Toxicity of copper-spiked sediments to Tubifex tubifex (Oligochaeta, Tubificidae): a comparison of the 28-day reproductive bioassay with a 6-month cohort experiment

Aquatic Toxicology 65 (2003) 253–265

Toxicity of copper-spiked sediments to Tubifex tubifex (Oligochaeta, Tubificidae): a comparison of the 28-day reproductive bioassay with a 6-month cohort experiment Andrea Pasteris a,∗ , Martina Vecchi b , Trefor B. Reynoldson c , Giuliano Bonomi b a

Centro Interdipartimentale di Ricerca per le Scienze Ambientali, Università di Bologna, Via Tombesi Dall’Ova 55, I-48100 Ravenna, Italy b Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Selmi 3, I-40126 Bologna, Italy c National Water Research Institute, Environment Canada, P.O. Box 115, Acadia University, Wolfville, Nova Scotia B0P 1 X0, Canada Received 30 September 2002; received in revised form 21 May 2003; accepted 21 May 2003

Abstract Results from a 28-day adult reproductive bioassay using the aquatic oligochaete Tubifex tubifex (Müller, 1774) are compared with life table statistics obtained from a 6-month experiment on cohorts of the same species. This was done by simultaneously performing the two tests on copper spiked sediments. Five concentrations and a control were tested. The 28-day bioassay was performed 3 times in succession. Several endpoints were considered for each test and LOEC, IC10 and IC50 were calculated. IC50 estimates for the number of young produced in the 28-day bioassay range from 81 to 107 mg/kg; IC50 estimates for different endpoints of the cohort experiment ranged from 88 to 106 mg/kg. The 28-day bioassay showed essentially the same sensitivity as the cohort experiment to copper. This suggests that the 28-day reproductive bioassay does provide information that is relevant in assessing long-term toxic effects at the population level. © 2003 Elsevier B.V. All rights reserved. Keywords: Bioassays; Toxicity testing; Sediment; Copper; Life tables; Tubifex tubifex

1. Introduction It is generally acknowledged that laboratory toxicity tests are an essential tool for the evaluation of the potential impact of chemicals on ecological systems and for a comprehensive assessment of contaminated environments (Chapman and Long, 1983; Long and Chapman, 1985; Rodriguez and Reynoldson, 1999). ∗ Corresponding author. Tel.: +39-0544-215126; fax: +39-0544-31204 E-mail address: [email protected] (A. Pasteris).

Yet results are often difficult to interpret ecologically, and the utility of laboratory tests in the prediction of the actual effects of toxicants on the ecosystems have been debated (Lamberson et al., 1992; Cairns et al., 1996; Chapman, 2002). This is especially true for several bioassays, performed under the very artificial environmental conditions commonly employed in routine toxicity testing. Bioassays must be rapid, simple, replicable, inexpensive and standardised, if they are to be of practical use in environmental assessment (Giesy and Hoke, 1990); however, since their ultimate goal is the pro-

0166-445X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0166-445X(03)00136-X

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tection of populations, communities and ecosystems from adverse effects of chemicals, ecological relevance is a critical issue. One way to enhance ecological realism in sediment toxicity testing, is the development of bioassays using whole sediments and true sediment-dwelling species, as opposed to adaptations of water-column assays that test elutriates or pore water using non-benthic species. Also, since organisms are generally chronically exposed to contaminated sediments, chronic bioassays that test sublethal endpoints are in most circumstances more realistic than acute lethal tests. While sediment bioassays with macrobenthic species usually consider survival or sometimes growth as endpoints, Reynoldson et al. (1991) proposed a test which measures reproduction of adult Tubifex tubifex (Müller, 1774) (Oligochaeta, Tubificidae) over 28 days, and a standardised protocol for this method has been published by ASTM (1994). The protocol has been applied, sometimes with minor modifications, in several studies (Reynoldson, 1994; Reynoldson et al., 1996; Chapman et al., 1999; Martinez-Madrid et al., 1999; Vecchi et al., 1999; Bettinetti and Provini, 2002). The bioassay is reasonably rapid, simple and inexpensive, and very little maintenance work is required during the test, thus it can be effectively applied to routine toxicity testing. The use of reproductive endpoints makes it potentially useful in the assessment of chronic effects on populations. However, the test cannot use demographic statistics as endpoints, because of its design, and its duration is short in comparison with the potential life span of the organism. As Lamberson et al. (1992) points out, toxicological endpoints must be demonstrably related to predictions of population growth, if the tests are to be used to protect populations of organisms. One powerful approach to achieve this linkage is to determine the sensitivity of population growth rate to changes in potential test endpoints, using established theoretical ecological demographic models. Life tables are one of the standard methods used by population ecologists to report age specific information on survival and fecundity (Begon and Mortimer, 1986). Life tables provide an analytical representation of the variation in the demographic traits as a function of the age of the individual and are the starting point for the estimation of several integrative and ecologically meaningful population parameters. The study of

cohorts, groups of individuals born at the same time, is the most reliable procedure for the construction of life tables. Ideally, the study of a cohort begins when individuals forming the cohort are born and ends when the last survivor dies. Over this period and at an appropriate frequency, the numbers of surviving individuals and new-borns are recorded. Laboratory experimentation on cohort cultures is arguably the best method for assessing the influence of environmental factors at the population level, allowing direct measurement of the effect of experimental treatments on life table statistics. There are, however, major practical problems in the application of the life table approach with aquatic oligochaetes. Oligochaetes are long-lived organisms with a potential life span of several years (Timm, 1984). Furthermore, if environmental conditions are favourable, tubificids breed continuously and their embryonic development is rapid in comparison to their life span (250 degree-days for T. tubifex; Bonacina et al., 1987). This requires the number of eggs laid by the cohort to be recorded frequently, e.g. weekly for T. tubifex at 23 ◦ C. A longer period between observations would result in embryos dying or hatching as young worms in the time between observations, and fecundity would be grossly underestimated. This combination of long duration and high frequency of observation makes it logistically difficult to conduct an ideal experiment on cohorts with tubificids. However, it is possible to conduct an experiment over several months, thus providing relevant information. Particularly λ, the finite growth rate at stable age distribution and r, the intrinsic rate of increase, can be adequately estimated from a partial life table. These two parameters are basically equivalent, since λ=er , and are possibly the most meaningful demographic parameters. Their value as endpoints in toxicity testing has been illustrated by Forbes and Calow (1999). Given the highly unrealistic assumptions required to define λ, it is generally of little value in predicting or describing actual population growth. However, it is a very effective measure of the potential of a population, since it takes into account both survival and fecundity over the whole life cycle of the species, and integrates in a single number the complex information contained in a life table. Theoretically estimation of λ, requires data collected over the whole life span of the study species. However, the influence of the oldest age classes on

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the value of λ is often very small. Consequently, estimates based on a partial life table are, for all practical purposes, identical to estimates based on the complete table, provided that data on a sufficient number of age classes are included in the computation. While for routine toxicity testing, experiments on cohorts, even when shortened to 6 months, are too long and expensive and likely impractical, they can be used as a benchmark for a shorter and less expensive test protocol such as the 28-day adult reproductive bioassay. The aim of this paper is to compare the endpoints from the 28-day adult reproductive bioassay proposed by Reynoldson et al. (1991) with life table statistics obtained from a 6-month experiment on cohorts. This was done by simultaneously performing the two tests on copper spiked sediments. If the results of the simpler test can reliably predict the results from experiments on cohorts, this would confirm that the shorter duration tests are capable of providing information that is relevant in assessing long-term toxic effects at the population level. 2. Methods 2.1. Stock cultures Laboratory stock cultures of T. tubifex have been established using worms collected from Lake Suviana, a reservoir located in the mountains of Appennino Tosco-Emiliano, N.E. Italy. Worms were reared in groups of approximately 30 individuals in circular glass containers (diameter: 11 cm; height: 6 cm), half filled with sand and then filled with aerated tap water. Frozen lettuce was put under the sand as food. Every week, each container was checked, the sand washed, the residual food removed and fresh lettuce added; cocoons were also removed and either discarded or placed in a separate container to start new stock cultures. Cultures where kept in the dark, at 23±1 ◦ C and discarded when 6 months old. 2.2. Soil collection, handling, spiking, and chemical analyses The experimental substrate was a terrestrial soil, collected from an uncultivated plot at Cà Bosco, near the city of Ravenna. This soil proved the most suit-

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Table 1 Chemical and physical properties of the C`a Bosco soil Silt+clay (%) SiO2 (%) TiO2 (%) Al2 O3 (%) Fe2 O3 (%) MnO2 (%) MgO (%) CaO (%) Na2 O (%) K2 O (%) P2 O5 (%)

62.5 51.75 0.41 10.25 3.47 0.08 2.68 12.86 1.67 2.24 0.2

TOC (%) Total N (mg/kg) Total P (mg/kg) Cr (mg/kg) Co (mg/kg) Ni (mg/kg) Cu (mg/kg) Zn (mg/kg) As (mg/kg) Cd (mg/kg) Pb (mg/kg)

1.03 906 1360 34 9 37 20 56 20 <1 14

able substrate among four previously tested, including two different aquatic sediments (Vecchi et al., 1999). The plot is located in NE Italy, in the River Po alluvial plane and has not been chemically treated for at least 10 years. Among the favourable characteristics of this substrate are low background concentrations of heavy metals and copper in particular (Table 1). Moreover, when this soil was copper spiked and used in bioassays, an adequate and consistent matching between target and measured concentration and a definite dose-response curve where obtained. Soil was collected with a spade; a superficial layer of 1–2 cm was scraped and discarded; then the soil was collected to a depth of 15 cm. This was dried in the laboratory at room temperature, manually ground using a mortar and dry sieved through 500 ␮m mesh to remove coarser particles. The standard protocol for the 28-day reproductive bioassay (Reynoldson et al., 1991; ASTM, 1994) prescribes 250 ␮m mesh for removal of confounding indigenous macrofauna, however, this is not required in the case of a terrestrial soil. Instead a 500 ␮m mesh was used, allowing much faster processing of the large amount of soil required for the experiment. The sieved soil was mixed 1:1 by volume with a commercial mineral water (Fonte Guizza, Acqua Minerale San Benedetto S.p.A. Scorzè, Italy; see Table 2). The resulting slurry was allowed to settle for 72 h and then the overlying water removed and discarded. After this treatment the soil had the appearance and consistency of wet sediment. Commercial mineral water was used to provide a standard water source, as a reliable source of good quality natural water with constant characteristics was unavailable. The 10 g/l copper stock solution used to spike the sediments was prepared by dissolving reagent grade

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Table 2 Chemical and physical properties of the mineral water used in the bioassays (Fonte Guizza, Acqua Minerale San Benedetto S.p.A. Scorz`e, Italy) pH Conductivity at 20 ◦ C (␮S/cm) Total solids at 180 ◦ C (mg/l) Na+ (mg/l) K+ (mg/l) Mg2+ (mg/l) Ca2+ (mg/l) HCO− 3 (mg/l) Cl− (mg/l) SO2− 4 (mg/l) NO− 3 (mg/l) P2 O5 (mg/l) SiO2 (mg/l)

7.67 401 250 7.1 1.1 31 46 296 2.8 4.6 7.5 <0.1 17

CuSO4 ·5H2 O in distilled water. Prior to spiking each batch of sediment, four 50 ml samples of wet sediment were weighed, oven dried at 80 ◦ C for 12 h and then re-weighed to estimate the dry weight/wet weight ratio. Spiking was performed by mixing the required weight of wet sediments (corresponding approximately to 1.5 l), with 2 l of commercial mineral water and the required volume of Cu stock solution in a 5-l glass jar. The contents of each jar were manually stirred for 5 min, using a stainless steel spoon. Jars were then placed in a refrigerator at 4 ◦ C for 14 days. Every 3 days stirring was repeated. The target nominal concentrations used were: 0 (control), 35, 50, 70, 100, 140 mg/kg. These concentrations were chosen on the basis of the results of a previous 28-day experiment on adults and cocoons (Vecchi et al., 1999). Spiking was started at day −16 (taking as 0 the day when the individuals of the cohort were first exposed to the spiked sediments) and repeated regularly every 28 days, so that freshly spiked sediments were available throughout the whole experiment. Control sediment was processed in the same way as the spiked sediment but with no added copper solution. Concentrations of copper in the whole sediments after spiking were determined with inductively coupled plasma-atomic emission spectroscopy (ICP-AES) on a multi-channel Jarrrell-Ash (Franklin, MA, USA) Atom Comp® 1100, after a two-step nitric acid–hydrochloric acid digestion (Mudroch, 1985).

2.3. 28-day reproductive bioassay The 28-day reproductive bioassay was performed according to the protocols described by Reynoldson et al. (1991) and ASTM (1994) with minor modifications. 2.3.1. Day −1 Each 250 ml bioassay beaker received 100 ml of spiked (or control) sediment, 100 ml of overlying water and 80 mg of powdered Tetramin® fish food (TetraWerke, Melle, Germany) suspended in 1 ml of water. The beakers were then placed in the dark, in the test incubator at 23±1 ◦ C to settle. Six replicated beakers were prepared for each nominal concentration, including the unspiked control; five were used in the bioassay and one, a blank containing no animals, supplied the sample for the analytical determination of copper concentration in the spiked sediments. 2.3.2. Day 0 The overlying water in each beaker was gently aerated for 1 h. Then temperature, pH, conductivity and dissolved oxygen were measured from the overlying water of each beaker. At each concentration a sample for the analytical determination of the copper concentration was taken from the ‘blank’ beaker. Sexually mature specimens were transferred from the stock culture to small Petri dishes, four per dish. When sufficient animals have been collected, each group of four was added to a bioassay beaker chosen at random; beakers were then returned to the incubator. All the beakers were kept in the incubator for 28 days. The overlying water was continuously and gently aerated. The beakers were examined every 2 or 3 days for loss of water due to evaporation and double distilled water was added to compensate if required. 2.3.3. Day 14 Temperature, pH, conductivity and dissolved oxygen were measured from the overlying water of each beaker. Beakers were otherwise left undisturbed until day 28. 2.3.4. Day 28 Beakers were removed from the test incubator and temperature, pH, conductivity, dissolved oxygen and

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ammonia were measured. The content of each beaker was individually wet sieved through a 500 and 250 ␮m mesh and the surviving adults were counted immediately. The residues from the two sieves were washed separately into two vials, preserved in 70% alcohol and examined later. Samples were enumerated with a dissecting microscope; cocoons were dissected to count embryos if their number was not visible through the wall. 2.3.5. Endpoints The endpoints measured were: number of surviving adults, number of laid cocoons (empty and full), number of embryos present inside the cocoons, and hatched young (‘large’ or ‘small’, if retained respectively by the 500 or by the 250 ␮m mesh). Total offspring was calculated as the sum of young and embryos. Embryos were counted although this endpoint was not used by Reynoldson et al. (1991) and ASTM (1994) to verify if and how much this would increase the sensitivity of the bioassay. The bioassay was performed 3 times in succession, with an interval of 4 weeks between the end of a repetition and the start of the next repetition, these dates corresponded to days −1, 55, and 111 of the long-term cohort experiment. 2.4. Long-term cohort experiment The protocol for the long-term experiment was developed so as to be comparable with the 28-day reproductive bioassay. 2.4.1. Day −5 Cocoons were removed from the stock cultures and discarded. This ensured that the cocoons from the stock culture used in the assay were 0–5 days old. 2.4.2. Day −2 Five replicate 250 ml beaker containing sediment, food and overlying water were prepared for the control and each nominal concentration, as described for the 28-day bioassay. The beakers were then placed in the test incubator together with the beakers used for the 28-day bioassay. Day −2 of the long-term experiment and day −1 of the first repetition of the 28-day bioassay were coincident and a total of eleven

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beaker was prepared for each nominal concentration which where then randomly chosen to be used for the long-term experiment, the 28-day bioassay or as the ‘blank’ for copper concentration confirmation. The position of each beaker in the incubator was chosen at random and maintained over the experimental period. 2.4.3. Day 0 The overlying water in each beaker was gently aerated for 1 h. Then temperature, pH, conductivity and dissolved oxygen were measured from the overlying water of each beaker. Cocoons were picked from the stock cultures and the number of embryos in each was determined by observation with a dissecting microscope. Groups of 4 cocoons, each containing 3, 4 or 5 embryos, to a total of 16 embryos were created. Each group was added to a test beaker chosen at random and each cocoon was placed in the sediment by pipette. Beakers were then returned to the incubators. Exposure of the individuals to the test sediment for the long-term experiment began one day after the 28-day bioassay. This 1 day difference allowed manageable sample processing. The beakers were kept in the same environmental conditions and subjected to the same handling as the beakers of the 28-day bioassay, including measurement of temperature, pH, conductivity and dissolved oxygen at day 14. 2.4.4. Day 28 Beakers were removed from the test incubator and temperature, pH, conductivity, dissolved oxygen and ammonia were measured in the overlying water. The content of each beaker was individually wet sieved through 500 and 250 ␮m mesh. Surviving individuals were counted and placed in a new beaker with water, food and freshly spiked sediment, to replace that lost by sieving. Measurements of temperature, pH, conductivity and dissolved oxygen were taken from the new beakers just before the introduction of the animals. No attempt was made of recording survival before day 28, since this may have stressed the embryos and newly hatched worms and possibly increased mortality. Furthermore, it is difficult to determine if an early developmental stage embryo is viable and impractical to enumerate older embryos inside the cocoons. At day 28 worms still appeared

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immature, and were left undisturbed for 2 more weeks. 2.4.5. Day 42 Beakers were removed from the test incubator and temperature; pH, conductivity and dissolved oxygen measured. The content of each beaker was individually wet sieved through 500 and 250 ␮m mesh. Surviving individuals were counted and transferred to a new beaker with water, food and the appropriate sediment. Measurements of temperature, pH, conductivity and dissolved oxygen were taken from the new beakers prior to the introduction of the animals. By this time the worms had attained sexual maturity and had begun reproducing. Cocoons from each beaker were preserved in 70% alcohol for later examination. Samples were enumerated with a dissecting microscope; cocoons were dissected to count embryos if the number was not visible through the wall. From this date on, cultures were sieved and checked weekly using the same procedure until day 168 (week 24). 2.4.6. Day 168 The standard weekly procedure was performed except worms were not transferred to new beakers. At the end of the experiment the survivors of each replicate were kept overnight in clean water without sediment or food, allowing the gut content to clear. Afterwards the worms of each replicate were put on a pre-weighed aluminium foil, oven dried at 80 ◦ C for 12 h and weighed to the nearest 0.1 mg. The weekly sieving of the sediment, required by the long-term experiment protocol, is the major difference between the two tests, where in the 28-day bioassay the worms and sediment are undisturbed for the entire period. The sediment processing was conducted to minimise the effects of this difference. As described, a new batch of sediment was spiked every 28 days and used in the long-term experiment and, when scheduled, in the 28-day reproductive bioassay. Using the freshly spiked sediment 26 beakers were prepared for each concentration; one provided the sample for the analysis of copper concentration, five were used for the replicates of the 28-day bioassay, and 20 for the replicates of the long-term experiment. Thus, four beakers were prepared for each experimental repli-

cate in the long-term experiment. One was used immediately as the test chamber and worms were transferred into it. The other beakers were placed in the same environmental conditions as the test organisms and used as test chambers in the following weeks, one for each week. This procedure was adopted to obtain a similar ageing of the spiked sediment in both tests. 2.4.7. Endpoints The number of worms found in each beaker after x weeks from the beginning of the experiment, i.e. the number of individuals surviving until the age of x weeks, will be referred as n(x). The number of embryos found in each beaker after x+1 weeks from the beginning of the experiment, i.e. the number of eggs laid when the individuals of the cohort had age between x and x+1 weeks will be referred as F(x). For each experimental replicate, the proportion of individuals surviving until the age of x weeks was computed as: l(x) = n(x)/n(0) where n(0) is the number of embryos used to start the replicate culture. For each experimental replicate, the mean number of new-borns produced by one individual surviving to age x, in the interval between age x and x+1 was computed as: b(x) = F(x)/n(x) All the values l(x) and b(x) for a replicate, ordered by x, form the life table of the replicate. Of the endpoints considered for the long-term experiment, two are selected values from the life table: l(4)

the proportion of individuals surviving for 4 weeks (28 days). l(24) the proportion of individuals surviving until the end of the experiment (24 weeks). Three endpoints are demographic statistics computed from the life table as a whole:

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x=24 x=0

F(x)


x=24

the total number of eggs laid by all the individuals in a replicate over the whole experiment.

F(x) the mean number of eggs laid, over n(4) the whole experiment, by each living individual at the end of the fourth week. This statistic is similar to the widely used R0 , the net reproductive rate,
which is computed as F(x)/n(0). Mortality over the first 4 weeks was high and displayed high intra-treatment variability at all tested concentrations including the control. After this mortality was very low in all but the two highest concentrations. Moreover, in the experimental conditions the worms are unable to breed until they are 4 weeks old. Thus, it is more meaningful to use fecundity of the individuals surviving past week 4. λ the finite growth rate at stable age distribution. The estimate of λ was obtained by means of the Euler–Lotka equation: x=0

x=x max 

l(x)b(x)λ−x = 1

x=0

The equation is implicit, and the final estimate of λ was obtained by recursive substitution of increasingly refined estimates, until the equation was verified. One more endpoint is based on weight data: w(24)

total biomass (dry weight) at the end of the experiment.

2.5. Statistics One-way ANOVA was used to test the overall significance of the effect of copper concentration on the endpoint values. Homogeneity of variances was tested by Bartlett’s test and the dependence of variances from means checked by plotting means versus standard deviations. When a deviation from ANOVA assumptions was found, the power transformation, which met most of the assumptions, was applied to the raw data. A treatment was excluded from the analysis when there

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was a 100% response in all five replicates, as these treatments have null variance, and a statistical test is not needed to detect difference from the control; furthermore, their inclusion in the ANOVA computation would violate the assumption of homogeneity of variance and strongly bias the estimate of within group variance. When ANOVA detected significant differences (α < 0.05), the Dunnett’s t-test at 0.05 significance level was used to identify the No Observed Effect Concentration (NOEC), which is the highest concentration not significantly different from the control and the Lowest Observed Effect Concentration (LOEC), which is the lowest concentration that is significantly different from the control. The copper concentration that would cause a 50% reduction of each endpoint (IC50) and the concentration that would cause a reduction of 10% (IC10) were estimated by fitting a logistic model to the data. The equation used was: y = b0 −

1 + (x/b1

b0 (log(9)/log(b 2 /b1 )) )

Where:y = endpoint value; x = copper concentration; b0 = expected endpoint value in absence of toxic effect; b1 = IC50; b2 = IC10. The logistic model can be written using a simpler equation, however this equation has the advantage that it includes both IC50 and IC10 as parameters. This allowed calculation of point estimates, standard error and confidence intervals for IC50 and IC10 by using the nonlinear regression procedure supplied by the software package Statistica for Windows (StatSoft, Inc., Tulsa, OK, USA).

3. Results The relationship between the six target nominal concentrations and actual measured concentrations are shown in Table 3. Measured concentrations are nearly always higher than the corresponding nominal concentration, possibly a consequence of the background levels, which are rather variable (CV = 37%) even though the soil used in the experiment was collected on a single occasion and homogenised as much as possible. The coefficient of variation of the six measured values at each nominal concentration tend to decrease with increasing concentration, mostly because

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Table 3 Measured copper concentrations after six successive spikings Nominal concentration (mg/kg)

0 35 50 70 100 140

Measured concentration (mg/kg) 1st spiking

2nd spiking

3rd spiking

4th spiking

5th spiking

6th spiking

Mean

Standard deviation

11.6 44.0 53.1 91.2 107.2 157.5

14.2 52.7 60.1 67.3 117.9 145.5

32.1 53.2 64.5 91.5 107.5 125.0

29.2 65.1 79.6 94.6 137.8 –

30.0 70.4 80.3 100.2 128.8 165.6

26.5 59.5 65.6 89.5 114.1 –

23.9 57.5 67.2 89.0 118.9 148.4

8.8 9.5 10.8 11.3 12.2 17.7

Fig. 1. Relationship between the measured copper concentration in the sediments and the measured endpoints in the 28-day bioassay: (a–c) number of cocoons laid respectively in the first, second and third repetition; (d–f) number of young; (g–i) number of embryos; (j–i) total offspring. Each point is the mean of five replicates, error bars are standard errors. The continuous line represents the logistic model fitted to the observed data.

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of the increased mean value at the denominator (highest CV = 17% at 35 mg/kg, lowest CV = 10% at 100 mg/kg). It is also noteworthy that the range of actual concentration measured at two adjacent nominal concentrations overlaps in some cases. The relationship between the measured concentration in the sediments and the endpoint values is shown in Fig. 1 for the 28-day bioassay and in Fig. 2 for the long-term experiment. In the latter, the worms at each nominal concentration were treated as being exposed to a constant copper concentration equivalent to the mean of the six measured values. However, since l(4) was determined at the end of the first month of experiment, only the first measured concentration was used with this endpoint. The estimates of λ were virtually identical whether the data collected over the whole experiment or the data collected over the first 4 month were included in

Fig. 2. Relationship between the measured copper concentration in the sediments and the measured endpoints in the long-term cohort experiment. See text (Section 2) for the meaning of each endpoint. Each point is the mean of five replicates, error bars are standard errors. The continuous line represents the logistic model fitted to the observed data.

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the computation. Thus, since the actual copper concentration in the sediment over the last 2 months of the experiment would not influence the observed values of λ, the concentration was computed as the mean of the first four measured values for this endpoint. LOECs, IC50s and other results of the logistic regression are reported in Table 4. Most endpoints, both of the 28-day bioassay and of the long-term experiment, have their LOEC at the nominal level of 100 mg/kg, corresponding to a measured concentration varying from 107 to 129 mg/kg. LOEC is at the nominal concentration of 70 mg/kg
for two endpoints of the long-term experiment ( F(x) and λ) and, only in the second repetition, for two endpoints of the 28-day bioassay (number of cocoons and number of young). Based on LOEC, the least sensitive endpoints are number of embryos (28-day bioassay) and l(4) (long-term experiment). In general the fit of the logistic model to the observed data (Figs. 1 and 2) seems acceptable, and the explained sum of squares is over 60% of the total for most endpoints. While in some cases data may suggest presence of hormesis, endpoint values for any copper concentration are never significantly higher than the control. On the whole use of the logistic model to estimate of IC50 and IC10 seems appropriate. Fig. 1a–c shows the effect of copper concentration on the number of laid cocoons for the three repetitions of the 28-day bioassay. The value expected in absence of toxic effect is fairly constant (range: 30.7–32.3). The mean of the three IC50 values is 116 mg/kg with a coefficient of variation of 12%. The width of the 95% confidence interval varies from 16 to 28% of the estimate in the three repetitions of the test. Number of young (Fig. 1d–f) appears slightly more sensitive than the previous endpoint; the mean of the three IC50 is 98 mg/kg and each IC50 is lower than the corresponding value for total cocoons. However, this endpoint seems less consistent. The CV among the three repetition is 15% and the width of the confidence intervals varies from 3 to 46% of the estimate. Moreover, the value expected in absence of toxic effect is much higher for the first repetition (130) than for the other (80 and 83). The estimated IC50 for the number of embryos in the first repetition (Fig. 1g) was outside the range of tested concentrations; in addition the sum of squares explained by regression was only 14% of the total sum

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Table 4 LOEC, IC50 and IC10 for all the endpoints considered in the two bioassays IC50 (mg/kg)

Nominal

Measured

Point estimate

95% confidence interval

Point estimate

95% confidence interval

28-day bioassay No of cocoons 1st repetition 2nd repetition 3rd repetition

100 70 100

107 92 129

125 100 123

107–142 89–110 113–133

78 77 97

57–99 65–89 81–112

68 77 90

No of young 1st repetition 2nd repetition 3rd repetition

100 70 100

107 92 129

107 81 105

106–108 62–100 96–114

106 52 87

100–111 30–74 69–105

66 72 83

No of embryos 1st repetition 2nd repetition 3rd repetition

–a 140 140

–a 125 166

–b 110 142

–b 102–118 130–154

–b 99 123

–b 94–105 110–136

14 65 75

Total offspring 1st repetition 2nd repetition 3rd repetition

100 100 100

107 108 129

131 104 122

108–154 95–114 112–132

91 88 96

71–111 77–99 80–112

58 75 84

Long-term experiment l(4) l(24) Total
weight
F(x) F(x)/n(4) λ

140 100 100 70 100 70

158 119 119 89 119 83

–b 97 88 94 101 106

–b 84–110 72–104 82–106 96–106 96–116

–b 80 63 73 86 87

–b 67–94 37–88 51–95 79–92 68–105

33 77 68 81 94 84

a b

IC10 (mg/kg)

ANOVA not significant. Estimated IC50 outside the range of tested concentrations, regression results considered as not reliable.

% of sum of squares explained by regression

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LOEC (mg/kg)

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of squares. As a consequence, the results of the regression were considered as not reliable. In the second and third repetition (Fig. 1h and i) the estimated IC50 was inside the range of tested concentrations and the percent of explained sum of squares was higher than in the first repetition, however, IC50 for this endpoint was higher than for the other endpoints. Results for total offspring (Fig. 1j–l) are fairly similar to results for the number of cocoons. Mean IC50 is 119 mg/kg, with a CV of 12%. The width of the confidence interval varies from 16 to 35% of the estimate. Since the long-term experiment was performed only once, a measure of inter-experiment repeatability is not available. The IC50 value for l(4) is very close to the highest tested concentration (Fig. 2a). While this estimate is an indication of a low sensitivity of the endpoint it cannot be considered as reliable, also because the explained sum of squares is only 33% of the total. IC50 for the other endpoints considered for the long-term experiment range from 88 to 106 mg/kg. This range is similar to the one defined by the three IC50 obtained for number of young in the 28-day bioassay. The sensitivity, as assessed by the point estimates
of the IC50, ranks in the order: total
weight > F (x) > l(24) > F(x)/n(4) > λ. However, the precision of the estimate, measured by the percent width of the confidence interval ranks in the order

F(x)/n(4) > λ > F(x) > l(24)>total weight. There is a fairly strong positive linear relation between the IC50 and the IC10 estimated for each endpoint. As a consequence of this relation, comparing the sensitivities of the different tested endpoints using IC10 instead of IC50 would lead to the same general picture. The most notable exception is number of cocoons in the first repetition of the 28-day bioassay that has one of the highest IC50 and an average IC10. To a lesser extent total offspring has a similar behaviour. However, IC50 are estimated with better precision than IC10, for all the endpoints except number of embryos in the second repetition.

4. Discussion We are unaware of published data on the relationship between measured and target nominal concentra-

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tions for repeated spiking of the same sediment. Thus, it is not possible to compare repeatability achieved in the present work with others’ experience. Certainly, in the long-term experiment, worms at each nominal concentration were not exposed to the same actual concentration throughout the experiment. Assuming that they were exposed to a constant level (i.e. the mean of the six observed values) is somewhat arbitrary; however, no other choice seemed possible. An alternative to spike a new batch of sediment every 28 days would have been to spike all the sediment required at the beginning of the experiment. Besides the practical difficulties of homogeneously spiking such a large amount of sediment, this option was discarded because we aimed at reproducing in the long-term experiment the same condition of exposure of the 28-day bioassay. In the present paper, LOECs have been reported since they are still widely used as measure of chronic toxicity. However, several authors have pointed out serious drawbacks in the use of LOEC, NOEC and similar measures (Hoekstra and Van Ewijk, 1993; Sims et al., 1997). LOEC is necessarily one of the test concentrations used. Therefore, if two tests are conducted under identical conditions but with different concentrations, they are bound to yield different LOECs and NOECs. In addition, since LOEC depends on the outcome of a statistical test, it is systematically affected by the power of the test itself. This means that endpoints with more intra-treatment variability tend to have higher LOECs and, that any effort to achieve a more reliable estimate, increasing the number of replicates or decreasing intra-treatment invariably leads to lower values of LOEC. Moreover, no statement of precision (e.g. confidence limits) can be obtained for the LOEC or NOEC. Because of these drawbacks, effect concentration (EC) and inhibition concentration (IC) are more appropriate measures of toxicity either acute or chronic. Here, comparisons among bioassays and endpoints are based mainly on IC50, which were in general estimated with better precision than IC10. Non-linear regression, in particular the logistic model, has been used by several authors to model concentration–response relationship and to estimate ICs or ECs (Scott-Fordsmand et al., 1997; Sims et al., 1997; Guilhermino et al., 1999; Pillard et al., 1999, 2000). Nonlinear regression methods, just like linear regression, assume that errors are normally distributed

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with homogeneous variance. Results of toxicity tests often fail to meet these assumptions. In particular, homogeneity of variance is unlikely, since the data scatter tends to decrease at higher concentrations, where response approaches 100%. To our knowledge, the practical effect of this on the reliability of the estimates of IC50 and its confidence interval has not been studied. While other different estimation methods (e.g. weighted regression, jackknife, bootstrap) may be useful, in the absence of a better alternative, we adopted the use of unweighted least-square regression as is customary in the context of IC50 estimation. If variances are heterogeneous, estimates may be improved using weighted least-squares. The principle is to weight the data so that those with larger variance are given less influence in the analysis. However, this approach was not adopted here because it was impossible to assign a proper weight to the observations at 140 mg/kg, when the observed value was 0 for all the five replicates and, as a consequence no estimate of variance was available. While λ is the most appealing endpoint from the standpoint of population dynamics theory, because of its interpretation as the potential long-term population growth rate, its practical use presented some problems, because of the unfavourable statistical properties. At the nominal concentration of 100 mg/kg the observed value was 0 for three replicates out of five, while the values for the remaining two replicates were similar to those observed at lower concentrations. This results in a markedly larger variance at 100 mg/kg in comparison to the other concentrations. Moreover, λ appeared less sensitive than the other endpoints of the long-term experiment with the exclusion of l(4). Enumeration of the embryos present inside the cocoons is required to measure two endpoints: the number of embryos, and total offspring. These results confirm that, while this increases the amount of work required to perform the 28-day bioassay, it does not improve sensitivity, as already pointed out by Vecchi et al. (1999). The number of embryos is the least sensitive among the endpoints in the 28-day bioassay and total offspring does not have any advantage over the other measured endpoints i.e. number of cocoons and number of young. Also consistent with the results of Vecchi et al. (1999) is the observation that l(4), survival of the cohort after 4 weeks, is insensitive. This suggests that

measures of adult reproduction in T. tubifex, are a more useful approach than early life stage survival.

5. Conclusions Each single cohort experiment on aquatic oligochaetes requires a large amount of work and several months to perform. As a consequence the data reported here are from a single experiment. Thus, these results cannot be generalised. However, the 28-day T. tubifex adult reproductive bioassay showed essentially the same sensitivity as the long-term cohort experiment to copper. This suggests that the 28-day bioassay does provide information that is relevant in assessing long-term toxic effects at the population level.

Acknowledgements We wish to thank Ugo Peruch for providing the Cà Bosco soil, Raffaella Ercolani and Roberta Vitali for assistance in the execution of the bioassays, and Jerry Rajkumar for conducting the chemical analyses.

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