Journulr?fSoilScience, 1993,44,97-110

Effects of time and temperature on the bioavailability of Cd and Pb from sludge-amended soils

P. S. H O O D A & B. .I. A L L O W A Y Environmental Science Unit, Geography Department, Queen Mary & Westfield College,

University of London, Mile End Road, London El 4NS, UK

S U M M A R Y

A pot experiment was conducted to compare the behaviour and bioavailability of Cd and Pb from two soils mixed with sewage sludge at three rates (0, 50 and 150 t ha-') and maintained at two contrasting ambient temperatures (15C and 25C) over a period of one year following the treatments. Ryegrass (Lolium perenne) accumulated Cd and Pb in the sewage sludge treated soils, although accumulation was significantly lower in the soils treated at the high rate ( 1 50 t ha- I ) compared to the low rate (50 t ha - I ) . Ryegrass grown in the warm environment (25C) accumulated significantly higher levels of Cd and Pb than that grown in cooler conditions (1 5OC). Samples of the soils spiked with nitrate salts of Cd and P b a t equivalent rates of metal loading resulted in the ryegrass accumulating much higher levels of both the metals than on the sludge treated soils.

Metal uptake by the ryegrass from the sludge treatments increased over successive harvests while that from metal salt treatments decreased. The observed trend of increasing plant metal uptake over time coincided with a trend of decreasing pH in the sludge treatments. However, the concentrations of Cd and Pb extracted by DTPA failed to predict the changes in plant metal uptake. The importance of sewage sludge as both a source and a sink of pollutant metals and the trend of increasing bioavailability over time shown by this experiment are discussed.

I N T R O D U C T I O N

The disposal of sewage sludge on agricultural land is increasing throughout the world. Although sewage sludge contains agronomically useful amounts of N and P, and has valuable soil condition- ing properties (Epstein, 1975; Chang et al., 1983), the concentrations of several potentially harmful contaminants commonly found in sludges limit the extent to which they can be applied to land. These contaminants include trace metals such as Cd, Cu, Cr, Ni, Pb and Zn and organic micro- pollutants such as polycyclic aromatic hydrocarbons and polychlorinated biphenyls. There is a significant risk that some of these contaminants will affect human health by accumulation in food crops. This is particularly important in the case of trace metals, such as Cd, which has a relatively high bioavailability. Of the factors known to control the bioavailability of sludge-borne trace metals, soil pH (Mahler et a/., 1980; Narwal et al., 1983; Eriksson, 1989; Alloway et a/., 1990; Jackson & Alloway, 1991), concentration of sludge-borne metals (Rappaport et al., 1988), organic matter (Maclean, 1976), and temperature appear to be most important.

Studies on the changes in the bioavailability of trace metals with time have generally shown that anomalously high concentrations of many metals remain available for several years after the appli- cation of sludges (McGrath, 1987; Alloway & Jackson, 1991). Several authors found that metal availability remained more or less the same (Chang et a/., 1982; McGrath, 1987), whereas others reported decreases after the last sludge application (Hinesly et al., 1979; Bidwell & Dowdy, 1987; Morel et a/., 1988). Some authors reported increases in availability with time, a t least for some

97

98 P. S . Hooda & B. J. Alloway elements, such as M o (Soon & Bates, 1985) and Ni (Korcak & Fanning, 1985). De Haan (1975) predicted that the availability of sludge-derived metals in most soils will eventually increase due to acidification by nitrification and leaching. However, it is now the normal practice to maintain the p H of sludged soils a t around 6.5. Several workers have also reported that the bioavailability of metals from soils spiked with metal salts is much greater than that of the equivalent amounts of mctals in sludged soils (Mahler et al., 1980; Korcak & Fanning, 1985). This is an important consideration because metal contamination from sewage sludge is often simulated by spiking with metals.

The fate of metals added to soils in sewage sludges will depend upon the chemical processes operative in the stabilization period after sludge application. It was therefore considered important to study changes in soil chemical parameters during this period and to assess thcir influence on bioavailability of the metals. The objectives of the investigations were: (i) to measure the changes over a twelve month period in soil pH and organic matter in the different treatments; (ii) to follow trends of bioaccumulation of Cd and Pb by ryegrass; (iii) to measure amounts of Cd and Pb extracted by DTPA; (iv) to compare the bioaccumulation of equivalent concentrations of Cd and Pb from sludged and metal-spiked soils; and (v) to assess the effects of markedly different ambient tcmpcraturcs on the chemistry and bioavailability of added metals.

M A T E R I A L S A N D M E T H O D S

Soil treutments Bulk samples of topsoil (0-1 5 cm) wcre collected from two sitcs on agricultural land in England and thcir propcrties are shown in Table I . Onc was a sandy soil from a farm near Thetford in the Breckland region of East Anglia. This was a brown sand of the Worlington/Methwold series developed on decalcified glacial drift overlying chalk. The other soil was sandy loam of the Bursledon series (gleyed brown earth) on drift over Eocene clay from a pasture near Brentwood in Essex. The bulk samples of soil were air dried, sieved (< 5 mm coarse nylon mesh) and thoroughly homogenised. The soils were mixed with the appropriate amounts of sludge or metal salt solutions and allowed to air dry before further grinding and sieving (< 2 mm). 4.6 kg of air-dry soil mixture was added to each PVC pot.

Table 1. Some physico-chemical properties of the soils and sludge

Variahlc Sandy loam Breckland sand Sludge

PH ( H P ) Organic matterJ (g kg I) LOI"(g kg I) Bulk dcnsity (kg m ') CEC (cmol, kg I ) Sand (g kg I) Silt (g kg I) Clay (gkg 7 FI ee Mn (pg g I ) Reducible Fc (pg g I )

Zil c u Pb Ni Cd

5.4 36. I 62.0

1.18 18.94

586 207 177 I18

I200

58.16 18.25 52.10 16.1 1 0.45

6.58 14.1 19.7

I .45 4.75

922 47

5 77

200 Total Metal (pg g I )

27.40 7.70

14.38 7.63 0.12

5.92 _.

556.5 0.63

~

1408 1031 706 259 40

,'Determined by the method of Walkley & Black "Loss on ignition at 400C.

Bioavailahility of Cd and Ph 99

A sample of anaerobically digested liquid sewage sludge was obtained from a sewage works in an urban area and mixed with the two soils a t proportions equivalent to the following field application rates: control (no sludge), 'low' sludge rate (50 t ha-') and a 'high' rate (150 t ha-'). Equivalent amounts of Cd and Pb nitrate solutions were added to other samples of soil to give the same low and high loadings of metals as in the sludge treatments. The resultant metal concentrations under the different treatments are given in Table 2.

Table 2. Cadmium and lead concentration (pg g- I) in the soils resulting from sewage sludge application and metal-salt spiking

Treatment

Sandy loam Breckland sand

Cd Pb Cd Pb

Sewage sludge application Control (0 t ha-') Low sludge (50 t ha ') High sludge (1 50 t ha I)

Metal-salt spiking Low spiking High spiking

0.45 53.1 0.12 13.7 2.02 77.13 2.00 42.5 5.66 131.82 5.62 97.1

2.43 82.1 2.04 42.6 6.07 142.2 5.85 100.4

In ordcr to investigate the effects of temperature, triplicate pots of each treatment were placed in a cool and a warm environment. The warm environment was provided in a greenhouse with the ambicnt temperature controlled at 25 f 3C but with daylight hours varying according to season. For the cool environment, another set of triplicate pots were placed in a growth chamber where the temperature was 15 & 2C with 12 h daylight.

Pot experiments The pot experiments commenced in January 1990 and on the first day (Day 0) each pot was saturated with deionised-distilled water and thereafter the pots were irrigated regularly in order to keep the soil moist during the experiments. Pots in the warm environment normally required irrigation twice a week but those in cool conditions needed irrigation only once a week. After an equilibration period of 1 month, ryegrass was sown as a test crop to compare the bioavailability of thc metals in different treatments. All experimental pots received a small startcr treatment of powdered compound fertilizer supplying small amounts of N, P and K. The first cut of ryegrass was made 6 weeks after sowing and thereafter the grass was cut a t intervals of 4 weeks. The harvcstcd ryegrass samples were washed with distilled water followed by a rinse in deionised-distilled water before drying at 65C.

Composite soil samples were taken at a depth of 0-7.5 cm from all the treatments on days 1 , 15, 30,60, 120,240, and 360. These soil samples were air dried and passed through a 2-mm mesh prior to analysis.

Soil analyses The total concentrations of trace metals in soil and sludge samples (i 2 mm) were determined in filtered extracts obtained from I -g samples of those materials which were digested in concentrated hydrofluoric and perchloric acids (Tessier et al., 1979). All samples were prepared and analysed in duplicate with reagent blanks. Subsamples of the certified reference materials, sludged soil BCR No. 143 and lake sediment IAEA SL-1, were used in all batches of samples analysed for quality control. Trace metal concentrations in all final solutions were determined by flame atomic absorption spcctrometry using an IL (Thermoelectron) S12 atomic absorption spectrophotometer with Smith Hieftje background correction.

I00 P . S. Hooda & B. J. Alloway The hydrous Mn oxide contents of the soils were determined after extraction of the soils with

hydroxylamine hydrochloride (Chao, 1972) and hydrous Fe oxide contents by extraction with sodium dithionite (Avery & Bascomb, 1974). The soil parameters measured were: texture by the pipette method (Day, 1965), pH in distilled water (1:2.5 w/v), bulk density (Avery & Bascomb, 1974), organic matter content, calcium carbonate equivalence and cation exchange capacity (Hesse, 1971). Data for thc soil and sludge physico-chemical parameters are presented in Table I . The soil samples taken from all the treatments at regular intervals during the experiments were analysed for pH, organic matter content and metals extracted by 0.005 M diethylenetriaminepentaacetic acid (DTPA) (Lindsay & Norvell, 1978).

Plant analyses For plant tissue analysis, only samples from the first, third, sixth and ninth harvests were analysed. The washed and dried samples were finely ground and stored in acid-washed sample bottles. Subsamples (0.5 g) of milled tissue wcrc digested in a concentrated HN0,-HCIO, (AristaR gradc, BDH Chemicals) mixture prior to determination of Cd and Pb by atomic absorption spectrometry. All of thc plant analyses were carried out in standard batch formats. Replicate anatyses of all the samples were made together with reagent blanks and the certified reference materials (CRMs) aquatic plant tissue BCR No. 60, olive leaves BCR No. 62 and tomato leaves NBS No. 1573 for quality control. Thc observed CRM valucs were within the certified ranges of the metals. For Cd, all the solutions were analysed first by flame atomic absorption spectrometry (FAAS), but those with Cd concentrations below the detection limit of FAAS were subsequently analysed by electro- thermal atomization atomic absorption spectrometry (ETAAS). The low concentrations of Pb in the ryegrass samples necessitated that all Pb analysis be made by ETAAS.

Statistical analyses Metal concentrations in plants and soils under different sewage sludge and metal salt spiking treatments were evaluated by the least significant difference (LSD) test. Nearly all the interactions were statistically significant a t PGO.01 or P

Bioavailahility of Cd and Ph

3

101

4-1

resulted in substantial increases in the concentrations of these metals in the ryegrass tissue relative to the controls. The amounts of both Cd and Pb accumulated in the plants on the spiked soils were several timcs higher than those in the sludge-treated soils (Tables 3 and 4). Similar differences in Cd and Zn accumulation in maize tissue were observed by Korcak & Fanning (1985) while invcstigating metal accumulation from sludged and metal-spiked soils. As shown in Tables 3 and 4, the differences in metal loadings between the high and low metal-spiked soils were closely reflected in the concen- trations of the metals in the ryegrass. In contrast to the spiking treatments, the concentrations of metals accumulated in the ryegrass on the sludge-treated soils were actually lower in the high sludge trcatments (150 t ha-') than in the low sludge treatment (50 t ha-') in both cool and warm environ- ments (Fig. 3). This could be due in part to a certain amount of dilution resulting from a slightly increased growth of grass a t the high rate of sludge. However, these findings would appear to support the conclusions of Corey et al. (1987) that the sewage sludge matrix acts as a major adsorptive tnedium for metals in sludged soils. The higher the amount of sludge applied, thc greater the total adsorptive capacity of the soil for trace metals, a t least in the short term. In the spiking treatments, where no additional adsorptive material was added to the soils, the different adsorptive capacitics of the soils themselves control the bioavailability of the metals. It is also important to note that no significant differences were found between the accumulation of Cd in ryegrass on either the sludge-treated Brcckland sand or the sandy loam (Table 3), but in the spiking treatments Cd uptake was significantly higher from the Breckland sand and Pb uptake greatest from the sandy loam (Table 4). Hence the sandy loam with greater clay and hydrous Mn and Fe oxide contents gives rise

102 P. S . Hooda & B. J . Alloway

4 1 I 1 I 0 I00 200 30 0 L

Residual period (d) I0

Fig. 2. Changes in soil pH resulting from sewage sludge application: (a) Breckland sand; (b) Sandy loam. 0 control, low sludge, A high sludge.

to a decrease in the availability of Cd and an increase in the availability of Pb relative to the Breckland sand.

The ejjects of temperature on metal uptake Thc data for Cd and Pb uptake by ryegrass when averaged over harvests, soils and treatments, showed that the difference in temperature between the cool ( 1 5 2C) and the warm (25 f 3C) environments significantly increased ( P < 0.01) their accumulation in plant tissue (Tables 3 and 4). The increment in uptake due to the increase in environmental temperature was slightly greater for Cd (77%) than Pb (65%). Furthermore, the averaged data for all treatments showed that the bioaccumulation of Cd in both the environments is higher on the Breckland sand than on the sandy loam (Table 3). Conversely, the uptake of Pb was greater from the sandy loam soil (Table 4).

As shown in Tables 3 and 4, the increase in temperature enhanced accumulation of both the metals by ryegrass in all treatments (including controls). However, the effect was more marked in metal-spiked than sludged soils. In warm conditions, the lower metal uptake from the high sludge treatment was not as pronounced as it was under cool conditions (Fig. 3). In a long-term field trial, Chang et ul. (1987) found soil temperature to be one of the major factors accounting for the variations in metal accumulation by crops.

The efiects oftime on metal uptake The Cd uptake data from the sludge treatments showed that there was a reduction in Cd accumu- lation in the third harvest (Fig. 4). In contrast, Pb uptake shows a steady and significant increase

Bioavailability of Cd and Ph 103 Table 3. Mean uptake of cadmium (pg g- DW) by ryegrass grown on sludged and metal-salt spiked soils under

two growing environmentsd

1st Harvest 3rd Harvest 6th Harvest 9th Harvest

Treatmcn 1 Coal Warm Cool Warm Cool Warm Cool Warm

Sandy loam soil Control 0.33 0.74 0.25 0.53 0.25 0.45 0.23 0.39 Low sludge 0.86 1.68 0.70 2.50 0.99 2.65 1.08 2.88 High sludge 0.60 1.47 0.55 2.81 0.87 2.75 1.05 2.76

High spiking 9.40 15.11 7.09 12.59 7.87 13.78 7.52 14.21 Low spiking 2.41 3.78 3.43 3.84 3.54 4.35 3.55 4.37

Brcckland sand Control 0.32 0.41 0.25 0.28 0.17 0.28 0.17 0.28 Low sludge 1.02 1.59 1.05 1.73 1.79 2.40 1.65 2.46 High sludge 0.83 1.30 0.77 2.64 0.89 2.70 1.08 2.85 Low spiking 4.45 6.78 3.27 5.66 3.91 4.54 3.72 4.58 High spiking 9.43 17.84 6.80 15.97 8.78 16.05 9.07 15.09

Cool and warm represent 15 & 2C and 25 hAll the treatments and their interactions were significantly different at PG0.05.

3C ambient temperature, respectively.

Table 4. Mean uptake of lead (pg g I DW) by ryegrass grown on sludged and metal-salt spiked soils under two growing environments

1st Harvest 3rd Harvest 6th Harvest 9th Harvest

Treatment Cool Warm Cool Warm Cool Warm Cool Warm

Sandy loam Control Low sludge High sludge Low spiking High spiking

Breckland sand Control Low sludge High sludgc Low spiking High spiking

0.193 0.240 0.273 0.519 0.218 0.409 0.731 1.174 2.056 2.279

0.092 0.128 0.170 0.348 0.120 0.315 0.652 0.957 1.603 1.924

0.252 0.294 0.357 0.762 0.274 0.762 0.627 1.297 1.426 2.115

0.101 0.166 0.192 0.347 0.097 0.365 0.464 1.144 1.424 1.861

0.189 0.272 0.430 0.909 0.413 0.918 0.700 1.189 1.413 2.005

0.098 0.176 0.253 0.435 0.232 0.456 0.473 1.332 1.315 1.804

0.197 0.212 0.489 1.014 0.537 0.999 0.663 1.315 1.287 2.171

0.107 0.202 0.364 0.420 0.251 0.454 0.540 1.277 1.360 2.006

All thc treatments and their interactions were significantly different at PG0.05

ovcr the successive harvests in relation to the first cut (Fig. 4). Although Cd accumulation data showed a significant decrease (PgO.01) over time in the controls and metal-spiked treatments (Table 3), Cd uptake from the sludge treatments increased over the residual period (Fig. 4). The rate of increase in Cd uptake over the time period was greater from the high sludge treatment than the low sludge treatment, but the concentrations of the metal were still lower from the high sludge treatments. Nevertheless, the magnitude of difference in Cd uptake between high and low sludge treatments was greater in the first cut than in subsequent cuts (Table 3).

104 P. S. Hooda & B. J, Alloway

I 5 0 s' a

3 100 - I Ul

a, Y

Q w

3 0 5 0

0 00 0.60 I-

0 50

s' a 0 4 0 - I Ul

3 0 3 0 a, Y

Y 2 0.20 n a

0.10

0.00 0 5 0 150

Sludge application rate (t ha-')

Fig. 3. Cadmium (a) and lead (b) uptake by ryegrass from the sludge-amended soils. W cool(loam), El warm- (loam), n cool (sand), H warm(sand).

As shown in Table 4 and Fig. 4, Pb uptake increased over time from all the treatments, except the high metal-spiked soils. It is important to recognise that the difference in the physiological age of the ryegrass sampled from the high metal-spiked soil treatments may influence the comparison of plant uptake data from different harvests on spiked and sludged treatments. As a result of resowing the ryegrass due to the phytotoxic effects of the high metal spikes, the grass was physiologically young while that on the other treatments was up to one year old. As with Cd, there was hardly any difference in Pb uptake from the two sludge treatments in the cuts of ryegrass taken a year after the application of the sewage sludge (Fig. 4). The results indicated that the accumulation of Cd and Pb from the sludge treatments over the residual time period was still gradually increasing. Other workers have also demonstrated the increased plant accumulation of sludge-borne metals with time after sludge application (Schaurer et al., 1980; Narwal et al., 1983), but Chang et al. (1982,1987) and Sanders et al. (1987) did not find this.

Accumulation ratios The mean accumulation ratios for Cd and Pb in different treatments are given in Table 5. The accumulation ratio is defined as the concentration of an element in plant tissue relative to its total concentration in the soil. The accumulation ratios in the control treatments show Cd to be between 200 and 440 times more readily accumulated than Pb. In the sludge and metal-spiking treatments, Cd tends to have accumulation ratios between 60 and 160 times greater than Pb. These higher accumulation ratios indicate that Cd is much more likely to accumulate in the food crops grown on

Bioavailability of Cd and Ph 105

100 t

0 2 4 6 8 10 Residual period (harvests)

Fig. 4. A comparison of Cd (a) and Pb (b) uptake by ryegrass from sandy soil amended with sludge and spiked with metal salts. --n-control, - x - low sludge, -A- low salt, -0- high sludge, -A- high salt.

contaminated soils than Pb. The accumulation ratios clearly show that the relative uptake of the metals decreases with sludge application rate. At the high sludge rate (150 t ha-') the accumulation ratio for Cd is 4 times lower than that for the low sludge application rate (50 t ha-'), whereas for Pb the factor is little over 2 (Table 5) . Accumulation ratios for the Cd and Pb treatments in the warm environmcnt are around twice as high as those in the cool environment.

DTPA extractability Partial extractions with the chelating agent DTPA have been used by many workers as a prediction of the amounts of metals which are bioavailable. The data in Tables 6 and 7 show that the concen- trations of DTPA-extractable Cd and Pb increased markedly as a result of the addition of the metals in both sewage sludge and metal spikes. However, DTPA-extractable Cd and Pb concentrations were more than twice as high in the metal salt-spiked soils compared to the equivalent sludged soils. Increased extraction of metals by DTPA with increasing rate of sewage sludge or metal salt additions is well documented in the literature (Schauer et al., 1980; Korcak & Fanning, 1985; Rappaport et al., 1988). DTPA extraction data from all the periodic samplings, averaged over treatments, showed that both the Cd and Pb were extracted in higher amounts from the sandy loam soil than from the Breckland sand (Tables 6 and 7). DTPA-extractable concentrations of Cd and Pb from all the treatments in the cool environment were slightly higher than those from the warm environment. Howevcr, the effects of temperature on metal extractability were not clear when compared within a

106 P. S. Hooda & B. J. Allowuy Table 5. Mean bioaccumulation ratios of Cd and Pb from different

sludge and metal-spiking treatments"

Sandy loam Breckland sand

Treatment Cd Pb Cd Pb

Cool environment Control 0.73 0.0036 2.66 0.006 Low sludge 0.42 0.0035 0.52 0.004 High sludge 0.10 0.0016 0.14 0.001 Low spiking 0.99 0.0089 2.18 0.015 High spiking 1.55 0.0144 1.59 0.016

Warm environment Control 1.62 0.0045 3.25 0.0093

High sludge 0.26 0.0031 0.23 0.0032 Low spiking 1.56 0.0143 3.32 0.0227 High spiking 2.49 0.0160 3.04 0.0191

Low sludge 0.82 0.0067 1.26 0.0081

~~

;'Mean bioaccumulation ratio is the ratio of plant conccntration ofan element to its total concentration in the soil.

trcaimcnt or for one of the soils. Thcre were no significant changes in DTPA-cxtractable Cd ovcr thc rcsidual period (Table 6) which departs from the trend of the data for Cd uptake by ryegrass. DTPA- cxtractablc Pb showcd a significant decreasc (P,

Tabl

e 6.

Cha

nges

in D

TPA

-ext

ract

able

Cd

(pg

g-')

as a

ffec

ted

by s

ludg

e app

licat

ion

and

met

al-s

alt s

piki

ng u

nder

two

ambi

ent t

empe

ratu

res"

Day

360

D

ay 2

40

Day

1

Day

15

Day

30

Day

60

Day

120

Tre

atm

entb

c

w

cw

c

w

cw

c

w

cw

c

w

T f?.

Low

slu

dge

0.94

0.

96

0.94

1.

10

1.11

1.

06

1.20

1.

10

1.02

1.

06

1.14

1.

21

1.31

1.

41

2

2.44

2.

16

=:

Low

spik

ing

2.42

2.

34

2.14

2.

28

2.39

2.

19

2.32

2.

26

2.24

2.

01

2.40

2.

32

2.36

2.

18

c % 2

Hig

h sl

udge

2.

08

1.88

2.

13

1.85

2.

17

2.10

2.

11

2.22

1.

93

2.23

2.

22

2.23

2.

31

2.10

2

Sand

y lo

am

Con

trol

0.

18

0.17

0.

18

0.18

0.

18

0.18

0.

19

0.19

0.

18

0.18

0.

18

0.18

0.

20

0.19

Hig

h sl

udge

2.

28

2.20

2.

25

2.25

2.

58

2.43

2.

59

2.71

2.

40

2.24

2.

40

2.77

Hig

h sp

ikin

g 6.

20

5.98

6.

01

5.97

6.

23

5.85

6.

39

6.20

6.

36

6.12

6.

37

6.08

6.

26

5.90

5

-. n s 4

Bre

ckla

nd s

and

Con

trol

0.

06

0.05

0.

06

0.05

0.

06

0.06

0.

06

0.06

0.

05

0.06

0.

06

0.06

0.

06

0.06

0.

99

0.82

Lo

w sl

udge

0.

89

0.90

0.

85

0.73

0.

79

0.81

0.

81

0.91

0.

70

0.74

0.

98

0.81

Low

spik

ing

1.74

1.

76

1.68

1.

67

1.69

1.

68

1.61

1.

64

1.65

1.

17

1.69

1.

59

1.64

1.

56

Hig

h sp

ikin

g 5.

06

5.03

5.

11

4.99

5.

05

4.99

4.

83

5.05

5.

02

4.97

4.

79

4.83

4.

94

5.03

"C an

d W

repr

esen

t coo

l (1 5

& 2

C) a

nd w

arm

(25

bAll t

he tr

eatm

ents

exce

pt ti

me,

tem

pera

ture

x tim

e, te

mpe

ratu

re x

trea

tmen

t and

tim

e x

trea

tmen

t wer

e si

gnifi

cant

ly di

ffer

ent a

t P