Algal growth inhibition test-feasibility and limitations for soil assessment

Chemosphere,

Pergamon

GROWTH

INHIBITION

No. 5, pp. 1069-1082, 1997 0 1997 Elsevier Science Ltd

All rightsreserved.Printedin Great Britain 0045-6535/97 $17.00+0.00

PII: SO045-6535(97)00173-2 ALGAL

Vol. 35,

TEST - FEASIBILITY

AND LIMITATIONS

FOR SOIL ASSESSMENT

K. Hund

Fraunhofer-Institut

f%r Umweltchemie

und Okotoxikologie,

(Receivedin Germany12 December

D-57392 Schmallenberg

1996; accepted 6 March 1997)

Abstract Unknown

toxic substances

including

chemical

assessment

in environmental

analyses

samples can be detected

and ecotoxicological

of aqueous extracts of environmental

In the presented

study the influence

and consequences

of nitrogen

and phosphorus

either stimulation

can be compensated

by preparing

in an uncontrollable compounds

present

for the test interpretation

sample Besides nitrogen and phosphorus stimulation

A commonly

applied

test

procedure

system

for the

samples is the growth inhibition test with algae.

of nutrients

investigated

nutrients

tests.

using a combined

in aqueous

extracts

on algal growth

are discussed. Depending

or inhibition can be observed

a second control (nutrient

was

on the concentration

The influence of these

control)

as reference

for the

&n-ther unknown nutrients seem to have an influence resulting

of growth. A test with autoclaved

extract showed that the stimulating

were already in the extract and were not released by microorganisms

during the test. A

stimulation of algal growth due to nutrients may mask the effects of toxic substances.

Therefore,

false

negative

containing

low

results

concentrations

can be obtained

of contaminants

when

using

the test

or high concentrations

these findings it should be considered

with

algae

for

samples

of unknown stimulatory substances.

According to

whether the chronic growth inhibition test with algae should/could

be replaced by an acute test when dealing with aqueous extracts of solids rich in nutrients. Possible test designs are already available

0 1997 Elsevier Science Ltd

Key words. biotest; soil assessment,

algae, nutrients

Introduction For soil assessments

ecotoxicological

of all bioavailable toxic substances.

analyses are of increasing importance, The spectrum

of chemically analyzed substances

their subjective selection based on the history of the area. However, not all contaminants account

may be known and metabolites

Ecotoxicological

with aqueous soil extracts

since they indicate the total

this an insufficient approach,

formed during biodegradation

tests therefore may give valuable additional information. are used to determine

the retention 1069

is limited due to

fbnction

since

are not taken into Aquatic test systems

of soils. Frequently

used and

1070 recommended

[I]

are

test

systems

immobilization

test with Duphnia

subspzcatus and the luminiscence

adapted

magnu,

from

chemical

the growth

and waste

inhibition

inhibition test with Vibrio fischeri

water

testing,

e.g

the

test with the alga Scenedesmus (Microtox

test)

The tests using

Duphnra magna and L’ibriojischerr as test organisms are acute tests, whereas the algal test is a chronic test All these tests have originally been developed soluble contaminants

for testing pure and water soluble substances

soil extracts contain additional components,

Apart from

e.g. macro and micro nutrients which

may influence the results. Due to the addition of organic material or inorganic nutrients the contents of easily bioavailable nutrients, esp. nitrogen and phosphorus higher than in untreated Therefore

synergistic

misinterpretation

soils. These additional

or antagonistic

in substrates from bioremediated

soils may be

nutrients may have an effect on the growth of algae

effects with toxic contaminants

may occur, which may lead to

of the test results.

Besides for the assessment

of soils the above mentioned

tests are also applied for the assessement

of

composts and wastes and at least in compost also high contents of nutrients can be measured The aim of the presented

study was to study the influence of nutrients on the results of the alga1 test and

to give recommendations

for the application of the assay Furthermore,

limitations of the test are shown

Material and Methods Environmental

samples

Soils.

agriculturally used soils

Soil characteristics

are listed in Table I

Table 1: Soil characterization

sand [%] silt [%] clay [%] PH

Cambisol

Luvisol

72.9 21 3 58 62

50 81.9 13 1 75

Chernozem 3.5 74.4 22 I 77

For the tests with algae the cambisol was amended with compost from biowaste in an environmentally relevant concentration

of 8 kg/m’ (compost fresh weight, 60 % dry weight)

Waste

biologically treated domestic waste before storage in a landfill

Compost.

biologically treated domestic biowaste or sewage sludge, comercially available

1071 Prenaration

of the aqueous extract

Samples were shaken with deionized

water (soil/water,

1 : 25;

soil-compost/water

1 : 10) for 24 hours at 4 “C in the dark. For the ecotoxicological

waste/water

aqueous phase obtained after centrifugation

compost/water,

determinations

the

(20,000 g; 20 min) was used.

Scenedesmus subsuicatus: growth inhibition test The test was carried out following the DIN-guideline the fluorescence

was determined

DIN 38412 part 33 [2]. As indicator for biomass

after 0, 24, 48 and 72 h. For evaluation the area below the growth

curves was calculated.

TWO controls were prepared. concentration

One control was performed

of 3.9 mg/L and a phosphorus

control serves as validity criterion.

according

concentration

to the guideline with a nitrogen

of 0.36 mg/L. The growth

In the case the N and P concentrations

than the values set in the guideline the nitrogen and phosphorus

rate of this

of the sample were higher

concentrations

of the second control

(nutrient control) were adjusted to the actual concentrations

of the respective

concentrations

the contents in the nutrient control were

in the sample were determined

photometrically;

sample. The N and P

adapted by adding NH4Cl and KHzPO4. Samples which had a lower nitrogen

and phosphorus

content than the standardized

mineral medium

requested in the guideline were adapted to this level. Dilution series were carried out with standardized mineral medium

containing

N and P. Samples

which had higher concentrations

compared

guideline were diluted with mineral medium containing respective increased concentrations In both set-ups micro nutrients were added as concentrate. samples contained the same concentrations was performed

This procedure

of nitrogen and phosphorus.

guaranteed,

to the

of N and P.

that the diluted

The addition of micronutrients

according to the guideline. Both controls were prepared in 6 replicates and samples in 3

replicates.

To study the effect of nitrogen guideline

for testing

chemicals

and phosphorus [3] with NH&l,

additional

tests were performed

according

NaNOX or KHzPOb as test substances.

to the

Incubation

conditions of the test according to this guideline and according to DIN 38412, part 33 [2] are the same.

Determination

of the colonv forming units (CFU) in aqueous extracts

Standard plate counts were used to determine the number of heterotrophic (medium: peptone

15 g/L, yeast extract 3 g/L+ NaCl 6 g/L, D(+)-Glucose

bacteria in aqueous extracts 1 g/L, agar-agar

incubation: 48 h, 28 “C). Besides fresh extracts autoclaved extracts (121 “C, 20 min) were used.

12 g/L,

1072 Results NitroPen- and phosphorus-concentrations Table 2 shows the concentrations environmental correspond extracts

samples

The

in the aqueous extracts of environmental

of DOC, nitrogen

values

vary

to high nutrient concentrations.

and phosphorus

considerably.

Except

for

samnles

in aqueous

extracts

the waste

of several

high DOC-values

The lowest nutrient contents were determined

in the waste

In most cases the amount of nitrate exceeds the amount of ammonium. With the exception of

the waste the nutrient concentrations

of all extracts

significantly

nitrogen concentrations

of 3.9 mg/l and phosphorus

Table 2. Concentrations

of DOC and nutrients in aqueous extracts of soil, waste and compost

DOC ImdU

NOx~-N

NHd+-N

I&L1

ImdU

concentrations

exceeds the test medium containing of 0 36 mg/l

Nt,,t,t

PO&-P

ImdU

ImdU

Soil 26 ‘)

94

02

96

07

17 l’

81

01

8.2

2.9

28 3’

4.5

01

46

I 1

Waste 51

27

04

31

0.5

510

05

0

05

0

1315

09

0

0.9

03

Compost 363

68

96

16.4

9.5

244

20

12

21 2

72

130

23

06

23 6

49

850

84

0.3

84 3

43

545

108

23

1102

55

670

178

23

201

13

’ Cambisol, ‘) Luvisol; ‘) Chernozem

1073 Influence of nitrogen and ohosnhorus

in the algal test according to the guideline

To investigate the influence of nitrogen on the growth of algae the test medium containing 3.9 mg N/L and 0.36 mg P/L was amended with additional N-concentrations (I” experiment) phosphorus

of 160, 100, 50, 25, 6.25, 3.125 mg/L

and 64.0, 50.0, 20.0, 12.5, 3.10, 0.8 mg/L (2nd experiment).

concentrations

For the experiments

of 80, 50, 25, 12.5, 3.125, 1.563 mg/L (l*’ experiment)

with

and 8.0, 6.25, 4.0,

1.56, 0.391, 0.1 mg/L (2”d experiment) were added.

The results of the nitrogen experiments N/L increasing growth

are presented in tables 3 - 5. Up to a concentration

of 25-50 mg

rates were obtained, whereas a further increase of this element resulted in an

inhibition. A maximum stimulation

of 36 % was obtained when the nitrogen concentration

was at least twice as high as in the standardized

in the test

mineral medium.

No significant difference was observed between samples with nitrogen added as m’

or as N03-.

Table 3 : Influence of additional ammonium on the growth of algae (1” test; by adding mineral medium the mentioned N-concentrations

in the set-ups were increased by 3.9 mg Nk) nitrogen concentration

[mg NH.+‘-N/L]

Control

3.125

6.25

25.0

50.0

100.0

160.0

448

503

476

413

369

340

243

standard deviation

19

10

32

16

19

14

46

inhibition [“A]

0

-12 ”

-6

8

18

24

46

mean value of the area below the growth curves

‘negative

values: stimulation of growth with respect to the control

Table 4: Influence of additional ammonium on the growth of algae (2nd test; by adding mineral medium the mentioned N-concentrations

in the set-ups were increased by 3.9 mg N/L) nitrogen concentration

mean value of the area below

[mg m’-N

/I]

Control

0.80

3.10

12.5

20.0

50.0

64.0

1095

1260

1494

1383

1436

1292

937

the growth curves standard deviation inhibition [%]

68

59

79

32

53

148

105

0

-15

-36

-26

-31

-18

14

1074 Table 5 : Influence of additional nitrate on the growth of algae (by adding mineral medium the mentioned N-concentrations

in the set-ups were increased by 3.9 mg N/L) nitrogen concentration

[mg N03--N /L]

Control

0.80

3:lO

12.5

20.0

50.0

64.0

1095

1188

1428

1459

1402

1214

1124

standard deviation

68

48

122

46

100

56

134

inhibition [%I

0

-8

-30

-33

-28

-11

-3

mean value of the area below the growth curves

Similar results were obtained for phosphorus

(Tab. 6 - 7) however, with much less pronounced

effects

Stimulator-y and inhibitory effects with a maximum of 14 % (6.25 mg P/L) and 18 % (80 mg P/L) were observed.

Table 6: Influence of additional phosphorus the mentioned P-concentrations

on the growth of algae (1”’test; by adding mineral medium

in the set-ups were increased by 0.36 mg P/L.) phosphorus

concentration

[mg/L]

Control

1.563

3.125

12.5

25.0

50.0

80.0

448

448

465

487

444

401

366

standard deviation

19

11

10

11

20

56

11

inhibition [%I

0

0

-4

-9

1

10

18

mean value of the area below the growth curves

Table 7: Influence of additional phosphorus the mentioned P-concentrations

on the growth of algae (2nd test; by adding mineral medium

in the set-ups were increased by 0.36 mg N/L) phosphorus

mean value of the area below

concentration

[mg/L]

Control

0.10

0.39

1.56

4.00

6.25

8.00

1095

1016

1042

1180

1168

1251

1182

68

13

27

43

26

98

26

0

7

5

-8

-7

-14

-8

the growth curves

standard deviation inhibition [w]

1075 In Table

8 the results

concentrations

of a combined

application

of the highest

ammonium

and phosphorus

are shown.

Table 8: Influence of additional ammonium and phosphorus

on the growth of algae (plus 3.6 mg NL

and 0.36 mg P/L by the addition of mineral medium)

T

ammonium concentration concentration

I

/ phosphorus

[mg/L]

Control

448

mean value of the area below

19 0

inhibition [%I

Although increased phosphorus combination

of phosphorus

concentrations

and nitrogen

seem to have a negligible effect on the growth of algae, a

(added as ammonium)

results in higher growth

rates than

ammonium as sole additional nutrient.

f&al test with soikomoost-extract: Table 9 shows the results concentrations

obtained

increased nutrient concentrations in two algal tests with soil/compost-extract.

of nitrogen and phosphorus

in the extract (test 1: NII,‘-N:

In both tests the

1.7 mg/L, N@--N: 7.9 mg/L;

P043--P: 1.1 mg/L; test 2 NkIi’-N: 0.1 mg/‘L, NO3‘-N: 37.8 mg/L; P043--P: 0.9 mg/L) were higher than in the test medium prepared

according

to the guideline. Therefore,

prepared, which contained the same content of nitrogen and phosphorus

additional

nutrient controls were

as the eluate.

1076

Table 9’ Algal test with soil/compost-extract extract concentration Contro

Nutrien

1

31.25

[n&L]

62.50

125.0

250.0

500.0

800 0

t control Test 1

mean value of the

454

634

522

544

621

657

746

741

standard deviation

14

14

17

12

1

31

18

7

inhibition [%I with

0

___

-15

-20

-37

-45

-65

-63

---

0

18

14

2

-4

-18

-17

area below the growth curves

respect to control inhibition [“%.I] with respect to nutrient control Test 2 mean value of the

1544

407

482

522

674

804

1468

2399

standard deviation

92

39

82

84

14

77

17

113

inhibition [%I with

0

___

69

66

56

48

5

-55

---

0

-19

-28

-66

-98

-261

-490

area below the growth curves

respect to control inhibition [%I with respect to nutrient control

Compared to the control prepared according to the guideline a stimulation up to 65 % was obtained in test 1. Even the lowest extract concentration control

a maximum

stimulation

resulted in a small stimulation. With respect to the nutrient

of 18 % was obtained

decreasing stimulation. For the lowest concentration test 2 with considerably

resulting

in

a slight growth inhibition of 18 % was measured

In

higher nitrogen concentrations

the validity control was observed

with decreasing

a strong inhibition of algal growth compared to

in all but the highest test concentration.

nutrient control a strong stimulation for all concentrations

concentrations

However

of the extract was obtained

compared

to the

1077 Algal test with waste extract: nutrient concentrations

according to guideline

In table 10 the results of the algae test with waste extract are presented. 3.1 mg/l and phosphorus

concentrations

medium were obtained. Therefore,

With nitrogen concentrations

of 0.5 mg/l nearly the same concentrations

of

as in the test

no nutrient control was prepared

Table 1O- Algal test with waste-extract extract concentration

[mL/L]

Control

31.25

62.50

125.0

250.0

500.0

800.0

342

470

486

520

591

680

784

standard deviation

26

3

4

5

16

12

8

inhibition [%]

0

-38

-42

-52

-73

-99

-129

mean value of the area below the growth curves

A strong stimulation of growth up to 129 % was obtained. This stimulation considerably maximum stimulation obtained in the test sets with additional nitrogen and phosphorus

exceeded the

(Table 3 - 8).

Algal test with natural and sterilized soilkomnost-extract To find out whether

extracted

microorganisms

play a role for the results, a soil/compost-extract

prepared. 500 ml of the extract were autoclaved. CFU/mL were determined, soil/compost-eluate

was

In the extract which had not been autoclaved 2.6 * lo6

whereas in the autoclaved extract no CFU could be detected. In the “normal“

nutrient concentrations

P/L were obtained, the concentrations

of 0 7 mg m’-N/L,

determined

50 mg NOS*-N/L and 3.0 mg P0,,3--

in the autoclaved

mg NOj--N/L and 2.8 mg P043--PL. In table 11 the results obtained with both extracts are shown.

eluate were 0.6 mg NII,‘-N/L,

52

1078 Table 11: Algal test with soil/compost-extract extract concentration Nutrient

31 25

62 50

125 0

[mL/L]

250 0

500.0

800 0

control compost/soil-extract mean value of the area below

1170

957

1102

976

1329

1916

2404

standard deviation

95

69

52

13

50

106

62

inhibition [%]

0

18

6

17

-14

-64

-106

the growth curves

autoclaved compost/soil-extract mean value of the area below

1183

1139

1172

1141

1224

2052

2422

standard deviation

76

19

143

9

11

133

11

inhibition [%]

0

4

I

4

-4

-73

-105

the growth curves

Both extracts resulted in a similar stimulation of algal growth

Discussion The biotest with algae is standardized standardized Therefore testing

for the assessment

mineral medium with defined

nutrient

of chemicals [4.3] and waste water [2] A

concentrations

is used for the dilution

in chemical testing only the toxic effect of the test substance

ftuther

to toxic substances

infavourable for the environment. the respective

additional

nutrients

are of interest.

Though nutrient concentrations

guidelines the investigation

is detected.

series

In waste water

Both substance

classes are

of waste water are not considered

in

of nutrient rich samples will detect both the effects of toxic

substances and nutrients At present the test with algae is also used in the assessment remediated substances

soils, sediments,

wastes,

composts),

of environmental

namely to detect

which may leach into the groundwater

samples (contaminated

unknown

toxic and bioavailable

For soils with high concentrations

the suitablility of the test has been proved in many cases, and it is recommended

[ 1,5,6,7]

To improve

investigations detected

the application

of contaminated

miniaturized

environmental

with the algal test [9]

contaminants

for the test organism,

contaminants

and therefore

test systems

were developed

samples have been reported

Such a non-response

and

of contaminants

for soil assessment [8]

Nevertheless,

where no effects could be

may be due to a low bioavailability

of the

or the test organism may not have the specific target site for the

reacts less sensitive

It is well known from the testing of pesticides

that

1079 organisms with a specific target site or a specific uptake mechanism for a substance have an increased sensitivity towards this chemical [lo]. For the algal test, which is a chronic test, nutrient concentrations response, Algal growth, which is used as toxicity parameter, environment,

Excessive

concentrations

growth

of nitrogen

may also result in decreasing as the composition

and phosphorus

[Ill.

However

according to the guideline. A stimulation

compared

the concentration

of each nutrient as well

when testing aqueous extracts of environmental

pattern in comparison

to the control prepared

can principally mask effects by toxic substances,

whereas an

of the toxicity. False negative or false positive results can partly

by the use of an additional nutrient control containing the same amounts of nitrogen and

as the eluate. Depending

on the content

of the nutrients

to the “original“ control are obtained (table 9). According

concentrations

[ 13,141. So far, the

study shows, however, that these extracts may contain high contents of nitrogen

inhibition can result in an overestimation

phosphorus

of inorganic nutrients

may influence the toxicity of contaminants

which may result in different growth

be compensated

for increased

high concentrations

growth rates [ 121. Furthermore

influence of nutrients has mostly not been considered

and phosphorus

is significantly affected by nutrients in the

of algae in lakes and rivers usually is an indicator

of the nutrients

samples. The presented

may be an additional reason for a non-

of nitrogen as well as of phosphorus

In environmental

higher or lower growth

rates

to the results of this study the

should be taken into account.

samples ammonium and nitrate are present as nitrogen sources. Usually the content of

nitrate exceeds the content of ammonium,

whereas the test media according to the guidelines contain

ammonia as nitrogen source. With respect to the presented results a differentiation of NO?--N and NHd*-N in the sample does not seem to be necessary

between the contents

when using Scenedesmus

.suh.sp~atu.s as test organism. It is sufficient to use only one nitrogen source for the control Besides nitrogen and phosphorus

additional stimulatory components

seem to be included in extracts of

soils, composts and wastes because stimulator-y effects could be observed even when taking into account the increased N- and P-concentrations

of the sample extracts (table 9 and 10). These components

may

originally be present in the extract, or they may be released as a result of microbial activity during the incubation

period, e.g. by degradation

autoclaved

extract shows that the presence

stimulation

of algal growth,

Growth stimulation

of easily available humic or fulvic acids. The experiment of microorganisms

but indicates that the stimulating

of Scenedesmus

with

does not seem to play a role for the compounds

are present

in the extract,

sp. by acetate was shown by Pearson et al. [ 151. This substance

may also be the reason for the stimulation of growth in the test with the extract of biologically treated waste (combination was determined which substances

of anaerobic and aerobic treatment).

in the leachate from an anaerobic are responsible

Acetate as one of the main organic products

digestion of a refuse mass [16]. As it is unknown

for the growth and whether they are of organic or inorganic nature

they cannot be taken into account. An uncontrollable

stimulation however can mask toxic effects.

1080 Within an assessment

of environmental

samples the above results clearly limit the suitability of the

growth inhibition test with algae for the detection of toxic substances in extracts of solids. However,

it cannot be concluded

environmental be detected

that tests with algae are generally inappropriate

samples. Toxic substances

for the testing of

which affect the special target site “photosynthesis“

with high sensitivity using this organism.

A compromise

could be the substitution

of the

chronic test by an acute test for the detection

of potential phytotoxicity.

suitable short-time tests exist, but modifications

might be necessary. One possible alternative may be the

determination

of the 02-production,

According

can only

to the literature

for which suitable devices are already available

The incubation

period is often no longer than 30 min and for herbicides the sensitivity seems to be at least as high as in the growth

inhibition test [ 171. The one generation

test with synchronized

cultures

of algae and a

maximum test duration of 24 h might also be a suitable alternative [ 181. In this test the increase of algal size is used as test parameter,

and the sensitivity corresponds

designs however there is a need for investigating

to the growth inhibition test. For both test

to which extent growth factors have an influence on

the results.

Conclusion From the results following conclusions ??

Environmental Dependent

samples can contain large amounts of the main nutrients nitrogen

on their concentration

algal growth rate in comparison ??

can be drawn: and phosphorus.

these main nutrients can result in a stimulation or a reduction of the to the control prepared according to the test guideline.

To minimize false positive or false negative results with respect to the toxicity of aqueous extracts of solids the concentration considered

of the main nutrients

nitrogen

and phosphorus

in the sample should be

A second control (nutrient control) as reference for the environmenal

samples should be

prepared. ??

Even when considering

the main nutrients further trace elements can cause a stimulation

growth in environmental

samples. Therefore, inhibition of algal growth seems to be a strong indicator

for toxic, bioavailable,

water soluble substances

samples. However,

is detected,

(nutrients vs. toxicants)

may occur. Therefore the suitability of the chronic test with algae seems to

To obtain information photosynthesis

of environmental

about substances

cannot be excluded.

if either no

effect or a stimulation

be questionable for the assessment ??

in environmental

toxic substances

of algal

as antagonistic

effects

samples with a high content of nutrients.

in extracts of solids that may have toxic effects on algae or

it may be in general more useful to replace the chronic growth test by an acute test

Possible test designs are already available

1081

Acknowlegdement The author thanks G. Host, R. Noker, G. Wodtke for technical assistance and W. Dott, A. Eisentrager, S. Riel3en for recommendation

of valuable literature.

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