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Toxicity Identification Evaluation of Lake Orta (Northern Italy) Sediments Using the Microtox System
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY ARTICLE NO. 0104
35, 231–235 (1996)
Toxicity Identification Evaluation of Lake Orta (Northern Italy) Sediments Using the Microtox System LICIA GUZZELLA,* CRISTINA BARTONE,* PHILIPPE ROSS,† GABRIELE TARTARI,‡
AND
HERBERT MUNTAU§
*Water Research Institute—CNR, via della Mornera 25, 20047 Brugherio, Milan, Italy; †The Citadel, Charleston, South Carolina; ‡Department of Biology, University of Milan, Milan, Italy; and §Italian Institute of Hydrobiology—CNR, Pallanza, Novara, Italy Received November 24, 1995
Pore waters extracted by centrifugation from Lake Orta (Northern Italy) sediments were studied with a modified Toxicity Identification Evaluation (TIE) procedure using the Microtox bacterial luminescence toxicity test system. The most toxic pore water samples were from stations near a rayon factory, known as a source of copper and ammonium discharges. The TIE manipulations used were filtration, EDTA chelation, and C18 solid-phase resin adsorption. The most effective treatments to remove toxicity were the EDTA and C18, indicating that both metals and nonpolar organic compounds contribute to the observed toxicity. © 1996 Academic Press
INTRODUCTION
Lake Orta is a warm, monomictic lake located near Novara in the subalpine region of Northern Italy (Fig. 1). The main physical characteristics of the lake are: surface area 4 18.2 km2, volume 4 1.3 km3, mean depth 4 71 m, maximum depth 4 143 m, actual mean residence time 4 10.7 years. Chemical contamination of the lake began in 1926 with massive discharges of ammonium sulfate and copper from a local rayon factory. All forms of aquatic life were greatly affected by this chemical pollution, and by 1929 the pelagic and benthic communities were virtually absent. Oxidation of ammonia led to a progressive accumulation of nitrates and a drastic acidification of the water column (Provini et al., 1986). Since the early 1950s, discharges from several electroplating plants on a tributary, the Lagna River, have added to the contamination of the lake through discharges of nickel and chromium. The discharges of copper and ammonia have continued virtually uninterrupted since the 1920s, but the introduction of a waste treatment system by the rayon factory in 1958, and improvements made to that system in 1980, have greatly reduced their concentrations in the waste stream discharged to the lake. In 1989 a recovery operation began, and 14,500 metric tons of calcium carbonate was added to the lake water, increasing the pH from 4 to 6, and decreasing ammonia nitrogen levels to 20 mg/liter. To evaluate the potential toxicity of contaminants ac-
cumulated in Lake Orta, a sediment investigation was undertaken in July, 1992. In this study, the toxicity of whole sediments and pore waters was evaluated using a seed germination/ root elongation test, a 48-h Ceriodaphnia dubia survival test, and the Microtox bacterial luminescence test. As a part of the present study, pore water extracts were collected and evaluated with the Microtox system using a procedure similar to the Toxicity Identification Evaluation (TIE) protocol for waste waters according to the United States EPA (US EPA 600-3-88/035). The TIE approach is based on the principle of sequential removal of various chemical fractions coupled with toxicity testing of the obtained fractions. When removal of a class of chemicals also removes toxicity, it is assumed that those compounds were responsible for some of the toxicity observed in the original sample; in the present study the USEPA Phase I approach was followed, except the aeration and pH adjustment manipulations were not done. These steps were eliminated because the TIE procedure was applied to testing pore water rather than waste waters, which imposed a limit on the volume of sample that could be obtained (<40 ml). The treatments chosen were filtration, metal chelation with EDTA, and passage through a C18 resin column. These treatments were thought most likely to yield information on compounds responsible for toxicity. In this model TIE protocol the toxicity of the initial extract and of the various treated pore water extracts was assessed using Vibrio fischeri in the Microtox test system. Microtox is a rapid, low-cost toxicity test which is used widely in screening tests for assessing sediment toxicity. Pore water, rather than whole sediment, was chosen for evaluation because the interstitial phase has been found to be representative of the bioavailable fraction of the contaminants of concern (i.e., metals and ammonia). In a study of the toxicity of cadmium in sediment to Daphnia magna, the free, or dissolved, uncomplexed cadmium was found by Schuytema et al. (1984) to be the toxic form of the metal, and the authors concluded that ‘‘cadmium adsorbed to the sediment had negligible toxicity.’’ According to Schuytema et al. (1984), the toxic effects of metal associated with sediments could best be predicted from concentrations of unbound metal in pore water. Furthermore, in a study of the toxicity of sediment copper to
231 0147-6513/96 $18.00 Copyright © 1996 by Academic Press All rights of reproduction in any form reserved.
232
GUZZELLA ET AL.
FIG. 1. The location of the sediment sampling stations in Lake Orta.
several organisms, including D. magna and Chironomus tentans, it was found that the toxicity of copper was attributable to soluble copper rather than unbound copper (Cairns et al., 1984). MATERIALS AND METHODS
The locations of the sediment sampling stations in Lake Orta are presented in Fig. 1. For the evaluation of pore water toxicity, surface samples were collected with a Ponar grab at stations 43, 56, and 32A, and vertical cores were taken at stations 45 and 13 (Table 1). The cores were divided into horizons and three strata were selected for analysis: 0–10 cm (recent deposits), 10–20 cm (intermediate-age deposits), and another 10-cm interval from a deeper layer (preindustrial deposits). The sediment samples were stored in darkness at 4°C until analysis. TABLE 1 Main Characteristics of the Sampling Stations Station
Sub-basin
Depth of station (m)
43 56 45 32 A 13
North North North Central South
89 58 143 90 47
Type of sample Grab Grab Core Grab Core
Sediment core horizons were dated on the basis of sedimentation rates measured by Provini and Gaggino (1985) using Cs137. Organic carbon content was determined in samples dried at 105°C, by oxidation with potassium dichromate after Jackson (1958), as modified by Gaudette et al. (1974) (Table 2). To separate pore water from the solid phase, approximately 100 g of each sediment sample was centrifuged at 4000g and 4°C for 45 min. The amount of pore water thus obtained varied from 22 to 42 ml, depending on the water content of the sediment (Table 2). Pore water toxicity was assessed with a Microtox Model 500 analyzer and lyophilized cultures of Vibrio fischeri NRRL-B11177, a rod-shaped, Gram-negative marine organism supplied by Microbics Corp. (Carlsbad, CA). In the Microtox system, the inhibition of bacterial light emission was measured in duplicate experiments at 15°C after 15 min of exposure to the pore water sample (Bulich and Isenberg 1981). The Microtox data acquisition software was then used to calculate the EC50 value, the percentage concentration of pore water causing a 50% reduction in bioluminescence. The modified TIE procedure is outlined in Fig. 2. The pore water samples were divided into five portions. The first aliquot was used to measure the toxicity of the untreated sample. The second was with Milli-Q water. The filtered sample was then tested for toxicity using the same procedure. For the third and fourth aliquots, respectively, 200 and 300 mg/liter of EDTA (sodium salt of ethylenediamine-tetraacetic acid) was added. The solutions thus obtained were used for the toxicity test. Prior to the experiment, the concentration/toxicity curve for EDTA was determined (EC50 4 402 mg/liter). Thus curve was then used to subtract the toxic effect of EDTA addition (10 and 30% of the inhibition for 200 and 300 mg/liter, EDTA, respectively). The addition of such high concentrations of EDTA is necessary to chelate the metal content in the salted medium (2% NaCl) used in the toxicity test. The fifth pore water aliquot was passed through a methanol-activated C18 resin column. The column run-off sample was then tested for toxicity, as was
TABLE 2 Dating, pH, Percentage of Water, and Organic Carbon Content of Samples
43 56 45 45 45 32 13 13 13
Station
Dating of sample
pH of pore water
% of water in sediment
% of org. carbon in sediment
(0–10 cm) (10–20 cm) (32–42 cm) A (0–10 cm) (10–20 cm) (50–60 cm)
1970–92 1970–92 1970–92 1948–70 1900–22 1961–92 1950–92 1908–50 1742–48
7.74 7.55 7.67 7.76 7.46 7.69 7.01 7.23 7.13
39.0 42.5 25.0 31.0 24.0 42.2 49.0 41.0 28.0
7.44 4.53 3.20 4.11 3.20 5.83 6.84 4.78 5.80
TOXICITY IDENTIFICATION EVALUATION OF LAKE ORTA SEDIMENTS
FIG. 2.
233
The scheme of the adopted Toxicity Identification Evaluation procedure.
the methanol eluate from the column, previously exchanged with Milli-Q water in a water bath at 45°C. Chemical characterization of the pre water samples, with the exception of metal concentrations, was performed after Mosello (1988). Ion contents were determined on 1.5-ml sample volumes by ion chromatography in a Dionex Model 210 with a conductometer detector and a Spectra Physics AS8780 autosampler. The concentrations of chloride, nitrate, and sulfate were determined using a Dionex AG4A-AS4A separation column with chemical suppression by AMMS micromembrane. Sodium, ammonium, potassium, magnesium, and calcium were measured with fast I–fast II Dionex separation columns, with chemical suppression by CMMS micromembrane. Eluant concentrations and fluxes were those indicated by Dionex. For the analysis of metals, the samples were evaporated to near dryness after adding 1 ml of Suprapure nitric acid. The residue was heated with an additional 1 ml of nitric acid and, after complete dissolution, the product was diluted with highpurity water to 10 ml. Chromium copper concentrations were determined by electrothermal atomic absorption spectrometry using a Perkin–Elmer Zeeman 3030 instrument equipped with a L’VOV platform and hollow cathodic lamps, applying ashing temperatures of 1200°C (Cu) and 1650°C (Cr), and atomization temperatures of 2300°C (Cu) and 2500°C (Cr). Calibration curves were established with the aid of freshly prepared standard solutions, using Merck Titrisol standards. RESULTS AND DISCUSSION
The results of the toxicity tests are reported in Table 3. For the untreated pore water samples (first column), no EC50 values could be calculated for the grab samples collected at Stations 43, 56, and 32A. The EC20 values (i.e., the percentage of sample causing 20% light inhibition), demonstrated greater toxicity for pore water from station 56. For the core samples,
those collected at station 13 indicated more toxic effects than those from station 45. In these cores, the superficial layers (0–20 cm) were more toxic than the deeper strata. These results can be explained by the proximity to the rayon factory discharge for station 13, and by the dating of the sediment core horizons for station 45. In fact, the 10 to 20-cm core horizon collected at station 45 was dated between 1948 and 1970, a period characterized by the highest water-column cuproammonium contamination (Baudo et al., 1989). When the initial pore water sample was filtered, this treatment had a detoxifying effect in all samples. This suggests that some of the toxicity in the initial sample may be associated with suspended rather than particulate matter. The addition of EDTA reduced toxicity on all pore water samples except the extract from station 32A. For the core segments collected at station 13, an addition of 300 mg/liter of EDTA was necessary to remove the observed toxicity, while in all other samples 200 mg/liter was sufficient. Passage through the C18 column removed toxicity in all samples, but no toxicity was found in most of the methanol eluates. This may indicate either that the organic compounds responsible for toxic effects were irreversibly bound to the C18 resin, or that methanol, commonly used for eluting C18 columns, was not efficient in removing all bound organic compounds. The only C18 eluate that demonstrated a toxic effect was the extract from station 32A. In this case, two hypotheses may be argued. First, toxic amounts of organic compounds discharged from a local factory may be present that can be eluted with methanol. Second, the absence of the inorganic matrix (i.e., the salt content) may be responsible for the toxicity of the compounds isolated by C18 extraction. Chemical analyses of the pore water samples are reported in Tables 2 and 4. The pH of all tested pore waters was near neutrality, while the organic carbon content of the whole sediments ranged from 3.2 to 7.44%. The highest organic carbon
234
GUZZELLA ET AL.
TABLE 3 EC50 and EC20 Values of Orta Pore Water Samples Chelation with EDTA Station
Whole
Filtered
EC50 EC20 56 EC50 EC20 45 (0–10 cm) EC50 EC20 45 (10–20 cm) EC50 EC20 45 (32–42 cm) EC50 EC20 32 A EC50 EC20 13 (0–10 cm) EC50 EC20 13 (10–20 cm) EC50 EC20 13 (50–60 cm) EC50 EC20
>100 68.3
>100 66.7
>100 39.7
>100 >100
42.5 16.3
100 17
220 ppm
300 ppm
After C18 SPE
C18 SPE extract
43
32.9 11.4
54.5 23.5
95.8 60
>100 90
>100 90
>100 90
>100 90
>100 90
>100 >100
>100 30
>100 60
>100 >100
>100 >100
>100 60
>100 80
>100 70
>100 >100
>100 90 >100 40
96.5 27.3
>100 70
>100 >100
100 80
>100 >100
>100 61.2
>100 90
>100 70
80 60
>100 >100
11.2 <5
13.1 6.1
40.2 14.2
17.7 <5
>100 60
>100 >100
>100 >100
19 6.3
43.9 16.2
37.8 <5
>100 70
>100 >100
>100 >100
40.9 16.5
49.1 18.1
35.5 <5
>100 >100
>100 59.6
>100 90
Note. SPE, solid-phase eluate.
values were observed in the station 43 grab sample and 0 to 10-cm stratum of the station 13 core, but there was no evidence of correlation between pore water toxicity and organic carbon content of the bulk sediment. No particular anomalies could be observed in the distribution of cations and anions (Table 4). High ammonia concentrations were observed in pore waters from samples collected at stations 45, 43, and 32A. This contamination was found even in the oldest horizon (32–42 cm) of the core from the deepest part of the lake (station 45). Although
Lake Orta has a well-documented history of loading with discharged ammonia, the ammonia in most samples could also be attributed to natural microbial degradation of nitrogenous organic compounds. By this mechanism, ammonia could accumulate to high concentrations under anaerobic conditions that occur in deep sediments (Berner, 1980). Concentrations of ammonia in excess of 10 mg/liter are often observed in pore water extracts (Ankley et al., 1990). Ankley et al. (1990) demonstrated that ammonia played a large role in determining pore
TABLE 4 Chemical Analyses of Orta Pore Water Samples Station 43 56 45 45 45 32 13 13 13
(0–10 cm) (10–20 cm) (32–42 cm) A (0–10 cm) (10–20 cm) (50–60 cm)
Na (mg/liter)
K (mg/liter)
Mg (mg/liter)
Ca (mg/liter)
Cl (mg/liter)
N-NO3 (mg/liter)
N-NH4 (mg/liter)
SO4 (mg/liter)
Cu (mg/liter)
Cr (mg/liter)
5.4 5.1 4.7 4.4 3.7 5.3 6.1 6.2 8.4
2.1 1.3 2.0 2.0 2.0 2.2 1.4 1.3 1.4
3.7 1.5 1.1 1.2 1.2 1.8 2.0 1.8 1.4
10.5 6.5 6.8 7.6 8.2 5.8 9.3 6.7 6.5
3.3 2.5 2.5 2.3 2.2 3.0 3.0 3.4 2.2
0.01 <0.01 0.02 0.01 0.02 0.01 0.03 0.03 0.03
8.69 3.24 9.86 10.26 10.70 7.42 2.44 3.78 2.47
0.1 0.2 2.2 1.5 0.5 0.6 3.2 1.6 0.5
0.25 0.14 0.17 0.07 0.10 1.11 0.39 0.13 0.08
0.260 0.020 0.027 0.013 0.003 0.270 0.150 0.022 0.013
TOXICITY IDENTIFICATION EVALUATION OF LAKE ORTA SEDIMENTS
water toxicity in Fox River (U.S.A.) sediment samples. In their study, the concentrations of ammonia were correlated with the toxicity observed in the 7-day chronic test with C. dubia. They suggested that the presence of ammonia could greatly complicate the results of sediment toxicity testing. High levels of ammonia could mask the effects of other toxicants, while low concentrations might interact with other sediment-associated contaminants in additive, synergistic, or antagonistic fashions. Therefore, when performing TIE evaluation on sediment samples with high concentrations of ammonia, Ankley et al. (1990) suggested the approach of using a test species that is not sensitive to ammonia (i.e., V. fisheri). In the present study, it was not possible to correlate the Microtox toxicity of pore water samples with the observed ammonia concentrations. Similarly, the detoxification effect obtained with the EDTA addition could not be related to pore water concentrations of the analyzed metals, most importantly copper and chromium. Of the possible metal analytes, copper was determined because it is most representative of the pollution produced by the rayon factory. This metal was present in greater concentrations in the most recent core horizons (0–10 cm) and particularly in the grab sample from station 32A, where there was a preferential deposition of suspended matter coming from the southern part of the lake. In a study of the spatial distribution of metals in Lake Orta sediments, Baudo et al. (1989) demonstrated a transport of metal-enriched fine particles to this part of the lake from inputs located further to the south. This was called a ‘‘focussing phenomenon’’ (Baudo et al., 1989). Chromium was considered to be the metal most representative of the discharge produced by the electroplating industries (Jackson, 1958). The higher concentrations observed for station 13 could be explained by the inflow from the Lagna, a river that collects waste waters from an important industrial area. Station 43 was also located near the inflow of a polluted river, while for station 32A the hypothesis of a selective accumulation of suspended matter should be considered. CONCLUSION
The modified Toxicity Identification Evaluation procedure used in the present study was especially useful in the evaluation of potentially toxic components in pore water samples from Lake Orta. Toxicity was attributed to three components: suspended solids, heavy metals, and nonpolar organic compounds. No evident correlations were demonstrated, however, between the results of the chemical analyses and those from the Microtox test. It might be argued either that the chemical analysis was inadequate (i.e., an insufficient number of ana-
235
lytes) for the characterization of the pore water samples, or that other interactions such as synergistic effects may influence the response of the Microtox test system. In the future more attention should be paid to the toxicological effects of sediment-associated contaminants. In fact, sediment/water interactions might make these chemicals more available to aquatic organisms, which could possibly interfere with lake recovery operations. ACKNOWLEDGMENTS The authors are grateful to Microbics Corp. (Carlsbad, CA), Instrumatic s.r.l. (Lainate, Milano, Italy), and R. Cardente and R. Cozzi of Exotox L.D.S. (Pregnana Milanese, Italy) for supporting this investigation through the availability of Microtox equipment, reagents, and expertise. They thank A. Lami, R. Baudo, and A. Ferrari of the Italian Institute of Hydrobiology (CNR-VerbaniaPallanza, Novara, Italy) for their assistance in collecting and processing the sediment samples. The participation of P. Ross was supported by a faculty Development Grant from Citadel Development Foundation.
REFERENCES Ankley, G. T., Katko, A., and Arthur, J. W. (1990). Identification of ammonia as an important sediment-associated toxicant in the lower Fox River and Green Bay, Wisconsin. Environ. Toxicol. Chem. 9, 313–322. Baudo, R., Amantini, L., Bo, F., Cenci, R., Hannaert, P., Iattanzio, A., Marengo, G., and Muntau, H. (1989). Spatial distribution patterns of metals in the surface sediments of Lake Orta (Italy). Sci. Total Environ. 87/88, 117–128. Berner, R. A. (1980). Early Diagenesis: A Theoretical Approach. Princeton University Press, Princeton, NJ. Bulich, A. A., and Isenberg, D. L. (1981). Use of the luminescent bacterial system for the rapid assessment of aquatic toxicity. Trans. Am. Inst. Soc. 20, 29–33. Cairns, M. A., Nebeker, A. V., Gakstatter, J. H., and Griffis, W. (1984). Toxicity of copper-spiked sediments to freshwater invertebrates. Environ. Toxicol. Chem. 3, 435–446. Gaudette, H. E., Flight, W. R., and Toner, L. (1974). An inexpensive titration method for the determination of organic carbon in recent sediments. J. Sediment Petrol. 44, 249–253. Jackson, M. L. (1958). Organic matter determinations for soils. In Soil Chemical Analyses, pp. 205–223. Prentice Hall, Englewood Cliffs, NJ. Mosello, R., Marchetto, A., and Tartari, G. A. (1988). Bulk and wet atmospheric deposition chemistry at Pallanza (N. Italy). Water Air Soil Pollution 42, 137–151. Provini, A., and Gaggino, G. F. (1985). Sediment as a record of copper pollution in Lake Orta. Verh. Int. Verein. Limnol. 22, 2390–2393. Provini, A., and Gaggino, G. F. (1986). Depth profiles of Cu, Cr and Zn in Lake Orta sediments (Northern Italy). In Sediments and Water Interactions (P. G. Sly, Ed.), pp. 167–174. Springer-Verlag, New York. Schuytema, G. S., Nelson, P. O., Malueg, K. W., Nebeker, A. V., Krawczyk, D. F., Ratcliff, A. K., and Gakstatter, J. H. (1984). Toxicity of cadmium in water and sediment to Daphnia magna. Environ. Toxicol. Chem. 3, 293– 308.
35, 231–235 (1996)
Toxicity Identification Evaluation of Lake Orta (Northern Italy) Sediments Using the Microtox System LICIA GUZZELLA,* CRISTINA BARTONE,* PHILIPPE ROSS,† GABRIELE TARTARI,‡
AND
HERBERT MUNTAU§
*Water Research Institute—CNR, via della Mornera 25, 20047 Brugherio, Milan, Italy; †The Citadel, Charleston, South Carolina; ‡Department of Biology, University of Milan, Milan, Italy; and §Italian Institute of Hydrobiology—CNR, Pallanza, Novara, Italy Received November 24, 1995
Pore waters extracted by centrifugation from Lake Orta (Northern Italy) sediments were studied with a modified Toxicity Identification Evaluation (TIE) procedure using the Microtox bacterial luminescence toxicity test system. The most toxic pore water samples were from stations near a rayon factory, known as a source of copper and ammonium discharges. The TIE manipulations used were filtration, EDTA chelation, and C18 solid-phase resin adsorption. The most effective treatments to remove toxicity were the EDTA and C18, indicating that both metals and nonpolar organic compounds contribute to the observed toxicity. © 1996 Academic Press
INTRODUCTION
Lake Orta is a warm, monomictic lake located near Novara in the subalpine region of Northern Italy (Fig. 1). The main physical characteristics of the lake are: surface area 4 18.2 km2, volume 4 1.3 km3, mean depth 4 71 m, maximum depth 4 143 m, actual mean residence time 4 10.7 years. Chemical contamination of the lake began in 1926 with massive discharges of ammonium sulfate and copper from a local rayon factory. All forms of aquatic life were greatly affected by this chemical pollution, and by 1929 the pelagic and benthic communities were virtually absent. Oxidation of ammonia led to a progressive accumulation of nitrates and a drastic acidification of the water column (Provini et al., 1986). Since the early 1950s, discharges from several electroplating plants on a tributary, the Lagna River, have added to the contamination of the lake through discharges of nickel and chromium. The discharges of copper and ammonia have continued virtually uninterrupted since the 1920s, but the introduction of a waste treatment system by the rayon factory in 1958, and improvements made to that system in 1980, have greatly reduced their concentrations in the waste stream discharged to the lake. In 1989 a recovery operation began, and 14,500 metric tons of calcium carbonate was added to the lake water, increasing the pH from 4 to 6, and decreasing ammonia nitrogen levels to 20 mg/liter. To evaluate the potential toxicity of contaminants ac-
cumulated in Lake Orta, a sediment investigation was undertaken in July, 1992. In this study, the toxicity of whole sediments and pore waters was evaluated using a seed germination/ root elongation test, a 48-h Ceriodaphnia dubia survival test, and the Microtox bacterial luminescence test. As a part of the present study, pore water extracts were collected and evaluated with the Microtox system using a procedure similar to the Toxicity Identification Evaluation (TIE) protocol for waste waters according to the United States EPA (US EPA 600-3-88/035). The TIE approach is based on the principle of sequential removal of various chemical fractions coupled with toxicity testing of the obtained fractions. When removal of a class of chemicals also removes toxicity, it is assumed that those compounds were responsible for some of the toxicity observed in the original sample; in the present study the USEPA Phase I approach was followed, except the aeration and pH adjustment manipulations were not done. These steps were eliminated because the TIE procedure was applied to testing pore water rather than waste waters, which imposed a limit on the volume of sample that could be obtained (<40 ml). The treatments chosen were filtration, metal chelation with EDTA, and passage through a C18 resin column. These treatments were thought most likely to yield information on compounds responsible for toxicity. In this model TIE protocol the toxicity of the initial extract and of the various treated pore water extracts was assessed using Vibrio fischeri in the Microtox test system. Microtox is a rapid, low-cost toxicity test which is used widely in screening tests for assessing sediment toxicity. Pore water, rather than whole sediment, was chosen for evaluation because the interstitial phase has been found to be representative of the bioavailable fraction of the contaminants of concern (i.e., metals and ammonia). In a study of the toxicity of cadmium in sediment to Daphnia magna, the free, or dissolved, uncomplexed cadmium was found by Schuytema et al. (1984) to be the toxic form of the metal, and the authors concluded that ‘‘cadmium adsorbed to the sediment had negligible toxicity.’’ According to Schuytema et al. (1984), the toxic effects of metal associated with sediments could best be predicted from concentrations of unbound metal in pore water. Furthermore, in a study of the toxicity of sediment copper to
231 0147-6513/96 $18.00 Copyright © 1996 by Academic Press All rights of reproduction in any form reserved.
232
GUZZELLA ET AL.
FIG. 1. The location of the sediment sampling stations in Lake Orta.
several organisms, including D. magna and Chironomus tentans, it was found that the toxicity of copper was attributable to soluble copper rather than unbound copper (Cairns et al., 1984). MATERIALS AND METHODS
The locations of the sediment sampling stations in Lake Orta are presented in Fig. 1. For the evaluation of pore water toxicity, surface samples were collected with a Ponar grab at stations 43, 56, and 32A, and vertical cores were taken at stations 45 and 13 (Table 1). The cores were divided into horizons and three strata were selected for analysis: 0–10 cm (recent deposits), 10–20 cm (intermediate-age deposits), and another 10-cm interval from a deeper layer (preindustrial deposits). The sediment samples were stored in darkness at 4°C until analysis. TABLE 1 Main Characteristics of the Sampling Stations Station
Sub-basin
Depth of station (m)
43 56 45 32 A 13
North North North Central South
89 58 143 90 47
Type of sample Grab Grab Core Grab Core
Sediment core horizons were dated on the basis of sedimentation rates measured by Provini and Gaggino (1985) using Cs137. Organic carbon content was determined in samples dried at 105°C, by oxidation with potassium dichromate after Jackson (1958), as modified by Gaudette et al. (1974) (Table 2). To separate pore water from the solid phase, approximately 100 g of each sediment sample was centrifuged at 4000g and 4°C for 45 min. The amount of pore water thus obtained varied from 22 to 42 ml, depending on the water content of the sediment (Table 2). Pore water toxicity was assessed with a Microtox Model 500 analyzer and lyophilized cultures of Vibrio fischeri NRRL-B11177, a rod-shaped, Gram-negative marine organism supplied by Microbics Corp. (Carlsbad, CA). In the Microtox system, the inhibition of bacterial light emission was measured in duplicate experiments at 15°C after 15 min of exposure to the pore water sample (Bulich and Isenberg 1981). The Microtox data acquisition software was then used to calculate the EC50 value, the percentage concentration of pore water causing a 50% reduction in bioluminescence. The modified TIE procedure is outlined in Fig. 2. The pore water samples were divided into five portions. The first aliquot was used to measure the toxicity of the untreated sample. The second was with Milli-Q water. The filtered sample was then tested for toxicity using the same procedure. For the third and fourth aliquots, respectively, 200 and 300 mg/liter of EDTA (sodium salt of ethylenediamine-tetraacetic acid) was added. The solutions thus obtained were used for the toxicity test. Prior to the experiment, the concentration/toxicity curve for EDTA was determined (EC50 4 402 mg/liter). Thus curve was then used to subtract the toxic effect of EDTA addition (10 and 30% of the inhibition for 200 and 300 mg/liter, EDTA, respectively). The addition of such high concentrations of EDTA is necessary to chelate the metal content in the salted medium (2% NaCl) used in the toxicity test. The fifth pore water aliquot was passed through a methanol-activated C18 resin column. The column run-off sample was then tested for toxicity, as was
TABLE 2 Dating, pH, Percentage of Water, and Organic Carbon Content of Samples
43 56 45 45 45 32 13 13 13
Station
Dating of sample
pH of pore water
% of water in sediment
% of org. carbon in sediment
(0–10 cm) (10–20 cm) (32–42 cm) A (0–10 cm) (10–20 cm) (50–60 cm)
1970–92 1970–92 1970–92 1948–70 1900–22 1961–92 1950–92 1908–50 1742–48
7.74 7.55 7.67 7.76 7.46 7.69 7.01 7.23 7.13
39.0 42.5 25.0 31.0 24.0 42.2 49.0 41.0 28.0
7.44 4.53 3.20 4.11 3.20 5.83 6.84 4.78 5.80
TOXICITY IDENTIFICATION EVALUATION OF LAKE ORTA SEDIMENTS
FIG. 2.
233
The scheme of the adopted Toxicity Identification Evaluation procedure.
the methanol eluate from the column, previously exchanged with Milli-Q water in a water bath at 45°C. Chemical characterization of the pre water samples, with the exception of metal concentrations, was performed after Mosello (1988). Ion contents were determined on 1.5-ml sample volumes by ion chromatography in a Dionex Model 210 with a conductometer detector and a Spectra Physics AS8780 autosampler. The concentrations of chloride, nitrate, and sulfate were determined using a Dionex AG4A-AS4A separation column with chemical suppression by AMMS micromembrane. Sodium, ammonium, potassium, magnesium, and calcium were measured with fast I–fast II Dionex separation columns, with chemical suppression by CMMS micromembrane. Eluant concentrations and fluxes were those indicated by Dionex. For the analysis of metals, the samples were evaporated to near dryness after adding 1 ml of Suprapure nitric acid. The residue was heated with an additional 1 ml of nitric acid and, after complete dissolution, the product was diluted with highpurity water to 10 ml. Chromium copper concentrations were determined by electrothermal atomic absorption spectrometry using a Perkin–Elmer Zeeman 3030 instrument equipped with a L’VOV platform and hollow cathodic lamps, applying ashing temperatures of 1200°C (Cu) and 1650°C (Cr), and atomization temperatures of 2300°C (Cu) and 2500°C (Cr). Calibration curves were established with the aid of freshly prepared standard solutions, using Merck Titrisol standards. RESULTS AND DISCUSSION
The results of the toxicity tests are reported in Table 3. For the untreated pore water samples (first column), no EC50 values could be calculated for the grab samples collected at Stations 43, 56, and 32A. The EC20 values (i.e., the percentage of sample causing 20% light inhibition), demonstrated greater toxicity for pore water from station 56. For the core samples,
those collected at station 13 indicated more toxic effects than those from station 45. In these cores, the superficial layers (0–20 cm) were more toxic than the deeper strata. These results can be explained by the proximity to the rayon factory discharge for station 13, and by the dating of the sediment core horizons for station 45. In fact, the 10 to 20-cm core horizon collected at station 45 was dated between 1948 and 1970, a period characterized by the highest water-column cuproammonium contamination (Baudo et al., 1989). When the initial pore water sample was filtered, this treatment had a detoxifying effect in all samples. This suggests that some of the toxicity in the initial sample may be associated with suspended rather than particulate matter. The addition of EDTA reduced toxicity on all pore water samples except the extract from station 32A. For the core segments collected at station 13, an addition of 300 mg/liter of EDTA was necessary to remove the observed toxicity, while in all other samples 200 mg/liter was sufficient. Passage through the C18 column removed toxicity in all samples, but no toxicity was found in most of the methanol eluates. This may indicate either that the organic compounds responsible for toxic effects were irreversibly bound to the C18 resin, or that methanol, commonly used for eluting C18 columns, was not efficient in removing all bound organic compounds. The only C18 eluate that demonstrated a toxic effect was the extract from station 32A. In this case, two hypotheses may be argued. First, toxic amounts of organic compounds discharged from a local factory may be present that can be eluted with methanol. Second, the absence of the inorganic matrix (i.e., the salt content) may be responsible for the toxicity of the compounds isolated by C18 extraction. Chemical analyses of the pore water samples are reported in Tables 2 and 4. The pH of all tested pore waters was near neutrality, while the organic carbon content of the whole sediments ranged from 3.2 to 7.44%. The highest organic carbon
234
GUZZELLA ET AL.
TABLE 3 EC50 and EC20 Values of Orta Pore Water Samples Chelation with EDTA Station
Whole
Filtered
EC50 EC20 56 EC50 EC20 45 (0–10 cm) EC50 EC20 45 (10–20 cm) EC50 EC20 45 (32–42 cm) EC50 EC20 32 A EC50 EC20 13 (0–10 cm) EC50 EC20 13 (10–20 cm) EC50 EC20 13 (50–60 cm) EC50 EC20
>100 68.3
>100 66.7
>100 39.7
>100 >100
42.5 16.3
100 17
220 ppm
300 ppm
After C18 SPE
C18 SPE extract
43
32.9 11.4
54.5 23.5
95.8 60
>100 90
>100 90
>100 90
>100 90
>100 90
>100 >100
>100 30
>100 60
>100 >100
>100 >100
>100 60
>100 80
>100 70
>100 >100
>100 90 >100 40
96.5 27.3
>100 70
>100 >100
100 80
>100 >100
>100 61.2
>100 90
>100 70
80 60
>100 >100
11.2 <5
13.1 6.1
40.2 14.2
17.7 <5
>100 60
>100 >100
>100 >100
19 6.3
43.9 16.2
37.8 <5
>100 70
>100 >100
>100 >100
40.9 16.5
49.1 18.1
35.5 <5
>100 >100
>100 59.6
>100 90
Note. SPE, solid-phase eluate.
values were observed in the station 43 grab sample and 0 to 10-cm stratum of the station 13 core, but there was no evidence of correlation between pore water toxicity and organic carbon content of the bulk sediment. No particular anomalies could be observed in the distribution of cations and anions (Table 4). High ammonia concentrations were observed in pore waters from samples collected at stations 45, 43, and 32A. This contamination was found even in the oldest horizon (32–42 cm) of the core from the deepest part of the lake (station 45). Although
Lake Orta has a well-documented history of loading with discharged ammonia, the ammonia in most samples could also be attributed to natural microbial degradation of nitrogenous organic compounds. By this mechanism, ammonia could accumulate to high concentrations under anaerobic conditions that occur in deep sediments (Berner, 1980). Concentrations of ammonia in excess of 10 mg/liter are often observed in pore water extracts (Ankley et al., 1990). Ankley et al. (1990) demonstrated that ammonia played a large role in determining pore
TABLE 4 Chemical Analyses of Orta Pore Water Samples Station 43 56 45 45 45 32 13 13 13
(0–10 cm) (10–20 cm) (32–42 cm) A (0–10 cm) (10–20 cm) (50–60 cm)
Na (mg/liter)
K (mg/liter)
Mg (mg/liter)
Ca (mg/liter)
Cl (mg/liter)
N-NO3 (mg/liter)
N-NH4 (mg/liter)
SO4 (mg/liter)
Cu (mg/liter)
Cr (mg/liter)
5.4 5.1 4.7 4.4 3.7 5.3 6.1 6.2 8.4
2.1 1.3 2.0 2.0 2.0 2.2 1.4 1.3 1.4
3.7 1.5 1.1 1.2 1.2 1.8 2.0 1.8 1.4
10.5 6.5 6.8 7.6 8.2 5.8 9.3 6.7 6.5
3.3 2.5 2.5 2.3 2.2 3.0 3.0 3.4 2.2
0.01 <0.01 0.02 0.01 0.02 0.01 0.03 0.03 0.03
8.69 3.24 9.86 10.26 10.70 7.42 2.44 3.78 2.47
0.1 0.2 2.2 1.5 0.5 0.6 3.2 1.6 0.5
0.25 0.14 0.17 0.07 0.10 1.11 0.39 0.13 0.08
0.260 0.020 0.027 0.013 0.003 0.270 0.150 0.022 0.013
TOXICITY IDENTIFICATION EVALUATION OF LAKE ORTA SEDIMENTS
water toxicity in Fox River (U.S.A.) sediment samples. In their study, the concentrations of ammonia were correlated with the toxicity observed in the 7-day chronic test with C. dubia. They suggested that the presence of ammonia could greatly complicate the results of sediment toxicity testing. High levels of ammonia could mask the effects of other toxicants, while low concentrations might interact with other sediment-associated contaminants in additive, synergistic, or antagonistic fashions. Therefore, when performing TIE evaluation on sediment samples with high concentrations of ammonia, Ankley et al. (1990) suggested the approach of using a test species that is not sensitive to ammonia (i.e., V. fisheri). In the present study, it was not possible to correlate the Microtox toxicity of pore water samples with the observed ammonia concentrations. Similarly, the detoxification effect obtained with the EDTA addition could not be related to pore water concentrations of the analyzed metals, most importantly copper and chromium. Of the possible metal analytes, copper was determined because it is most representative of the pollution produced by the rayon factory. This metal was present in greater concentrations in the most recent core horizons (0–10 cm) and particularly in the grab sample from station 32A, where there was a preferential deposition of suspended matter coming from the southern part of the lake. In a study of the spatial distribution of metals in Lake Orta sediments, Baudo et al. (1989) demonstrated a transport of metal-enriched fine particles to this part of the lake from inputs located further to the south. This was called a ‘‘focussing phenomenon’’ (Baudo et al., 1989). Chromium was considered to be the metal most representative of the discharge produced by the electroplating industries (Jackson, 1958). The higher concentrations observed for station 13 could be explained by the inflow from the Lagna, a river that collects waste waters from an important industrial area. Station 43 was also located near the inflow of a polluted river, while for station 32A the hypothesis of a selective accumulation of suspended matter should be considered. CONCLUSION
The modified Toxicity Identification Evaluation procedure used in the present study was especially useful in the evaluation of potentially toxic components in pore water samples from Lake Orta. Toxicity was attributed to three components: suspended solids, heavy metals, and nonpolar organic compounds. No evident correlations were demonstrated, however, between the results of the chemical analyses and those from the Microtox test. It might be argued either that the chemical analysis was inadequate (i.e., an insufficient number of ana-
235
lytes) for the characterization of the pore water samples, or that other interactions such as synergistic effects may influence the response of the Microtox test system. In the future more attention should be paid to the toxicological effects of sediment-associated contaminants. In fact, sediment/water interactions might make these chemicals more available to aquatic organisms, which could possibly interfere with lake recovery operations. ACKNOWLEDGMENTS The authors are grateful to Microbics Corp. (Carlsbad, CA), Instrumatic s.r.l. (Lainate, Milano, Italy), and R. Cardente and R. Cozzi of Exotox L.D.S. (Pregnana Milanese, Italy) for supporting this investigation through the availability of Microtox equipment, reagents, and expertise. They thank A. Lami, R. Baudo, and A. Ferrari of the Italian Institute of Hydrobiology (CNR-VerbaniaPallanza, Novara, Italy) for their assistance in collecting and processing the sediment samples. The participation of P. Ross was supported by a faculty Development Grant from Citadel Development Foundation.
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