Soil heavy metal concentrations, microbial biomass and enzyme activities in a contaminated grassland ecosystem

PII:

SOIL, HEAVY METAL BIOMASS AND CONTAMINATED

Soil Bid. Biochent. Vol. 29, No. 2. pp. 179%190. 1997 _) 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain SOO38-0717(96)00297-0 0038-07 I7/97 s 17.00 + 0.00

CONCENTRATIONS, MICROBIAL ENZYME ACTIVITIES IN A GRASSLAND ECOSYSTEM

ROMAN G. KUPERMAN’*t

and MARGARET

M. CARREIRO’

‘Argonne National Laboratory, Environmental Assessment Division, 9700 South Cass Avenue, Argonne, :Illinois 60439, U.S.A. and ‘The Louis Calder Center, Fordham University, 53 Whippoorwill Road, Box K, Armonk, New York 10504, U.S.A. (Accepted 5 October 1996) Summary-Soil enzyme activities and microbial biomass were measured in a grassland ecosystem with a wide range of heavy metal concentrations ranging from 7.2 to 48.1 mmol kg-’ (As, Cd, Cr, Cu, Ni, Pb and Zn) in portions of the U.S. Army’s Aberdeen Proving Ground, Maryland, U.S.A. Total and fluorescein diacetate active (FDA) fungal biomass, FDA-active bacterial biomass, substrate-induced respiration (SIR), the activity of N-acetylglucosaminidase, p-glucosidase, endocellulase, and acid and alkaline phosphatases were also measured. Most measures of microbial biomass were lower in polluted soils. Significant .reductions (lO- to 50-fold) in the activities of all enzymes closely paralleled the increase in heavy metal concentrations. These results demonstrate that heavy metal contamination of soil has adversely alfected the abundance and activity of microorganisms involved in organic matter decomposition and nutrient cycling in this site. 0 1997 Elsevier Science Ltd

INTRODUCTION Military operations at Army bases often release toxic chemicals into the environment. At the Aberdeen Proving Ground (APG) in Maryland, open burning and open detonation are used to dis-

pose of chemical agents, bulk chemical wastes and high explosives. These disposal methods contaminate the soil with :nutrients and heavy metals, which can affect soil biota and the ecological processes these organisms regulate. To address the increasing concern about environmental quality at Army bases and to understand! more fully the ecological effects of soil contamination, ecotoxicological methods must be expanded beyond existing standard phytotoxicity and earthworm toxicity tests (U.S. Environmental Protection Agency, 1988). A more comprehensive approach to ecological risk assessment should include the quantification of soil biota and critical, biologically-mediated soil processes because these biom and processes are important in determining overall ecosystem function. The relative effects of heavy-metal-contamination of soils on ecosystemlevel nutrient cycling processes can be determined by comparing microbial biomass and activity, rates of organic matter degradation and subsequent nutrient release in contaminated

and less contaminated communities. Comparative studies have reported reductions in microbial biomass or soil enzyme activities for acidic forest soils exposed to long-term atmospheric deposition of metals from smelters (Tyler and Westman, 1979; Freedman and Hutchinson, 1980; Nordgren et al., 1986; Baath, 1989), for agricultural soils amended with metal-containing sewage sludge (Fliessbach et al., 1994; McGrath et al., 1993, and under controlled experimental conditions (Duxbury, 1981; Brynhildsen et al., 1988). However, little or no information is available on the effects of heavy metals on microbial biomass and carbon-, nitrogen-, and phosphorus-acquiring enzymes in grassland soils with low organic matter content (Bardgett er al., 1994). Our purpose was to determine if military waste disposal operations, which result in localized deposition of chemicals, are adversely affecting microbial activities in the APG’s grassland soils. Specifically, we aimed to determine whether relationships exist among altered edaphic conditions and microbial biomass, soil respiration and the activities of five extracellular enzymes involved in the breakdown of organic matter.

MATERIALSAND

*Present address: U.S.

Army Edgewood Research, Development, and Engineering Center, SCBRD RTL E3220, 5101 Hoaldley Rd, Aberdeen Proving Ground, MD 21010-5423,1J.S.A. tAuthor for correspondence.

METHODS

Site description and soil collection

The study site was dominated by a number of graminoids, including Andropogon virginicus. 179

180

R. G. Kuperman and M. M. Carreiro

Agrostis perennans oligantha, Arthraxon hispidus, Aristida oligantha and Phragmites australis. Species

lists and community characteristics for different locations of the study site were reported in Hlohowskyj et al. (1996). Soils were sampled at the Toxic Burning Pits (TBP) area of J-Field on APG. J-Field, which is nearly flat and is covered by open fields, woods and nontidal marshes, encompasses about 186 ha at the southern end of the Gunpowder Neck Peninsula (39” 20’N, 76” 18’W), which projects into the Chesapeake Bay. Soil at J-Field is dominated by Elkton silt loam and Sassafras loam (Perkins and Winant, 1927). The TBP area is an open field located on approximately 3.6 ha in the southern portion of JField. Between the late 1940s and 1980s the pits were used to dispose of chemical agents, bulk chemical wastes, high explosives, nerve agents, incapacitating agents, blister agents and chlorinated solvents. Methods used for disposal included open burning and open detonation (Nemeth, 1989). The pits were maintained by pushing burned soil and ash toward an adjacent area referred to as the “pushout” area (PA). The present study was conducted in OctoberNovember, 1994, along survey grids located in the PA near the TBP and in the local background (LB) area approximately 30 m from the TBP. The location of sampling points in the TBP area was reported in Kuperman (1996). A reference site (with soil characteristics similar to those at J-Field) was selected in Gunpowder Falls State Park, 7 km west of J-Field. Each survey grid measured 10 x 10 m. Soil cores were collected from five randomly selected quadrats (1 x 1 m) in each survey grid (n = 5 per grid) using a 5 cm-dia soil corer to a depth of 10 cm. Soil cores were placed in a cooler as collected, and stored at 2-4°C until shipped for analyses. For al1 subsequent chemical analyses soils from each core were analyzed separately, and were not pooled. The biomass of above-ground vegetation was determined by collecting plants inside 0.1 mz quadrats (33 x 33 cm) at the same sampling locations. Soil chemistry

Total As, Cd, Cr, Cu, Pb, Ni, and Zn were extracted by nitric-perchloric acid digestion and quantified by inductively coupled plasma atomic emission spectroscopy (North Centra1 Region [NCR], 1988). Potassium, calcium and magnesium were detected by using the ammonium acetate extraction method, whereas phosphorus was detected by means of the Bray Pl method (NCR, 1988). Organic matter (OM) content was determined by loss on ignition. Soil pH,,,, was determined by adding water to soil (1:l mixture) and measuring after 10 min.

Enzyme assays

Soil cores were stored in a freezer at -20°C until they were assayed in January, 1995. The cores were then thawed at room temperature for 2 h and placed in the refrigerator at 4°C during subsequent handling. Al1 soil samples were processed and assayed during 2 days (10 and 11 January). Samples did not remain in the refrigerator for longer than 10 h before being assayed. The potential activities of the following soil extracellular enzymes were quantified for each individual soil core: N-acetylglucosaminidase (EC 3.2.1.30), /?1,4,-glucosidase (EC 3.2.1.21), B- 1,Cendoglucanase (endocellulase) (EC 3.2.1.4), acid phosphatase (EC 3.1.3.2), and alkaline phosphatase (EC 3.1.3.1). Assays were made at 25°C by using soil slurries (10 g field moist, 8.5 g dry soil) suspended in 150 ml acetate buffer (50mM, pH 5). The only exceptions were the samples assayed for alkaline phosphatase activity. These slurries were suspended in 100 mM tris buffer at pH 9.5. Incubation times for these assays were: /I-glucosidase and acid phosphatase 5 h, alkaline phosphatase and N-acetylglucosaminidase 6 h and endocellulase 9 h. The activities of al1 the extracellular enzymes (except endocellulase) were measured by using the spectrophotometric method of Sinsabaugh and Linkins (1990). This method uses substrates bound to the chromogen, p-nitrophenol @NP). These substrates were pNP-P-D-glucopyranoside, pNP-N-acetThe and pNP-phosphate. ylglucosaminide endocellulase assay is a viscometric method that uses carboxymethylcellulose (CMC) as a substrate (Aimin and Eriksson, 1967). Three replicate subsamples were taken from each slurry for the pNP assays, along with two replicates for the endocellulase assay. Enzyme activity for the pNP assays is expressed as pmol substrate converted g-’ dry wt (dry wt) h-’ soil and g-’ ash-free dry wt. Endocellulase activity is expressed as viscometric units g-i dry wt h-’ and g-’ ash-free dry wt. Total phosphatase activity was obtained by summing the mean activities for acid phosphatase and alkaline phosphatase for each soil sample. These assays permitted comparison of potential enzyme abundance and activity among sites in this study and should not be interpreted as actual Nt situ activities at the time of collection. Microbial biomass

Active bacterial and fungal biomass were estimated by determining the numbers, diameters and lengths of FDA-stained bacteria, and fungal hyphae in soil-agar films using epifluorescent microscopy (Soderstrom, 1977; Ingham and Klein, 1984). Total fungal biomass was estimated by measuring the length and diameter of al1 fungal hyphae in soilagar films by means of phase-contrast microscopy (Ingham and Klein, 1984). Substrate-induced respir-

Enzyme activities in heavy metal-contaminated soil ation (SIR) is an index of active microbial biomass (Beare et al., 1990; Hopkins et al., 1994). SIR was measured at 2:!“C by using a soil-respiration measuring system with continuous gas flow (Cheng and Coleman, 1989; Cheng and Virginia, 1993). The soil-respiration measuring system used an incubation chamber, an air flow control unit (LI-COR’H‘ model LI-670) an air flow measuring unit, and a CO* analyzer (LI-COR‘H model LI-6251). A 15 g subsample of fresh mixed soil was placed in a 125 ml Erlenmeyer flask. Glucose solution was then added to each flask to achieve a final concentration of 8 mg g-’ dry wt soil and at the same time, to raise the soil water content to near holding capacity. When the rate of CO* evolution from the soil sample becarne constant at an air flow rate of 180 ml min-’ (approximately 40 min), the COz evolution rate was recorded as the SIR. Data analyses

Statistical comparisons among the three sites were made using ANOVA. Heavy metal concentrations, enzyme activities, microbial biomass and plant standing crop biomass data were log-transformed before analysis to stabilize the variante. Bacterial numbers were square-root transformed. The Fisher PLSD test was used to determine the significante (95% level) between al1 possible pairs of the three site groups (n = five soil core replicates per site). Correlation analyses were performed to

Table 1, Concentration

Cd mg kg-’ mmo: kg-’ Cr mg k& mmol kg-’

determine relationships among edaphic enzyme activities and microbial biomass.

Aboveground vegetation

Significant differences (P < 0.0001) in plant standing crop biomass were found among the three sites. Mean vegetation biomass in the reference, LB and PA sites were, respectively, 676.1, 155.9 and 22.6 g m-*. At the PA site, only three plant species were found. In contrast, five plant species were collected at the LB site and seven at the reference site. Soil heavy metal content and other edaphic factors

Combined concentrations of seven heavy metals (mmol kg-’ dry wt soil) were 22.5 times greater in the PA and 4.4 times greater in the LB site than in the reference site (Table 1). Total mean heavy metal concentrations for the reference, LB and PA sites were, respectively, 1.75, 7.69 and 39.50 mmol kg-’ dry wt soil. The order of abundance of these heavy metals (mmol kg-’ dry wt soil) in both the PA and LB site was Zn>Cu>Pb>Cr>Ni>As>Cd. The order of abundance in the reference site was Zn > Cr > Ni > Cu > Pb > As > Cd. Values of other edaphic factors also varied significantly among the different sites (Table 2). The concentrations of Hf for the LB (pH 5.9) and PA (pH 8.2) soils were 1.8

Reference site

Local background site

Pushout area

P value

4.8 (0.17)” 0.064 (0.002)

5.64 (0.46)“ 0.075 (0.006)

15.18 (4.38)b 0.203 (0.059)

0.0006

0.20 (0.0)“ 0.002 (0.0)

0.88 (0.14)b 0.008 (0.001)

3.64 (0.86)’ 0.032 (0.007)

0.000 1

23.4 (0.4)” 0.45 (0.008)

41.8 (2.4+’ 0.804 (0.046)

143.4 (24.7)F 2.757 (0.337)

0.000 1

11.0 (0.6)” 0.173 (0.009)

123.6 (8.2)’ 1.945 (0.129)

569.0 (143.4)’ 8.955 (2.077)

0.0001

19.4 (1.6)” 0.094 (0.008)

250.0 (22.5)b 1.207 (0.109)

1340.0 (285.8)’ 6.467 (1.167)

0.0001

ll.4 (0.4)” 0.194 (0.007)

14.6 (0.8)h 0.249 (0.014)

22.8 (2.5)’ 0.388 (0.034)

0.0001

50.8 (5.2)” 0.777 (0.080)

222.2 (7.7)b 3.399 (0.118)

1352.6 (260.1)’ 20.688 (2.775)

0.000 1

121.0 (6.8) 1.754 (0.088)

658.7 (28.2)h 7.686 (0.272)

3446.6 (244.9)’ 39.491 (3.243)

0.0001

CU

mg kg;-’

mmol kg-’ Pb mg kg-’ mmol kg-’ Ni mg kg-’ mmol kg-’ Zn

mg kg,-’

mmol kg-’ Total heavy metals mg kg-’ mmol kg-’

factors,

RESULTS

of heavy metals in wils of three grassland sites near or in Aberdeen Proving Ground, Maryland. U.S.A.

Heavy Inetal AS mg kg-’ mmol kg-’

181

Values are averages (n = live per site) with standard errors in parentheses. Means ijq the same row with the same superscript are not significantly different at P = 0.05 (Fisher PLSD test) P values and results of the Fisher PLSD test are the same for both mg kg-’ and mmol kg-’ concentrati,Jns.

182

R. G. Kuperman and M. M. Carreiro Table 2. Edaphic characteristics of three grassland sites near or in Aberdeen Proving Ground, Maryland, U.S.A.

Edaphic factor

Reference site

Local background site

Pushout area

P value

PH Organic matter (X) CEC (meq 100 g-’ dry wt) Ca (mg kg-‘) Mg (mg kg-‘) P (mg kg- ) R (mg kg?

5.6 (0.06)” 4.22 (0.19)” 6.8 (0.4)” 664.0 (32.2)’ 130.5 (4.4)” 12.4 (2.2)” 63.9 (6.0)d

5.9 (0.08)b 1.66 (0.14)s 3.2 (0.2)s 474.0 (28.7)b 83.5 (7.4)ï 5.4 (0.4)s 38.6 (2.3)b

8.2 (0.1)’ 1.38 (0.12)’ 9.8 (0.7)’ 407.0 (53.9)b 933.0 (84.4)b 53.0 (3.1)c 53.6 (4.4)”

0.0001 0.0001 0.0001 0.0018 0.0001 0.0001 0.0060

Values are averages (n = tìve per site) with standard errors in parentheses. Means in the same row with the same superscript are not significantly different at P = 0.05 (Fisher PLSD test).

and 390 times greater

than

the reference

site soils

(pH 5.6). Organic matter content decreased from 4.22% in the reference site to 1.38% in the PA site. Calcium concentration was highest in the reference site and lowest in the PA site. However, several factors (cation exchange capacity (CEC), and Mg, P and K concentrations) were highest in the PA site and lowest in the LB site. Total heavy metal concentrations covaried strongly with pH (Pearson correlation coefficient r = 0.9589; P = 0.0001) and moderately, but significantly, with percent OM [r = 0.6365; P = 0.01; Fig. 1 (A) and (B)]. Relationships of heavy metals to other edaphic factors were either weakly linear or nonexistent. Unlike for many other soil types, CEC and percent OM in these samples were not correlated with each other, but CEC correlated moderately wel1 with magnesium (r = 0.8531; P = 0.0001) and phosphorus (r = 0.8293;

centrations ranging from 9 to 12 mm01 (7501109 mg) kg-’ dry wt soil [Fig. 3 (A)-(E)]. However, activities of alkaline phosphatase actually increased between 30 and 48 mmol kg-’ dry wt soil (3723 mg kg-‘) and approached the activities measured in the reference site. The other four enzymes showed very low but detectable activities between 30 and 48 mmol total heavy metals kg-‘. Microbial biomass

Most estimates of microbial biomass were depressed in the LB and PA sites relative to the refer-

P = 0.0001). Enzyme activities

Compared with the reference site, the activities of carbon-, nitrogenand phosphorus-acquiring enzymes were significantly lower in the LB and PA sites [Table 3; Fig. 2 (A) and (C)l. Enzyme activities are expressed on a g-’ dry wt soil basis [Fig. 2 (A) and (C)] and also on a g-’ ash-free dry wt soil basis [Fig. 2 (B) and (D)] to adjust for differences in total soil activity due to the lower OM content found in the heavy-metal-exposed sites. On a g-’ ash-free dry wt soil basis, /I-glucosidase activity was greatly depressed by conditions in the heavy-metal-contaminated sites. /?-glucosidase activity in the PA site was only 3.3% that of the reference control site. Acid phosphatase activity in the PA site was 7.8%, Nacetylglucosaminidase was 10.8%, endocellulase was 18.9%, and alkaline phosphatase was 91.6% of the activities measured in the reference site. Enzyme activities correlated significantly and positively with Ca and percent OM, and except for alkaline phosphatase, negatively, but non-linearly, with pH and total heavy metal concentration (Table 4). Plots of enzyme activity VS total heavy metal content indicate that enzyme activities in these sites approach zero at total heavy metal con-

5,e.. 28

4-0 :

.;

3..

0,2. ò = 8’

??

.

:!

0

.

10

20

30

.

.

??

40

50

Total heavy metals (mmol kg -’ dw soil) Fig. 1. Relationships be.tween (A) soil pH and (B) organic matter content and concentrations of total heavy metals in soils of three grassland sites near or in the Aberdeen Proving Ground.

Enzyme activities Tdble

3.

in heavy metal-contaminated soil

183

Activities of carbon-. nitrogen- and phosphorus-acquiring enzymes in soils of three grasstand sites near of in Aberdeen Proving Ground, Maryland, U.S.A.

Enzyme

Reference site

Local background site

Pushout area

P value

Endocellulase’

88.7 (16.9)ï 1912.0 (384)”

16.30 (4.9+ 769.0 (158.0)“b

9.2 (4.0)b 361.0 (121.0)b

0.0004 0.006

f3-glucosidase+

0.629 (0.32)” 13.567 (0.931)

0.089 (0.023)b 4.191 (0.973)b

0.012 (0.010)’ 0.445 (0.282)’

0.0001 0.0001

N-acetylglucosaminidase+

0.268 (0.020)” 5.785 (0.507)”

0.065 (0.015)b 3.044 (0446)b

0.016 (0.004)L 0.623 (0.094)’

0.0001 0.0001

Acid phosjahatase’

1.782 (0.069)” 38.814 (3.565)”

0.402 (0.080)b 19.098 (2.039)h

0.081 (0.041)’ 3.011 (1.142)’

0.0001 0.000 I

Alkaline phosphatase+

0.296 (0.029)” 6.439 (0.875)”

0.118 (0.020j’ 5.914 (0.817)”

0.145 (0.048)b 5.901 (1.843)”

0.04 0.69

Total phosphatase’

2.078 (0.092)” 45.253 (4.302)’

0.520 (0.094)’ 25.002 (2.376)”

0.227 (0.081)’ 8.913 (2.540)b

0.0001 0.0001

‘Enzyme activitiy is expressed as viscometric units g-’ dry wt hh’ (first row) and g-’ ash-free dry wt hh’ (second row) wil. ‘Enzyme activity is expressed as micromoles substrate converted g-’ dry wt hh’ (first row) and g-’ ash-free dry wt h-’ (second row) soil. Values are averages (n = five per site) with standard errors in parentheses. Means in the same row with the same superscript are not significantly different at P = 0.05 (Fisher PLSD).

ence site (Table 5). Mean FDA-active fungal length and fungal biomass, however, were approximately 40% greater in the LB than they were in the reference site, but these differences were not statistically significant. FDA-active bacterial biomass in the LB and PA sites was 70 and 19%, respectively, of the biomass found in the reference site. Total fungal length and fungal biomass were reduced by approximately 50% in the LB site and by 85% in the PA site compared to the reference control site. SIR in the LB and PA sites was also only 32 and 23%, respectively, of the values found in the reference site. The ratios of FDA-active bacterial-to-FDAactive fungal biornass were greatest at the PA site (4.6) and least at the LB site (0.8). The FDA-active fungal biomass was 2.7%, 7.2% and 1.2% of total fungal biomass in the reference, LB and PA sites, respectively. Good correspondence was observed between some components of microbial biomass and enzyme activity, and between microbial biomass and total heavy metal concentration (Table 6). Values for the Pearson correlation coefficient were highest between the activities of al1 enzymes (except for alkaline phosphatase) and SIR and total fungal length. FDA-active fungal length did not correlate with any of the enzyme activities measured. A high degree of correlation existed between the total heavy metal concentration and FDA-active bacterial biomass, the total (bacterial and fungal) FDA-active biomass, the total fungal length, and SIR (Table 6). Of al1 the microbial biomass indicators measured, .FDA-active fungal length exhibited the least correlation with total heavy metal concentration.

DISCUSSION

Relationships among edaphic factors, microbial biomass and enzyme activities

Our study demonstrated that changes in soil conditions caused by disposal activities in the area have had a large, negative effect on soil microbes and extracellular enzyme activities. Both fungal and bacterial biomass decreased by as much as an order of magnitude, as did the activities of four out of five extracellular enzymes involved in the breakdown of organic carbon, nitrogen and phosphorus. Another independent measure of total microbial biomass, the SIR technique, also revealed that microbial biomass had been severely depressed in the affected sites. Therefore, it can be inferred that the processes mediated by microbes, namely, OM decomposition and nutrient cycling, have been severely reduced by military operations in this grassland. The mixture of different heavy metals and their concentration range in this study are most similar to the concentrations observed near the Gusum and Ronskar foundries in Sweden and near the smelter in Sudbury, Canada (Baath, 1989). However, the soils in these studies were very acidic forest humus, high in organic matter, while the grassland soils in this study ranged from acidic to alkaline and were much lower in OM content. Consequently, the relative effects of heavy metals on microbial biomass and enzyme activities between these other sites and those in this study are expected to differ in severity. At the Gusum sites, which had a total heavy metal content close to that of the PA site in this study, amylase activity was depressed by 25% (Ebregt and Boldewijn, 1977) phosphatase by 30%

R. G. Kuperman and M. M. Carreiro

184

B.

A.

.?! Y0 .?J

100

100

80

80

60

60

40

40 20 0

0 BG

NA

EC

E 0

100

100

àj

80

80

60

60

40

40

20

20

n

OG

EC

NA

ACP

ALP

TP

0

0 ACP

ALP

TP

Enzymes

Enzymes

dry weight basis

ash-free dry weight basis Reference site Local background site Pushout area

Fig. 2. Relative activities of (A and B) carbon- and nitrogen-acquiring soil enzymes, and (C and D) phosphorus-acquiring soil enzymes calculated on (A and C) a soil dry weight basis and (B and D) an ash-free dry weight basis in sites with varying concentrations of heavy metals. Percentages were calculated from average activity values (n = five per site); means with the same letter are not significantly different at P = 0.05 (Fisher PLSD test). BG = B-glucosidase; EC = endocellulase; NA = N-acetylglucosaminidase; ACP = acid phosphatase; ALP = alkaline phosphatase; and TP = total phosphatase.

and

urease

by

35%

(Tyler,

1974). Total

fungal

length and FDA fungal length were reduced by 40 and 35%, respectively (Nordgren et al., 1983). Near Ronskar, where combined soil Cu, Pb, Zn, and As concentrations were 6500 mg kg-‘, no statistically significant effect of heavy metals on phosphatase activity or total and FDA-active fungal lengths was observed (Nordgren et al.,1986). However, SIR correlated negatively with heavy metals at Ronskar (Nordgren et al., 1988). Tyler and Westman (1979) found that at some distance from the Ronskar foundry, where total soil heavy metal concentrations of only 239 mg kg-’ were measured, phosphatase and urease activities were reduced by 28%. Near Sudbury, phosphatase activity was reduced by 69% in an area containing 2600 mg Cu kg-’ and 1900 mg Ni kg-’ compared to an uncontaminated

nickel of with combined copper and site 500 mg kg-’ (Freedman and Hutchinson, 1980). Microbial biomass and most enzyme activities were proportionately lower in our study than they were in Sweden and Canada. A partial explanation is the different heavy metal ratios and the lower OM content of the soils in the contaminated sites in this grassland area (1-4%) than in the forest humus s&ls in Sweden (77%) and Canada. Low OM content can reduce the soil’s capacity to sustain microbial growth and to immobilize heavy metals. (Duxbury, 1985; Tyler et al., 1989; Gadd, 1993). Quantifying the relative contributions of the percent of OM, pH, calcium, and total heavy metal concentrations to the reduction in soil microbial biomass and enzyme activities in these sites is difficult because these factors covaried significantly. The site with the highest heavy metal concentrations

Enzyme activities in heavy metal-contaminated

soil

185

Table 4. Pearson correlation coefficients between enzyme activities and edaphic factors (P values in parentheses) Organic matter (%)

Calcium (mg kg-‘)

Total heavy metals (mmol kg-‘)

Endocellu lase

0.885 (0.0001)

0.641 (0.0002)

-0.566 (0.028)

-0.574 (0.01)

P-glucosidase

0.954 (0.0001)

0.823 (0.01)

-0.673 (0.006)

-0.643 (0.025)

N-acetylglucosaminidase

0.932 (0.0001)

0.844 (0.000l)

-0.709 (0.003)

-0.678 (0.006)

Acid phosphatase

0.946 (0.0001)

0.817 (0.0002)

-0.712 (0.003)

-0.693 (0.004)

Alkaline phosphatase

0.713 (0.003)

0.665 (0.007)

-0.254 (0.361)

-0.331 (0.228)

Total phosphatase

0.941 (0.0001)

0.819 (0.0002)

-0.674 (0.006)

-0.666 (0.007)

Enzyme

also had the lowest OM and calcium content and the highest pH; conversely, the site with the lowest heavy metal concentrations had the highest OM and calcium content and the lowest pH. Decreased OM content alo:ne could reduce microbial populations in soils, a,ssuming similar carbon quality. In addition, the ~011’s OM can bind heavy metals (Tyler et al., 1989; Gadd, 1993), thus mitigating the negative effects of these metals on microbial growth and enzyme function. The lower OM content in the two contaminated sites compared to the reference site may have further suppressed enzyme activity by heavy metals by reducing microbial growth. The lower OM content of the soil probably results from the reduced plant biomass in the affected areas. Expression of microbial biomass and enzyme activities on a g-’ ash-free dry wt basis can adjust for the differences in soil OM content among the sites. A large reduction in microbial biomass and enzyme activities among sites can stil1 be detected even when they are expressed on a g-’ ash-free dry wt basis. As in the present study, a 1977 study by Ebregt and Boldewijn found that Ca was negatively correlated with heavy metals in soils near Gusum. Ebregt and Boldewijn suggested that the negative correlation was clue to Ca replacement by heavy metals in soils. They also noted a positive correlation between CaL and amylase activity but offered no possible cause-and-effect explanation. This suggests that a siz,eable proportion of the reduction in microbial biomass and enzyme activities in the contaminated sites can be attributed to the two remaining environmental variables that covaried significantly: increased heavy metal concentrations and soil pH. Our data set does not allow US to determine whether the increased heavy metal content or the increased soil pH was primarily responsible for the related decrease in microbial biomass and enzyme SBB 2912.-D

PH

activity in the two contaminated sites. Either factor could produce a reduction in microbial biomass, and it is wel1 known that pH interacts with metals to enhance or diminish their toxicity (Starkey, 1973; Gadd and Griffiths, 1980; Bewley and Stotzky, 1983; Gadd and White, 1985; Collins and Stotzky, 1992; Gadd, 1993). In the most contaminated site, which had a pH of 8.4, FDA-active fungal biomass was reduced proportionately more than FDA-active bacterial biomass. Bardgett et al. (1994) also observed a greater sensitivity of fungal biomass relative to bacterial biomass in pasture soils exposed to heavy metals (Cu, Cr, As). Fungi are generally known to be more abundant in acidic soils (Griffin, 1972; BZth et al., 1978, 1979, 1980a,b, 1984; Wainwright, 1979, 1980; Lettl, 1981; Bewley and Parkinson, 1984). Other grassland soils with circumneutral pH contain FDA-active and total fungal lengths similar to those measured in the acidic reference site in this study (Elliott et al., 1988; Ingham et al., 1989). In comparing the reference site with the moderately contaminated site, the pH differente was not great (pH 5.6 VS 5.9), yet total fungal biomass, FDA-active bacterial biomass, and SIR decreased by 45, 30, and 68%, respectively. Nordgren et al. (1983) also observed that pH correlated positively with heavy metals at Gusum and found that fungal biomass did not respond to liming of control soils, which had low heavy metal content. This suggests that the very high heavy metal concentrations in the PA site with alkaline pH may have been primarily responsible for the decrease in the microbial biomass. Because the total heavy metal concentration correlated positively and significantly with soil pH, it is possible that the heavy metal deposition itself was responsible for the marked pH rise in the most contaminated site. If the metals were deposited as oxides, they could directly cause the pH increase in these soils (Nordgren et al., 1983; Baath, 1989). But

186

R. G. Kuperman and M. M. Carreiro A. &glucosidase

2.

0

15. . ‘0

10.

6.

T

C. Acid phosphatase

L B

50:

8

r

12 D. Alkaline phosphatase 1

.

Total heavy metals (mmoles kg -’ dw soil)

E. N-acetylglucosaminidase .

%

.

4

’ .

0

10

20

30

40

50

Total heavy metals (mmoles kg -’dw soil) Fig. 3. Relationships between relative activities of soil enzymes and concentrations of total heavy metals in soils of three grassland sites near or in the Aberdeen Proving Ground.

the low acidity in the contaminated soils could also be due in part to the low plant biomass measured there. Less root production and fewer rhizosphere microbes result in less organic acid production that would lower soil pH. To summarize, heavy metal contamination and pH are often confounding variables in many studies. Soil microcosm studies are needed to complement this field study if mechanisms of microbial suppression in this system are to be clarified.

Activities of four of the five enzymes assayed were severely reduced as heavy metal concentrations increased. /I-glucosidase activity was the most sensitive to soil conditions in the contaminated sites and its activity was depressed by 69 and 96% on an ash-free dry wt basis. This strongiy suppressive effect on /Sglucosidase activity differs from observations by (1) Tyler (1974) near Ronskar in which /3-glucosidase activity was the least sensitive to copper and zinc contamination; and (2) Eivazi and

Enzyme activities

in heavy metal-contaminated soil

187

Table 5. Microbial biomass in soils of three sites containing different levels of heavy metals near or in Aberdeen Proving Ground, Maryland, U.S.A. Microbial biomass category FDA-active bacterial biomass* FDA-active fungal length’ FDA-active fungal biomass’ Total fuga1 length’ Total fungal biomass’ SIR* Ratio of FDA-active bacterial-to-fungal biomass

Reference site

Local background site

Pushout area

P value

15.4 (3.1)”

10.9 (1.6)”

2.9 (0.3)b

0.0001

4.70 (0.63)” 9.1 (1.6)’

6.57 (2.01)” 13.2 (4.0)’

0.33 (0.28)b 0.6 (0.5)b

0.0003 0.0005

175.5 (24.8)” 335.3 (61.3)” 1.9 (0.10)’ 1.69

90.9 (15.0)b 182.9 (30.1)” 0.60 (0.05)b 0.82

27.0 (5.6)’ 49.0 (10.2)b 0.42 (0.05)b 4.59

0.0001 0.0001 0.0001

*pg g-’ dry wt soil. +rn hyphae g-’ dry wt r,oil. *Substrate-induced respiration; flg CO* g-’ dry wt soil min-‘. Values are averages (n = five per site) with standard errors in parentheses. Means in the same row with the same superscript are not significantly different at P = 0.05 (Fisher PLSD).

Tabatabai (1990) in which /?-glucosidase activity was inhibited by only 2-32% even in the presence of heavy metal concentrations of 25 mmol kg-’ soil. Also, /&glucosidase activity was not significantly depressed in apple orchard soils amended with up to 2000 mg Cu kg-‘, although microbial biomass carbon was reduced by 20-33% at levels of 500 mg Cu kg-’ (Aoyama et al., 1993). These differences in /?-glucosidase activity can perhaps be accounted for by differences in soil conditions between this and other sites and by possible synergistic interactions among the seven major heavy metal contaminants in the grassland zrites in this study. Endocellulase and N-acetylglucosaminidase activities were not measured in other cited studies of heavy metal effects on soil processes. Therefore, activities of these enzymes cannot be compared with other studies. The activity of alkaline phosphatase proved to be the most robust in the contaminated sites. Possible explanations for the activity pattern of alkaline phosphatase in these soils are more complex than for the other four ‘enzymes, since it did not correlate with any single edaphic factor. First, rates of alkaline phosphatase activity were 83 and 69% lower than acid phosphatase in the low pH reference and LB sites, respectively (alkaline-to-acid phosphatase ratios of 0.166 and 0.294). However, in the high pH contaminated site, although total phosphatase activity was depressed, alkaline phosphatase activity

was nearly double that of acid phosphatase (alkaline-to-acid phosphatase ratio of 1.79). It is not surprising that relative alkaline phosphatase and acid phosphatase activities correlate wel1 with environmental pH, and this phenomenon has been observed in other soil habitats (Halstead, 1964; Haynes and Swift, 1988). Also, alkaline phosphatase activity, even on an ash-free dry wt basis, continued to rise as total heavy metal concentrations in the most contaminated site (pH 8.0-8.5) increased from 30 to 48 mmol kg-’ dry wt. This pattern of increased activity was unexpected, but the removal of enzymes from clays and organic matter, and their mobilization into soil solution by divalent cations, or even the heavy metals themselves, may explain the increase in potential activity measured (Ladd and Butler, 1970; Ladd, 1972; Tipping et al., 1988; Wetzel, 1991). Also, the concentration of magnesium, which can stimulate alkaline phosphatase activity by causing allosteric changes in the enzyme (Linden et al., 1977; Chrost, 1991), was greatest in the PA site and could have been partially responsible for increasing the activity of this enzyme. Alternatively, the increased alkaline phosphatase activities at high metal concentrations could reflect a change in the composition of the microbial community. Such changes may result from the environmental selection of strains resistant to the toxicity of heavy metals (Dick, 1991; Fliessbach et al., 1994). The increase in alkaline

Table 6. Pearson correlation coefficients for enzyme activities, total heavy metals and various measures of microbial biomass Enzyme Endocellulase /?-glucosidase N-acetylglucosaminidase Acid phosphatase Alkaline phosphatase Total phosphatase Total heavy metals

FDA bacterial biomass

FDA fungal length

Total FDA biomass

Total fungal length

SIR

0.477 0.627” 0.619”

0.212 0.185 0.219

0.412 0.468 0.484

0.757”’ 0.836”” 0.872””

0.791”’ 0.975”” 0.961 ***

0.687” 0.439 0.674”

0.222 -0.071 0.191

0.525 0.192 0.497

0.835”” 0.554’ 0.821”’

0.982”” 0.780”’ 0.982””

-0.732”

-0.636”

-0.825”’

'P< 0.05; "P
-0.760”’

??

-0.659”

188

R. G.

Kuperman and M. M. Carreiro

phosphatase activity can also be related to the nearly lO-fold increase in P concentrations in the PA site. This increase could have resulted from hydrolysis of chemical warfare agents disposed of at this site. Some hydrolysis products of GB (isopropyl methylphosphono-fluoridate) and VX (methylphosphonothioic acid) under alkaline soil conditions are P-containing compounds (Nemeth, 1989). Relationships

between

microbial

biomass

and enzyme

activities

Several microbial indicators (SIR, total fungal length and biomass, and FDA bacterial biomass) were correlated with enzyme activities to a statistically significant extent (Table 6). These correlations are consistent with the hypothesis that the decrease in enzyme activities is caused primarily by direct suppression of microbial growth by soil conditions in these contaminated sites. Alternatively, a portion of the reduction in enzyme activity may possibly have been caused by direct interactions between enzymes and heavy metals (Gadd, 1993). SIR correlated better with enzyme activity than did the other microbial indices. The estimates obtained by direct microscopic methods for determining fungal and bacterial biomass did not correlate as wel1 with enzyme activity as did SIR for several possible reasons. The sampling scales of the SIR and enzyme assays are more similarly matched, and, therefore, integrate the activities of similarsized pools of microbes. Approximately the same amount of soil per sample was used for the enzyme and SIR assays. Much smaller amounts of soil were subsampled and scanned with the microscopic methods. Therefore, the microscopic methods are likely to provide less reliable estimates of mean biomass because the effective sample size is smaller. This is of particular concern when soil samples contain very low microbial biomass, as these soils did. This problem is accentuated for the FDA-active biomass, which was appreciably lower than total biomass in these samples, and correlated poorly with al1 the enzymes assayed. One would also expect poorer correlations between FDA-active biomass and enzymes for biologica1 reasons. FDA-active biomass represents the relatively smal1 portion of the total biomass that responds to current environmental conditions, such as rainfall, whereas enzymes, due to their potential for persisting in soils, may reflect the activity of microbes that grew in the past as wel1 as those active when sampled. Total fungal length and biomass correlated significantly with most of the enzymes, since total fungi represent both living hyphae and dead fungal cells that accrued over a longer interval of time than did the FDA-active fungi. Therefore, in some systems total microbial biomass may match the tempora1 scale over which soil enzymes are

active better than active biomass, which depends on recent variability in weather conditions occurring at the site. In conclusion, this study shows that soil contamination at J-Field, APG, had a detrimental effect on soil microorganisms and enzyme activities. It appears that high heavy metal concentrations are primarily responsible for these observed reductions. Integration of microbial biomass and extracellular enzyme activity measurements into ecological risk assessment procedures would permit direct assessment of negative impacts on the structure and function of soil communities and ecosystem processes. In future ecotoxicological studies of soils where low microbial biomass is suspected, we recommend the use of SIR or direct microscopic estimations of total fungal and total bacterial biomass because they are more integrative of microbial growth over time than is FDA-active biomass. Acknowledgements-We are grateful to Elaine R. Ingham for assistance in determining microbial biomass, Christopher Dunn and Robert Van Lonkhuyzen for help with the vegetation survey, Deborah Repert for help with the enzyme assays, and Research Extension Analytical Laboratory-Ohio Agricultural Research and Development Center for chemical analyses of soils. This work was supported under a military interdepartmental purchase request from the U.S. Department of Defense, Directorate of Safety, Health, and Environment, Aberdeen Proving Ground, Maryland, through a U.S. Department of Energy contract, W-31-109-Eng-38.

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