The efficiency of two standardized procedures for extracting mutagenic chemicals from soils

Chemosphere,Vol.16,No.6,pp Printed ~n G r e a t B r i t a i n

1243-1255,1987

THE EFFICIENCY OF TWO STANDARDIZED CHEMICALS

L. A. Maggard,

0045-6535/87 $ 3 . 0 0 + .O0 Pergamon Journals Ltd.

PROCEDURES FOR EXTRACTING MUTAGENIC FROM SOILS

K. W. Brown and K. C. Donnelly

Soil and Crop Sciences Department Texas A&M University, College Station, Texas

77843

ABSTRACT A major technical limitation to monitoring the fate of mutagenic compounds in soil is the lack of an established extraction procedure. This study was conducted to evaluate the efflciency of the blender extraction procedure for extracting benzo(a)pyrene (BAP) or 2-nitrofluorene (2NF) from a Weswood silt loam or a Bastrop clay loam soil. Extracted residues were evaluated using the Salmonella/microsome mutagenicity assay and high performance liquid chromatography (HPLC) to quantify recovery of the two chemicals from soil. In addition, a limited study using only the mutagenicity assay was conducted to compare the efficiency of the Blender and Soxhlet extraction procedures for recovering organic mutagens from both soils amended with a wood preserving bottom sediment waste. Extracted residues were dissolved in dimethylsulfoxide to concentrations that could be detected in the bioassay. Over all treatment levels and for both soils, the extraction efficiency of the blender procedure for BAP was greater than 80% for 12 of 16 treatments as measured using both bioassay and HPLC analysis; while, for 2NF the extraction efficiency was greater than 90% for all 15 treatments as measured by HPLC, and greater than 80% for twelve of 15 treatments as measured by bioassay. These results indicate that blender extraction provided efficient recovery of the pure compounds tested. When the efficiency of the blender and Soxhlet procedure for extracting the wood-preserving bottom sediment and waste amended soil were compared, significantly greater levels of mutagenic activity were detected in the fractions extracted using the blender extraction than were detected using the Soxhlet extraction. INTRODUCTION

Industrial primary

activities

products,

materials

contain

group

agents

of

effects

intermediate genotoxic which

mutagenic,

wastes

must

carcinogenic,

genotoxic

effects

disposal

have

incineration.

to

of

products,

highly

quantities or

specific

at subtoxic be disposed

waste

These for

nucleic

of properly

teratogenic

agents

generations.

The

landfilling, techniques

of waste

1243

materials

Many

of

constitute acids

these

either

hazardous

a relatively

and

produce

as

small

deleterious

(I).

to prevent and

most

deep-well

hazardous

materials.

concentrations

or

these

of

compounds

future

included Each

large

chemicals.

are

in genetic elements

Hazardous

generate

to

exposure

prevent

common

disposal

the

methods

injection,

of the populus

deep-sea

may result

transmission

of hazardous dumping,

in the

to of

waste or

intentional

1244

or

accidental

needed

to quantify

of hazardous The soil

release

of

obstacle

which

(2,3,4,). extracting

a

compounds.

et

wide

The

associated

waste

phenolic

during

Polytron

al.

nature

proportion

treatments

and

to

be

technique 14C-BAP

the fate and

extracting

organic soil.

oil

from

extraction

are

transport

phenols,

method was

to

are soil

suitable and

estimate

found

from

data

contaminated

anilines,

was

also

mutagens Some

method

for

neutral

the

to work well

14C

to

soil

and

for

at

intra-laboratory benzene

6 hr extraction

hazard on soil

(GC) for phenolics.

a

solids

BAP

A

technique apparatus

and was determined

from

between

was

soil

hexane

greater

g soil

with

to be 85 to

acetone

and water.

Bulman

with

the

treatments.

as in the biologically

effective

of

Soxhlet

active

et al.

than

the

(8)

using

Because

to soil was found to be substantial.

BAP/50

preserving

of quantification basis.

in

to extract

a wood

(BAP) from soil. labeled

initiated.

more

procedure

soils

the methods

distilled

partitioning

mg

from

and by gas chromatography

precision

a subsequent

2.5

phenolics

inter-

sorption

biologically

significantly

and

benzo[a]pyrene

bound

the

chloride Soxhlet extraction

utilized

further

14C

with

an

extracted

in the sterilized

appear

procedures

contaminated

including

for PNAs

(7)

with

of BAP,

of

than

so that

neutral

procedure

variable

on

al.

(8)

homogenizer

hydrophobic

great

et

for recovering

et

Extraction

extracting

dry

extraction

the

(PNAs)

by HPLC highly

a 16 hr exposure

Bulman

this

but

a methylene

compounds

Shabad

90% efficient

the

was and

PNA

by

for

hydrocarbons

poor

and

developed

use

of

a

compounds

specimens,

Analysis

showed

in soils

for efficiently

efficiency

of

(6) used

aromatic

site.

Results

soil.

with oil (5).

Baty and Perket

plant

the

and monitoring

developed

variety

heavily contaminated

polynuclear

the (5)

intended

with

activity

procedures

al.

to

can be monitored.

to assessing

evaluate

Warner

chemicals

of mutagenic

chemicals

established

is a major

available

genotoxic

levels

organic

lack

of

0.25

mg

BAP/50

of

14C-BAP

soil and,

therefore,

found

Soxhlet

Polytron

of the

In their study,

Binding

the

a

technique

g

soil

was

as

did not

extraction

for

recovering

from soil.

Camoni

et

efficiently percent

al.

extract

recovery

When

some

L),

a

5-8%

chloride

compounds

found is

also

included

both study

this

mutagenic

were

of

is

TCDD

the

best

procedures

the Soxhlet was

compounds

with

deviation extracted was

methanol

for

preparing

and Blender

from soil.

McGill

extractant

to evaluate

15

followed

the

time

and oil soil

techniques efficiency

and

Brown

(2)

for have

contaminated

samples

for

these

two

chloride

soil. was

The

90.1

chloride

(I0)

to mean

+ 4.4.

(3 x 0.3

indicated

that

extracting

mutagenic

shown

methylene

soil.

that The

mutagenicity

using methylene of

from samples

solvent

Rowell in

soil

using methylene

Donnelly efficient

by methylene

(TCDD)

treated

a second

the most

wastes.

for

for

recovered.

consistently

in hazardous

interim

of

extraction

standard

of the samples

further

published

used

2,3,7,8-tetrachloro-dibenzo-p-dioxin

and

methylene

chloride

(9)

chloride.

procedures

USEPA

(11)

testing

and

The objective for

recovering

1245

MATERIALS Benzo(a)pyrene compounds

to

(BAP)

Salmonella/microsome nonpolar a

mutagen

complex

may

plant

waste contained Two

selected soils

are

method

the

in

Soil

pH

The

of other

Procedure

biological each

were

The

Bastrop

heat

soils

soils,

sterility

oven-dried

degradation

soil

days.

was

the

from

for

90 min

then

brought

respectively,

with

to

at

field

treatment

the

section

levels of

Salmonella/microsome 50

~g/g

and

for

the

(3).

Dg/g,

test

samples for

the

ammonium

saturation

using

results,

were the

the

To

50g

prevent

samples

of

consecutive

for the Weswood

soils

selected pure

glass

and

using a 1.4 N

103.5 kPa on three

All

a

fraction

rm~ sieve.

and 22%

were

Bastrop

wet

were

and

checked

for

addition.

to fall within

chemical

BAP,

the

soil

treatment

dose

2NF,

the

soil

treatment

dose

for

and

by

were measured

4

loam (Udic

by

organic

a

18%

The

measured

suspension

water.

wood

determined

was

Detroit, MI) before chemical

curve

For

while

and

capacity,

g soil

dose-response

bioassay 500

50

the

121°C

was

the

the

through

deionized

agar (Difco,

Three

sieved

influencing

sterile

by

and potassium)

and

soils

sterilized

were

lO0°c

by plating on nutrient

mutagenic

pg/g,

in

at

from

a

practices

the Weswood

matter)

(soil:water)

a waste

agents.

clay

management

distribution

1:1

of from

(13).

of

determined

acting

In addition,

preservative

prior

(organic

estimated

indirect

mutagen.

and a Bastrop

was

a

was

as

properties size

carbon

(phosphorous

Soil Application

an

mutagenic

in the Ames

collected

organics

and

known

representative was

creosote

textures

in

content

nutrients

It

Ustochrept)

Particle

measured

solution at pH 4.2.

soil

and

capacity

as

response

of

as

soils.

chemical

Total

ammonium acetate

Each

soil and

exchange was

selected

solvent extractable

(15).

nitrogen

representative

was

loam (Fluventic

1 (14,10).

Day

Cation

(18).

concentrations

is

contaminated

Physical

selected

of a polar direct-acting

sediment

in

different

Table

of

(16).

(17).

BAP

percent

silt

study.

given

electrode

(12).

were

both give a positive

pentachlorophenol

representing

technique

combustion

because

bottom

utilizing

(2NF)

soils

found

a Weswood

for

pipette

be

twenty-seven

soils,

Paleustalf),

bioassay

preserving

which

preserving

2-nitrofluorene

from

and 2NF is representative

wood

material

and

be extracted

AND METHODS

in

the

levels

the Ames

were

levels

were

5 25

~g/g, 250 ug/g and 2500 ~g/g. The

chemicals

Triplicate treament levels

samples level

on

the

on

were

mixed

of

Weswood

the

Bastrop

aseptically

Bastrop soil

soil

at

soil,

were

with

all

and

the

three

duplicate

incubated

soil

using

treatment samples

a

levels at

in the dark at 20°C

the

glass and

stirring of

other

the

two

rod. medium

treatment

for i d or 7 d prior

to

extraction. The

wood

preserving

Weswood

soil

at

moistened amended

to soil

to extraction.

bottom-sediment

a solvent

field were

capacity collected

extractable prior

waste organics

to waste

immediately

was

thoroughly

level

of 3%.

addition.

Three

after

waste

mixed

with

5.5

The unsterilized samples

application

of waste

and stored

kg

of

the

soil was and waste

at O°C prior

1246

Extraction Procedures The wood-preserving at

field

capacity,

blender

the

s.

This

extraction

the

solvent

added

process

remained

The

of

was

filter

residues

or 2NF were extracted

Brown

with

and

Methylene

Six

in a Waring

chloride

transferred

to a

from soil (maintained

chloride

(4).

and mixed

were

combined,

on a Brinkman-Bucci glass

a Waring

of methylene for 30

for 15 s each, or until

extractions

tared

using

volumes

blender

repeated at least two more times,

and taken to dryness

were

methylene

Donnelly

to 25 g of the sample

colorless.

a #42 Whatman

40°C.

BAP,

18% moisture)

procedures

(150 mL) were

through

sediment,

approximately

following

chloride

at

bottom

culture

filtered

Rotary Evaporator

tube

with

methylene

chloride and dried under a stream of nitrogen. The

wood

methylene soil/waste capacity

Extracts

was

then

taken

also

extracted

extraction

6

from the Weswood

method.

The

soil using

homogenized

waste

into a cellulose extraction thimble and placed

A heating mantle with

used. A

was

Soxhlet

was weighed

unit.

were

sediment

the

extractor.

chloride

extractor

bottom

with

mixture

Soxhlet

methylene the

preserving

chloride

Refrigerated h

solvent

to

round

coolant

extraction

dryness

and

was

bottom

flasks

circulated

period

with

partitioned

into

in a 200 mL

containing 200 mL

through

6

condensers

flushings/h

acid,

or

base,

was

and

of

used. neutral

fractions according to the scheme by Brown et al. (13).

Salmonella/Microsome Mutagenicity Bioassay The

Salmonella/microsome

measure in

the mutagenic

dimethyl

each

Salmonella

et

al.

37°C. was

The

enzyme

obtained

according

(19).

strain

control,

that

tested.

TA98

activation

in two

Bionetics

used

Ames

and

et

to demonstrate (Sigma,

Dr.

B.

was

used

to

serially diluted

1:99,

N.

were

incubated

1254-induced SC).

experiments

sterility

1:9,

each extract

cultures

al.

(19)

Samples were

of by

al.

and

were

Ames,

1:999)

of

tested with

University

of

the same as those described by Ames

KS) and

(Charleston,

by

independent

solvent,

bacterial

et

(original,

supplied

Aroclor

Ames

samples.

used were

Lenexa,

system,

by

Dilutions

(kindly

overnight

i0 Dg 2-aminoanthracene

solvent control.

be

The procedures

recommendations

plates

described

concentrations

2 (KC Biological,

from Litton

to

Positive,

positive was

except

Broth No.

four

could

CA).

as

of the extracted

that

level

Berkeley,

(19)

duplicate

so

typhimurium

California,

Nutrient

potential

sulfoxide

soil-treatment

bioassay

$9

grown with

fraction

The $9 microsomal

(19).

All

in

i0

shaking

samples

from

mL

Oxoid

for 16 h at rat

livers,

mix was prepared were

tested

in the standard plate incorporation

controls

were

included

in

each

test.

on

assay The

the functioning of the metabolic activation system, St. Louis,

MO).

Dimethyl

sulfoxide

was

used

as the

All reagents and extracts were tested for sterility.

Chemical Analysis The model

recovery 100-10

Chromatograph. Partisil-10

of

BAP

and

2NF

spectrophotometer

ODS-2

The

from

chromatography

column,

to .01 mm micro-particle

4.6 [mn ID silica

soils

coupled mode X

gel, was

was

with was

also a

An

packed with

isocratic

by HPLC using a Hitachi

model

reversed-phase

250 rmm long, used.

evaluated

Beckman

334

Gradient

partition.

A

octadecylsilane

Liquid Whatman bonded

mobile phase composition of

1247

80/20

and

70/30

efficient

acetonitrile

peak

detection was

following

1

d

incubation. 3390A

ratio

serial

BAP

and

injections

were made

of each

respectively,

were

5 ~i,

Solvent

and

provided

ultraviolet

for BAP and 2NF controls and

sample

residue

quantification

Integrator.

2NF,

introduction

dilution

Chromatographic

Reporting

for

volumes

Triplicate

concentration

Packard

water

Injection

at 0.254 n~n.

for the highest

Hewlett

to

resolution.

of

blanks

the

extracted peak

from soil

area

and untreated

was

by

a

soil extracts

were also analyzed. Statistical analysis

of

analyses

variance

of the bioassay

procedures;

mean

data

included

separation was

the SAS

general

linear models and

by Duncan's Multiple

Range

test at

the 0.05 level (20).

RESULTS AND DISCUSSION

Extraction of Pure Chemical There the

were

blender

(Table

Two

the

low

of

to

it

these BAP

one

mutagenic

also

numbers.

the

Thus,

the mutagenic

for

nineteen

treatments

the Weswood

In a

soil

a

soil

where and

potential

of

22

to

between

2NF amended

soil.

30%,

was

which

a significant

the mean

replicate

control,

difference

of

than the BAP control.

the

possibly

of soil binding were

was

study

little of

or

bioassay of

by as much

no

7

due

day to

At

incubation

soil

not apparent

was

binding.

It

regardless

at the higher

curve for BAP is relatively

biological

variability

approximately

as 50%.

was

to the Bastrop

Thus,

significance

by

Brown

25%,

et

al.

although

it appears

that

between (13),

individual the blender

recovery of BAP from soil.

8

extraction

of

the bacterial However,

extract

treatments

to the soil may have been constant,

deviation

slightly

Only

that although

selected

year

differed

be

3).

BAP

bound

there

of the blender

appeared

the

soil

of the BAP or

replicate

the medium dose

the slope of the dose-response

that

standard

occasionally

difference

than

the

the effects

three

total

(Table

soil,

because

likely

efficiency

indicate

to

of chemical

extraction provided efficient

analysis

between soil

incubation

dose

Weswood

However,

is

experiments

the

applied.

displayed

The

differences BAP-amended

day

low

the amount

levels.

level,

the

the the

less

that

the dose

dose

of

the soil extract was higher and lower, respectively,

dose

significantly appears

of

included

soil where

from Soil

significant

extract

2).

observed

no

less

for recovering

than

that

twenty-one

of

BAP

treatments

mutagenicity

deviation

appreciable

treatments,

over

differences it

is

as

not

3 years

that

using

exhibit

2NF and

biological

a

significant

the extract

of the

from the control 2NF by more than for

2NF

occurred between

likely

compound 2 NF from

measured

did

of the control

only one soil extract varied standard

the polar

the

(13).

These

the mutagenic

blender

results

response

extraction

in

provided

efficient recovery of the 2NF spike from the soil. A comparison or

Bastrop

At

the

low

technique analysis

soils dose

of

of

averaged of

the

the efficiency

as measured

low

BAP

to either

greater dose

than of

of

the blender

extraction

using HPLC and bioassay

BAP

soil,

is provided

the extraction

efficiency

There

to

100%. as

of BAP and 2NF from Weswood

techniques

four

of

appears five

be

replicates

some

in Table 4.

as measured problem

yielded

in

greater

by either the

HPLC

than

200%

1248

recovery,

although

It is suspected was

magnified

bioassay

level

due

in

as

to

either

level

the

low

results

efficient

of

extraction

efficiency soil

low

recovery

BAP,

appeared

and limited

observed

treatment either

probably

not

recovery

extraction

appeared

for

BAP

level,

an

efficiencies

to

provide

were

the

for

efficiencies

(Table 4).

both

efficient

observed

treatment

indicating

observed

using

in the

Low recovery

medium

recovery

factor

were

evaluated

curve.

at

technique,

interfering

error which

its expression

higher

measurement

when

efficiencies

of

was

in Table

4 also

polar

compound

the 2NF

from

of

for

that

At the

both

soils

HPLC and bioassay

recovery

selected

of

BAP

replicates

to

for

than

provide

2NF

soil

that

from was

the blender

soil.

As

greater

extraction

measured

than

95%

by

for

from

within

one

80%.

more

replicate, Thus,

the

when

consistent

average

evaluated

recovery

of

2NF

within

HPLC,

from

the

indicated a

recovery

using

provided HPLC,

all replicates

In addition, although the bioassay results

75%

greater

indicate

either

levels.

efficiency

procedure

same using

good

than 200% (Table 4).

of the BAP dose-response

Thus,

treatment

level

was

greater

level.

within

treatment

the

was

caused by a small weighing

tested

were

technique.

presented

recovery

all

slope

HPLC,

soil

recovery

the medium treatment The

of

blender

However,

At

particles

measurement

techniques,

level

Bastrop

been

of compound

using

soil.

the

clay

treatment

soil.

low dose

determined

in

replicates

results may have

the

the Weswood

adsorption

using

these

to

five bioassay

to the relatively

observed

high

that

due

efficiencies,

were

only one of

the

the

soil

each

blender than

was

compared

for

observed with the non-polar BAP. The

efficiencies

recovering amended

The

soil

for

expression

The results

in

5

of

This may

was

results

standard These

Soxhlet

results

fractions

is possible

While

to

that inhibit

these

when

reduced

reflect

can

the

using

indicate

results

do

that

and

be

mutagens

potential

used

mutagenic all

three

obtained

using

mutagenicity increased

of

residue

crude,

to

blender base,

differ

extraction

and

neutral

extracted

twice

that

from

the

of the acid

the blender procedure did not

present

extraction of

the

compare

the

which may have

in the acid

fraction

indicate that there was fractions

extracted

from

soil. to

potential fractions the the

blender

of of

the

procedure

to

the

from waste amended soil. crude,

the

blender

extraction obtained

obtained

base,

and

waste-amended

fractions

that was

residue removed

the activity

appear

the

fraction

approximately

the Soxhlet

mutagenic

also

the

the additional or dilute

the

acid

preserving waste or waste-

that

for the recovery of organic mutagens

that

waste,

greater The

may

5

extraction

the

significantly

extraction

Table

indicate

of

extraction.

served

in

in

were

to the soil, such as pentachlorophenol,

equivalent amounts of the waste and waste-amended The

of a wood

the

of the bacterial

the

procedures

indicate

mutagens

procedure

obtained

difference

Table

potential

bound

extraction

amounts

in

organic

the blender

organics

a greater

significant

of

from the waste.

extract

of the waste.

presented

mutagenic

using

extracted

efficiently

no

the

and Soxhlet

from equivalent

recovery

However,

waste-amended

allowed

blender

results

efficient

fractions.

fraction

the

organic mutagens

soil.

achieved

of

using

neutral

soil

was

than

the

Soxhlet

using

the

Soxhlet

this

procedure.

It

from the soil using the Soxhlet procedure

of mutagenic significantly

chemicals

present

numerically,

in the residue.

it is impossible

to

1249

determine

if these

extracted

using

assay

differences

either

when

tested

levels

of

mutagenic

blender

or Soxhlet

are also biologically

procedure

at

equal

yielded

a strong

levels.

Thus,

dose

compounds

extraction

were

detected

in

significant. positive

That

is, all fractions

response

in

the

the

results

indicate

that

the

residue

obtained

using

Salmonella appreciable either

the

evaluated,

the

procedure.

CONCLUSIONS Results

indicated

blender

extraction

provided

efficient

For the soil

and

was

by

that

to

of

the

adsorption

BAP may

of

However,

Bulman

et

level

5

(8)

~g

influenced

of polynuclear

Problems

have

alleviated

been

bioassay,

antagonistic

low soil-treatment Although of

mutagenic

was

toxic

bound

to

interactions

the

blender from

significantly

soil

extraction

individual

which

is impossible for

the

used

can occur

to determine

matter

low

that

the

in the Weswood

errors

associated

constituents observed organic

extract.

appeared

techniques

appeared

which

be

used,

analysis

the

the

at

that

a

dose

adsorption

carbon

content

As measured

of may

by

to be magnified

effective

considerable from soil.

the

at the

the

that as much

in

the as

the

was

accurate

of

possible

when of

mixture,

it

However,

samples,

which of by

the

number

results.

concentration is

for

observed

environmental

been

mutagenic

infinite

or waste-soil

Regardless

indicate

may have

efficient

variation

the

using the

results

extracted

be

Given

comparable

mixture,

extracted

These

to

the most

extraction. indicate

of

of a waste

compounds

increased

achieve

mixture

appears

waste

provided

to

waste-soil

alone.

soil,

mutagenic

be

or

as pentachlorophenol,

expression

the components

results

waste

such

technique

procedure

should

of

greater

from

between

provide

to

and/or

level,

level of benzo(a)pyrene

solvent

that of the waste

to extract

to

the

soil (21)

increasing

when analysis

if this effect occurred.

binding

fraction, a

compounds

were

appear is

to

al.

from the waste-soil

blender

monitoring

in

than

for

the

of

procedure

Soxhlet

acid

purposes

procedures

employed

while

techniques

interactions

soil

solvent,

soil.

matrix

dose

analytical

treatment

the

fractions

fraction

the

allowing

Thus,

of

and

greater of

constituents.

two

and/or

most

of the acid

constituents the

BAP et

with

concentrating

sample

to organic

and/or

at the low and medium

further

mutagens

potential

that

of

limited

lower

to determine

Means

increases

extracting

levels.

both

extraction

blender

by

handling,

binding

the

was

the

the

by binding

addition,

hydrocarbons

observed

at

it is difficult

maximum In

as

for BAP at a dose level of 5 ~g/g

from

possible,

preparation,

of BAP,

soil.

aromatic

the soil.

the

been

is

and soils

sensitivity

interference

have

observed

BAP/g

chloride

especially

It

waste,

from soil and waste-amended

of 25 ~g/g soil,

background

due to sample

al.

of

methylene

analyte.

the low concentration

compounds,

compounds

of mutagens,

level

high

of

utilizing

low concentrations

HPLC

types

of mutagenic

for 2NF at a dose due

soil.

the

technique, recovery

concentration

with

for

both

extraction the

extract

extracting

a

greater weight of waste or soil if necessary. ACKNOWLEDGEMENT Contribution Station,

Texas.

the USEPA.

of Texas This

Agricultural

work was

funded

Experiment

Station,

Texas

in part by Cooperative

A&M University,

Agreement

College

CR-807701-01

with

1250

Table

i.

Physical and chemical properties soils.

of the Weswood and Bastrop

Parameter

Weswood

Bastrop

Sand (%)

10.6

34.7

Silt (%)

62.1

33.8

Clay (%)

27.2

30.3

USDA Classification

silt loam

clay loam

Organic Matter (%)

1.4

1.0

CEC (moles/kg)

19.6

27.4 6.9

pH

7.7

N (mg/kg)

70.0

160.0

P (mg/kg)

94.0

25.0

K (mg/kg)

312.0

200.0

Ca (mg/kg)

4,000.0

2,920.0

Mg (mg/kg)

485.0

385.0

% Moisture (w/w) (wilting pont)

12.0

6.0

(field capacity)

18.0

22.0

(saturated)

33.0

25.0

Bulk Density (Mg m -3) (wilting point)

1.48

1.67

(field capacity)

1.44

1.49

(oven-dry) Extractable Hydrocarbon (%)

1.49

1.76

0.057

0.229

2 3

1

1

2 3

I

1

7

I

1

7

I

I

303 +

501 +

77(a) 85(a)

532 +

34(a)

34(a)

23(a)

23(a)

23(a)

422 +

336 +

336 +

501 +

501 +

37(a)

17(a)

65(a)

46(a)

76(a)

34(a)

49(a)

32(a)

17(b)

54(a)

22(a)

483 + I07(a)

347 +

373 +

362 +

524 +

351 +

416 +

546 +

174 +

498 + 129(a)

669 +

451 +

61(b)

292 +

88(a)

Soil + BAP (Mean + SD)

424 + ]96(a)

343 +

424 + 196(a)*

Control BAP (Mean + SD)?

36(a)

55(a)

37(a)

44(a)

85(a)

51(a)

43(a)

23(a)

59(a)

472 + 147(a)

720 +

598 +

628 +

519 +

589 +

330 +

502 +

529 +

561 +

Control BAP (Mean + SD)

421 + 91(a)

ND

607 + 45(a)

478 + 24(a)

520 + 15(a)

383 + 21(a)

403 + 20(b)

279 + 20(a)

442 + 50(a)

ND

596 + 83(a)

640 + 61(a)

Soil + BAP (Mean + SD)

are not

Bastrop

Standard error of all the means = half the square root of mean square error = 56. Mean responses with the same letter between chemical control and soil plus chemical significantly different at the 0.05 level. ND Not determined.

? *

High

Medium

3

1

7

1 2

1

Low

Replication

i

Incubation (d)

Weswood

E f f i c i e n c y of e x t r a c t i n g BAP from W e s w o o d or Bastrop soil following i d soil incubation time (3 replications) or 7 d soil incubation time as m e a s u r e d using S. t y p h i m u r i u m strain TA98.

Dose

Table 2.

50 %m

1

3

i

1

2 3

i

I

7

i

1

7

1 2

I

+ 498a

+ 142a

+ 438a

93a

93a

3030 + 136a

2764 +

2764 +

93a

+ 438a

2764 ~

2669

2772 + 142a

2772

2669

1747 + 398a

1747 + 398a

2694

2694 + 4 9 8 a *

Control 2NF (Mean + SD)t

Weswood

54b

+ 374b

+

+ b

+ 616b

+ 360a

2942

+ 109a

2756 + 136a

2527 + 171b

2519 ~ 167b

2079

2876

2748 + 245a

2099

1012 + 180b

1316 + 267b

2186

2289

Soil + 2NF (Mean + SD)

*

+

33a

28a

+ 358a

+ l17a

+ 377a

+ 362a

error = 56. plus chemical

2820

2725

2722 ~ 801b

2400 + 159b

2706

2663

2255 + 137a

2164

2343 +

Control 2NF (Mean + SD)

are

59b

94a

+ 113a

2454 + 286b

ND

2654 + 257a

3495 J 180a

2633

ND

2408 + 329b

80a

+ 130a 2573 +

2211

NDt

1903 +

2208 +

Soil + 2NF (Mean + SD)

not

Bastrop

from W e s w o o d or Bastrop soil following I d soil incubation time incubation time as m e a s u r e d using S. t y p h i m u r i u m strain TA98.

Standard error of all the means = half the square root of m e a n square Mean responses with the same letter between chemical control and soil significantly different at the 0.05 level. ND Not determined.

High

Medium

2 3

1

7

1

i

Low

Replication

Efficiency of e x t r a c t i n g 2NF (3 r e p l i c a t i o n s ) or 7 d soil

Incubation (d)

3.

Dose

Table

bo Ln

High

Medium

Low

High

71 131 229 110 83 70 108 111 82 85 81 75 79 99 104 91 91 i00

(%)

(%)

98 658 245 56 43 62 84 I01 99 278 90 I01 99 100 I00 105 104 100

Bioassay

HPLC

210 216 ND 91 51 103 94 I01 ND 107 125 ND 95 98 ND 99 I00 ND

(%)

HPLC

114 113 ND 85 68 74 80 84 ND 94 88 ND 97 89 ND 128 97 ND

(~)

Bioassay

Bastrop

for the three

Percent recovery, as measured by HPLC, is calculated from the mean of three control and three sample peak area values. Percent recovery, as measured by the bioassay, is calculated from the mean of four measurements of mutagenic activity per sample from duplicate plates in two independent experiments. ND Not determined.

t

2NF

1 2 3 1 2 3 I 2 3 1 2 3 I 2 3 i 2 3

Low

BAP

Medium

Replication

Dose

Weswood

Extraction efficiency of BAP and 2NF from Weswood and Bastrop soils replications of extractions following I d soil incubation time.t

Chemical

Table 4.

L254

Table 5.

Extract

Comparisons of the mutagenic activity, as measured by S. typhimurium TA98, with metabolic activation, for Waring blender and Soxhlet extracts of wood preserving bottom sediment waste and of Weswood soil treated with the waste.

Extraction Method

Solvent Extractable Organics (mg/g)

Waste

War[ng blender

270

Soil/Waste

Waring blender

135

Waste

Soxhlet

590

Soil/Waste

Soxhlet

271

Crude

Fraction Acid Base Neutral (Histidine-Positive Revertants/Plate)

181 + 14a*

140 + 25b

152 + 23b

215 + 33a

169 + 25a

361 + 26a

243 + 69a

155 + 28a

121 + 17b

149 + 24b

109 + 19b

124 + 35b

129 + 30b

122 + 42b

*

Mean responses with the same letter (within fraction) are not significantly at the 0.05 level. Mutagenicity of Weswood soil extracts is provided in Brown et al. (1984).

different

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of

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Brown, K. W., L. E. Deuel, Jr., and J. C. Thomas, "Soil Disposal Final report to U.S. Environmental Protection Agency. Cincinnati,

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5.

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