The influence of nonylphenol on life-history of the earthworm Dendrobaena octaedra Savigny: linking effects from the individual- to the population-level

ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 58 (2004) 147–159

Rapid Communication

The influence of nonylphenol on life-history of the earthworm Dendrobaena octaedra Savigny: linking effects from the individual- to the population-level T.H. Widarto,a,b M. Holmstrup,c and V.E. Forbesa,* a

Department of Life Sciences and Chemistry, Roskilde University, Universitetsvej 1, PO Box 260, Roskilde DK-4000, Denmark b Department of Biology, Bogor Agricultural University, Indonesia c Department of Terrestrial Ecology, National Environmental Research Institute, Denmark Received 5 November 2003; received in revised form 8 March 2004; accepted 18 March 2004

Abstract We conducted a study to look at the effects of nonylphenol (NP) on the life-history of the parthenogenetic earthworm, Dendrobaena octaedra. During a 196-day study, we observed that the growth rate of juveniles and the percentage of worms producing cocoons were the only traits significantly affected by NP, while the total number of cocoons produced was marginally affected. Despite some fairly large changes in the average values of individual life-history traits caused by NP, the effects were difficult to detect statistically due to large interindividual variability. A declining trend was observed for population growth rate (l) with increasing NP concentration, but the decline was not statistically significant. The percent reduction in l was less than the percent reduction in the most sensitive life-history trait (fecundity). An elasticity analysis showed that l was more sensitive to changes in survival than to changes in reproductive traits. However, neither juvenile nor adult survival were affected by NP, and decomposition analysis showed that the minor changes in l were mainly caused by effects of NP on time to first reproduction, time between reproduction events and fecundity. The present study suggests that extrapolation from laboratory studies to population effects in the field may be greatly enhanced by combining ecotoxicological and demographic methods. r 2004 Elsevier Inc. All rights reserved. Keywords: Dendrobaena octaedra; Nonylphenol; Population growth rate; Decomposition and elasticity analysis

1. Introduction Nonylphenol (NP) is an intermediate breakdown product of NP ethoxylates used as nonionic surfactants for detergents, emulsifiers, lubricants, oil additives, pesticides, herbicides, and toiletries (Nimrod and Benson, 1996). Because of its estrogenic features (e.g., Purdom et al., 1994; White et al., 1994; Jobling et al., 1996; Yadetie et al., 1999), its high toxicity (McLeese et al., 1981; Granmo et al., 1989), its persistence under anaerobic conditions (Maguire, 1999), and its tendency to bioaccumulate (Ekelund et al., 1990; Johnson et al., 1998), some European countries have banned the use of alkyl phenol ethoxylates in household cleaning products and industrial cleaning applications (Renner, 1997). Nonylphenol mostly reaches the terrestrial environment *Corresponding author. Fax: +45-46-74-30-11. E-mail address: [email protected] (V.E. Forbes). 0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.03.006

as sludge produced by sewage treatment facilities. Some sludge is dumped into the soil as fertilizer to improve its quality. Since the concentration of NP has been determined to reach relatively high concentrations of between 22 and 4000 mg NP/kg dry-weight sludge (Bennie, 1999), examination of its potential effects on soil dwelling organisms is relevant. Earthworms are very important organisms in soil due to their improvement of soil structure and because they contribute to the breakdown of organic matter and release of plant nutrients. Dendrobaena octaedra is an epigeic and eurytopic earthworm living in coniferous forests of Canada, northern United States, and northern Europe where it is an important primary decomposer (Edwards and Bohlen, 1996). The species is an obligatory parthenogenetic worm (Terhivuo and Saura, 1996) with an apomictic mode of meiosis (Omodeo, 1955). Most of the available information on the effects of toxic chemicals on invertebrates is biased toward

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measures of individual survival during short-term exposures to high concentrations or individual growth and reproduction during long-term exposures to low concentrations (e.g., Calow et al., 1997). However, more studies have been conducted recently to look at the effects on population dynamics since an important aim of ecotoxicology is to assess effects of chemicals on populations (e.g., Munns et al., 1997; Walthall and Stark, 1997; Hansen et al., 1999; Moe et al., 2001; Kuhn et al., 2001). Determining the extent to which responses to toxicants measured at the individual level are indicative of responses measured at the population level is a key issue in this context. The relationship between individual and population responses is neither simple nor linear. However, the application of life-history models can lead to valuable insights into these relationships (Calow et al., 1997; Hansen et al., 1999; Forbes and Calow, 1999). The life-history traits of D. octaedra exposed to sublethal concentrations of NP were measured under laboratory conditions. Survival, growth, time to first reproduction, cocoon production, time between production of cocoons, cocoon incubation time, and cocoon viability were investigated by following newly hatched juveniles kept in contaminated soil. The aims of our study were to determine which individual trait was the most/least sensitive to NP, to interpret the effects on individual traits in relation to effects on population growth rate (l) and to identify which trait(s) was (were) mainly responsible for the effects on l: The effects on l were analyzed using a two-stage life-cycle model (Calow and Sibly, 1990). In addition, the relative sensitivity (i.e., elasticity) of l to changes in each of the life-history traits was examined.

2. Materials and methods 2.1. Test animals This experiment used a laboratory culture of D. octaedra from a Danish population originally collected in a coniferous forest near Silkeborg, Denmark. Cocoons collected from the culture were incubated in Petri dishes layered with wet filter paper. Undeveloped cocoons (yellow color) were incubated at 20
C, whereas developed cocoons (red color) were incubated at 15
C. The incubation of the developed cocoons at the low temperature was used to slow down their development so that they would hatch at the same time as the undeveloped cocoons. After hatching, the juvenile worms were placed in a Petri dish and maintained at 5
C until enough individuals were obtained to initiate the experiment. As an obligatory parthenogenetic lumbricid, D. octaedra produces cocoons without prior exchange of

sperm. Clones are therefore common in nature but high genetic heterogeneity has been reported, and D. octaedra has the highest clonal diversity among all parthenogenetic lumbricids in northern Europe (Terhivuo and Saura, 2003). The worms that founded the laboratory culture used in the present study originated from several locations. Some genetic variation was therefore present in the material used in this study even though genetically identical siblings may also have occurred. Adult females produce cocoons, one at a time, and the cocoons each contain a single juvenile. 2.2. Soil preparation Light-textured, sandy loam agricultural soil from Askov Experimental Station, Denmark, was used for the experiment. The soil was sieved through 2-mm mesh and consisted of 38.4% coarse sand (200–2000 mm), 23.6% fine sand (63–200 mm), 10% coarse silt (20– 63 mm), 12.3% fine silt (2–20 mm), and 13% clay (o2 mm). The soil contained 2.8% humus and 1.6% organic carbon, had a pH of 6.2, a density of 1.135 g/ cm3 dry soil and a total cation exchange capacity of 8.14 meq/100 g soil (Sverdrup et al., 2002). Prior to use in the experiment the soil was dried and defaunated at 80
C and then sieved through 1-mm mesh. The soil was contaminated with NP (Aldrich, Cat. No. 29.005.8, 100% pure) dissolved in acetone (J.T. Barker, Hayward, CA, HPLC quality) before being mixed with the soil. A stock solution was made corresponding to the highest test concentration. The stock solution was diluted with acetone to make the remaining concentrations. To contaminate the soil, 252 mL solution was added and mixed thoroughly into 1.2 kg of soil. Pure acetone was used for the control soil. The solvent was evaporated for 24 h under a fume hood. The dried contaminated soils were then put in plastic bags and stored in a deep freezer (20
C) until use. Cow dung that was dried at 80
C, ground, and sieved was given to the animals as food. One part of manure was mixed with approximately 3 parts distilled water. The mixture was stirred into 30 g of the soil in a plastic container (4.5 cm high, 7 cm diameter) just prior to the start of the experiment. The dry soil was wetted with 21% distilled water (by weight), which corresponded to approximately 50% of the water-holding capacity. 2.3. Experimental design Based on a range finding test using juveniles, six sublethal concentrations including a control were selected, namely 0, 10, 20, 30, 40, and 50 mg NP/kg dry weight soil. Ten replicates were used for each concentration. In each replicate, one juvenile of 1–7 days old was put into the wet soil with added cow dung as food. All plastic containers were covered with six-holed

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caps and then put in a large plastic box. To reduce the possible evaporation of water from the soil, wet tissue papers were placed in the bottom of the box before it was covered. The tissue papers were kept wet so that no addition of water to the soil was needed until the next soil change. The experiment was conducted in a constant-temperature room at 15
C. The worms were checked, weighed, and placed into new containers with fresh contaminated substrate every 2–4 weeks. Mortality, fresh body weight, number of cocoons produced, and cocoon development time were recorded. These end points were checked each time the substrate was changed. The worms were gently removed from the soil and rinsed carefully in tap water. They were then placed, individually, in a wet and clean plastic container in which they were allowed to empty their guts. Until the second census time, worms were allowed to empty their guts (which can be visually discerned through inspection of the body surface) for 3 h, and for the remainder of the experiment they were given 24 h to ensure as much elimination of their gut contents as possible. Thereafter, they were blotted dry with tissue paper, weighed and then transferred into new substrate. The cocoons were collected by wet sieving the soil through a 1-mm mesh. After they were rinsed in tap water, the cocoons produced by replicate worms from the same concentration were pooled and put in multiwell dishes (4 mm diameter and 1 mm depth) filled with wet tissue papers. Cocoons were incubated until hatching in a constant temperature room at 15
C. The numbers of hatching cocoons in each treatment were recorded weekly. The observations were stopped after no cocoons hatched during 3 consecutive weeks. 2.4. Population growth, elasticity, and decomposition analysis 2.4.1. Population growth (l) We calculated l for each treatment by fitting the lifehistory data to a two-stage model (Calow and Sibly, 1990): 1 ¼ nSj ltj þ Sa lta ; where n is the number of hatched cocoons per worm per week, Sj represents juvenile survival (the probability that a juvenile survives from birth to first reproduction), Sa represents adult survival (i.e., the probability that an adult survives between census days), tj is the time to first reproduction, and ta is time between production of cocoons, which was estimated from the period between census dates divided by the number of cocoons produced during the census period (set here as the time between censuses). The effects of NP on population growth rate were analyzed by calculating 95% confidence intervals on l following the analytical method of Sibly et al. (2000).

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The confidence intervals were calculated from the total variance in l; which is the sum of the variance contributions from each of the five life-history traits. Differences between population growth rates were deemed significant if there was no overlap between the confidence intervals. 2.4.2. Elasticity and decomposition analysis Elasticity analysis was carried out to determine the relative sensitivity of population growth rate (l) to changes in each of the life-history traits, i.e., number of cocoons per worm per week, time to first reproduction, time between production of cocoons, juvenile survival, and adult survival. To measure the elasticity we employed the equation ea ¼ ða=lÞðdl=daÞ; where a is a life-history trait and dl=da is the sensitivity of l to changes in the particular life-history trait, a (Caswell, 2001). The sensitivity dl=da was calculated based on Eqs. (3)–(7) in Forbes et al. (2001). In this case, n is the number of cocoons per week, tj is time to first reproduction, Sj is juvenile survivorship from birth to first cocoon production, and Sa is adult survivorship between time units, and we define T ¼ nSj tj þ Sa ta ltj ta : Decomposition analysis was conducted to determine how much each life-history trait contributed to the mean changes in l: The contributions of treatment effects on each life-history trait to the effect on l were calculated following Caswell (1989) and Levin et al. (1996). 2.5. Determination of NP concentration in soil Prior to analyses, NP in the soil was extracted as follows: 4 g of soil sample (wet weight) was weighed directly in the extraction tubes in which 100 mL of internal standard (4-t-octylphenol) was added (Cis ¼ 5:24  103 mg/mL). Fifteen milliliters of acetone:dichloromethane (1:1 v/v) was then added. The extraction used programmable Microwave Assisted Extraction set up for 10-min extraction with ramping for ambient temperature until 110
C. This temperature was held for 30 min followed by cooling for 30–60 min. After the cooling period, 15 mL of solvent was transferred to a glass tube and evaporated using a gentle stream of dry air. Another 15 mL of acetone:dichloromethane (1:1 v/v) was added to the tubes and extracted for the second time by the same procedure as above. This second extraction was pooled with the first one and dried again. The extract was redissolved in 2 mL of acetonitrile, and 3 g of anhydrous Na2SO4 was then added to the extract to remove any remaining water. The solution of extracted NP was transferred to a gas chromatography vial via a Pasteur pipette containing cotton as a filter. The amount of 100 ml BSA (bistrimethylsilyl-acetamide) was added to the vial, which was then sealed and placed in the oven for 2 h at 80
C. The GC-MS (HP5890-5971A, Hewlett Packard

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Corporation, Palo Alto, California) settings were as follows: acquisition mode, SIM (selective-ion monitoring); injection volume, 4 mL; injection chamber temperature, 280
C; transfer line temperature, 250
C; solvent delay, 5 min; detection ions, 292, 263, 235, 221, 193, 179, 278. Temperature program: initial temperature was 100
C held for 1 min and increased at a rate of 7
C/ min. After the final temperature reached 300
C, the sample was cooled immediately with total time needed: 29.57 min.

Table 1 Comparison of nominal and measured concentrations of NP in the experimental treatments (soil samples had been frozen for approximately one and a half years prior to analysis) Nominal concentration of NP (mg NP/kg dw)

Measured concentration of NP (mg NP/kg dw)

0 10 20 30 40 50

0 9.9071.8 25.3072.1 34.00711.1 36.7079.1 57.20718.4

2.6. Statistical analysis Two-way ANOVA was employed to test differences in body weight as an effect of NP and time, and one-way ANOVA was used to test for differences in time to first reproduction, time between production of cocoons, number of cocoons produced, hatch time and cocoon viability as a function of NP concentration. Tukey’s pairwise comparison tests were performed when ANOVAs were significant. Trends in mean total cocoon production, time to first reproduction, and population growth rate (l) as a function of NP concentration were analyzed by regression. To test for differences in the proportion of worms producing cocoons we used a oneway nonparametric ANOVA (Kruskal–Wallis test). Prior to this analysis, the proportion was transformed to ranks separately for each census day. Differences in l between pairs of NP treatments were considered significant if the 95% confidence limits did not overlap. The effects were considered significant when Pp0:05; and considered marginally significant when 0:05oPp0:10: All statistical analyses were conducted with SYSTAT Version 9.

10, 10, and 7 worms (replicates) in the respective exposure treatments. 3.3. Growth

The analysis showed that the actual NP concentrations in the soil were in fairly good agreement with nominal concentrations (Table 1). Based on the response factor graph (RFG), we found a linear relationship between the internal standard (t-octylphenol) and the NP in the soil (r ¼ 0:998).

The worms were raised from juveniles of the same initial size and age, and growth was estimated as the increase in fresh body weight. The worms grew steadily until 155 days of exposure. Thereafter growth rates increased markedly, and worms gained more than 50 mg in 2 weeks. Until 110 days of exposure, body weight was affected significantly by NP (0:01oPo0:04), except for 76 days of exposure, for which the effect was marginally significant (P ¼ 0:07). After this period, no significant effects were observed (0:3oPo0:4). For all worms pooled, mean fresh body weight increased significantly from an average of 5 to 281 mg over the course of the experiment (Fig. 1). On the last census day, most worms showed a decline in body weight. Since worms produced cocoons at different times, we compared the body weight of those producing and not producing cocoons in each treatment separately. When the cocoons were produced for the first time, after 110 days of exposure, the body weight difference was not obvious between worms with and without cocoons (Fig. 2a). As exposure time increased, this difference became more apparent (Figs. 2b–d). Among worms without cocoons, there was a tendency that as NP concentration increased, fresh body weight declined. The differences between treatments, however, were not significant in all census periods. The increase in body weight was less for worms with than without cocoons (time  cocoon, Po0:01).

3.2. Survival

3.4. Time to first reproduction

Juvenile and adult survival were not affected by NP. Out of 10 replicates (with the exception of a few worms lost accidentally), there was no mortality during the juvenile period in the control or in any of the NPcontaminated soils. After 196 days of exposure, there remained 8 worms (replicates) in the control and 9, 9,

Since the worms were not checked daily, time to first reproduction was estimated by adding the number of days just before cocoons were observed for the first time with half the census period (i.e., last census day prior to cocoon appearance minus first census day at which a cocoon was observed divided by two).

3. Results 3.1. Actual soil concentration

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400 Control 10 mg NP/kg dw 20 mg NP/kg dw 30 mg NP/kg dw 40 mg NP/kg dw 50 mg NP/kg dw

Fresh body weight (mg)

350 300 250 200 150 100 50 0 0

20

40

60

80

100

120

140

160

180

200

Time (days) Fig. 1. The growth of D. octaedra expressed as fresh body weight (mean7SEM) during 196 days of exposure to NP.

After 110 days of exposure

400

Without cocoon With cocoon

Fresh body weight (mg)

Fresh body weight (mg)

400 350 300 250 200

300 250 200 150

100

100 0

20

30

40

0

50

After 155 days of exposure

400 Without cocoon With cocoon

350 300 250 200 150

20

30

40

50

After 169 days of exposure

350 300 250 200 Without cocoon With cocoon 150 100

100 0

(c)

10

Soil NP concentration (mg/kg dry weight)

(b)

Fresh body weight (mg)

Fresh body weight (mg)

10

Soil NP concentration (mg/kg dry weight) 400

Without cocoon With cocoon

350

150

(a)

After 141 days of exposure

10

20

30

40

0

50

Soil NP concentration (mg/kg dry weight)

(d)

10

20

30

40

50

Soil NP concentration (mg/kg dry weight)

Fig. 2. Fresh body weight of individual D. octaedra producing and not producing cocoons after (a) 110 days, (b) 141 days, (c) 155 days, and (d) 169 days of exposure. The lines were trend lines fitted through the means.

There appeared to be a delay in time to first reproduction with increasing NP concentration, however this effect was not statistically significant (P ¼ 0:721) (Fig. 3). The worms produced cocoons as

early as 123731 days on average in the control (the shortest), whereas the worms in the highest concentration produced cocoons after 152736 days on average.

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152 200

0.7 0.6

Cocoons/worm/day

Time (days)

180

160

140

120

0.5 0.4

Control 10 mg NP/kg dw 20 mg NP/kg dw 30 mg NP/kg dw 40 mg NP/kg dw 50 mg NP/kg dw

0.3 0.2 0.1 0.0

100 0

10

20

30

40

50

Soil NP concentration (mg/kg dry weight)

110

141

155

169

196

Census day (days)

(a) 50

Fig. 3. Estimated time to first reproduction of D. octaedra exposed to NP (mean7SEM). The line was a trend line fitted by regression.

3.5. Cocoon production and time between production of cocoons Cocoons were found for the first time after the worms were exposed to NP for 110 days (34 days after the previous census) in all concentrations. The average number of cocoons produced per worm per day during the 34-day period was 2.673.2 cocoons in the control and 0.00470.01 cocoons per day in the highest concentration. However, the effects of NP on the number of cocoons produced during each census (analyzed separately) were not significant even though the differences in mean number of cocoons, especially between the control and the highest concentration, were large (Fig. 4a). Fig. 4a shows that cocoon production per worm per day increased with time. It peaked during the fourth census period (after 169 days of exposure) in the control, 20 and 40 mg NP/kg, and tended to decline thereafter. In the other concentrations cocoon production showed a tendency to increase until the end of the experiment. There was a marginally significant decline in the total number of cocoons produced per worm with increasing NP concentration (P ¼ 0:066). Though variability among worms was high, for about 103 days of production time worms produced an average of 30 cocoons per individual (0.3070.14 cocoons/day) in the control and this declined to 14.9 cocoons per individual (0.1470.13 cocoons/day) in the highest concentration (Fig. 4b). By the end of the experiment, only one worm did not produce cocoons in the 30 mg NP/kg treatment and one worm in the 50 mg NP/kg treatment. All remaining worms produced at least one cocoon during the 196-day experiment. Since females can only produce one cocoon at a time, time between production of cocoons (ta ) was calculated by dividing the length of the census period by the number of cocoons produced within that period. From

Number of cocoons

40

30

20

10

0

0

(b)

10

20

30

40

50

Soil NP concentration (mg/kg dry weight)

Fig. 4. (a) Mean number of cocoons produced per worm per day in each census period during 196 days of exposure to NP Error bars=SEM. (b) Total number of cocoons produced per individual worm during 196 days of exposure to NP (open circles represent individual cocoon production; filled circles represent mean production). The line was a trend line fitted by regression.

four periods of collection we calculated the average of each individual worm as ta : Average ta of individuals in the highest concentration was two times longer than in the control (ANOVA showed that the differences, however, were not significant, P ¼ 0:17). 3.6. Proportion of reproductive worms By the first census date, 50% of the worms in the control had produced cocoons compared to 14% in the highest concentration. Until the third census, the proportion of worms producing cocoons in the control remained higher than that in the other treatments. By the fourth census, the proportion of worms producing cocoons in all treatments reached more than 80% except for 30 mg NP/kg and 50 mg NP/kg. For all census days pooled, the Kruskal–Wallis test showed that the proportion of individual worms producing cocoons decreased as the concentration of NP increased (P ¼ 0:001).

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153

Fig. 5. Mean hatching time7SD from four different batches of cocoons after being incubated in 15
C. Error bars=SEM.

110

All cocoons produced in each treatment over each census period (starting with the second census) were incubated at 15
C and followed until most of them hatched (Fig. 5). There was no pattern observed in the effect of NP on hatching time. The variability in hatching time within and among treatments was very high. For the four census periods pooled, the average time needed for cocoons to hatch was not affected by NP (P ¼ 0:834). The cocoons hatched as early as 84.4 days at 30 mg NP/kg and the latest one hatched after 103 days at 10 mg NP/kg. In the control, the average hatching time was 90.8 days. Average cocoon viability varied within and among treatments (Fig. 6). The lowest viability was 59% of cocoons found in 10 mg NP/kg. Two of the batches of cocoons produced at 30 mg NP/kg and 50 mg NP/kg had a 100% hatching success. Although viability appeared to show an increasing trend with concentration, the effect of NP on this trait was not significant (P ¼ 0:79). For all treatments but the control, the lowest viability was observed on the last census date compared to the other three census dates (data not shown).

100

3.8. Effect on population growth Mean population growth rate (l) showed a declining trend with increasing NP concentration (r ¼ 0:844; P ¼ 0:03) (Fig. 7). Using the regression equation for the relationship: l ¼ 1:173  0:001 NP

Viability (%)

3.7. Hatch time and viability

90

80

70

60

50 0

10

20

30

40

50

Soil NP concentration (mg/kg dry weight) Fig. 6. Average hatching success of cocoons collected from four census days (open circles represent average of replicates; filled circles represent the mean of four census dates). The line was a trend line fitted by regression.

and solving for l ¼ 1 gives a value of 173 mg NP/kg (i.e., approximately three and a half times higher than the highest concentration used), suggesting that any concentration higher than this would have led to population extinction under the conditions of this experiment. However, comparison of 95% confidence limits around l indicated that differences among pairs of NP treatments were not significant due to high variability among individuals within treatments. 3.8.1. Elasticity and decomposition analysis The elasticity analysis performed on the two-stage model showed that l was most sensitive to changes in

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respectively), and the remainder (37%) was contributed by time between reproduction events.

Population growth rate (week)-1

1.30 1.25

1.20

4. Discussion

1.15

This study shows that juvenile and adult D. octaedra survived, grew, and reproduced during a prolonged exposure to NP in the concentration range of 0–50 mg NP/kg dry soil over more than 6 months under laboratory conditions. Individual growth rate and the percentage of worms producing cocoons were the only traits significantly affected by NP, while the total number of cocoons produced was marginally affected. Despite some rather large differences in mean values of traits among treatments, these effects were difficult to detect statistically due to interindividual variability. Of the measured individual-level traits, fecundity was the most sensitive to NP and was reduced by 21–46% compared to control worms (although this effect was only marginally significant). Average time to first reproduction and time between reproduction events, though not statistically significant, were delayed in exposed worms by 6–23% and 38–105%, respectively; whereas adult and juvenile survival were not affected at all. We did not detect any significant decrease in population growth rate (l) over the range of NP concentrations tested. The decline in l was only between 3.4% and 6.5%, indicating that, when measured as percentage reduction, effects of toxicants at the individual level were ameliorated at the population level.

1.10

1.05 1.00 0

10

20

30

40

50

Concentration (mg/kg) Fig. 7. Population growth rate (l) of D. octaedra exposed to NP. Error bars represent 95% confidence intervals. The line was a trend line fitted by regression.

1.0

0.8 tj n Sj Sa ta

Elasticity

0.6

0.4

0.2

0.0

0

10

20

30

40

50

Soil NP concentration (mg/kg dry weight) Fig. 8. Elasticity of time to first reproduction (tj ), time between production of cocoons (ta ), juvenile survival (Sj ) and adult survival (Sa ) against NP.

juvenile survival (which was not affected by NP), and the sensitivity of l to this trait was several times higher than to the other traits (Fig. 8). Population growth rate was moderately sensitive to changes in adult survival, and relatively insensitive to changes in time to first reproduction, fecundity, and time between reproduction events. For all traits, the elasticity was generally unchanged as a function of NP exposure. As there were no effects of NP on juvenile and adult survival, their contribution to the changes in mean population growth rate (l) was essentially zero, and changes were mainly caused by effects of NP on reproduction, i.e., time to first reproduction, time between reproduction events, and fecundity. Based on decomposition analysis, contributions of the traits to changes in l varied among treatments (Table 2). The average contribution of time to first reproduction and fecundity to the changes in l were equal (32% and 31%,

4.1. Survival and growth rate Effects on body growth rate suggest that the response of juveniles to NP is more sensitive than that of adults. We observed that growth of juveniles, until an age of 110–117 days, was the trait most negatively affected by NP. Negative effects of NP on growth have also been recorded in various aquatic animals such as midges (Bettinetti et al., 2002), amphipods (Brown et al., 1999), freshwater sponges (Hill et al., 2002), and fish (Dreze et al., 2000). In the present study, growth of the adults as a whole seemed not to be affected by NP. However, when we separated adult worms into those producing cocoons and those not producing cocoons we found that the latter group appeared to be more affected than the former. The greater sensitivity of juveniles compared to adults has been observed in earthworms for many other toxicants (Spurgeon and Hopkin, 1996; Booth and O’Halloran, 2001), suggesting that early development in the life of the earthworms is rather sensitive to pollutants. The effects of NP on juvenile growth and survival in this study could be slightly underestimated since the exposure was started with juveniles at 1–7 days of age.

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Table 2 Percentage changes of individual traits and population growth rate (l) and proportional contribution of individual traits (%) to changes in mean population growth rate (l) (based on Caswell, 1996; Levin et al., 1996) Concentration (mg NP/kg dw)

0 10 20 30 40 50

% Changes of

% Contribution of

l

tj

n

ta

tj

n

ta

4.60 3.38 3.75 5.44 6.51

15.46 6.14 14.38 14.97 23.43

37.91 22.81 21.57 30.08 46.36

38.36 53.45 40.40 50.89 105.80

34.20 19.95 40.75 34.83 29.55

39.47 30.34 24.88 31.95 30.60

26.33 49.71 34.37 33.22 39.85

The toxicity of NP has been recorded for various animals from sponges to mammals (e.g., Fry et al., 1987; White et al., 1994; Guillette et al., 1994; Raloff, 1996; de Jager et al., 1999; Lussier et al., 2000; Mann and Bidwell, 2001). Calow (1991) and Gibbs et al. (1996) suggested that in order to deal with the effects of stressors (including toxicants), animals must allocate some of the energy obtained from food that would otherwise be used for maintenance, growth and reproduction (Klok and deRoos, 1996). Therefore, we suggest that the negative effect of NP on the growth of juveniles observed in this study may be due to the cost of detoxification, and that there is in turn less energy allocated to growth. In adults, the cost is expressed as a reduced proportion of worms producing cocoons. Effects of NP differed depending on whether or not worms were producing cocoons. Only after 110 days of exposure, did the differences in body weight between worms not producing cocoons and worms producing cocoons become obvious, with the first group having a higher growth rate than the second. These differences can be interpreted as the amount of energy allocated for producing cocoons. In the first group, the effect of NP was exhibited by a declining trend in body weight as concentration increased, while the second group showed no effect on body weight. However, in the second group, a clear effect of NP was observed on fecundity (the total average cocoon production). Similar trade-offs between growth and reproduction have been observed to occur in the earthworm, Eisenia andrei (van Gestel et al., 1992; van Gestel and Hoogerwerf, 2001). Donker et al. (1993) suggested that energy spent for resisting pollutants would reduce not only growth and differentiation but also impact other important lifehistory traits, such as cocoon production, time to sexual maturity, and generation time. Other studies have found either an enhancement of growth due to NP or no effect of NP. Hansen et al. (1999) observed a stimulation of growth in the polychaete Capitella sp. I exposed to NP-spiked sedi-

ment. In the lowest concentration (14 mg/g dry mass of sediment) worms grew larger than in the control, but growth was reduced at higher concentrations. Another positive effect of NP was displayed by Caenorhabditis elegans (Nematode) that showed increased growth until a concentration of 66 mg/L (Hoss et al., 2002); with no adverse effects on growth in the highest concentration (235.2 mg/l) that would rarely be found in a natural habitat. The findings from our study suggest that worms may have acclimated to NP and were then able to make up for the lower growth rate they showed when first exposed as young juveniles. From 110 days of exposure until the end of the experiment, the fresh body weight of treated worms did not differ significantly from control worms. It is interesting to note that at the end the experiment the worms maintained in the highest concentration gained some weight while the others lost weight. This may be due to inadequate amounts of food in the soil. For the last 27 days, the worms only had 2 g of food. For the (larger) worms in the control this amount of food was not enough to maintain growth. In contrast, for (the smaller) worms kept in the highest concentration, 2 g of food was still sufficient to maintain their growth rate. 4.2. Reproductive aspects Among several reproductive aspects examined in this study, only the fecundity and the percentage of cocoonproducing worms seemed to be significantly affected by NP. Several of the traits (e.g., tj ) showed relatively large declines in their average value with increasing NP exposure. However, due to the large variability among individuals these effects were not statistically significant. Other studies on other species of invertebrates have shown different results in different reproductive traits. Studies by Brown et al. (1999) suggested that, after exposure to NP, the amphipod Corophium volutator increased female fertility whereas sex ratio was not affected. Bechmann (1999) studied the marine copepod Tisbe battagliai in two life-table

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experiments but did not see any significant effect of NP on the percentage of females, either in parent (P) or offspring (F1). However, she did find that NP reduced the number of egg-producing females. Hansen et al. (1999) found negative effects of NP on brood size, volume specific fecundity, and time to first reproduction in Capitella in the highest concentration (174 mg/g dry mass of sediment). In the nematode C. elegans, NP enhanced the number of offspring per worm (Hoss et al., 2002). 4.3. NP as an endocrine-disrupting compound Many studies have suggested that the estrogenic features of NP disrupt the endocrine system of various animals. It acts by mimicking natural (endogenous) estrogen (E2), binds to and activates the estrogen receptor (ER) rendering estrogenic effects inside the body of vertebrates (e.g., White et al., 1994; Yadetie et al., 1999). In invertebrates Hood et al. (2000) detected the presence of ER in two free-living nematode species Panagrellus redivivus and C. elegans after exposing them to dieldrin, NP and toxaphene, indicating that both species may have estrogen-like hormones. It is unclear if such endocrine effects were brought about by NP in D. octaedra given that relatively little is known about the endocrine control of reproduction in earthworms (Takahama et al., 1998; Fujino et al., 1999) and that our study was not designed to examine mechanisms of endocrine disruption. In Nereis (a marine polychaete), a neuropeptide hormone secreted by the brain may be involved in regulating body growth and reproduction simultaneously. Its presence during early development promotes somatic growth while inhibiting oocyte growth (Highnam and Hill, 1978). The decreasing concentration of this hormone toward maturation may explain the change in energy allocation between growth and reproduction with age. This is consistent with our results in which nonfertile worms were consistently larger than fertile worms, showing a clear trade-off between somatic growth and cocoon production. In addition, the proportion of nonfertile worms was somewhat larger in the groups exposed to the highest NP concentrations. These questions should be further investigated in relation to endocrine-disrupting effects of NP. It should be noted that this study was designed to maintain relatively constant exposure of worms by replacing the contaminated soil every 2–4 weeks. This may overestimate exposure compared to field conditions in which degradation of NP added to surface soil in sewage sludge would occur. Half-lives of NP in soil have been reported to be around 37 days (Jacobsen et al., 2004). However, rates of degradation may vary widely, depending on the properties of the sludge–soil mixture (Hesselsoe et al., 2001).

4.4. Population growth (l) Population growth rate is a better measure of responses to toxicants than are individual-level effects because it integrates potentially complex interactions among life-history traits and provides a more relevant measure of ecological impact (Forbes and Calow, 1999). The present study showed that mean population growth rate tended to decline with increasing NP concentration, despite a lack of effect on juvenile or adult survival or on time to first reproduction. Worm fecundity was significantly reduced by NP exposure, and this effect could be attributed to both a reduction in juvenile growth rate and to a reduction in the number of worms producing cocoons. The regression equation suggested that extinction would occur when a population of D. octaedra is exposed to a concentration greater than 173 mg NP/kg. However, an initial range-finding test showed that juveniles did not survive 100 mg NP/kg. In the field worms would rarely be expected to encounter NP in such a high concentration. Lozano (2002), however, found NP in the range 6.4–69 mg NP/kg dry weight in soil samples at the former Akzo Nobel plant in Mo¨lndal, Sweden. Assuming that maximum concentrations of 4000 mg NP/kg in biosolids or sludge can be reached (Bennie, 1999) and 12 tons dry weight of sludge per hectare are mixed homogenously into 15 cm depth of a loam soil with density 1.5 g/cm3 (Tchobanoglous and Burton, 1991), a final soil concentration of approximately 21 mg NP/kg could be expected to occur. Such an exposure concentration is in the range of concentrations used in the present study. Krogh et al. (1996) reported a 10% reduction in reproduction of the earthworm Apporectodea caliginosa at 3.4 mg NP/kg when NP was added directly to soil in a 21-day study. In contrast, their later study showed that 56-day reproduction of Eisenia fetida was not affected at a maximum concentration of 46 mg NP/kg when the NP was added to soil as sludge (Jensen and Krogh, 1997). An elasticity analysis was used to explore the sensitivity of l to changes in the life-cycle traits contributing to it (Caswell, 1989). Elasticity measures the proportional change in l resulting from a proportional change in a given life-history trait, holding all other traits constant. If a trait has a high elasticity, then relatively small proportional changes in the trait will have a relatively large influence on l: The life-cycle of D. octaedra under the laboratory conditions used in the present study was such that the elasticity of juvenile survival was higher than the other traits. Thus, a given impact on juvenile survival would be expected to have much greater consequences for population dynamics than would a similar proportional impact on adult survival, fecundity, or timing of reproduction.

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Whereas elasticity analyses can be used to ask what would happen to l if a life-history trait changed, such an analysis does not provide mechanistic insight into observed effects on l in a given experimental setting. Thus a decomposition analysis was used to quantify the relative contribution of NP effects on the individual lifehistory traits to the observed effects on l (Caswell, 1989). Since no mortality was observed during the experiment, the contribution of juvenile and adult survival were essentially zero, despite the fact that juvenile survival had a high elasticity. On the contrary, the decomposition analysis shows that the observed decline in mean l was driven largely by effects on time to first reproduction and fecundity (each trait explained on average 32% and 31% of the effect on l) and time between reproduction events (explained on average 37% of the effect on l). The high sensitivity of reproductive performance to toxicant exposure has been recorded in many studies as noted above. However, such reproductive effects do not always result in population-level impacts. Kamenga et al. (1996) found that the reproductive period of the nematode, Plectus acuminatus, was the most sensitive trait (reduced by 45%) to cadmium exposure, but that such a reduction did not lead to a detectable effect on population growth rate, whereas rather small effects of cadmium on time to maturity had a much greater influence on population growth rate. Hansen et al. (1999) demonstrated that stimulatory effects of NP on reproduction of the polychaete, Capitella sp. I observed in the lowest exposure concentration (14 mg/g) had no significant effect on population growth rate. Studies that quantify the relationship between effects of a toxicant(s) on individual-level responses and on population dynamics demonstrate the value of integrating demographic analyses into ecotoxicological investigations. Comprehensive studies of life-history effects have not been common in earthworm ecotoxicological studies. The present study suggests that extrapolation from laboratory studies to population effects in the field may be greatly enhanced by combining ecotoxicological and demographic methods.

Acknowledgments This study was supported financially by the QUE (Quality for Undergraduate Education) Project under World Bank Loan Scheme to the Indonesian Government as a doctoral stipend to T.H.W. Most of the work was conducted at the Ecotoxicology Laboratory, Department of Biology and Chemistry, Roskilde University, Denmark, and Department of Terrestrial Ecology (TERI), National Environmental Research Institute, Denmark. The authors thank P.H. Krogh and L. Birkso

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who provided logistical support during the work at TERI. We also thank to Z. Gavor, K.K. Jacobsen, E. J^rgensen, T. S^rensen, and L. Maarup for helping with laboratory work.

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