Deliverable reference number and title: D.2.3.1 Report …nomiracle.jrc.ec.europa.eu/Documents/PublicDeliverables/D.2.3... · 2.4 System Specific Training Set of Chemicals ... The

Front page for deliverables Project no. 003956 Project acronym NOMIRACLE Project title Novel Methods for Integrated Risk Assessment of

Cumulative Stressors in Europe Instrument IP Thematic Priority 1.1.6.3, ‘Global Change and Ecosystems’ Topic VII.1.1.a, ‘Development of risk assessment

methodologies’ Deliverable reference number and title: D.2.3.1 Report on Validated experimental procedures for the determination of compound turnover in water and sediments Due date of deliverable: October 31, 2005 Actual submission date: October 31, 2005 Start date of project: 1 November 2004 Duration: 5 years Organisation name of lead contractor for this deliverable: ECT Oekotoxikologie GmbH

Revision [draft, 1, 2, …]: Final Report

Project co-funded by the European Commission within the Sixth Framework Programme (2002-2006) Dissemination Level

PU Public X PP Restricted to other programme participants (including the Commission Services) RE Restricted to a group specified by the consortium (including the Commission Services) CO Confidential, only for members of the consortium (including the Commission Services)

Authors and their organisation: Michael Meller, Christiane Elste, Thomas Junker & Thomas Knacker ECT Oekotoxikologie GmbH, Boettgerstr. 2-14, D-65439 Floersheim Germany (Partner 26) Deliverable no: D.2.3.1

Nature: Report

Dissemination level: PU

Date of delivery: October 31, 2005

Status: Final Report Date of publishing: October 31, 2005

Reviewed by (name and period): Sabcho Dimitrov, LMC, October 19, 2005 - October 24, 2005 Gerrit Schüürmann, UFZ, October 19, 2005 - October 31, 2005

D.2.3.1 Page 3/25

EּCּT Oekotoxikologie GmbH

Contents

Page

1 Introduction....................................................................................................................................................4

2 Material and Methods...................................................................................................................................6

2.1 Microbial Inoculum................................................................................................................................ 6

2.2 Sediment ............................................................................................................................................... 7

2.3 Overlying Water .................................................................................................................................... 8

2.4 System Specific Training Set of Chemicals......................................................................................... 8

2.5 Test System .......................................................................................................................................... 9

2.6 Development and Validation of Test System and Experimental Procedures.................................. 12

3 Discussion and Results ............................................................................................................................ 14

3.1 Influence of ATU on the Biodegradation of Aniline ........................................................................... 14

3.2 Biodegradation of Aniline and Benzoic Acid in the Presence of Suspended Sediment Solids ...... 15

3.3 Influence of Sediment Pre-Treatments on the Respiration Rate of the Artificial Sediment ............ 17

3.4 Biodegradation of Aniline in a Water-Sediment System................................................................... 18

3.5 Comparison of the Biodegradation of Aniline in Water-Only, Suspension of Sediment Solids and

the Water-Sediment System ............................................................................................................................ 21

4 Conclusion and Outlook........................................................................................................................... 22

5 References .................................................................................................................................................. 24

D.2.3.1 Page 4/25


1 Introduction The environmental fate of a compound is, inter alia, determined by transformation and

distribution processes which, in turn, are strongly dependant on the specific environmental

conditions. In general, there are three major approaches for experimental environmental fate

studies. Field tests allow for the clarification of a substances behaviour under realistic

conditions, whereas laboratory simulation (e.g. OECD guideline 308, 2002b) and screening

tests (e.g. OECD guideline 301, 1992a) display only a certain detail of the entire scenario. Since

field tests are quite costly, time consuming and deliver usually very complex data, their

realisation is usually limited to a small number of cases. In this light, laboratory tests are the

normally applied tools for the investigation of individual environmental processes, providing high

comparability and reproducibility due to standardised test conditions (Löffler et al. 2004).

Numerous laboratory test systems have been established allowing for the investigation of a

chemical’s fate under a variation of relevant environmental conditions in terrestrial, aquatic and

other scenarios (Brodsky et al. 1997, Hill et al. 1994, Freitag et al. 1982, Freitag et al. 1985).

Respective standardised test procedures are provided by several organisations and institutions,

i.e. OECD 2005 (Tab. 1) and SETAC 1995. They are an important part of the risk assessment

of environmental relevant chemicals.

Tab. 1: Available OECD Guidelines for the testing of the degradation of chemicals in terrestrial and aquatic laboratory test systems

Test OECD Guideline No. and Reference

Ready biodegradability (several methods) 301 (1992a) Inherent biodegradability (several methods) 302 (1981b, 1981c, 1992c) Simulation tests – aerobic sewage treatment A: Activated Sludge units B: Biofilms

303 (2001)

Inherent biodegradability in soil 304 (1981a) Biodegradability in seawater 306 (1992b) Aerobic and anaerobic transformation in soil 307 (2002a) Aerobic and anaerobic transformation in aquatic sediment systems 308 (2002b) Aerobic mineralization in surface water – simulation biodegradation test

309 (2004a)

Besides these experimental approaches, the prediction of the metabolic fate of chemicals using

mathematical models is attracting more interest in recent years. Currently available models

were developed mainly for more simple compounds (e.g. non-ionised organic chemicals). Most

of the underlying experimental data were generated in qualitative form (e.g. ready vs. not ready

biodegradable), mostly according to OECD guideline 301 C or D. These methods represent

D.2.3.1 Page 5/25


standardised laboratory screening tests, conducted under aerobic conditions, in which the test

system is inoculated with microorganisms derived from domestic sewage, activated sludge or

secondary effluent. The biodegradation of high concentrations of the test substance is

measured by non-specific parameters like Dissolved Organic Carbon (DOC), Biochemical

Oxygen Demand (BOD) or CO2 production.

As a consequence, the prediction of outdoor system half-lives by mathematical models is

hampered by the lack of quantitative or at least semi-quantitative biodegradation rates that

apply to more realistic conditions. Available data sets are typically related to water-only systems

and not to soils or sediments, where sorption, ageing, sequestration, and cross coupling may

affect the bioavailability, transformation and degradation. To overcome these shortcomings, the

performance of existing experimental methods to determine the biodegradation of chemicals in

water-sediment systems was evaluated by the partners of WP 2.3 at the NOMIRACLE-kick off

meeting in Barcelona. In order to ensure a high degree of comparability of experimental data

from water-only (OECD 301) and water-sediment studies it was agreed to develop a new water-

sediment test system. The design of the new test system should be close to the OECD

Guideline 301 C (MITI I). The newly developed method will be used to generate data on the

biodegradation of the selected test set of compounds within NOMIRACLE. Furthermore the

method will be used to provide experimental data to other partners of WP 2.3 in order to train

their existing models or to develop new models to predict the biodegradation of chemicals in

water-sediment systems.

D.2.3.1 Page 6/25


2 Material and Methods

2.1 Microbial Inoculum

To ensure the presence of a great variety of degrading organisms in the tests, a mixed inoculum

according to OECD guideline 301 C (MITI I, OECD 1992a) was established in the laboratory.

The MITI-inoculum was derived from microbial populations from ten sites in the Rhein-Main

region, Germany.

Fig. 1 Sampling sites for the MITI-inoculum in the Rhein-Main region in Germany

The sampling sites are located mainly in a region where a variety of chemicals (e.g. pesticides,

industrial and household chemicals, human and veterinary pharmaceuticals) are used and

discharged. Sewage treatment works, rivers and lakes of different sizes in diverse areas (rural,

urban, industrial) were selected as sampling sites.

D.2.3.1 Page 7/25


Tab. 2: Characterisation of sampling sites for the MITI-inoculum

Sewage Treatment Plants Location Size of STP (Population Equivalent) STP Trebur-Geinsheim city of Trebur 5.500 [1] STP Flörsheim city of Flörsheim 90.000 [2] STP Wiesbaden Mitte city of Wiesbaden 350.000 [3] STP Niederrad city of Frankfurt/M. 1.350.000 [4] Lakes Location Description Mönchswaldsee near city of

Kelsterbach size: 15.4 ha mean depth: approx. 17 m trophic status (2003): mesotrophic [5]

Oberwaldsee near city of Mörfelden-Walldorf

size: 1.39 ha mean depth: approx. 2 m trophic status (2002/2003): mesotrophic [5]

Steinrodsee near city of Weiterstadt-Gräfenhausen

size: 7.40 ha mean depth: 2.37 m trophic status (2003): polytrophic 1 [5]

Rivers Sampling Site Description Main at the confluence

of the Wickerbach near the city of Flörsheim

biological water quality (2000): moderately polluted [5] [6]

Wickerbach near the effluent of the STP Flörsheim

small tributary of the River Main biological water quality (2000): moderately polluted [5] [6]

Schwarzbach near the city of Trebur

small tributary of the River Rhein biological water quality (2000): critically polluted [5] [6]

[1], [2], [3], [4], HMULV 2005; [5] HLUG 2003, [6] classifications according to the German water quality classification scheme

2.2 Sediment

In order to ensure a high degree of standardisation an artificial sediment based on OECD

Guideline 218 (2004b) modified according to Egeler et al. 1997 and Meller et al. 1998 was used.

It deviated from the formulated sediment recommended by the OECD Guideline by a significant

lower organic content and slightly higher sand and clay fractions (Tab. 3).

D.2.3.1 Page 8/25


Tab. 3: Composition of the artificial sediment

Constituent

Characteristics % of sediment dry weight

Peat Sphagnum moss peat, no visible plant remains, finely ground (particle size ≤ 1 mm) and air dried 2 ± 0.5

Quartz sand Grain size: > 50% of the particles should be in the range of 50-200 µm 76

Kaolinite clay Kaolinite content ≥ 30% 22 Organic carbon 1 ± 0.2 [1]

Calcium carbonate CaCO3, pulverised, chemically pure, in addition to dry sediment 0.05 − 0.1

Water Conductivity ≤ 10 µS/cm, in addition to dry sediment 30 − 50 [1] Egeler 2005 (pers. comm.)

2.3 Overlying Water

Mineral medium according to OECD 301 C (1992a) was used as overlying water in the tests. Tab. 4: Composition of the final mineral medium according to OECD 301 C (OECD 1992a) in mg/L

KH2PO4 25.5 MgSO4*7 H2O 67.5

K2HPO4 65.25 CaCl2 82.5

Na2HPO4*12 H2O 133.8 FeCl3*6 H2O 0.75

NH4Cl 5.1

2.4 System Specific Training Set of Chemicals

The substances listed in Tab. 5 were used to develop and to characterise the new

biodegradation water-sediment test system. Aniline as well as benzoic acid were selected, since

both substances are well known as ready biodegradable (Reuschenbach 2000) and are

recommended by OECD 301 (1992a) as reference compounds.

D.2.3.1 Page 9/25


Tab. 5: Identification and selected properties of the test substances

Aniline Benzoic Acid CAS-No.: 62-53-3 [2] 65-85-0 [3]

Solubility: in water: 34 g/L (25 °C) [2] in water: 2.9 g/L [3]

Empirical formula: C6H7N [2] C7H6O2 [3]

Structural formula:

[3]

Molecular weight: 93.61 g/mol [2] 122.12 g/mol [3]

ThOD (NH3) in mg/mg [1]: 2.405 1.965 [1] The theoretical oxygen demand (without nitrification) per mg test substance was calculated according to OECD 301 (OECD 1992a); [2] Koch 1995; [3] Merck 2005.

2.5 Test System

The equipment to perform standardised OECD 301 water-only test is commercially available

(e.g. the OxiTop®-system of WTW, D-82362 Weilheim, Germany). The OxiTop®-system

determines manometric changes, which occur when oxygen is consumed to transform organic

carbon into carbon dioxide. In the closed system the carbon dioxide is trapped by an absorbent

(e.g. soda lime). A decrease in pressure is used by the OxiTop®-system for calculating the

biochemical oxygen demand (BOD) based on the following equation (WTW 1998).

)()(2

0

2 OpTT

VVV

TROMW

LmgBOD m

Fl

Flges

m

∆⋅⎟⎟⎠

⎞⎜⎜⎝

⎛+

−⋅

⋅=⎥⎦

⎤⎢⎣⎡ α [Equation 1]

where: MW(O2) molecular weight (32000 mg/mol)

R gas constant (83.144 L·mbar/mol·K)

T0 reference temperature (273.15 K)

Tm actual temperature [K]

Vges volume of test vessel [mL]

VFl volume of test solution [mL]

α Bunsenscher absorption coefficient (0.03103)

∆p(O2) difference of oxygen partial pressure [mbar]

D.2.3.1 Page 10/25


After correction with the oxygen uptake of a blank inoculum (a control test system without test

substance) the BOD values of the test solutions are expressed as the percentage

biodegradation according to OECD guideline 301 (1992a):

[ ][ ] [ ] %100

///% ⋅

⋅=

LmgCmgmgThODLmgBODThOD corr [Equation 2]

where: ThOD theoretical oxygen demand

C concentration of test substance

The theoretical oxygen demand of an organic compound can be calculated according to OECD

guideline 301 (1992a) based on its empirical formula (CcHhClclNnNanaOoPpSs):

( )

MW

onapsnclhc

mgmgThODNH

⎥⎦⎤

⎢⎣⎡ −+++−−+⋅

=⎥⎦

⎤⎢⎣

⎡ 21

2533

21216

3 [Equation 3]

where: c number of C-atoms per molecule

h number of H-atoms per molecule

cl number of Cl-atoms per molecule

n number of N-atoms per molecule

na number of Na-atoms per molecule

o number of O-atoms per molecule

p number of P-atoms per molecule

s number of S-atoms per molecule

When nitrification occurs during the degradation of nitrogen containing organic compounds the

theoretical oxygen demand is calculated according to:

( )

MW

onapsnclhc

mgmgThODNO

⎥⎦⎤

⎢⎣⎡ −++++−+⋅

=⎥⎦

⎤⎢⎣

⎡ 21

253

25

21216

3 [Equation 4]

D.2.3.1 Page 11/25


In order to meet the goal to integrate the compartment sediment into the OECD 301 approach

the standardised OxiTop®-system was modified (Fig. 2). Household preserving jars were used

as bottom parts of the test system, since they are a well proven simple and cost effective tool

for vacuum processing. Suitable lids and seals made of material not permeable for air (e.g.

butyl-rubber) ensure that the test vessels are air-tight (Platen & Wirtz 1999). Lids described by

Platen & Wirtz (1999) for the measurement of the respiration activity of soils were used in the

water-sediment system after they were equipped with two sample ports in order to enable the

sampling of overlying water during the test (e.g. DOC samples).

sediment

overlying waterstirring device

lid with screwconnection forpressure sensor

OxiTop® C pressure sensor

gas tight sealmade of butyl-rubber

stainless steel meshto generate a turbulent water flow

sample port fore.g. DOC samples

1.5 L – test vessel

lid-clip

magnetic stirrer

CO2-trap

sediment








lid-clip

magnetic stirrer

CO2-trap

sediment








lid-clip

magnetic stirrer sediment








lid-clip

sediment








lid-clip

magnetic stirrer

CO2-trap

Fig. 2: Image of a water-sediment test system

To ensure a sufficient oxygen flux into the water the test solution of the standardised water-only

OxiTop®-system is stirred vigorously using a conventional magnetic stirrer. To avoid an

excessive resuspension of sediment solids in a stratified water-sediment system the speed of

stirring the water in the water-sediment system was reduced and the shaft of a specific stirring

device (Fig. 2) was fixed on the inside of the modified lid.

D.2.3.1 Page 12/25


The oxygen flux from the headspace into the water body might be limited by the reduced speed

of stirring the overlying water (see section 3.4). In order to enhance the oxygen flux, a turbulent

water flow was generated by introducing a kind of “wave trap” into the rotating water body. For

this intended purpose a stainless steel mesh which submerged into the overlying water was

fixed at the shaft of the stirring device (Fig. 2).

Since a leakage of the closed system would complicate the interpretation of degradation

kinetics all materials used in this modified OxiTop®-system were not permeable for air and the

pressure-tightness of the system was checked prior to the experiments.

2.6 Development and Validation of Test System and Experimental Procedures

Several experiments using different methods were performed in order to adapt the test

conditions of the standardised OECD 301 water-only tests to the water-sediment test system:

• To investigate the influence of nitrification inhibitor allyl thiourea (ATU) on the

degradation of aniline, experiments were performed using the standardised OECD 301

water-only test method.

• To investigate the influence of components of the selected artificial sediment on the

degradation of aniline and benzoic acid, the standardised OECD 301 water-only test

method was used. The sediment components were added to the test solutions and the

solutions were stirred during the entire exposure period resulting in suspensions of

sediment solids (suspended solids method).

• The influence of sediment pre-treatments on the respiration rate of the artificial

sediment were investigated using the suspended solids method and the stratified water-

sediment system. For this purpose the artificial sediment or single components of it

were treated in different ways prior to the usage of the sediment in the tests:

Sterilisation (autoclaved for 20 min at 121°C);

Storage/ageing (at least 7 days at 4°C);

Conditioning: the sediment or its components was conditioned for at least 7 days.

For this purpose the sediment was topped with mineral medium (sediment-water

volume ratio: 1 : 3.5 – 4.5) mixed with inoculum (30 mg dry weight/L) and was

incubated under the same conditions, which prevail in the subsequent test.

Immediately prior to set-up of the test vessels, the supernatant was removed and

discarded.

D.2.3.1 Page 13/25


• The developed experimental procedures were tested and validated using aniline as a

test substance in the stratified water-sediment system.

Tab. 6: Overview of the exposure conditions in the tests using the water-only system, the suspended solids method and the water-sediment system

Water-Only System

Suspended Solids Method

Water-Sediment System

Medium / overlying water: mineral medium mineral medium mineral medium Type of sediment solids: - peat-kaolin mixture artificial sediment Concentration of test substance in mg/L medium or overlying water:

100 100 100

Concentration of ATU [3]: aniline: 10 mg/L aniline: 10 mg/L benzoic acid: none

aniline: 10 mg/L

Volume of test vessel: 500 mL 500 mL 1646.5 mL Volume of test medium/overlying water per test vessel [4]:

170 mL 120 mL 450 mL

Amount of sediment per test vessel:

- 6.6 g peat-kaolin (dw) [2]

170 g (fw) corresponding to 116.4 g (dw)

Sediment-water volume ratio: - 1 : 4 [1] [2] 1 : 4 Amount of inoculum as suspended solids:

30 mg/L (dw) 30 mg/L (dw) 30 mg/L (dw)

Temperature: 25°C 20 - 25°C; kept constant within a range of ± 1°C during a single test.

25 ± 1°C

Light intensity: complete darkness complete darkness complete darkness pH of test solution: 7 ± 1 7 ± 1 7 ± 1 Adjustment of pH in the test solutions:

none none none

Measuring interval 84 – 112 min 112 min 112 min dw: dry weight; fw: fresh weight; [2] representing a fraction of 2% peat (dw) and 22 % kaolin (dw) of the total amount of sediment (dw) based on an assumed sediment-water ratio of 1:4; [3] nitrification inhibitor allyl thiourea; [4] the volume of test medium/overlying water to be used in a single test was selected based on the expected oxygen demand. In order to compensate for the oxygen demand of the test solution the volume of the air in the remaining headspace above the water surface was choosen to be large enough to contain oxygen in excess. Since the BOD is primarily dependent on the concentration of the test substance in the test solution and the expression of the results as % biodegradation (%ThOD) was normalised to the test substance concentration (see Equation 2) an influence of the different volumes on the results can be excluded.

D.2.3.1 Page 14/25


3 Discussion and Results

3.1 Influence of ATU on the Biodegradation of Aniline

The principle of the manometric measurement is based on the fact that under the consumption

of oxygen the carbon of organic compounds is oxidised to CO2, the formed CO2 is trapped and

the pressure in the closed system decreases. However, additional oxygen may be consumed

when nitrifying bacteria are involved in the biodegradation of nitrogen-containing compounds or

organic material. This leads to additional manometric changes and to more complicated

pressure and BOD curves (Reuschenbach 2000). Nitrification may also occur in sediment-water

systems using artificial sediment (Liebig et al. 2004). In order to facilitate the interpretation of

degradation data nitrifying processes related to the organic material of the artificial sediment or

related to the biodegradation of nitrogen-containing compounds should be prevented. To meet

this goal, allyl thiourea (ATU) was used as a known nitrification inhibitor (Reuschenbach 2000).

In several water-only tests it was confirmed that ATU had no statistically significant influence on

the biodegradation of aniline. An example of a degradation curve is presented in Fig. 3.

Test Substance: Aniline

-10

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12

time [d]

% T

hOD

ATU (+) ATU (+) ATU (-) ATU (-) Fig. 3: Biodegradation of aniline in the presence and absence of 10 mg/L nitrification inhibitor allyl thiourea (ATU) in a water-only test

D.2.3.1 Page 15/25


3.2 Biodegradation of Aniline and Benzoic Acid in the Presence of Suspended Sediment Solids

The selected artificial sediment contains peat as organic material, which might be used as a

carbon and energy resource by the microbial inoculum. The biodegradation of the peat may

increase the background respiration of the bacteria in a way that one may not be able to

differentiate between the degradation of test substance and peat. Reuschenbach (2000)

reported background values in the controls of 35.0 ± 11.1 mg O2/L after 28 days in 22 water-

only tests. Compared to Reuschenbach (2000) in our experiments with suspensions of

suspended peat and kaolin a significant higher biological oxygen demand in the controls was

observed. After 17 days BOD-values in the controls of 63 – 123 mg O2/L were measured (Fig.

4).


-40

0

40

80

120

160

200

240

280

320

360

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

time [d]

BO

D [m

g/L]

Control (aged) Control (sterilised) 100 mg/L (aged) 100 mg/L (sterilised)

Fig. 4: Biodegradation of aniline: biological oxygen demand (BOD) curves of the controls and the treatment (100 mg/L) in the presence of suspended peat and kaolin solids. The used sediment components were pre-treated in different ways (aged vs. sterilised).

However, despite the high BOD-values observed in the controls, it was possible to differentiate

between background respiration and degradation of the test substances (Fig. 4). Thus it could

be demonstrated that a distinct biodegradation of aniline (Fig. 5) and benzoic acid (Fig. 6) in the

presence of suspended sediment solids could be determined.

D.2.3.1 Page 16/25



0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

time [d]

% T

hOD

aged sterilised Fig. 5: Biodegradation of aniline in the presence of suspended peat and kaolin solids. The used sediment components were pre-treated in different ways (aged vs. sterilised).

Test Substance: Benzoic Acid

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8time [d]

% T

hOD

aged conditioned Fig. 6: Biodegradation of benzoic acid in the presence of suspended peat and kaolin solids (mean-values; n = 2). The used sediment fractions were pre-treated in different ways (aged vs conditioned).

The results of the tests with aniline (Fig. 4) and benzoic acid (BOD-curves not presented here)

indicated differences in the BOD of the suspended sediment components depending on their

treatment prior to the start of the test (aged, sterilised or conditioned; for a description of the

D.2.3.1 Page 17/25


different pre-treatments see section 2.6). Since the test design did not include sufficient

replication (n ≤ 2) these differences could not be confirmed statistically. However, a potential

difference was investigated in later experiments using the stratified water-sediment system.

3.3 Influence of Sediment Pre-Treatments on the Respiration Rate of the Artificial Sediment

The results of the tests in the presence of suspended sediment solids indicated differences in

the BOD of the sediment components depending on their treatment prior to the start of the test

(Fig. 4). In order to investigate the influence of the sediment pre-treatment on the respiration

rate of the artificial sediment in the stratified water-sediment system, two pre-treatments were

tested (conditioning vs. aged; see section 2.6). Following a temperature adaptation phase of

approximately one to two days, the stratified water-sediment systems showed a linear increase

of the BOD (Fig. 7). The respiration rate of each sediment was derived from the slope of the

linear range of the BOD curves. It was calculated using a least square linear regression (Tab.

7). No statistically significant differences could be determined (t-test, p = 0.05, two-sided)

between the mean respiration rates of the sediments. Therefore an influence of the sediment

pre-treatment on the BOD in the stratified system could be excluded.

-20

-10

0

10

20

30

40

50

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

time [d]

BO

D [m

g/L]

conditioned aged Fig. 7: Biological oxygen demand (BOD) curves of the artificial sediment in stratified control (inoculum blank) water-sediment systems (mean-values; n = 4). The used sediments were pre-treated in different ways (conditioned vs. aged).

D.2.3.1 Page 18/25


Tab. 7: Respiration rate of the artificial sediment in stratified control (inoculum blank) water-sediment systems dependant on the sediment pre-treatment

Sediment Pre-treatment

Replicate Respiration rate [mg BOD L-1 d-1]

[1]

Regression coefficient

(r2) [1]

Mean Respiration rate [mg BOD L-1 d-1]

SD

4°C A 3.517809 0.941351 4°C B 4.644387 0.945383 4°C C 3.682610 0.937653 4°C D 3.898178 0.953476 3.935746 0.430794

conditioned A 3.714857 0.943779 conditioned B 4.249187 0.952677 conditioned C 3.898178 0.932298 conditioned D 3.047331 0.869192 3.727388 0.437058

SD: Standard Deviation; [1] regression coefficient of the least square fit used to determine the respiration rates.

3.4 Biodegradation of Aniline in a Water-Sediment System

Taking into account the results and experiences from the pre-tests the developed experimental

procedures were tested and validated using aniline as a system specific training chemical in the

stratified water-sediment system (Fig. 8). Following a lag-phase of approximately 1.8 days

(degradation < 10%) the aniline was degraded exponentially. After approximately 8 - 9 days a

plateau was achieved and after 10 days the biodegradation was > 60%.

The measurement of the oxygen content in the overlying water of a separate test vessel during

the first four days of the experiment showed that the overlying water did not remain aerobic

during the entire exponential phase of degradation. In this phase, the oxygen consumption of

the microbial community exceeded the supply of oxygen from the headspace above the water

body. As a consequence of the observed oxygen depletion the BOD curves were less steep.

Even though a rapid recovery of the oxygen content could be observed after the reduced

steepness of the curve, the slope of the BOD-curves remained reduced. This phenomenon was

also reported for water-only tests by Storhas and co-workers (2000). It can be assumed that the

oxygen content of the overlying water gave a limit for the microbial growth and aerobic

metabolism during the exponential phase.

The oxygen flux from the headspace into the water body is diffusion driven. It can be enforced

by an appropriate movement of the water, which for example enlarges the gas exchange

D.2.3.1 Page 19/25


surface area. In order to avoid an excessive resuspension of sediment solids the speed of

stirring the water in the water-sediment system is limited. Therefore, the movement of the water

body in the water-sediment system is much lower than in water-only tests. This fact might

explain that in the stratified water-sediment system the “kink” of the BOD curves of aniline

occurred already at a degradation of approximately 35% of the ThOD, whereas in the water-

only tests it was observed at a degradation of approximately 60% of the ThOD (Fig. 3).


0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10

time [d]

% T

hOD

0

1

2

3

4

5

6

7

8

9

10

oxyg

en c

onte

nt [m

g/L]

conditioned aged oxygen content Fig. 8: Biodegradation of aniline in stratified water-sediment systems (Co: control (n = 4), C1: treatment (n = 2)). The used sediments were pre-treated in different ways (conditioned with inoculum or stored at 4°C). Oxygen content in the overlying water was measured in a separate test vessel.

However, assuming a first-order kinetic the data from the relatively short exponential phase

could be used to derive the biodegradation rate constant (k) and the ultimate half-life (t1/2) of

aniline in the water-sediment system (Fig. 9 and Tab. 8). As it was demonstrated for the

controls (see section 3.3) no significant differences between the two tested sediment pre-

treatments could be determined based on the kinetic parameters of the degradation curves

(Tab. 8).

D.2.3.1 Page 20/25


Test Substance: Anilinefirst-order kinetic

y = 0.6954x - 1.1288R2 = 0.9995

y = 0.7677x - 1.3065R2 = 0.995

y = 0.7912x - 1.4753R2 = 0.9826

y = 0.7045x - 1.2887R2 = 0.9965

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

1.5 2.0 2.5 3.0

time [d]

ln(C

(t0)/C

(t))

C1 (aged) C1 (aged) C1 (conditioned) C1 (conditioned)Linear (C1 (aged)) Linear (C1 (aged)) Linear (C1 (conditioned)) Linear (C1 (conditioned))

Fig. 9: Biodegradation of aniline in the water-sediment system assuming a first-order kinetic. Derivation of the biodegradation rate constant using the data of the exponential phase. The slope of the regression line representing the rate constant.

Tab. 8: Parameters of the biodegradation of aniline in the water-sediment systems assuming a first-order kinetic.

Replicate Lag-Period [d] [1]

Degradation [%]

Duration[d]

k [1/d]

t1/2 [d]

C1 (aged) 1.7 60.9 12.7 0.6954 1.00 C1 (aged) 1.7 62.2 12.7 0.7677 0.90 C1 (conditioned) 1.9 70.7 12.7 0.7912 0.88 C1 (conditioned) 1.9 65.3 12.7 0.7045 0.98 Mean 1.8 64.8 - 0.7397 0.94 SD 0.1 4.4 - 0.0470 0.06

SD: standard deviation; k: first-order rate constant; t1/2: ultimate half-life calculated using the equation

(t1/2 = 0.693/k); [1] the duration of the lag period was defined as the time prior to the exponential phase

with a degradation < 10%

D.2.3.1 Page 21/25


3.5 Comparison of the Biodegradation of Aniline in Water-Only, Suspension of Sediment Solids and the Water-Sediment System

In order to compare the degradation of aniline in the various experimental approaches the

degradation rate constants and the ultimate half-lives assuming a first-order kinetic were

calculated using the data of the exponential phase. For a further description of the degradation

curves, the duration of the lag-period and the degree of degradation at the plateau were

estimated (Tab. 9).

Tab. 9: Parameters of the biodegradation of aniline in water-only, suspension of sediment solids and water-sediment systems assuming a first-order kinetic (mean values with standard deviation in brackets).

Test Substance

Test System

Lag-Period[d] [1]

Degree of Degradation

at the plateau[%]

Duration [d]

k [1/d]

t1/2 [d]

n

4.6 81.8 13.7 1.6094 0.44 4 Aniline water-only (0.4) (7.1) (0.2305) (0.06) 2.6 91.5 11.0 1.6290 0.43 4 Aniline water-only

(0.1) (1.8) (0.2270) (0.07) 3.1 87.2 17.0 1.2001) 0.58 2 Aniline suspended

solids (0.8) (1.0) (0.1910) (0.09) 1.8 64.8 12.7 0.7397 0.94 4 Aniline water-

sediment (0.1) (4.4) (0.0470) (0.06) n: number of replicates; k: first-order rate constant; t1/2: ultimate half-life calculated using the equation (t1/2 = 0.693/k); [1] the duration of the lag period was defined as the time prior to the exponential phase with a degradation < 10%


0.0

0.5

1.0

1.5

2.0

water-only water-only suspended solids water-sediment

k [1

/d] a

nd t 1

/2 [d

]

first-order rate constantshalf lives

Fig. 10: Biodegradation of aniline in the water-sediment system: first-order rate constants and ultimate half-lives (mean values ± standard deviations)

D.2.3.1 Page 22/25


The comparison of the parameters describing the degradation of aniline in the different test

systems showed that in the presence of sediment particles the biodegradation was slower than

in the water-only tests (Fig. 10). A statistically significant difference between the mean rate

constants of aniline from water-only and water-sediment tests could be determined (Welch-t test

for inhomogeneous variances, p = 0.05, two-sided). However, the observed difference in the

fate of aniline has to be confirmed in experiments which run under the identical conditions in

parallel (e.g. using the same microbial inoculum).

4 Conclusion and Outlook

With the intention to enable the comparison of experimental data from water-only (OECD 301)

and water-sediment systems a sediment compartment was integrated into the OECD 301

approach. To ensure a high degree of standardisation, an artificial sediment was used in the

developed water-sediment system. The test system and experimental procedures were tested

and validated using aniline as a system specific training chemical.

It could be observed that the overlying water did not remain aerobic during the entire

exponential phase of degradation. It can be assumed that the oxygen content of the overlying

water gave a limit for the microbial growth and aerobic metabolism during the exponential

phase. However, assuming a first-order kinetic the data from the relatively short exponential

phase could be used to derive the biodegradation rate constant and the ultimate half-life of

aniline in the water-sediment system.

In order to optimise the test performance and the degradation curves, the oxygen depletion in

the overlying water during the exponential degradation phase should be minimised in future

experiments. Currently, two approaches are tested to meet this goal: 1) enhancement of the

oxygen flux into the water body; 2) reduction of the test substance concentration to reduce the

bacterial oxygen demand.

It can be expected that the developed water-sediment system will be applicable for chemicals

which are suspected to be not highly volatile or highly toxic to microorganisms. In the case of

highly volatile substances, where phase partitioning between the three media sediment, water

and air would lead to an excessive and irreversible stripping of the compound into the

D.2.3.1 Page 23/25


headspace, the substance would neither be bioavailable for microbial degradation in overlying

water nor in sediment. Highly toxic chemicals may inhibit the microbial activity and therefore a

distinction between inhibition of inoculum and persistence of test compound may be impossible.

In this case low test concentrations should be employed, necessitating the use of the stringent

and sensitive closed bottle test or the use of C14-labelled material (OECD 1992a).

In analogy to OECD guideline 301 (OECD 1992a) substances, which are poorly soluble in

water, can be applied to the overlying water of the water-sediment system either directly on a

weight or volume basis or using a solvent or emulsifier. The used solvent or emulsifier should

neither be toxic to bacteria nor biodegradable. Assuming that most poorly soluble compounds

show a tendency to adsorb to sediment particles, the test substance can be spiked alternatively

directly into the sediment using standardised methods (e.g. OECD 2004b). Spiking of sediment

may also be considered in testing of highly adsorbing chemicals.

The experiences and the data gained from the experiments so far showed that the developed

water-sediment test system can be considered as ready for use for the generation of

biodegradation kinetics within NOMIRACLE.

D.2.3.1 Page 24/25


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