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Assessment of the toxicity of River Po sediments with Ceriodaphnia dubia
Aquatic Toxicology 47 (2000) 191 – 202 www.elsevier.com/locate/aquatox
Assessment of the toxicity of River Po sediments with Ceriodaphnia dubia L. Vigano`* Water Research Institute, CNR, 20047 Brugherio, Milan, Italy Received 8 January 1999; accepted 16 March 1999
Abstract A recent interdisciplinary project aimed at the assessment of the quality of the River Po focusing primarily on the bed sediments of the principal Italian river. In such a moderate/low polluted environment, the identification of sites at risk using a number of toxicological tools was considered of great interest. The present study examines in detail the information which was obtained from two of the toxicological approaches adopted in that project, that is, the 7-day whole-sediment test with Ceriodaphnia dubia and the acute toxicity test (48 h) with the same crustacean conducted on an organic-solvent extract of the sediment fine material ( B 63 mm). These toxicity tests were applied to composite sediment samples collected in summer and winter low-flow conditions, downstream from the main tributaries of the River Po. Summer and winter sediments seemed to be characterized by different qualities, as suggested by their oxygen demands and C/N ratios, whereas they were indistinguishable for the contents of organic carbon and fine material. For some reaches of the River Po, the 7-day tests showed that the summer sediments cause chronic toxic stress, whereas the winter sediments enhance reproduction and growth. Apart from these seasonal differences, it was found that the biomass production (reproduction + somatic growth) of C. dubia was negatively correlated to the fraction of sediment organic carbon contributed by fine material ( B 63 mm). The results of the extract tests showed that the toxic potentials of sediments vary along the River Po, identifying some critical sites. In contrast with whole-sediment results, it was found that the toxic potentials, reported as sediment toxic units (TUs), are relatively independent from seasons and substantially stable, particularly along the larger potamon section. The concurrent examination of biomass production and sediment TUs confirmed the sites at risk and demonstrated that the biomass production of C. dubia is inhibited by the increase of sediment TUs. This examination also demonstrated that some sediments, particularly of the winter survey, have high toxic potentials which, however, were not effective. Apparently this was a result of different source and quality of sediment organic matter, which may affect either the bioavailability of chemicals or the nutritional value of fine particles. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Daphnid growth; Fine material; Organic carbon; Sediment extract; Toxic unit; Whole sediment test
E-mail address: [email protected] (L. Vigano`) 0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 9 9 ) 0 0 0 1 9 - 3
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1. Introduction The adsorption of chemicals onto the organic particles of sediments is an important process through which a lot of contaminants can reach much higher concentrations in the bottom-sediment compartment than in the upper water column. Contaminated sediments can be a sink but often a long-term source of toxicants which can produce adverse effects to aquatic life (Landrum and Robbins, 1990; Burton and Scott, 1992). In large bodies of water, toxic effects can rarely approach the acute levels since the dilution capacity often reduces the concentrations of chemical inputs at or below the chronically effective levels. In such environments, dilution combined with a limited bioavailability of toxicants, make even sediments rarely responsible for acute toxic effects. Consequently, the adoption of sensitive sublethal endpoints is generally required to identify the presence of toxicity and assess the potential effects to aquatic life of a given area. Chronic toxicity tests and biomarkers applied to whole sediment assays can be two important tools of investigation (Vigano` et al., 1995, 1998; Burton et al., 1996). The River Po is the largest watercourse in Italy. It drains an area of ca 70 000 km2 and receives from the different tributaries and sub-basins variable and complex mixtures of contaminants of both industrial, agricultural and urban origins (Marchetti et al., 1994; Pagnotta et al., 1995). A new monitoring program has recently been undertaken to assess the quality of the River Po. This monitoring program has largely focused on the bottom sediments of the river environment assuming that, in conjunction with the riverine community, they can be the most representative compartment to accomplish the objective of a quality assessment. Composite sediment samples were collected downstream from the confluences of principal tributaries and compared with an ambient control sediment sampled from the upper reach of the River Po. To investigate two potentially different conditions of the river sediment environment, both a summer and a winter lowflow surveys were undertaken. River sediments have concurrently been examined for several
chemical and toxicological indicators whose sensitivity and potential for discriminating sites at risk are still being examined and compared. A comprehensive report on this project will soon be published. In this context, the present study discusses in detail the results which have been obtained from two of the toxicological approaches adopted in the Po sediment project. They are the 7-day whole sediment test with Ceriodaphnia dubia and the acute test (48 h) conducted with the same crustacean on an organic-solvent extract of the sediment fine material (B 63 mm). The three main objectives of this study were: (a) to evaluate the potential and thus the usefulness of these two approaches for identifying the toxic risk present along the River Po; (b) to investigate whether sediment properties may affect the daphnid responses; and (c) to obtain greater insights for the quality assessment process, also evaluating less conventional daphnids endpoints.
2. Materials and methods
2.1. Area of study and sediment collection Ten reaches of the River Po were chosen to describe the quality of the watercourse. The first was located ca 50 km from the springs of the river (Casalgrasso, TO), and the sediments collected from this area, hereafter indicated as Up Turin, served as an ambient control, being in the upper course of the river and thus before it receives important loads of contaminants. The other nine reaches were located downstream the main tributaries of the River Po. As shown in Fig. 1, these tributaries were: the Dora Riparia, Dora Baltea, Sesia, Tanaro, Ticino, Lambro, Adda, Oglio and Panaro. The River Panaro is not important per se but because it is the last tributary of the River Po. The corresponding downstream reach is therefore representative of the entire watershed of the Italian river, before it flows into the Adriatic Sea. Hereafter, each of the nine sediments and corresponding river stretch will be identified by the name of the tributary, e.g., Sesia sediment. At each sampling area, the sediment collection started downstream from the completion of the
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mixing process between the main river and the tributary, thereby assuming that where River Po waters are homogeneous also bottom sediments are correspondingly in homogeneous conditions. The observation of completely mixed waters, usually made after some kilometers from the confluences, was based on several cross-section profiles obtained for conductivity, as detailed in a previous study (Vigano` et al., 1996). At each sampling area sediment collection usually terminated before the confluence of the next tributary. Generally the extension of the sampling areas increased going toward the Po delta, that is from about 1 – 2 km in the upper tract, where the River Po has a mean annual flow of ca 70 m3 s − 1, to 20 – 40 km of the lower/final reaches, where the river has a mean annual flow of ca 1500 m3 s − 1. A Ponar grab sampler was used to collect composite samples of bottom river sediments. Water depth at sedimentation sites varied between 0.5 and 5 m, with an average value of approx. 1.5 m. Both summer and winter sediment collections were completed within a few days and in stable low flow conditions. No substantial change of the
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location of the natural deposition areas and thus of sampling sites were observed in the two surveys. Composite samples were stored at 4°C for a few days before the 7-day tests. Sediments and the corresponding fine fractions were analyzed for nitrogen and organic carbon contents (Carlo Erba C:N:S analyzer). The results of these measurements are listed in Table 1. The contents of fine materials, also reported in Table 1, were determined by a sedimentation technique (Day, 1965; Gee and Bauder, 1986). To this aim, 25-g aliquots of freeze-dried sediment samples were first dispersed in 0.5 l of distilled water with Na-hexametaphosphate as a dispersant (25 ml of a solution containing 33 g (NaPO3)6 l − 1 + 7 g Na2CO3 l − 1), and then poured through a 0.1-mm sieve into a sedimentation cylinder (Esenwein). According to Stokes’s Law (20°C and 2.7 g cm − 3 as mean particle density), samples of the sediment suspension were taken from the cylinder at given time intervals and depth so that fine particles characterized by different settling velocities were collected (pipet method) and dry weight determined. Since freeze-drying is suspected to alter particle-
Fig. 1. Map showing the watershed of the River Po and its main tributaries downstream from which the composite sediment samples were collected.
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Table 1 Composition of sediment samples collected along the River Po during summer and winter low-flow conditionsa Sediment
Up Turin D. Riparia D. Baltea Sesia Tanaro Ticino Lambro Adda Oglio Panaro
Summer
Winter
Fine fraction (%)
N (%)
Org. C (%)
C/N
Fine fraction (%)
N (%)
Org. C (%)
C/N
12.0 21.2 69.8 46.6 47.4 37.0 58.8 32.8 56.1 50.7
0.25 0.25 0.16 0.16 0.16 0.16 0.26 0.21 0.23 0.19
2.73 2.29 1.30 1.48 1.38 1.37 2.07 1.76 1.91 1.62
10.92 9.16 8.13 9.25 8.63 8.56 7.96 8.38 8.30 8.53
26.6 28.0 23.5 45.2 66.5 37.2 47.6 40.8 58.4 48.7
0.20 0.37 0.21 0.20 0.16 0.14 0.34 0.18 0.20 0.13
1.64 2.59 1.72 1.55 1.27 1.17 2.29 1.37 1.50 1.14
8.20 7.00 8.19 7.75 7.94 8.36 6.74 7.61 7.50 8.77
The contents of fine material (B63 mm) and the results of the elemental analyses carried out on the sediment fine fractions are reported. Sediments were collected upstream from the city of Turin (ambient control; Up Turin) and downstream from the confluences of nine main tributaries of the River Po. a
size distribution, for five out of the ten sediments of the winter series a comparison was made between fresh and freeze-dried samples. The amounts of fine material determined with the sedimentation technique gave the following results for fresh and freeze-dried samples, respectively: D. Riparia 22.2%, 21.6%; Sesia 40.4%, 39.4%; Ticino 31.6%, 33.0%; Adda 34.6%, 36%; Panaro 47.0%, 43.0%. According to this comparison (although based on B 50-mm instead of B63-mm particles), we felt reasonably confident of the results which can be obtained using freeze-dried samples.
2.2. Sediment extraction Since smaller particles bind xenobiotics much more efficiently than coarse fraction of sediment, only fine fraction was taken into account for xenobiotic extraction and acute testing. The fine fraction (B 63 mm) of freeze-dried sediments was separated by sieving and extracted for 8 h in a Soxhlet apparatus using a mixture (1:1) of n-hexane and acetone (60 g fine material; 200 ml solvent mixture). The extract was reduced to small volume by rotary evaporation and an aliquot transferred to DMSO obtaining a final equivalent concentration of 6 g of sediment fine fraction per ml. This kind of extract obtained from the ten sediments of the River Po and a procedural blank prepared through the same procedure but without sediment, were acutely tested with C. dubia.
2.3. Toxicity tests C. dubia is routinely cultured at our laboratory and, as described in previous papers, culturing is based on a partially synthetic medium (hardness 150 mg CaCO3 l − 1) (Vigano`, 1991) and a diet composed of Selenastrum capricornutum and Saccharomyces cere6isiae (Vigano` et al., 1996), fortified with three vitamins provided at final concentrations of: B1 75 ng l − 1, B12 1 ng l − 1and H 0.75 ng l − 1(Cowgill et al., 1985). The 7-day tests on whole sediments were conducted according to the ASTM procedure (ASTM, 1994). In brief, each sediment was tested in ten replicates prepared in 50-ml beakers, using 5 ml of sediment and 20 ml of semisynthetic water (1:4). One C. dubia less than 24 h old was added to each beaker after a few-hour settling period. Probably because of the adoption of a partially different diet from ASTM proposal, feeding had to be modified to meet the validity criteria of the test (80% survival and 15 young/female). For external nutrition procedure, applied during overlying water renewals and young count and removal, nominal S. capricornutum and Saccharomyces cere6isiae densities were kept at 300 000 cells ml − 1 for the entire duration of the test. On the contrary, to minimize the additional loads of cells and organic matter to test beakers (containing sediment), nominal algae and yeast cell concentrations in overlying water were gradually increased from 100 000 cells ml − 1
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at the start of the test, to 200 000 on days 1, 2 and 3, to 250 000 cells ml − 1 on days 4, 5 and 6. Clean river sand was used as a substrate for control treatment. At 7 days the body length of each test organism was measured using a light microscope and an ocular micrometer. The acute toxicity of the sediment extracts was evaluated conducting 48-h toxicity tests on C. dubia. Sediment extracts were used according to the original quantities of fine material extracted and the toxicity results were examined in terms of equivalent grams (of fine material extracted) per liter of test solution. Hereafter these toxicity results will simply be reported in terms of g l − 1. Extracts were tested at a minimum of five different concentrations, usually ranging between 20 and 1.25 g l − 1. Ten C. dubia less than 24 h old were randomly distributed to each of the 20-ml solutions of sediment extracts prepared in 50-ml beakers. Tests were conducted at 2591°C, under dim light conditions, and dilution water was the same semisynthetic medium used for both laboratory culture and 7-day tests (Vigano`, 1991). The procedural blanks showed 48hEC50 values close to 1.2% (v/v) so the background contributions to the acute toxic effects of sediment extracts was assumed to be negligible. In fact, at the equivalent concentration of 20 g l − 1, usually the highest of the five-concentration series, the corresponding solvent concentration was 0.3% (below the 48hEC1 of 0.7%).
2.4. Statistics Results of reproduction and growth from the 7-day whole sediment tests on C. dubia were first examined for normal distribution and homogeneity of variance using x 2-test and Bartlett’s test, respectively. The two series of results of the summer survey passed both the tests (P =0.01) and were examined by one-way ANOVA. Then each treatment group was compared to the ambient control (Up Turin sediment) using the Bonferroni test. This was possible because ambient and laboratory controls were first compared using Student t-test and found to be not significantly different; this result was observed for both the surveys on the River Po (data not shown). Also for the
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results of the winter survey the same procedure was followed. In this case, however, fertility results failed Bartletts’ homogeneity test and the nonparametric Wilcoxon rank sum test was used to compare each treatment to the ambient control (Up Turin). On the contrary, growth results passed normality and homogeneity tests and both ANOVA and Bonferroni analyses could be used. With regard to acute toxicity tests, 48hEC50 values and 95% confidence intervals were calculated with probit analysis. All calculations were performed using Toxstat 3.5 computer package (West and Gulley, 1991).
3. Results
3.1. Whole sediment tests In no case did River Po sediments cause significant mortality of C. dubia. The only exception was observed for the winter survey when the Lambro sediment killed all the organisms after 24 h of exposure (Table 2). Although amplified by the static-renewal procedure of the laboratory test, this result can be attributed to the high ammonia concentration of porewater, which in this sediment reached 72 mg l − 1 (N-NH4), the highest value observed in the study (G. Tartari and S. Valsecchi, Water Research Institute, Milan, personal communication), and the high oxygen demand of the sediment material which lowered DO to almost zero level during the first 24 h of the test. No similar event could be observed in the summer survey when the sediment quality of the Lambro area, as on average of the entire River Po, seemed to be different from that of the winter period (see below). Neither summer nor winter whole-sediment tests revealed significant inhibition of fertility when compared to ambient control (Up Turin) (Table 2). On the contrary, winter sediments collected from the D. Riparia, D. Baltea and Adda stretches significantly enhanced reproduction of C. dubia. The measurement of body growth gave more information on daphnid response, so that the D. Baltea, Tanaro, Oglio and Panaro summer sediments significantly reduced the final body
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Table 2 Results of the 7-day toxicity tests conducted with Ceriodaphnia dubia on whole sediment samples of the River Poa Sediment
Up Turin D. Riparia D.Baltea Sesia Tanaro Ticino Lambro Adda Oglio Panaro
Summer
Winter
Neonates/female
Body length (mm)
Neonates/female
Body length (mm)
19.195.6 20.1 9 6.8 14.6 95.3 19.0 95.4 16.3 9 4.8 21.6 94.8 18.9 93.7 19.1 9 3.4 12.8 9 6.0 15.8 94.9
0.936 9 0.040 0.9269 0.034 0.8809 0.038* 0.8959 0.061 0.8609 0.058* 0.9159 0.050 0.8939 0.024 0.8889 0.060 0.8799 0.041* 0.8819 0.029*
18.5 93.5 30.2 93.1* 23.1 91.7* 21.6 93.3 17.7 94.8 19.4 93.5 –b 27.4 95.6* 24.8 9 9.4 17.1 9 3.0
0.936 9 0.039 1.009 9 0.025* 0.978 9 0.027* 0.968 9 0.026 0.941 9 0.034 0.949 9 0.039 –b 0.961 90.033 0.980 9 0.045* 0.915 90.029
a
Composite sediment samples were collected in summer and winter low-flow conditions, upstream the city of Turin (ambient control; Up Turin) and downstream from the confluences of nine main tributaries of the River Po. Both young productions and body lengths are reported as mean values ( 9 S.D.) of treatment group. b The winter sediment of the Lambro reach caused the complete mortality of C. dubia. * Significantly different from ambient control value (PB0.05).
lengths of the test organisms (Table 2). On the other hand, some winter sediments confirmed their positive effects, determining also a growth enhancement of daphnids. In fact, C. dubia exposed to the D. Riparia, D. Baltea and Oglio winter sediments were larger than those of ambient control (Table 2).
4. Discussion The reproductive output of C. dubia exposed to whole sediments in the 7-day tests provided no
3.2. Sediment extract tests The results of the 48-h toxicity tests showed a significant variation in the levels of contamination along the River Po (Fig. 2). The D. Riparia and Lambro areas were the most contaminated followed by Adda and Oglio, all showing 48hEC50s lower than 10 g l − 1. On the other extreme, the sediments collected downstream from the confluence of the River Ticino were consistently the least toxic. This trend appears substantially stable for both the surveys, whereas, as illustrated in Fig. 2, the toxicities of the sediments collected from the upper reaches were more variable. Particularly the Up Turin sediment showed the greatest variability, as the 48hEC50 was 6.2 g l − 1 in summer and 24.7 g l − 1 in winter, i.e. comparable to the most toxic and the least toxic downstream areas, respectively.
Fig. 2. Toxicities of the extracts of the sediment fine materials ( B63 mm) to Ceriodaphnia dubia. The results, reported as 48hEC50 with 95% confidence intervals, are in terms of equivalent grams of fine material extracted per liter of test solution (see text). Sediments were collected along the River Po in summer (gray bars) and winter (black bars) low-flow conditions, upstream from the city of Turin (ambient control; Up Turin) and downstream from the confluences of nine tributaries.
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significant evidence of a toxic risk along the River Po, both for winter and summer low-flow conditions. The reproductive response showed, on the contrary, that a stimulation could even be effective at three stretches of the winter riverine environment (D. Riparia, D. Baltea, Adda). The adoption of the parameter carapace length was more informative. It showed significant growth inhibition, and thereby the risk of chronic toxicity at four areas of the summer riverine environment (D. Baltea, Tanaro, Oglio, Panaro). The higher neonate productions observed for D. Riparia- and D. Baltea-winter sediments were also confirmed by the correspondingly higher growth rates of test organisms. Consistent with this general picture of a moderate/low level of toxic risk, the River Po environment showed only one case of markedly harmful conditions which were observed in winter for the Lambro stretch. The advantage of using growth to detect chronic toxicity with C. dubia and, generally, daphnids is seemingly the result of two factors which, although not completely distinguishable, can be identified as: (a) the responsiveness of the parameter itself; and (b) the relatively low variability of growth data. The first aspect was documented in previous studies, most conducted on Daphnia magna, and both with organics, trace metals and toxicant mixtures (Geiger et al., 1980; Winner, 1981; Van Leeuwen et al., 1987; Vigano` et al., 1996). The second factor is equally or even more important to determine the usefulness of size as an indicator of stress. In the present study, in fact, the reproduction data show a mean coefficient of variation of ca 24% against a corresponding value as low as 4% for the growth data. Such a higher quality of growth data allow the minor differences from control to be statistically detected. This favorable property of growth data is also confirmed by previous data sets as those found, for example, by Knight and Waller (1987). Besides these observations, the results obtained from the 7-day tests suggest that the summer and winter benthic environments of the River Po are characterized by different conditions and likely problems. In fact, the summer sediments can cause chronic toxic stress, whereas the winter
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Fig. 3. (a) Spatial trends of the C/N ratios of sediment fine fraction ( B63 mm). (b) Spatial trends of the mean oxygen concentrations measured during the 7-day whole-sediment tests with Ceriodaphnia dubia. Sediments were collected along the River Po in summer and winter low-flow conditions, upstream from the city of Turin (Up Turin) and downstream from the confluences of nine tributaries.
sediments can have a nutritional value which enhances fertility and/or growth. Both the mean concentrations of dissolved oxygen (mg l − 1) measured during the 7-day tests, and the C/N ratios of sediment fine particles, confirm that summer and winter benthic environments are significantly different. The spatial trends of the C/N ratios along the River Po are shown in Fig. 3a. These results demonstrate that the fine material of summer sediments has on average a significantly higher C/N ratio than winter sediments (8.89 0.8 vs. 7.89 0.6; PB 0.00). This difference might be explained by a higher contribution of plant debris from both aquatic macrophytes and terrestrial plants occurring during the most favorable sum-
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mer period. Aquatic macrophytes, high cellulose and lignine particles and, in general, progressively more decomposed materials (of autochthonous and allochthonous origins) show increasingly higher C/N ratios (ca 12 – 20 and over) (Wetzel, 1983; Santiago et al., 1994; Allan, 1995). In summer, emergent macrophytes (Cyperus sp.) and sometimes also submersed macrophytes were abundant in sedimentation areas of the River Po, whereas they were virtually absent in winter. A similar trend was recently observed, for example, in the Detroit River (Lovett-Doust et al., 1997). Low C/N ratios (ca 4 – 8) characterize, on the other hand, the particulate matter which originates from wastewater treatment plants (Santiago et al., 1994) and the micro community of periphyton (Allan, 1995). The ‘absence’ of macrophyte and terrestrial plant residuals combined with slower mineralization rates which are typical of the winter period should favor the presence of particulate matter characterized by lower C/N ratios, particularly in those stretches which are exposed to heavier wastewater inputs. This is the case of both the D. Riparia and Lambro reaches which are exposed to high and direct loads of effluent discharges (Marchetti et al., 1994; G. Barbiero, Water Research Institute, Rome, personal communication). Accordingly, their winter sediments showed the lowest C/N ratios of the entire River Po. Santiago et al. (1994) found a similar behavior of the C/N ratio in both suspended and bed sediments of the River Rhone. Consistent with all these observations, the mean concentrations of dissolved oxygen, recorded during the 7-day tests, showed two clearly distinct patterns (Fig. 3b), so that the winter sediments demonstrated on average the highest oxygen consumption (5.09 1.1 mg O2 l − 1 in winter versus 6.390.2 mg O2 l − 1 in summer; PB0.00). In this context, it is of particular interest to mention that neither the organic carbon of whole sediment, its contribution from fine particles, nor the content of fine material were significantly different in the two seasons. Thus, the distinction between summer and winter sediments seems to be the result of qualitative (compositional) rather than quantitative aspects of the
organic matter, as might be described by a C/N ratio. On these grounds, an attempt was made to understand how the sediment properties could explain the variability in response of C. dubia. With this aim in view, both growth and fertility data were first converted to a common biomass basis and then integrated into a final single value. This was accomplished, for the growth data, calculating the mean somatic weight of a female from the regression with body length found by Anderson and Benke (1994) and, for the fertility data, multiplying the mean number of young produced in a treatment by the mean dry weight of an egg (0.75 mg) which in turn was measured for C. dubia by the same authors. In this way, the responses of C. dubia exposed to River Po sediments could be examined through a single term of biomass production obtained as the sum of the estimates of reproductive and somatic components. Since the organic content of sediments is a key factor for the accumulation of organic chemicals and hence the contamination of sediments (Karickhoff et al., 1979; Kukkonen and Landrum, 1996), it was explored whether the variability of biomass production among treatments could be explained by the sediment contents of organic carbon. It was found that the organic carbon of whole sediment was not descriptive of biomass variation (data not shown). On the contrary, the organic content contributed by the fine material, and more precisely, its concentration reported on a whole sediment basis, was a better descriptor. In other words, such a fraction of total organic carbon due to sediment fine material was found to be a first reasonably good descriptor of C. dubia responses. With the only exception of four sediments which sustained relatively higher biomass values, Fig. 4 shows that the biomass production of C. dubia is inversely correlated with the organic carbon contributed by the sediment particles smaller than 63 mm (r= − 0.72; PB 0.00). To understand the responses of C. dubia, further important information was obtained from the toxicities of the sediment extracts. Taking into account both the sediment content of fine material
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Fig. 4. Relationship between the mean biomass production of Ceriodaphnia dubia (mg per female) exposed to River Po sediments and the sediment contents of organic carbon (%) contributed by the fine material ( B 63 mm).
and the toxicity of its solvent-extractable contaminants (48hEC50) a convenient estimate of the toxic potential of each sediment could be obtained in terms of toxic units (sediment content of fine material/48hEC50 of fine-material extract= whole sediment TUs). For both the summer and winter surveys, these kinds of results are illustrated in Fig. 5, which shows the toxic potentials estimated for the different stretches of the River Po. Besides
Fig. 5. Potential toxic units estimated for the River Po sediments. These values are based on the amount of fine material ( B 63 mm) present in each sediment and the toxicity of the organic-solvent extractable contaminants of such fraction. Results are reported for both the summer (clear bars) and winter low-flow surveys.
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Fig. 6. Relationship between the mean biomass productions of Ceriodaphnia dubia exposed to the summer sediments of the River Po and their toxic potentials reported as toxic units (see text). UP, Up Turin; DR, Dora Riparia; DB, Dora Baltea; SE, Sesia; TA, Tanaro; TI, Ticino; LA, Lambro; AD, Adda; OG, Oglio; PA, Panaro.
the few exceptions of the most upstream areas, where TU variation was however limited to a factor of 2, most riverine environments showed no substantial change between the summer and winter surveys. Fig. 5 clearly demonstrates the two extreme conditions of the Ticino and Lambro stretches, which show the least and highest toxic potentials of the River Po, respectively. Also Up Turin demonstrates very low toxic potential, confirming its role as an ambient control. It can be mentioned that some previous studies (Vigano` et al. 1995, 1998, 1999) as well as the preliminary results on sediment contents of, for example, NNH4 and extractable organic halides (EOX) are consistent with the picture obtained for toxic potentials (Fig. 5) (G. Tartari and S. Valsecchi, Water Research Institute, Milan, personal communication; A. Lopez, Water Research Institute, Bari, personal communication). The availability of the sediment extract toxicities allowed us to examine whether the different levels of biomass production of C. dubia could be explained by the TUs of sediments. To better understand this relationship it is useful to first examine the summer sediments only. As shown in Fig. 6, most of the summer samples demonstrate
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that the biomass production of C. dubia is inhibited by the increase of sediment TUs (r= − 0.79; P =0.02). However, both the Lambro and Dora Riparia sediments do not meet this relationship, suggesting that their high toxic potentials are substantially unavailable to exert toxic effects. These two sediments could sustain a relatively high biomass production, at levels comparable to those of sediments with least toxic contents (TUs), such as Up Turin, Sesia and Ticino. The inclusion of the winter-survey results modified the relationship as illustrated in Fig. 7. The contemporary analysis of the two-survey results increased, on one hand, the number of cases characterized by a low toxic potential and high biomass production, confirming what we can call the ‘basic relationship’ of Fig. 6, but, on the other hand, also provided further examples of sediments which, despite a relatively high toxic potential, did not inhibit the biomass production of C. dubia.
Fig. 7. Relationship between the mean biomass production of Ceriodaphnia dubia and the sediment toxic potentials. Results are from both the summer and winter low-flow surveys. The empty and filled circles distinguish the sediments with a fine material characterized by a C/N ratio higher than 8 () and lower than 8 ( ), respectively (abbreviations as in Fig. 6).
These latter cases increased the limited number of outliers whose (organic-solvent extractable) contaminants seem to be largely or completely unavailable. In the first group and precisely in the upper-left portion of the graph, we can consistently find both the Ticino and the Up Turin sediment samples (no effect). In the lower-central position there are the four sediments which were identified as toxic by growth inhibition, and in the upper-right position (eutrophic) there are those sediments which, despite similar or even higher toxic potentials (TUs), showed either unaffected or much higher biomass productions. Some preliminary results on the macroinvertebrates of the Ticino and Lambro reaches may be significant at this point. It has been observed that the standard biotic indexes cannot distinguish the summer macroinvertebrates communities of the two reaches, that is, when the 7-day tests show equivalent biomass production (Fig. 7). In winter, when the Lambro sediment caused the complete mortality of C. dubia, the macroinvertebrates of the Lambro reach show a significant impairment compared to the Ticino reach (A. Buffagni, Water Research Institute, Milan, personal communication). In this case, interstitial ammonia and sediment oxygen demand seem to play a prominent role in affecting organisms. A number of previous studies pointed out that the toxic effects of sediments and, in general, the bioavailability of sediment-bound organic chemicals can be modified by the content of organic matter as well as its source and composition (DeWitt et al., 1992; Suedel et al., 1993; Boese et al., 1995; Standley, 1997). It was also suggested that the sediments dominated by ‘anthropogenic carbon’, as are often the most polluted, may have a greater affinity for contaminants than those containing ‘natural organic carbon’ (Lake et al., 1990). It may be of interest to note that those sediments which meet the basic relationship of Fig. 7 (empty circles) have a fine material characterized by a C/N ratio higher than 8, whereas those sediments which do not meet that basic relationship (filled circles) have C/N ratios lower than 8. Apart from the validity of such a numerical limit which is not proposed in this discussion, this simple observation seem to confirm that the
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composition of the organic matter may influence the toxicity of sediments to C. dubia. The higher biomass productions induced by the ‘eutrophic’ sediments of Fig. 7 are probably the results of the combination of a low bioavailability of toxicants and a generic increase of food availability from sediment fine particles. As documented in further studies, daphnids can feed on naturally occurring particulate matter and microorganisms (Gophen et al., 1974; Arruda et al., 1983; Vigano` et al., 1996; Stewart and Konetsky, 1998), and low C/N ratios generally characterize organic matter with higher nutritional value (Allan, 1995). The interaction between toxicity and organic matter composition seems to be particularly evident for the Oglio and Adda sediments which, although showing the same relatively high toxic potentials in both summer and winter samples (Fig. 7), caused markedly different responses of C. dubia, and apparently as a result of only the quality change of the sediment organic fraction.
5. Conclusions Based on the results obtained testing the sediments of the Po river, the main findings of the present study can be summarized as follows: (1) The summer and winter sediments of the River Po seem to be characterized by a different quality of the organic matter and particularly of the fine-grain fraction; this difference seems to be related with the toxic effects of whole sediments to C. dubia. (2) The 7-day tests on C. dubia have shown that the summer sediments of four areas can cause chronic toxic stress; no similar effects were observed for the winter sediments some of which could even enhance the biomass production of test organisms. (3) Besides the seasonal differences, much of the daphnid biomass production appeared to be inversely correlated with the sediment content of fine organic matter, thus confirming the role of fine particles in determining the sediment properties. (4) The toxicities of sediment extracts identified some critical sites along the River Po and sug-
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gested that the toxic potentials should be relatively stable in the two low-flow conditions. (5) The bioavailability of such toxic potentials, and thereby the effects on C. dubia, vary in apparent relationship with the source and quality of fine-grain organic matter. Source and quality seem to be partially controlled by season and may be even more important in determining toxicity than the amount of the fine organic matter itself. (6) The combined application of both wholesediment and extract toxicity tests seems to provide a better description of sediment problems than when singly applied and with more conventional endpoints.
Acknowledgements The author is grateful to Luisa Patrolecco for the elemental analyses of sediments, the Ecology Section of the University of Milan for sediment extracts preparation, Luciano Previtali and Giorgio Caresano for technical assistance, and Romano Pagnotta, Roberto Negro, Roberto Spaggiari, Luigi Rampa, Riccardo Rossini, and many other people, not least the boat crews, who helped in many important ways the realization of the River Po sediment project.
References Anderson, D.H., Benke, A.C., 1994. Growth and reproduction of the cladoceran Ceriodaphnia dubia from a forested floodplain swamp. Limnol. Oceanogr. 39, 1517 – 1527. Allan, J.D., 1995. Herbivory. In: Stream Ecology, Structure and Function of Running Waters. Chapman and Hall, London, pp. 187 – 203. Arruda, J.A., Marzolf, G.R., Faulk, R.T., 1983. The role of suspended sediments in the nutrition of zooplankton in turbid reservoirs. Ecology 64, 1225 – 1235. ASTM, 1994. Standard guide for conducting sediment toxicity tests with freshwater invertebrates, E 1383. American Society for Testing and Materials, Philadelphia, PA, Annual Book of ASTM Standards, vol. 11.04, pp. 1196 – 1225. Boese, B.L., Windsor, M., Lee, H. II, Echols, S., Pelletier, J., Randall, R., 1995. PCB congeners and hexachlorbenzene biota sediment accumulation factors for Macoma nasuta exposed to sediments with different total organic carbon contents. Environ. Toxicol. Chem. 14, 303 – 310.
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Burton, G.A., Scott, K.J., 1992. Sediment toxicity evaluations: their niche in ecological assessments. Environ. Sci. Technol. 26, 2068 – 2075. Burton, G.A., Ingersoll, C.G., Burnett, L.C., Henry, M., Hinman, M.L., Klaine, S.J., Lamdrum, P.F., Ross, P., Tuchman, M., 1996. A comparison of sediment toxicity test methods at three Great Lake areas of concern. J. Great Lakes Res. 22, 495–511. Cowgill, U.M., Keating, K.I., Takahashi, I.T., 1985. Fecundity and longevity of Ceriodaphnia dubia/affinis in relation to diet at two different temperatures. J. Crust. Biol. 5, 420 – 429. Day, P.R., 1965. Particle fractionation and particle-size analysis. In: Black, C.A. (Ed.), Methods of Soil Analysis, Part I. American Society of Agronomy, Madison, WI, pp. 545– 567. DeWitt, T.H., Ozretich, R.J., Swartz, R.C., Lamberson, J.O., Schults, D.W., Ditsworth, G.R., Jones, K.P., Hoselton, L., Smith, L.M., 1992. The influence of organic matter quality on the toxicity and partitioning of sediment-associated fluoranthene. Environ. Toxicol. Chem. 11, 197–208. Gee, G.W., Bauder, J.W., 1986. Particle-size analysis. In: Klute, A. (Ed.), Methods of Soil Analysis, Part I, Physical and Mineralogical Methods, 2nd ed. American Society of Agronomy, Madison, WI, pp. 383–411. Geiger, J.G., Buikema, A.L., Cairns, J., 1980. A tentative seven-day test for predicting effects of stress on population of Daphnia pulex. In: Eaton, J.G., Parrish, P.R., Hendricks, A.C. II (Eds.), Aquatic Toxicology, 3rd Symposium ASTM STP 707. American Society for Testing and Materials, Philadelphia, PA, pp. 13–26. Gophen, M., Cavari, B.Z., Berman, T., 1974. Zooplankton feeding on differentially labelled algae and bacteria. Nature 247, 393 – 394. Karickhoff, S.W., Brown, D.S., Scott, T.A., 1979. Sorption of hydrophobic pollutants on natural sediments. Wat. Res. 13, 241 – 248. Knight, J.T., Waller, W.T., 1987. Incorporating Daphnia magna into the seven-day Ceriodaphnia effluent toxicity test method. Environ. Toxicol. Chem. 6, 635–645. Kukkonen, J., Landrum, P.F., 1996. Distribution of organic carbon and organic xenobiotics among different particlesize fractions in sediments. Chemosphere 32, 1063–1076. Lake, J.L., Rubinstein, N.I., Lee, H. II, Lake, C.A., Heltshe, J., Pavignano, S., 1990. Equilibrium partitioning and bioaccumulation of sediment-associated contaminants by infaunal organisms. Environ. Toxicol. Chem. 9, 1095– 1106. Landrum, P.F., Robbins, J.A., 1990. Bioavailability of sediment-associated contaminants to benthic invertebrates. In: Baudo, R., Giesy, J.P, Muntau, H. (Eds.), Sediments: Chemistry and Toxicity of in-place Pollutants. Lewis, Chelsea, MI, pp. 237–263. Lovett-Doust, J., Lovett-Doust, L., Biernacki, M., Mal, T.K., Lazar, R., 1997. Organic contaminants in submersed
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macrophytes drifting in the Detroit River. Can. J. Fish. Aquat. Sci. 54, 2417 – 2427. Marchetti, R., Passino, R., Pagnotta, R., 1994. The Po River basin (Italy): problems and water management policies. Eur. Wat. Pollut. Contam. 4, 26 – 33. Pagnotta, R., Caggiati, G., Piazza, D., Ferrari, F., 1995. Il controllo della qualita` delle acque superficiali del bacino padano: situazione attuale e prospettive future (Abstract in English). Inquinamento 4, 8 – 14. Santiago, S., Thomas, R.L., Larbaigt, G., Corvi, C., Rossel, D., Tarradellas, J., Gregor, D.J., McCarthy, L., Vernet, J.P., 1994. Nutrient, heavy metal and organic pollutant composition of suspended and bed sediments in the Rhone River. Aquat. Sci. 56, 220 – 242. Standley, L.J., 1997. Effect of sedimentary organic matter composition on the partitioning and bioavailability of dieldrin to the oligochaete Lumbriculus 6ariegatus. Environ. Sci. Technol. 31, 2577 – 2583. Stewart, A.J., Konetsky, B.K., 1998. Longevity and reproduction of Ceriodaphnia dubia in receiving waters. Environ. Toxicol. Chem. 17, 1165 – 1171. Suedel, B.C., Rodgers, J.H., Clifford, P.A., 1993. Bioavailability of fluoranthene in freshwater sediment toxicity tests. Environ. Toxicol. Chem. 12, 155 – 165. Van Leeuwen, C.J., Niebeck, G., Rijkeboer, M., 1987. Effects of chemical stress on the population dynamics of Daphnia magna: a comparison of two test procedures. Ecotoxicol. Environ. Saf. 14, 1 – 11. Vigano`, L., 1991. Suitability of commercially available spring waters as standard medium to culture Daphnia magna. Bull. Environ. Contam. Toxicol. 47, 775 – 782. Vigano`, L., Arillo, A., De Flora, S., Lazorchak, J., 1995. Evaluation of microsomal and cytosolic biomarkers in a seven-day larval trout sediment toxicity test. Aquat. Toxicol. 31, 189 – 202. Vigano`, L., Bassi, A., Garino, A., 1996. Toxicity evaluation of waters from a tributary of the River Po using the 7-day Ceriodaphnia dubia test. Ecotoxicol. Environ. Saf. 35, 199 – 208. Vigano`, L., Arillo, A., Falugi, C., Melodia, F., 1998. Histochemical and biochemical markers in trout larvae exposed to river sediments. Chemosphere 37, 2797 – 2807. Vigano`, L., Barbiero, G., Buffagni, A., Mingazzini, M., Pagnotta, R., 1999. Assessment of the alterations of the aquatic environment downstream from a polluted tributary of the River Po (Italy). Aquat. Ecosystem Health Manage. (in press). West Inc., and Gulley, D.D., 1991. Toxstat 3.5. Western Ecosystem Technology, Cheyenne, WY. Wetzel, R.G., 1983. Limnology. CBS College/Saunders, Philadelphia, PA 767 pp. Winner, R.W., 1981. A comparison of body length, brood size and longevity as indices of chronic copper and zinc stresses in Daphnia magna. Environ. Pollut., A 26, 33 – 37.
Assessment of the toxicity of River Po sediments with Ceriodaphnia dubia L. Vigano`* Water Research Institute, CNR, 20047 Brugherio, Milan, Italy Received 8 January 1999; accepted 16 March 1999
Abstract A recent interdisciplinary project aimed at the assessment of the quality of the River Po focusing primarily on the bed sediments of the principal Italian river. In such a moderate/low polluted environment, the identification of sites at risk using a number of toxicological tools was considered of great interest. The present study examines in detail the information which was obtained from two of the toxicological approaches adopted in that project, that is, the 7-day whole-sediment test with Ceriodaphnia dubia and the acute toxicity test (48 h) with the same crustacean conducted on an organic-solvent extract of the sediment fine material ( B 63 mm). These toxicity tests were applied to composite sediment samples collected in summer and winter low-flow conditions, downstream from the main tributaries of the River Po. Summer and winter sediments seemed to be characterized by different qualities, as suggested by their oxygen demands and C/N ratios, whereas they were indistinguishable for the contents of organic carbon and fine material. For some reaches of the River Po, the 7-day tests showed that the summer sediments cause chronic toxic stress, whereas the winter sediments enhance reproduction and growth. Apart from these seasonal differences, it was found that the biomass production (reproduction + somatic growth) of C. dubia was negatively correlated to the fraction of sediment organic carbon contributed by fine material ( B 63 mm). The results of the extract tests showed that the toxic potentials of sediments vary along the River Po, identifying some critical sites. In contrast with whole-sediment results, it was found that the toxic potentials, reported as sediment toxic units (TUs), are relatively independent from seasons and substantially stable, particularly along the larger potamon section. The concurrent examination of biomass production and sediment TUs confirmed the sites at risk and demonstrated that the biomass production of C. dubia is inhibited by the increase of sediment TUs. This examination also demonstrated that some sediments, particularly of the winter survey, have high toxic potentials which, however, were not effective. Apparently this was a result of different source and quality of sediment organic matter, which may affect either the bioavailability of chemicals or the nutritional value of fine particles. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Daphnid growth; Fine material; Organic carbon; Sediment extract; Toxic unit; Whole sediment test
E-mail address: [email protected] (L. Vigano`) 0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 9 9 ) 0 0 0 1 9 - 3
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1. Introduction The adsorption of chemicals onto the organic particles of sediments is an important process through which a lot of contaminants can reach much higher concentrations in the bottom-sediment compartment than in the upper water column. Contaminated sediments can be a sink but often a long-term source of toxicants which can produce adverse effects to aquatic life (Landrum and Robbins, 1990; Burton and Scott, 1992). In large bodies of water, toxic effects can rarely approach the acute levels since the dilution capacity often reduces the concentrations of chemical inputs at or below the chronically effective levels. In such environments, dilution combined with a limited bioavailability of toxicants, make even sediments rarely responsible for acute toxic effects. Consequently, the adoption of sensitive sublethal endpoints is generally required to identify the presence of toxicity and assess the potential effects to aquatic life of a given area. Chronic toxicity tests and biomarkers applied to whole sediment assays can be two important tools of investigation (Vigano` et al., 1995, 1998; Burton et al., 1996). The River Po is the largest watercourse in Italy. It drains an area of ca 70 000 km2 and receives from the different tributaries and sub-basins variable and complex mixtures of contaminants of both industrial, agricultural and urban origins (Marchetti et al., 1994; Pagnotta et al., 1995). A new monitoring program has recently been undertaken to assess the quality of the River Po. This monitoring program has largely focused on the bottom sediments of the river environment assuming that, in conjunction with the riverine community, they can be the most representative compartment to accomplish the objective of a quality assessment. Composite sediment samples were collected downstream from the confluences of principal tributaries and compared with an ambient control sediment sampled from the upper reach of the River Po. To investigate two potentially different conditions of the river sediment environment, both a summer and a winter lowflow surveys were undertaken. River sediments have concurrently been examined for several
chemical and toxicological indicators whose sensitivity and potential for discriminating sites at risk are still being examined and compared. A comprehensive report on this project will soon be published. In this context, the present study discusses in detail the results which have been obtained from two of the toxicological approaches adopted in the Po sediment project. They are the 7-day whole sediment test with Ceriodaphnia dubia and the acute test (48 h) conducted with the same crustacean on an organic-solvent extract of the sediment fine material (B 63 mm). The three main objectives of this study were: (a) to evaluate the potential and thus the usefulness of these two approaches for identifying the toxic risk present along the River Po; (b) to investigate whether sediment properties may affect the daphnid responses; and (c) to obtain greater insights for the quality assessment process, also evaluating less conventional daphnids endpoints.
2. Materials and methods
2.1. Area of study and sediment collection Ten reaches of the River Po were chosen to describe the quality of the watercourse. The first was located ca 50 km from the springs of the river (Casalgrasso, TO), and the sediments collected from this area, hereafter indicated as Up Turin, served as an ambient control, being in the upper course of the river and thus before it receives important loads of contaminants. The other nine reaches were located downstream the main tributaries of the River Po. As shown in Fig. 1, these tributaries were: the Dora Riparia, Dora Baltea, Sesia, Tanaro, Ticino, Lambro, Adda, Oglio and Panaro. The River Panaro is not important per se but because it is the last tributary of the River Po. The corresponding downstream reach is therefore representative of the entire watershed of the Italian river, before it flows into the Adriatic Sea. Hereafter, each of the nine sediments and corresponding river stretch will be identified by the name of the tributary, e.g., Sesia sediment. At each sampling area, the sediment collection started downstream from the completion of the
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mixing process between the main river and the tributary, thereby assuming that where River Po waters are homogeneous also bottom sediments are correspondingly in homogeneous conditions. The observation of completely mixed waters, usually made after some kilometers from the confluences, was based on several cross-section profiles obtained for conductivity, as detailed in a previous study (Vigano` et al., 1996). At each sampling area sediment collection usually terminated before the confluence of the next tributary. Generally the extension of the sampling areas increased going toward the Po delta, that is from about 1 – 2 km in the upper tract, where the River Po has a mean annual flow of ca 70 m3 s − 1, to 20 – 40 km of the lower/final reaches, where the river has a mean annual flow of ca 1500 m3 s − 1. A Ponar grab sampler was used to collect composite samples of bottom river sediments. Water depth at sedimentation sites varied between 0.5 and 5 m, with an average value of approx. 1.5 m. Both summer and winter sediment collections were completed within a few days and in stable low flow conditions. No substantial change of the
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location of the natural deposition areas and thus of sampling sites were observed in the two surveys. Composite samples were stored at 4°C for a few days before the 7-day tests. Sediments and the corresponding fine fractions were analyzed for nitrogen and organic carbon contents (Carlo Erba C:N:S analyzer). The results of these measurements are listed in Table 1. The contents of fine materials, also reported in Table 1, were determined by a sedimentation technique (Day, 1965; Gee and Bauder, 1986). To this aim, 25-g aliquots of freeze-dried sediment samples were first dispersed in 0.5 l of distilled water with Na-hexametaphosphate as a dispersant (25 ml of a solution containing 33 g (NaPO3)6 l − 1 + 7 g Na2CO3 l − 1), and then poured through a 0.1-mm sieve into a sedimentation cylinder (Esenwein). According to Stokes’s Law (20°C and 2.7 g cm − 3 as mean particle density), samples of the sediment suspension were taken from the cylinder at given time intervals and depth so that fine particles characterized by different settling velocities were collected (pipet method) and dry weight determined. Since freeze-drying is suspected to alter particle-
Fig. 1. Map showing the watershed of the River Po and its main tributaries downstream from which the composite sediment samples were collected.
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Table 1 Composition of sediment samples collected along the River Po during summer and winter low-flow conditionsa Sediment
Up Turin D. Riparia D. Baltea Sesia Tanaro Ticino Lambro Adda Oglio Panaro
Summer
Winter
Fine fraction (%)
N (%)
Org. C (%)
C/N
Fine fraction (%)
N (%)
Org. C (%)
C/N
12.0 21.2 69.8 46.6 47.4 37.0 58.8 32.8 56.1 50.7
0.25 0.25 0.16 0.16 0.16 0.16 0.26 0.21 0.23 0.19
2.73 2.29 1.30 1.48 1.38 1.37 2.07 1.76 1.91 1.62
10.92 9.16 8.13 9.25 8.63 8.56 7.96 8.38 8.30 8.53
26.6 28.0 23.5 45.2 66.5 37.2 47.6 40.8 58.4 48.7
0.20 0.37 0.21 0.20 0.16 0.14 0.34 0.18 0.20 0.13
1.64 2.59 1.72 1.55 1.27 1.17 2.29 1.37 1.50 1.14
8.20 7.00 8.19 7.75 7.94 8.36 6.74 7.61 7.50 8.77
The contents of fine material (B63 mm) and the results of the elemental analyses carried out on the sediment fine fractions are reported. Sediments were collected upstream from the city of Turin (ambient control; Up Turin) and downstream from the confluences of nine main tributaries of the River Po. a
size distribution, for five out of the ten sediments of the winter series a comparison was made between fresh and freeze-dried samples. The amounts of fine material determined with the sedimentation technique gave the following results for fresh and freeze-dried samples, respectively: D. Riparia 22.2%, 21.6%; Sesia 40.4%, 39.4%; Ticino 31.6%, 33.0%; Adda 34.6%, 36%; Panaro 47.0%, 43.0%. According to this comparison (although based on B 50-mm instead of B63-mm particles), we felt reasonably confident of the results which can be obtained using freeze-dried samples.
2.2. Sediment extraction Since smaller particles bind xenobiotics much more efficiently than coarse fraction of sediment, only fine fraction was taken into account for xenobiotic extraction and acute testing. The fine fraction (B 63 mm) of freeze-dried sediments was separated by sieving and extracted for 8 h in a Soxhlet apparatus using a mixture (1:1) of n-hexane and acetone (60 g fine material; 200 ml solvent mixture). The extract was reduced to small volume by rotary evaporation and an aliquot transferred to DMSO obtaining a final equivalent concentration of 6 g of sediment fine fraction per ml. This kind of extract obtained from the ten sediments of the River Po and a procedural blank prepared through the same procedure but without sediment, were acutely tested with C. dubia.
2.3. Toxicity tests C. dubia is routinely cultured at our laboratory and, as described in previous papers, culturing is based on a partially synthetic medium (hardness 150 mg CaCO3 l − 1) (Vigano`, 1991) and a diet composed of Selenastrum capricornutum and Saccharomyces cere6isiae (Vigano` et al., 1996), fortified with three vitamins provided at final concentrations of: B1 75 ng l − 1, B12 1 ng l − 1and H 0.75 ng l − 1(Cowgill et al., 1985). The 7-day tests on whole sediments were conducted according to the ASTM procedure (ASTM, 1994). In brief, each sediment was tested in ten replicates prepared in 50-ml beakers, using 5 ml of sediment and 20 ml of semisynthetic water (1:4). One C. dubia less than 24 h old was added to each beaker after a few-hour settling period. Probably because of the adoption of a partially different diet from ASTM proposal, feeding had to be modified to meet the validity criteria of the test (80% survival and 15 young/female). For external nutrition procedure, applied during overlying water renewals and young count and removal, nominal S. capricornutum and Saccharomyces cere6isiae densities were kept at 300 000 cells ml − 1 for the entire duration of the test. On the contrary, to minimize the additional loads of cells and organic matter to test beakers (containing sediment), nominal algae and yeast cell concentrations in overlying water were gradually increased from 100 000 cells ml − 1
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at the start of the test, to 200 000 on days 1, 2 and 3, to 250 000 cells ml − 1 on days 4, 5 and 6. Clean river sand was used as a substrate for control treatment. At 7 days the body length of each test organism was measured using a light microscope and an ocular micrometer. The acute toxicity of the sediment extracts was evaluated conducting 48-h toxicity tests on C. dubia. Sediment extracts were used according to the original quantities of fine material extracted and the toxicity results were examined in terms of equivalent grams (of fine material extracted) per liter of test solution. Hereafter these toxicity results will simply be reported in terms of g l − 1. Extracts were tested at a minimum of five different concentrations, usually ranging between 20 and 1.25 g l − 1. Ten C. dubia less than 24 h old were randomly distributed to each of the 20-ml solutions of sediment extracts prepared in 50-ml beakers. Tests were conducted at 2591°C, under dim light conditions, and dilution water was the same semisynthetic medium used for both laboratory culture and 7-day tests (Vigano`, 1991). The procedural blanks showed 48hEC50 values close to 1.2% (v/v) so the background contributions to the acute toxic effects of sediment extracts was assumed to be negligible. In fact, at the equivalent concentration of 20 g l − 1, usually the highest of the five-concentration series, the corresponding solvent concentration was 0.3% (below the 48hEC1 of 0.7%).
2.4. Statistics Results of reproduction and growth from the 7-day whole sediment tests on C. dubia were first examined for normal distribution and homogeneity of variance using x 2-test and Bartlett’s test, respectively. The two series of results of the summer survey passed both the tests (P =0.01) and were examined by one-way ANOVA. Then each treatment group was compared to the ambient control (Up Turin sediment) using the Bonferroni test. This was possible because ambient and laboratory controls were first compared using Student t-test and found to be not significantly different; this result was observed for both the surveys on the River Po (data not shown). Also for the
195
results of the winter survey the same procedure was followed. In this case, however, fertility results failed Bartletts’ homogeneity test and the nonparametric Wilcoxon rank sum test was used to compare each treatment to the ambient control (Up Turin). On the contrary, growth results passed normality and homogeneity tests and both ANOVA and Bonferroni analyses could be used. With regard to acute toxicity tests, 48hEC50 values and 95% confidence intervals were calculated with probit analysis. All calculations were performed using Toxstat 3.5 computer package (West and Gulley, 1991).
3. Results
3.1. Whole sediment tests In no case did River Po sediments cause significant mortality of C. dubia. The only exception was observed for the winter survey when the Lambro sediment killed all the organisms after 24 h of exposure (Table 2). Although amplified by the static-renewal procedure of the laboratory test, this result can be attributed to the high ammonia concentration of porewater, which in this sediment reached 72 mg l − 1 (N-NH4), the highest value observed in the study (G. Tartari and S. Valsecchi, Water Research Institute, Milan, personal communication), and the high oxygen demand of the sediment material which lowered DO to almost zero level during the first 24 h of the test. No similar event could be observed in the summer survey when the sediment quality of the Lambro area, as on average of the entire River Po, seemed to be different from that of the winter period (see below). Neither summer nor winter whole-sediment tests revealed significant inhibition of fertility when compared to ambient control (Up Turin) (Table 2). On the contrary, winter sediments collected from the D. Riparia, D. Baltea and Adda stretches significantly enhanced reproduction of C. dubia. The measurement of body growth gave more information on daphnid response, so that the D. Baltea, Tanaro, Oglio and Panaro summer sediments significantly reduced the final body
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Table 2 Results of the 7-day toxicity tests conducted with Ceriodaphnia dubia on whole sediment samples of the River Poa Sediment
Up Turin D. Riparia D.Baltea Sesia Tanaro Ticino Lambro Adda Oglio Panaro
Summer
Winter
Neonates/female
Body length (mm)
Neonates/female
Body length (mm)
19.195.6 20.1 9 6.8 14.6 95.3 19.0 95.4 16.3 9 4.8 21.6 94.8 18.9 93.7 19.1 9 3.4 12.8 9 6.0 15.8 94.9
0.936 9 0.040 0.9269 0.034 0.8809 0.038* 0.8959 0.061 0.8609 0.058* 0.9159 0.050 0.8939 0.024 0.8889 0.060 0.8799 0.041* 0.8819 0.029*
18.5 93.5 30.2 93.1* 23.1 91.7* 21.6 93.3 17.7 94.8 19.4 93.5 –b 27.4 95.6* 24.8 9 9.4 17.1 9 3.0
0.936 9 0.039 1.009 9 0.025* 0.978 9 0.027* 0.968 9 0.026 0.941 9 0.034 0.949 9 0.039 –b 0.961 90.033 0.980 9 0.045* 0.915 90.029
a
Composite sediment samples were collected in summer and winter low-flow conditions, upstream the city of Turin (ambient control; Up Turin) and downstream from the confluences of nine main tributaries of the River Po. Both young productions and body lengths are reported as mean values ( 9 S.D.) of treatment group. b The winter sediment of the Lambro reach caused the complete mortality of C. dubia. * Significantly different from ambient control value (PB0.05).
lengths of the test organisms (Table 2). On the other hand, some winter sediments confirmed their positive effects, determining also a growth enhancement of daphnids. In fact, C. dubia exposed to the D. Riparia, D. Baltea and Oglio winter sediments were larger than those of ambient control (Table 2).
4. Discussion The reproductive output of C. dubia exposed to whole sediments in the 7-day tests provided no
3.2. Sediment extract tests The results of the 48-h toxicity tests showed a significant variation in the levels of contamination along the River Po (Fig. 2). The D. Riparia and Lambro areas were the most contaminated followed by Adda and Oglio, all showing 48hEC50s lower than 10 g l − 1. On the other extreme, the sediments collected downstream from the confluence of the River Ticino were consistently the least toxic. This trend appears substantially stable for both the surveys, whereas, as illustrated in Fig. 2, the toxicities of the sediments collected from the upper reaches were more variable. Particularly the Up Turin sediment showed the greatest variability, as the 48hEC50 was 6.2 g l − 1 in summer and 24.7 g l − 1 in winter, i.e. comparable to the most toxic and the least toxic downstream areas, respectively.
Fig. 2. Toxicities of the extracts of the sediment fine materials ( B63 mm) to Ceriodaphnia dubia. The results, reported as 48hEC50 with 95% confidence intervals, are in terms of equivalent grams of fine material extracted per liter of test solution (see text). Sediments were collected along the River Po in summer (gray bars) and winter (black bars) low-flow conditions, upstream from the city of Turin (ambient control; Up Turin) and downstream from the confluences of nine tributaries.
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significant evidence of a toxic risk along the River Po, both for winter and summer low-flow conditions. The reproductive response showed, on the contrary, that a stimulation could even be effective at three stretches of the winter riverine environment (D. Riparia, D. Baltea, Adda). The adoption of the parameter carapace length was more informative. It showed significant growth inhibition, and thereby the risk of chronic toxicity at four areas of the summer riverine environment (D. Baltea, Tanaro, Oglio, Panaro). The higher neonate productions observed for D. Riparia- and D. Baltea-winter sediments were also confirmed by the correspondingly higher growth rates of test organisms. Consistent with this general picture of a moderate/low level of toxic risk, the River Po environment showed only one case of markedly harmful conditions which were observed in winter for the Lambro stretch. The advantage of using growth to detect chronic toxicity with C. dubia and, generally, daphnids is seemingly the result of two factors which, although not completely distinguishable, can be identified as: (a) the responsiveness of the parameter itself; and (b) the relatively low variability of growth data. The first aspect was documented in previous studies, most conducted on Daphnia magna, and both with organics, trace metals and toxicant mixtures (Geiger et al., 1980; Winner, 1981; Van Leeuwen et al., 1987; Vigano` et al., 1996). The second factor is equally or even more important to determine the usefulness of size as an indicator of stress. In the present study, in fact, the reproduction data show a mean coefficient of variation of ca 24% against a corresponding value as low as 4% for the growth data. Such a higher quality of growth data allow the minor differences from control to be statistically detected. This favorable property of growth data is also confirmed by previous data sets as those found, for example, by Knight and Waller (1987). Besides these observations, the results obtained from the 7-day tests suggest that the summer and winter benthic environments of the River Po are characterized by different conditions and likely problems. In fact, the summer sediments can cause chronic toxic stress, whereas the winter
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Fig. 3. (a) Spatial trends of the C/N ratios of sediment fine fraction ( B63 mm). (b) Spatial trends of the mean oxygen concentrations measured during the 7-day whole-sediment tests with Ceriodaphnia dubia. Sediments were collected along the River Po in summer and winter low-flow conditions, upstream from the city of Turin (Up Turin) and downstream from the confluences of nine tributaries.
sediments can have a nutritional value which enhances fertility and/or growth. Both the mean concentrations of dissolved oxygen (mg l − 1) measured during the 7-day tests, and the C/N ratios of sediment fine particles, confirm that summer and winter benthic environments are significantly different. The spatial trends of the C/N ratios along the River Po are shown in Fig. 3a. These results demonstrate that the fine material of summer sediments has on average a significantly higher C/N ratio than winter sediments (8.89 0.8 vs. 7.89 0.6; PB 0.00). This difference might be explained by a higher contribution of plant debris from both aquatic macrophytes and terrestrial plants occurring during the most favorable sum-
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mer period. Aquatic macrophytes, high cellulose and lignine particles and, in general, progressively more decomposed materials (of autochthonous and allochthonous origins) show increasingly higher C/N ratios (ca 12 – 20 and over) (Wetzel, 1983; Santiago et al., 1994; Allan, 1995). In summer, emergent macrophytes (Cyperus sp.) and sometimes also submersed macrophytes were abundant in sedimentation areas of the River Po, whereas they were virtually absent in winter. A similar trend was recently observed, for example, in the Detroit River (Lovett-Doust et al., 1997). Low C/N ratios (ca 4 – 8) characterize, on the other hand, the particulate matter which originates from wastewater treatment plants (Santiago et al., 1994) and the micro community of periphyton (Allan, 1995). The ‘absence’ of macrophyte and terrestrial plant residuals combined with slower mineralization rates which are typical of the winter period should favor the presence of particulate matter characterized by lower C/N ratios, particularly in those stretches which are exposed to heavier wastewater inputs. This is the case of both the D. Riparia and Lambro reaches which are exposed to high and direct loads of effluent discharges (Marchetti et al., 1994; G. Barbiero, Water Research Institute, Rome, personal communication). Accordingly, their winter sediments showed the lowest C/N ratios of the entire River Po. Santiago et al. (1994) found a similar behavior of the C/N ratio in both suspended and bed sediments of the River Rhone. Consistent with all these observations, the mean concentrations of dissolved oxygen, recorded during the 7-day tests, showed two clearly distinct patterns (Fig. 3b), so that the winter sediments demonstrated on average the highest oxygen consumption (5.09 1.1 mg O2 l − 1 in winter versus 6.390.2 mg O2 l − 1 in summer; PB0.00). In this context, it is of particular interest to mention that neither the organic carbon of whole sediment, its contribution from fine particles, nor the content of fine material were significantly different in the two seasons. Thus, the distinction between summer and winter sediments seems to be the result of qualitative (compositional) rather than quantitative aspects of the
organic matter, as might be described by a C/N ratio. On these grounds, an attempt was made to understand how the sediment properties could explain the variability in response of C. dubia. With this aim in view, both growth and fertility data were first converted to a common biomass basis and then integrated into a final single value. This was accomplished, for the growth data, calculating the mean somatic weight of a female from the regression with body length found by Anderson and Benke (1994) and, for the fertility data, multiplying the mean number of young produced in a treatment by the mean dry weight of an egg (0.75 mg) which in turn was measured for C. dubia by the same authors. In this way, the responses of C. dubia exposed to River Po sediments could be examined through a single term of biomass production obtained as the sum of the estimates of reproductive and somatic components. Since the organic content of sediments is a key factor for the accumulation of organic chemicals and hence the contamination of sediments (Karickhoff et al., 1979; Kukkonen and Landrum, 1996), it was explored whether the variability of biomass production among treatments could be explained by the sediment contents of organic carbon. It was found that the organic carbon of whole sediment was not descriptive of biomass variation (data not shown). On the contrary, the organic content contributed by the fine material, and more precisely, its concentration reported on a whole sediment basis, was a better descriptor. In other words, such a fraction of total organic carbon due to sediment fine material was found to be a first reasonably good descriptor of C. dubia responses. With the only exception of four sediments which sustained relatively higher biomass values, Fig. 4 shows that the biomass production of C. dubia is inversely correlated with the organic carbon contributed by the sediment particles smaller than 63 mm (r= − 0.72; PB 0.00). To understand the responses of C. dubia, further important information was obtained from the toxicities of the sediment extracts. Taking into account both the sediment content of fine material
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Fig. 4. Relationship between the mean biomass production of Ceriodaphnia dubia (mg per female) exposed to River Po sediments and the sediment contents of organic carbon (%) contributed by the fine material ( B 63 mm).
and the toxicity of its solvent-extractable contaminants (48hEC50) a convenient estimate of the toxic potential of each sediment could be obtained in terms of toxic units (sediment content of fine material/48hEC50 of fine-material extract= whole sediment TUs). For both the summer and winter surveys, these kinds of results are illustrated in Fig. 5, which shows the toxic potentials estimated for the different stretches of the River Po. Besides
Fig. 5. Potential toxic units estimated for the River Po sediments. These values are based on the amount of fine material ( B 63 mm) present in each sediment and the toxicity of the organic-solvent extractable contaminants of such fraction. Results are reported for both the summer (clear bars) and winter low-flow surveys.
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Fig. 6. Relationship between the mean biomass productions of Ceriodaphnia dubia exposed to the summer sediments of the River Po and their toxic potentials reported as toxic units (see text). UP, Up Turin; DR, Dora Riparia; DB, Dora Baltea; SE, Sesia; TA, Tanaro; TI, Ticino; LA, Lambro; AD, Adda; OG, Oglio; PA, Panaro.
the few exceptions of the most upstream areas, where TU variation was however limited to a factor of 2, most riverine environments showed no substantial change between the summer and winter surveys. Fig. 5 clearly demonstrates the two extreme conditions of the Ticino and Lambro stretches, which show the least and highest toxic potentials of the River Po, respectively. Also Up Turin demonstrates very low toxic potential, confirming its role as an ambient control. It can be mentioned that some previous studies (Vigano` et al. 1995, 1998, 1999) as well as the preliminary results on sediment contents of, for example, NNH4 and extractable organic halides (EOX) are consistent with the picture obtained for toxic potentials (Fig. 5) (G. Tartari and S. Valsecchi, Water Research Institute, Milan, personal communication; A. Lopez, Water Research Institute, Bari, personal communication). The availability of the sediment extract toxicities allowed us to examine whether the different levels of biomass production of C. dubia could be explained by the TUs of sediments. To better understand this relationship it is useful to first examine the summer sediments only. As shown in Fig. 6, most of the summer samples demonstrate
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that the biomass production of C. dubia is inhibited by the increase of sediment TUs (r= − 0.79; P =0.02). However, both the Lambro and Dora Riparia sediments do not meet this relationship, suggesting that their high toxic potentials are substantially unavailable to exert toxic effects. These two sediments could sustain a relatively high biomass production, at levels comparable to those of sediments with least toxic contents (TUs), such as Up Turin, Sesia and Ticino. The inclusion of the winter-survey results modified the relationship as illustrated in Fig. 7. The contemporary analysis of the two-survey results increased, on one hand, the number of cases characterized by a low toxic potential and high biomass production, confirming what we can call the ‘basic relationship’ of Fig. 6, but, on the other hand, also provided further examples of sediments which, despite a relatively high toxic potential, did not inhibit the biomass production of C. dubia.
Fig. 7. Relationship between the mean biomass production of Ceriodaphnia dubia and the sediment toxic potentials. Results are from both the summer and winter low-flow surveys. The empty and filled circles distinguish the sediments with a fine material characterized by a C/N ratio higher than 8 () and lower than 8 ( ), respectively (abbreviations as in Fig. 6).
These latter cases increased the limited number of outliers whose (organic-solvent extractable) contaminants seem to be largely or completely unavailable. In the first group and precisely in the upper-left portion of the graph, we can consistently find both the Ticino and the Up Turin sediment samples (no effect). In the lower-central position there are the four sediments which were identified as toxic by growth inhibition, and in the upper-right position (eutrophic) there are those sediments which, despite similar or even higher toxic potentials (TUs), showed either unaffected or much higher biomass productions. Some preliminary results on the macroinvertebrates of the Ticino and Lambro reaches may be significant at this point. It has been observed that the standard biotic indexes cannot distinguish the summer macroinvertebrates communities of the two reaches, that is, when the 7-day tests show equivalent biomass production (Fig. 7). In winter, when the Lambro sediment caused the complete mortality of C. dubia, the macroinvertebrates of the Lambro reach show a significant impairment compared to the Ticino reach (A. Buffagni, Water Research Institute, Milan, personal communication). In this case, interstitial ammonia and sediment oxygen demand seem to play a prominent role in affecting organisms. A number of previous studies pointed out that the toxic effects of sediments and, in general, the bioavailability of sediment-bound organic chemicals can be modified by the content of organic matter as well as its source and composition (DeWitt et al., 1992; Suedel et al., 1993; Boese et al., 1995; Standley, 1997). It was also suggested that the sediments dominated by ‘anthropogenic carbon’, as are often the most polluted, may have a greater affinity for contaminants than those containing ‘natural organic carbon’ (Lake et al., 1990). It may be of interest to note that those sediments which meet the basic relationship of Fig. 7 (empty circles) have a fine material characterized by a C/N ratio higher than 8, whereas those sediments which do not meet that basic relationship (filled circles) have C/N ratios lower than 8. Apart from the validity of such a numerical limit which is not proposed in this discussion, this simple observation seem to confirm that the
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composition of the organic matter may influence the toxicity of sediments to C. dubia. The higher biomass productions induced by the ‘eutrophic’ sediments of Fig. 7 are probably the results of the combination of a low bioavailability of toxicants and a generic increase of food availability from sediment fine particles. As documented in further studies, daphnids can feed on naturally occurring particulate matter and microorganisms (Gophen et al., 1974; Arruda et al., 1983; Vigano` et al., 1996; Stewart and Konetsky, 1998), and low C/N ratios generally characterize organic matter with higher nutritional value (Allan, 1995). The interaction between toxicity and organic matter composition seems to be particularly evident for the Oglio and Adda sediments which, although showing the same relatively high toxic potentials in both summer and winter samples (Fig. 7), caused markedly different responses of C. dubia, and apparently as a result of only the quality change of the sediment organic fraction.
5. Conclusions Based on the results obtained testing the sediments of the Po river, the main findings of the present study can be summarized as follows: (1) The summer and winter sediments of the River Po seem to be characterized by a different quality of the organic matter and particularly of the fine-grain fraction; this difference seems to be related with the toxic effects of whole sediments to C. dubia. (2) The 7-day tests on C. dubia have shown that the summer sediments of four areas can cause chronic toxic stress; no similar effects were observed for the winter sediments some of which could even enhance the biomass production of test organisms. (3) Besides the seasonal differences, much of the daphnid biomass production appeared to be inversely correlated with the sediment content of fine organic matter, thus confirming the role of fine particles in determining the sediment properties. (4) The toxicities of sediment extracts identified some critical sites along the River Po and sug-
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gested that the toxic potentials should be relatively stable in the two low-flow conditions. (5) The bioavailability of such toxic potentials, and thereby the effects on C. dubia, vary in apparent relationship with the source and quality of fine-grain organic matter. Source and quality seem to be partially controlled by season and may be even more important in determining toxicity than the amount of the fine organic matter itself. (6) The combined application of both wholesediment and extract toxicity tests seems to provide a better description of sediment problems than when singly applied and with more conventional endpoints.
Acknowledgements The author is grateful to Luisa Patrolecco for the elemental analyses of sediments, the Ecology Section of the University of Milan for sediment extracts preparation, Luciano Previtali and Giorgio Caresano for technical assistance, and Romano Pagnotta, Roberto Negro, Roberto Spaggiari, Luigi Rampa, Riccardo Rossini, and many other people, not least the boat crews, who helped in many important ways the realization of the River Po sediment project.
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