Assessment of the alterations of the aquatic environment downstream from a polluted tributary of the river Po (Italy)

Assessment of the alterations of the aquatic environment downstream from a polluted tributary of the river Po (Italy)

Aquatic Ecosystem Health and Management 2 (1999) 55–69 Assessment of the alterations of the aquatic environment downst...

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Aquatic Ecosystem Health and Management 2 (1999) 55–69

Assessment of the alterations of the aquatic environment downstream from a polluted tributary of the river Po (Italy) L. Vigano` a,*, G. Barbiero b, A. Buffagni a, M. Mingazzini a, R. Pagnotta a a

Water Research Institute, CNR, 20047 Brugherio, Milan, Italy Water Research Institute, CNR, via Reno 1, 00198 Rome, Italy


Abstract A comprehensive evaluation of the results of many recent and ongoing studies has been performed in an attempt to assess which stressors are acting and what alterations could be derived for the aquatic organisms of the river Po downstream from one of its tributaries, the river Lambro. With this aim in view, this stretch of the river Po was compared with the immediate upper reach which is characterized by an overall good quality. The comparison was based on four categories of indicators, that is, watershed stress, physical-habitat alteration, and in particular chemical exposure and biological responses both of which were thoroughly examined. The reach located downstream of the river Lambro was found to be contaminated by a complex mixture of pollutants that were detected at low/moderate concentration values. This makes the area representative of what can be considered as a ‘grey’ level of pollution, that is the most frequent condition of watercourses worldwide. Nevertheless, the effects derived from such moderate/low levels of contamination for aquatic life are difficult to assess. Most of the indicators were found to be changed downstream of the river Lambro and different kinds of alterations were observed in both resident species and laboratory-test organisms. Although more information is necessary to understand less evident impairments as, for example, of the fish community, it can be concluded that a continuous interaction between chronic toxicity and trophic enrichment is the process that characterizes this riverine environment and affects the community. On the grounds of these findings, some suggestions are also proposed pertaining to ‘grey’ areas and their investigation. q 1999 Elsevier Science Ltd and AEHMS. All rights reserved. Keywords: Chemical and biological indicators; Chronic toxicity; Trophic enrichment; Community changes

1. Introduction The aquatic community and its species composition are largely determined by the combined effects of chemical, physical and hydrological factors. Significant variations of any of these factors can induce a response at the organism level (organism being the smallest unit interacting with the environment) and such alteration may propagate across several levels

* Corresponding author.

of ecological organisation, through populations up to the ecosystem. It is now accepted that the ecological integrity is the result of simultaneous occurrence of chemical, physical and biological integrity. Altering the processes associated with any of these components can have an impact on the quality of the aquatic ecosystem. Appropriate sets of indicators are, therefore, of paramount importance to describe, for example, existing or emerging problems, status and trends of any system component, or to meet management objectives related to the ecological

1463-4988/99/$20.00 q 1999 Elsevier Science Ltd and AEHMS. All rights reserved. PII: S1463-498 8(99)00011-1


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Fig. 1. Map (a) showing the entire basin of the river Po and the sub-basins of the river Ticino (dotted area, left side) and of the river Lambro (dashed area, right side). According to the Italian literature, the sub-basins 6B and 7B of the river Po watershed have been taken into account (Istituto di Ricerca Sulle Acque (IRSA), 1977). The map enlargement (b) details the portion of the middle river Po comprised between 270 and 330 km from the springs of the Italian river. This portion comprises the TI reach (270 to 305 km) and the LA reach (305 to 330 km) which are located downstream from the confluences of the river Ticino and Lambro, respectively. The final shaded portion of both the TI and LA stretches represents the approximate area of collection of organisms, water and sediment samples.

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health (Risser et al., 1984; Plafkin et al., 1989; Karr, 1993). The complexity of water quality assessment and the corresponding limitations of current approaches become more evident for those areas which show intermediate levels of alteration which are rarely the response to a single stressor. Indeed, this represents the most common environmental condition and in these areas characterized by this level of ‘grey’ quality, the aquatic community is typically exposed to chronic and sublethal stresses. In one such ‘grey’ area of the river Po, which is located downstream from the confluence of one of its tributaries, the river Lambro, several studies are under way or have recently been completed. This tract of the principal Italian river is exposed to a complex mixture of anthropogenic pollutants originating from the domestic, industrial and agricultural activities. This makes the area of the middle reach of the river Po a typical case study which can be considered representative of many water courses. Most of the above mentioned studies compared this grey area to that located immediately upstream and which receives the waters of the river Ticino, another important and only lightly polluted tributary of the middle river Po (Fig. 1). As the overall good quality of this upstream stretch (TI) has been and is being confirmed in many studies (see later), TI will be used as a reference site to estimate the degree of alteration observed in the river Po environment downstream from the confluence of the river Lambro (LA). Available results do not allow us to clearly distinguish the changes deriving for the river Po, as a result of the immission of the river Ticino. However, the aforementioned studies suggest that this tributary, whose watershed is partially protected as a river park (ca. 970 km 2), often improves the quality of the river Po, making the TI area one of the best of the entire Po watercourse, comparable to the upper tract of the river with regard to several chemical-exposure indicators. These characteristics should permit a sound comparison between the TI and LA stretches of the river Po, and the assessment, as well, of the alterations caused by the river Lambro to the “TI condition” of the river Po. The average flow of the middle river Po is approximately 750 m 3 s 21 at the TI stretch (downstream of the confluence of the Ticino) and 820 m 3 s 21 at the LA stretch (downstream of the confluence of the Lambro). The mean flows of

57 3


the two tributaries are about 200 m s for the river Ticino and 70 m 3 s 21 for the river Lambro. Fig. 1 shows the location of the two stretches of the main river whose approximate lengths are 35 and 25 km for the TI and LA reach, respectively. The data examined in this study were selected from the above mentioned recent and current studies according to two fundamental criteria. The first criterion to be met was that of being produced according to either standard procedures (e.g. US Environmental Protection Agency (USEPA), 1978; American Standards for Testing and Materials (ASTM), 1994; International Standards Organization (ISO), 1984), or methods described in recently published or accepted international studies (e.g. Benke, 1993; Mingazzini, 1993; Camusso et al., 1994; Vigano` et al., 1998b). The second criterion was that of being obtained from organisms, water or sediment samples collected from the final sections of the TI and LA stretches (Fig. 1), where the mixing processes are complete, so that the results could be accepted as representative of the river Po environment downstream from the two tributaries. The concurrent evaluation of all the selected data was aimed at two main objectives: first, to identify the stressors which are acting in the aquatic environment of the LA area; second, to investigate what kind of effects, if any, might derive from such stressors for the aquatic community of the LA reach. A third but minor objective of the present study was to make a first selection of the most effective indicators which can provide evidence of grey-area alterations. Because of the variety of potential stressors, many indicators have been examined and organized into four groups. These are: (a) watershed stress, (b) physical habitat alteration, (c) chemical exposure and (d) biological response and exposure. These groups represent four reference categories for the assessment of the ecological quality and, although not equally studied, they should provide a preliminary picture describing the problems of this area of the river Po.

2. Watershed stress indicators Despite the fact that the basin of the river Lambro accounts for only 6% of the total watershed area of the river Po, it contributes about 30% of the total


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Table 1 Indicators of watershed stress in the sub-basins of the river Ticino and Lambro. In section (a) demographic and soil use descriptors are reported; In section (b) the amounts of pesticides and fertilizers applied in the two sub-basins are also reported. Loads are the estimation of phosphorous and nitrogen quantities potentially released into the tributaries. From the Italian literature, only the sub-basins 6B and 7B of the river Po watershed have been taken into account for present calculations (Instituto di Ricerca Sulle Acque (IRSA), 1977) Parameter

River Ticino Basin

River Lambro Basin

(a) Demographic parameters Basin area (km 2) Resident population density (indiv. km 22) Cultivated area (km 2; % of subbasin) Total equivalent population Density of total equivalent population (n km 22) Industrial equivalent population Density of industrial equivalents (n km 22)

3352 229

3953 1186

1875 (56)

1803 (46)

4095 1222

19 874 000 5028

1900 567

12 228 000 3093

(b) P,N and pesticide loadings Pesticides application (kg × 10 3) N 1 P fertilizers application (kg × 10 3) Sources of P and N loads (kg × 10 3) Resident population Industrial activities Cattle farming Cultivated areas Noncultivated areas Total of P and N loads

2853 64.9

2463 64




257 52 85 113 15 522

1730 1107 426 2995 295 6553

1570 313 163 109 22 2177

equivalent population (resident 1 industrial equivalent population) of the Po watershed, which amounts to 62.7 × 10 6 (Barbiero and Giuliano, 1997). This is due to the high densities of both the resident population (1,186 individuals km 22) and the industrial activities (3093 industrial population equivalents km 22; Barbiero and Giuliano, 1997) which characterize the Lambro basin (Table 1). Markedly less populated and industrialized is the slightly smaller basin of the river Ticino so that, although accounting for a fraction of the Po watershed area which is similar to that of the river Lambro, it contributes only 0.006 of the total equivalent population. The river Ticino basin shows densities of resident population and of total and industrial equivalents which are substantially similar to the mean values of the river Po basin which, in turn, are 227 individuals km 22, 818 total equivalents km 22 and

N 10 544 7536 902 2886 430 22 300

554 industrial equivalents km 22, respectively. These values and similar ones for the Ticino basin vary between about 15 to 25% of the corresponding values for the Lambro watershed (Table 1). In addition to resident population, chemical, foodprocessing, paper-pulp and textile industries are the main responsible parties (90% of industrial equivalents) for the load of pollutants transported by the river Lambro to the river Po. In the Ticino basin, practically the same percentage of industrial equivalents is contributed by food processing, chemical, paper-pulp, textile and leather tanning industries, but all these industrial activities account for only 0.016% of the industrial load of the Lambro watershed. Both the sub-basins show important agricultural activities which are comparable to the mean agricultural usage of the Po catchment (44%). The Ticino

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Table 2 Representative physical- and chemical-indicator values which were found significantly modified in the LA reach of the river Po when compared to the TI reach, located immediately upstream. Results are organized in (a) water and (b) sediment compartment. They were obtained from both monitoring studies and surveys, and most are mean values or ranges which have been selected for being highly representative of the area of study. Single-cell comments describe the parameter change in moving from the TI to the LA reach Parameter

TI reach

LA reach


(a) Water compartment Temperature Dissolved oxygen (mg l 21) Hardness (mg l 21 as CaCo3) Conductivity (mS cm 21) Orthophosphate (mg P–PO4I 21) Total ammonia (mg N–NH3l 21) Nitrite (mg N–NO21 21) MBAS (mg l 21) Malathion (ng l 21) a 2,4 D (ng l 21) a Atrazine (ng l 21) a Simazine (ng l 21) a Molinate (ng l 21) a Ni dissolved (mg l 21) Zn dissolved (mg l 21)

Approximate increase 18C Mean decrease 0.3–1.5 168 176 345 367 34 80 0.09 0.53 0.03 0.05 27 46 nd b 55 nd b 50 40 90 32 86 50 254 1.3 2.1 4.0 10.1

Annoni et al., 1996 Guzzi et al., 1997 Vigano` et al., 1994a Tartari et al., 1997 Tartari et al., 1997 Tartari et al., 1997 Tartari et al., 1997 Guzzella et al., 1997 Guzzella et al., 1997 Guzzella et al., 1997 Guzzella et al., 1997 Ciceri et al., 1991 Ciceri et al., 1991 Pagnotta and Passino, 1997 Pagnotta and Passino, 1997

(b) Sediment compartment Sediment , 63 mm material (%) Sediment organic carbon (%) Sediment total N (%) Total ammonia in pore water (mg N–NH3l 21) c PAH (ng g 21 whole sediment) PCB (ng g 21 whole sediment) EOX (mg Cl kg 21 whole sediment) Zn (mg g 21 , 63 mm fraction) Cu (mg g 21 , 63 mm fraction) Cd (mg g 21 , 63 mm fraction) Pb (mg g 21 , 63 mm fraction) Hg (mg g 21 , 63 mm fraction)

33 0.6–0.9 0.07–0.1 7–10

42–54 1.7–1.9 0.2–0.23 39–72

Vigano`, unpublished data L. Patrolecco, p.c. d L. Patrolecco, p.c. G. Tartari, S. Valsechi, p.c.

15 4.8 36–116 145 47 0.316 19 0.092

179 58.9 197–1144 239 90 0.630 53 0.286

Vigano` et al., 1998a S. Galassi, p.c. A. Lopez, p.c. M. Camusso, p.c. M. Camusso, p.c. M. Camusso, p.c. M. Camusso, p.c. M. Camusso, p.c.


Examples taken from a series of measurements. Here nd denotes values not detected. c Pore-water concentrations of ammonia are preliminary results and thus only indicative. d p.c. denotes personal communication. b

basin has a prevalent agricultural character and about 56% of its area is used for crop cultivations and thereby exposed to “agrochemical” stressors. However, the cultivated areas of the two sub-basins have substantially the same extension and hence an almost equivalent application of fertilizers and pesticides (Table 1).

Consistent with these general features, agriculture (as cultivated soils) and resident population are the main sources of N and P in the Ticino basin, while resident population and industrial activities have this role in the Lambro basin. The latter basin has total N and P loads 3 to 4 times those estimated for the Ticino area (Table 1).


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3. Indicators of physical habitat alteration The physical habitat of this area of study is poorly characterized. Water temperature is usually higher in the river Lambro and thus its behaviour and mixing in the receiving Po water could be traced using this parameter as shown for conductivity (see later). Available results do not allow us, however, to describe the temperature increase of the river Po water along the downstream stretch satisfactorily, nor to evaluate the final change of the main river upon completion of the mixing process (Annoni et al., 1996). Nevertheless, these results suggest that the residual increase of water temperature should be # 18C. Bed sediment geomorphology of the river Po is modified by the load of suspended solids transported by the river Lambro, whose mean value was 12 mg l 21 according to Queirazza et al. (1992) and 46.7 mg l 21 according to Pettine et al. (1996). A recent monitoring program examining the bottom sediments of the main river provides some useful data by comparing the grain-size compositions of TI and LA area. These results suggest that the LA habitat of bottom sediments is on average more homogeneous than the upstream one, or to be more precise, is shifted towards finer grain-size particles (33% of , 63 mm fraction in the TI reach vs 42 to 54% in the LA reach; Table 2).

4. Indicators of chemical exposure Conductivity, hardness, dissolved oxygen and nutrient concentrations of the river Po are all significantly modified in the LA area, although changes of these water-quality variables showed different amplitudes in different time periods depending respectively on flows and conditions of the two rivers. In particular, conductivity and dissolved oxygen (DO) characterize the ‘new water quality’ of the main river which shows, at the completion of the mixing processes, a slightly higher conductivity (mean value 345 vs 367 mS cm 21 in the TI and LA tracts), hardness (168 vs 176 mg l 21 as CaCO3) and lower oxygen concentration (Vigano` et al., 1996; Annoni et al., 1996; Tartari et al., 1997). The mean annual concentration of DO of the LA reach is consistently lower than in the TI stretch with a mean decrease

ranging between 0.3 and 1.5 mg l 21, over a seven year monitoring period (Guzzi et al., 1997). The immission of the river Lambro makes higher concentrations available in the river Po environment of both N and P in their different chemical forms. Phosphorus, which controls the trophic potential of the watercourse (Mingazzini, 1993, 1997a), increases from 34 to 80 mg P–PO4 l 21. Amongst nitrogen compounds, ammonia shows a significant increase which has recently been assessed at 6 fold the upstream level, 0.09 vs 0.53 mg N–NH3 l 21 (Tartari et al., 1997). Both these ammonia concentrations (and estimated unionized fractions) are lower than those recommended for a healthy cyprinid fish habitat (Svobodova et al., 1993) and in water quality criteria (WQC) for aquatic life (USEPA, 1986). The relatively high content in organic matter of the Lambro waters, measured as both dissolved organic carbon (DOC) (8.6 ^ 3.4 mg C l 21) and particulate organic carbon (POC) (17 ^ 4.5%) (Queirazza et al., 1992), is reflected in a corresponding increase of both organic C and total N contents of bottom sediments of the LA stretch, which show twice the levels measured in the TI area (Table 2; L. Patrolecco, Water Research Institute, Rome, personal communication) and even more by the sharp increase of interstitial ammonia concentration (Table 2; G. Tartari and S. Valsecchi, Water Research Institute, Milan, personal communication) which largely results from degradation and ammonification processes of organic matter (Ankley et al., 1990). Similarly to what was found by Botermans and Admiral (1989) in the river Rhine, no significant nitrification seems to take place in LA area as suggested by the limited nitrite increase (Table 2) and the absence of nitrate variation. All these inorganic and organic nutrients change the trophic condition or potential of the main river, while pore-water ammonia concentration can account for a marked quality change of the sediment environment and toxic potential (Ankley et al., 1990; Schubauer-Berigan and Ankley, 1991; Whiteman et al., 1996). With regard to many other potentially hazardous chemicals, some recent results can be mentioned. Large applications of various pesticides have been identified in both reaches of the river Po. Generally, LA water concentration values are higher than those of the TI stretch (Table 2). Insecticides and herbicides

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are the most common pesticides and there is a net contribution of both from the Lambro watershed. As summarized in Table 2, certain compounds, for example, the organophosphorous insecticide malathion, are undetectable in the TI area, and others, for e.g. the herbicides atrazine and molinate, increase downstream from the river Lambro immission (Ciceri et al., 1991; Guzzella et al., 1997). Particularly triazinic and carbamate herbicides show seasonal time trends of water concentration with maximum values in springtime when they are used in corn and rice cultivations. Some early-summer peaks were also observed (Ciceri et al., 1991; Guzzella et al., 1997). Apart from the relatively short springtime maximum, when some toxic effects are to be expected (see later), pesticide concentrations are low or undetectable, showing values which are below the corresponding WQC and recommended maximum levels for protection of aquatic organisms (Nowell and Resek, 1994). The same springtime maximum of herbicide compounds (partially paralleled by organophosphorous insecticides), has also been found at the closure of the river Po basin (Galassi et al., 1992) before it empties into the Adriatic Sea, and seems to be a widespread problem affecting most of the Po catchment. The Lambro tributary represents a significant source of many trace metals. Among them Zn, Pb, Ni, Cu, Cr, As and Cd seem to be, in decreasing order, the most important (Pettine et al., 1996; Pagnotta and Passino, 1997). Depending on several factors (e.g. organic content of suspended solids and dissolved oxygen concentration), results suggest that for most of these metals solid transport is more important than dissolved, so that with the limited exception of Zn and Ni (Table 2), the increase of metal concentrations in LA water was found, on average, to be negligible (Pagnotta and Passino, 1997). Accordingly, the latest analytical results (M. Camusso, Water Research Institute, Milan, personal communication) confirm that, with regard to some trace metals (Hg, Pb, Cd, Cu, Zn), the river Po sediment is the compartment where quality is more markedly changed by the river Lambro (Table 2). Water concentrations of trace metals in LA stretch are lower than WQC (USEPA, 1986), while sediment values are comparable, with few differences for specific metals, to those found in moderately-polluted or polluted sites of rivers, lakes


and estuaries (Verbrugge et al., 1991; Bryan and Langston, 1992; Teil et al., 1996; Feng et al., 1998). According to Long and MacDonald (1992), Hg, Zn and to a lesser extent Cu and Pb, are present in LA sediments at concentrations which may possibly affect the aquatic life. Methylene-blue-active substances (MBAS) and organo halides (Adsorbable OX or Extractable OX; Lopez et al., 1995) are examples of gross indicators of chemical exposure which have been measured in the area of study. Anionic surfactants such as linear alkylbenzene sulfonates (LAS) and their residuals, that is, the most important fraction detected as MBAS (Gerike et al., 1989), again show the role of the Lambro tributary in changing the quality of Po water. The LA section has about twice the MBAS concentration observed in the TI stretch (27 ^ 37 mg l 21 vs 46 ^ 32 mg l 21; Guzzella et al., 1997). Similar levels of MBAS were observed in the river Rhine and other German watercourses exposed to anthropogenic contamination (Gerike et al., 1989). The water concentration of organo halides (as adsorbable OX) was found, on average, to be less significantly modified (Guzzella et al., 1997). The level of OX, probably because of the lipophilic nature of many of the compounds transported by the river Lambro, again reveals a significant impairment to the sediment compartment of the river Po. The LA reach shows 5– 10 fold the TI level of extractable OX (A. Lopez, Water Research Institute, Bari, personal communication; Table 2). These data suggest that, besides the contribution from PCBs (see later) which together with chlorinated pesticides generally account for a small fraction of OX (5–15% of extractable organically-bound chloride, EOCI), LA sediments could be a source to aquatic organisms of several OX contaminants which though present at relatively high concentrations are not currently studied (Wesen et al., 1990; Asplund and Grimvall, 1991). The Po sediment compartment demonstrates that the river Lambro is a source to the river Po of both PAHs and PCBs (Table 2) which increases the low levels of contaminations observable in the TI area several times over (Vigano` et al., 1998a; S. Galassi, University of Milan, personal communication). Despite this increase, the concentrations in LA sediment of these two classes of hazardous chemicals remain comparable to moderate/low polluted river

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Table 3 Summary of the most significant biological results obtained from both laboratory and field studies undertaken in the two stretches of the river Po located downstream from two tributaries, the river Ticino (TI) and the river Lambro (LA). Results are from monitoring studies and surveys, and for most of them, significance have been statistically validated. Single-cell comments describe the alteration of a given parameter in organisms exposed to the LA reach as compared to the TI reach. Results are organized into: (a) standard laboratory tests, (b) macroinvertebrate response, (c) fish and fish metabolism Parameter

TI reach

LA reach


(a) Standard laboratory tests Algal growth potential (mg l 21 d.w.) Mean increase of algal growth inhibition 14-day test (%) Ceriodaphnia dubia 7-day test C. dubia sediment extract 48 hEC50 (g of , 63 mm fraction l 21) C. dubia 7-day whole sediment test biomass output (mg female 21) AHH, EROD, UDPGT activities trout larvae 7-day whole sediment test Trout larvae 7-day sediment test Trout larvae with apoptotic-like events (%) 7-day sediment test Skeleton calcification trout larvae (% of control) 7-day sediment test DNA adducts in trout larvae (n 10 28 nucleotides) 7-day sediment test

11 25 14 Growth enhancement and inhibition 20–22.8 4.1–4.3

Mingazzini, 1997a Mingazzini, 1997a Vigano` et al., 1996 Vigano`, unpublished data




ne a

Biotransformation Induction

Vigano` et al., 1995a

ne a ne a 71

Growth inhibition 66 18

Vigano` et al., 1998b Vigano` et al., 1998b Vigano` et al., 1998b



S. De Flora, A. Izzotti p.c. b

5.2 49.6 7.1 2 56 6.5 18.6 0.330 2 34 424 1 93 1 309 8.6

4.6 41.1 4.6

Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished Buffagni, unpublished

(b) Macroinvertebrate responses Average score per taxon (ASPT) Biological monitoring working party score (BMWPS) Extended biotic index (EBI) Ephemeroptera relative abundance variation (%) Heptagenia sulphurea (indiv. m 22) Heptagenia coerulans (indiv. m 22) H. coerulans production (g m 22 y 21) Trichoptera relative abundance variation (%) Hydropsychidae (indiv. m 22) Gammaridae 1 Asellidae relative abundance variation (%) Hirudinea relative abundance variation (%) Naididae (Oligochaeta) (indiv. m 22)

1.6 5 0.046 280


(c) Fish and fish metabolism Ames test on fish bile extract Activation of promutagen Trp–P–2 EROD activity Barbus plebejus UDPGT and GST in feral Lipid content of feral cyprinid muscle PCBs in muscle of feral cyprinids a b

Here ne denotes no effect. p.c. denotes personal communication.

ne a ne a 9.9


Mutagen ( 1 S9) Activation 37.5 Enhancement up to 2 fold increase 13 469

De Flora et al., 1993 De Flora et al., 1993 Vigano` et al., 1998a Vigano` et al., 1998a S. Focardi, S. Aurigi, p.c. S. Focardi, S. Aurigi, p.c.

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sites (Santiago et al., 1994; Teil et al., 1996). With regard to PCBs, Stackelberg (1997) has confirmed that the most frequent detection and highest concentrations in river sediments are in the most heavily urbanized and populated areas, and that simple indicators of land usage (urban, agriculture, forest) can be predictive of the presence of contaminants in riverbed sediments.

5. Biological response and exposure The trophic status of the river Po is markedly affected by the river Lambro. The algal growth potential of the two riverwaters has recently been determined by the Raphidocelis subcapitata (formerly Selenastrum capricornutum) algal assay, and the results, expressed as mg dry weight of the algal biomass produced per liter of riverwater, pointed out an extremely high enrichment potential of the Lambro waters (Mingazzini, 1997a). On the basis of a oneyear monitoring study, a mean value of 196 mg l 21, with low seasonal variations (CV 25%) was calculated for the river Lambro, while much lower and more variable values were observed for the TI reach of the river Po (11 mg l 21, CV 77%). An increase of the trophic potential by more than twice the TI level has been evaluated for the LA area of the main river, which is characterized by a mean value of 25 mg l 21 together with a low amplitude of seasonal variations (CV 54%). According to the algal assay productivity classification (Miller et al., 1974), the Po river waters, which range from a low- to high-productivity in the upstream stretch, are impaired to a stable ‘very high productivity’ class downstream from the confluence of the Lambro river (Mingazzini, 1997a). The role played by the trophic level as an important factor controlling the magnitude of toxic effects was highlighted by the results obtained with enrichmentalgal assays conducted on water samples taken from the end section of the river Po (Mingazzini, 1993) as well as from the TI and LA areas (Mingazzini, 1997a, 1997b). In these studies it was shown that the sensitivity of the watersystem to toxic pollutants increases in low nutrient conditions. On the basis of 14-day algal assays, the same author has also documented the presence in the TI area of water-borne toxicity which becomes occasionally effective as a result of


the lower trophic status combined with the seasonal contamination events. On the contrary, the LA area shows constantly effective toxicity to R. subcapitata (on average 1 14%) in spite of the higher trophic level of the river stretch (Mingazzini, 1997a; Table 3). The increased trophic potential of the LA reach was also documented by a study undertaken with the 7-day test on Ceriodaphnia dubia which occasionally showed growth enhancement of test organisms, thereby confirming that additional amounts of organic “extra food” (most likely particulate organic matter and microorganisms) can be available downstream from the Lambro inflow (Vigano` et al., 1996). This same study also suggested that the potential for trophic stimulation continuously interacts with an average condition of (low) chronic toxicity which in turn seems to be occasionally present in water samples taken from the same area. This interaction between trophic and toxic effects is further supported by some recent results obtained by testing sediment toxicity on C. dubia. Although the LA-organic solvent sediment extract contained 5-fold more toxic material than the TI-sediment extract (Table 3), this toxic potential seems to be largely unexpressed, and in exposing C. dubia to TI and LA whole sediments (7-day tests with the same samples used for the extracts) almost equivalent biomass outputs (growth 1 neonates) have been observed (Table 3) (Vigano`, unpublished data). The organic chemicals responsible for the toxic potential and the aforementioned trace metals seem not to be bioavailable to exert toxic effects, even with the contribution of LA-interstitial ammonia (Table 2). However, it may also be argued that the equivalent biomass outputs and thus the poor exploitation of the LA ‘extra food’ is the result of an ‘energy cost’ due to a moderate toxic effect. These are the results observed with sediment samples collected in summer. In winter, the toxic potential evaluated with sediment extract is the same, whilst in contrast, the effects of whole sediment are markedly worse. Higher interstitial ammonia concentration combined with higher oxygen demand of sediment material caused the complete mortality of C. dubia exposed to winter samples of whole sediment. Although amplified by the static-renewal procedure of the laboratory test, more such critical condition of the winter period


L. Vigano` et al. / Aquatic Ecosystem Health and Management 2 (1999) 55–69

Fig. 2. Time trends of acute toxicity observed testing daily water samples of the river Lambro during 1994 springtime toxic event (April 26 to May 14). Untreated water samples were tested full strength (100%) on Ceriodaphnia dubia (24, 48 and, 72 h) and rainbow trout fry (Oncorhynchus mykiss) (48 h). Results are reported as a percentage of dead organisms.

seems to be confirmed by the responses of resident macroinvertebrates (see later). Laboratory toxicity tests on algae and daphnids generally show that the highest toxic effects occur in springtime, probably because of run-off and transport of pesticides to watercourses, although the corresponding effects on the aquatic community are still to be investigated. This spring phenomenon seems to affect large portions of the entire river Po basin as the highest toxicity has simultaneously been found in the upstream stretch of the river Po, in the river Lambro (which becomes acutely toxic, Fig. 2), in the downstream stretch of the river Po (where toxicity is slightly higher than upstream), and over the same time period, at the closure of the river Po basin (Mingazzini and Berri, 1991; Galassi et al., 1992; Guzzella and Mingazzini, 1994; Vigano` et al., 1996; Mingazzini, 1997a). While these studies did not find any direct relationship between specific pesticides and toxicity, they provided several indirect signs of evidence. Significant correlations were found, for example, between the toxicity of river water extracts, tested on R. subcapitata (growth inhibition) and

Daphnia magna (mortality), and the total level of herbicides (atrazine, molinate, alachlor, etc.) and insecticides (phorate, diazinon, dimethyl parathion, etc.). In addition, several untreated water samples collected from the river Lambro in springtime caused the complete mortality of C. dubia but had no appreciable effect on rainbow trout (Oncorhynchus mykiss) (Fig. 2). These peculiar results suggest that the run-off of either organophosphorus or carbamate insecticides are highly toxic to invertebrates but less toxic to fish. Besides this extreme yet temporary event, the interaction between trophic enrichment and toxicity seems to be the leading process in the LA area. Accordingly, some recent results suggest that the sensitivity of a species and the capability of taking advantage of the increased trophic condition (e.g. by detritivores), are two important factors which combine to affect the community of the LA area. Macroinvertebrates and fish organisms provide some interesting examples of such two-factor interaction. Obtaining comparable and representative samples of the macroinvertebrate community in a river as large as the Po is critical and may explain some

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inconsistencies of published results regarding the health of the LA benthic community (Battegazzore et al., 1992, 1997; Galli et al., 1996). Nevertheless, some recent applications of standard biotic indexes provided evidence that the LA-community of macroinvertebrates is moderately impaired showing, on average, lower index values than that of the TI reach. Three standard approaches, extended biotic index (EBI; Woodiwiss, 1964; modified by Ghetti, 1986), biological monitoring working party score (BMWPS) and average score per taxon (ASPT, Armitage et al., 1983) gave index values of 7.1, 49.6 and 5.2 for the TI area and 6.6, 41.1 and 4.6 for the LA area, respectively (Table 3; Buffagni, unpublished data). The most critical condition for LA macroinvertebrates was observed in winter, while improvement was found in summer when the two communities were indistinguishable. This is in accordance with that found for some pollutants, e.g. ammonia in pore water and C. dubia toxicity tests (on water and sediments) which showed that in summer the lowest impairment occurs and the organisms more efficiently exploit ‘extra food’ (Vigano` et al., 1996; G. Tartari and S. Valsecchi, Water Research Institute, Milan, personal communication; Vigano`, unpublished data). In addition to standard biotic indexes which aim at giving an overall judgment of the entire benthic community, the examination of single groups of organisms and their relative abundances seem to be a sensitive approach to detect community changes of ‘grey’ areas. It has been observed, for example, that the most sensitive groups, mayflies (Ephemeroptera) and caddiesflies (Trichoptera) exhibited reduced abundance in the LA stretch ( 2 56 and 2 34% respectively), while tolerant crustaceans (Gammaridae and Asellidae) and leeches (Hirudinea) increased ( 1 93 and 1 309%, respectively). The response of single selected taxa can supply further evidence of the alteration of the LA benthic community. For instance, the mayflies Heptagenia coerulans Rostock and Heptagenia sulphurea (Mu¨ller), along with the caddisflies Hydropsychidae, show a clear contraction of their populations, while the oligochaete worms Naididae strongly increased (Table 3). Although less clearly defined, other taxa, such as Chironomidae, seem to take advantage of the LA environmental condition. A recent study further suggests that the estimation of benthos secondary production can be a


useful tool to evaluate taxa responses, production being the most comprehensive representation of ‘success’ of a taxon (Benke, 1993). Upon estimating the annual secondary production of H. coerulans in the TI and LA areas, it was found that not only was the abundance of this mayfly species strongly reduced in the LA stretch but its production was severely impaired, being reduced from 0.330 to 0.046 g m 22 per year (Buffagni, unpublished data). With regard to fish organisms, many exposure indicators consistently show the presence of chemical stressors in the LA reach, thereby confirming that the quality of the river Po worsens after the inflow of the river Lambro, although the increased trophic condition again interacts with the effects of toxic chemicals. The alterations of both phase I and phase II enzyme activities, as well as of other biomarkers, have been observed exposing hatchery-reared fish or their larvae to both dissolved (Vigano` et al., 1994a) and sediment-associated contaminants (Vigano` et al., 1995a, 1995b). Consistent results have recently been observed in feral cyprinid populations of TI and LA areas (Vigano` et al., 1998a) confirming that both PAHs and PCBs are bioavailable to the fish community, and specifically to LA fish via both trophic transfer and direct uptake from water (De Flora et al., 1993; Vigano` et al., 1994b). Cyprinids show muscle concentrations of PCBs up to 6 fold higher in LA than in TI reaches (Table 3; S. Focardi and S. Aurigi, University of Siena, personal communication). Recent 7-day tests on trout larvae exposed to TI and LA sediments have shown that the latter markedly depressed the calcification process of fish skeleton, induced MFO enzyme activities and the presence of apoptotic-like cells (Table 3; Vigano` et al., 1998b). The threat potential for the fish community of the LA area is further supported by other results. The mutagenicity of bile from fish exposed to river Po water was assayed and a clear dose-related mutagenicity was found only in fish exposed downstream of the river Lambro. This study also showed that this material was capable of activating heterocyclic amine to mutagen compounds (De Flora et al., 1993). The presence of a genotoxic risk in this area has been confirmed in tests of the sediment compartment. In preliminary tests it was shown that twice the number of DNA adducts have been found in trout larvae


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exposed to LA sediment as against those exposed to TI sediment (Table 3) (S. De Flora and A. Izzotti, University of Genoa, personal communication). White et al. (1998) have recently demonstrated the accumulation of genotoxins in macroinvertebrates and fishes of the St Lawrence river. In apparent contrast with such a range of alterations and stressors, the mean lipid content of LA-cyprinids is up to twice that of the TI specimens (Table 3). This result suggests that those invertebrates whose production can take advantage of LA changes, represent, in turn, an increased source of food (and contaminants) for some species of fish, notably for carnivorous fish feeding in soft-sediment habitat (Dabrowska et al., 1996; West et al., 1997; White et al., 1998; S. Focardi and S. Aurigi, University of Siena, personal communication). A similar increase in the lipid level (and of some contaminants) was found while comparing the fish (Rutilus rutilus) collected in the river Seine upstream and downstream the polluting loads from Paris (Blanchard et al., 1997). However, it should be born in mind that such an increase of lipid reserve is not necessarily an indicator of fish health. In fact, this same increase was observed, for example, in fish (Catostomus commersoni) living downstream from a pulp and paper mill. These fishes also exhibited several metabolic alterations (including monooxygenase enzyme alterations), delayed age and increased size at maturity, smaller gonads and low fecundity (Hodson et al., 1992; Gagnon et al., 1995). More information on resident fish is necessary to assess whether pathological lesions, alterations of fecundity, growth or other less evident effects are threatening the fish community of the LA area.

6. Conclusions In the present study, four categories of indicators have been examined in an attempt to assess the hazard to which the aquatic community of the river Po is exposed, especially those alterations deriving from the confluence of the river Lambro. The several indicators of watershed stress have described two markedly different conditions within the TI and LA drainage areas. As they are subject to almost equivalent agricultural usage and hence to similar related variables such as the application of

pesticides, fertilizers and nutrient release, the great differences between the two sub-basins are in terms of industrial development and population density. In this study, these watershed indicators describe two extreme and thus easily distinguishable conditions which generally seem to be reliable predictors of river-quality alterations. The use of watershed indicators can help to identify those areas where stressors are present and quality problems are more likely, although chemical and biological alterations observed in the present study seem to be much less severe than could be expected from watershed indictors. The levels of most of the chemicals identified in the area of study were found to be increased in the LA area but at all times below those concentrations which are individually known to be protective of aquatic organisms (e.g. no-observed effect concentrations, WQC, etc). Many studies demonstrated, however, that the toxicity of mixtures of sub-lethal and subchronic concentrations of chemicals are additive or partially additive (Konemann, 1981; Hermens et al., 1984; Spehar and Fiandt, 1986; European Inland Fisheries Advisory Commission (EIFAC), 1987). Accordingly, it would be prudent to assume that combined effects of multiple sub-chronic doses are active in the LA area and this is even more realistic if we consider that the chemicals identified and found in modified form in this area of the river Po represent only a small fraction of the whole Lambro input. The water toxicity of the LA area is sublethal and chronic, and as shown in a previous study (Vigano` et al., 1996), it is dependent on the dilution ratio between the river Lambro and the river Po, in such a way that when dilution prevents toxicity the eutrophic potential becomes (more) evident. In addition, algal enrichment assays showed that the increase of nutrient availability can significantly reduce toxic effects (Mingazzini, 1997b). Such results suggest that when a preliminary assessment of hazard potential is carried out, this continuous combination between nutrient and toxic effect can act as a confounding factor. Hazard assessment could be misleading, particularly for the water compartment of ‘grey’ areas, so that long-term monitoring programs and chronic-toxicity tests on more species are necessary. Available data on the LA area demonstrate that the sediment compartment, when compared to the water column, has higher concentrations of many chemicals and is the compartment

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where quality is more markedly impaired. This characteristic suggests that sediments may be more suitable to assess the hazard existing at different sites. However, the confounding interaction between nutrient and toxic effects also seems to be effective in whole sediment tests. For both water column and sediment, the selection of grouping parameters and extraction-techniques (coupled with toxicity tests) could be more effective at least to evaluate the hazard potential of a given area. Besides the different approaches which might be used to evaluate the hazard potential of an area, assessment of the bioavailability of chemical stressors and their effects on resident organisms remain the primary objectives. As documented in this study, a number of toxicity tests and field studies can provide important information for these purposes. Wholesample tests, field cageing, abundance and secondary production of benthic macroinvertebrates and biomarkers in feral fish are some examples. In the LA area, the sedimentation of particulate matter transported by the river Lambro combined with an increased ammonia concentration and oxygen demand of sediment compartment reduce habitat suitability and perhaps patchiness, as well. However, the results obtained from both the laboratory and field studies suggest that taxa, owing to different sensitivities and trophic niche, show different vulnerabilities and interactions with the LA environment. The higher incidence of DNA adducts, the inhibition of calcification and growth in fish larvae, the reduction of both Ephemeroptera and Trichoptera abundances, are all effects of the toxic stressors threatening the aquatic community of the LA reach. On the contrary, the increase of algal growth potential, Naididae abundance, and lipid content of fish muscle are biological responses to the increased trophic condition of the LA environment. Invertebrates which can withstand toxic stressors and take advantage of nutrient load, represent an abundant source of (lipophilic) (geno) toxic substances to carnivorous fish, which are exposed thereby to trophic transfer. Although more information is necessary to describe the community of the river Po and characterize the subtle and less evident impairments of LA organisms, both laboratory and field results show that the nutrient enrichment and toxic stressors of this ‘grey’ area interact, affecting the aquatic community. The inter-


action of these two factors is more easily evaluated in laboratory tests, for example, algal assays, than in the field studies where the modifications of physical habitat can also be effective. A battery of multitrophic biological indicators, that is characterized by different relationships with environmental trophic changes, should provide a greater insight to quality assessment. Acknowledgements The authors are grateful to all those colleagues who made the results of their unpublished and ongoing studies available. They are also indebted to L. Previtali, L. Marescotti and E. Comin for technical assistance. We thank Dr. S. Lawrence, Technical editor of AEHM for her assistance which improved the clarity of the article. References Ankley, G.T., Katko, A., Arthur, J.W., 1990. Identification of ammonia as important sediment-associated toxicant in the lower Fox river and Green Bay. Wisconsin. Environ. Toxicol. Chem. 9, 313–322. Annoni, P., Maran, S., Tartari, G., Previtali, L., Guzzi, L., Martinotti, W., 1996. Processi di miscelamento del Lambro nel Po. Rapporti Tecnici Ist. Ric. Acque R/147. CNR, Rome (Abstract in English), p. 109. Armitage, P.D., Moss, D., Wright, J.F., Furse, M.T., 1983. The performance of a new biological water quality score system based on macroinvertebrates over a wide range of unpolluted running water sites. Wat. Res. 17, 333–347. Asplund, G., Grimvall, A., 1991. Organohalogens in nature. Environ. Sci. Technol. 25, 1347–1350. American Standards for Testing and Materials (ASTM), 1994. Standard guide for conducting sediment toxicity tests with freshwater invertebrates. American standards for testing and materials standard Guide E 1383, Annual Book of ASTM Standards, vol. 11.04, Philadelphia, PA. Barbiero, G., Giuliano, G., 1997. Analisi dei fattori di generazione dell’inquinamento nel bacino del Lambro, In: Nodo Lambro-Po: Trasporto di inquinanti ed effetti biologici, Quad. Ist. Ric. Acque, CNR, Rome, (Abstract in English), 102, pp. 20–47. Battegazzore, M., Petersen, R.C., Moretti, G., Rossaro, B., 1992. An evaluation of the environmental quality of the river Po using benthic macroinvertebrates. Arch. fur Hydrobiologie 125, 175– 206. Battegazzore, M., Galli, R., Paoletti, A., Rossaro, B., 1997. Indagine sui macroinvertebrati bentonici, In: Nodo Lambro-Po: Trasporto di inquinanti ed effetti biologici, Quad. Ist. Ric. Acque, CNR, Rome, (Abstract in English), 102, pp. 277–297.


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