We thank the ‘De NayerSchool’ in Mechelen, Professor Dr De Ley and Pro-fessor Dr E. Kuhn (KULeuven) for support and advicein the MT analysis, Dr K. De Mol, P. Parren and R.Van Houdt (KULeuven) for practical help. We are alsoindebted to Dr J. Postma, M. Buckert-de Jong and DrC. Davids (University of Amsterdam) for help in themetal analyses. The manuscript was greatly improvedthanks to the comments of Professor Dr R. Mercx, DrF. Volkaert (KUL), Dr A. Gerhardt (Lund University,Sweden) and Dr P. Mitchell (Staffordshire University).

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(Manuscript accepted 15 August 1997)

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