Environmental Risk Assessment of Antimicrobials

Hans-Christian Holten Lützhøft

Section of Environmental ChemistryDepartment of Analytical and Pharmaceutical Chemistry

The Royal Danish School of Pharmacy

Demand from the European Community (EMEA, 1998)

Antimicrobial discharge can be expected after medication due to limited effluent treatment at the fish farm resulting in possible exposure to the environment

Why Bother?

Environmental Occurrence

Overview of Performed Tasks

Outline Main objective The antimicrobials Environmental fate Environmental effects Exposure scenarios Environmental risk assessment

Main Objective Assess the aquatic environmental risk associated with

application of antimicrobials in freshwater fish farming

The Antimicrobials

Physical Chemical Properties

Physical Chemical Properties

Application Treat infections among fish in fish farming

Enteric red mouth disease Furunculosis Vibriosis

Fry mortality syndrome

FLU, OXA, SAFSDZ/TMPAMX, OTC

AMX, OTC

OXA and SDZ/TMP are commercially available as medicated feed

Consumption in Danish Fish Farming, kg

Year OXAa SDZa TMPa AMXb OTCb Otherb Total1994 700 1,000 200 6 94 132 2,1321995 906 1,241 248 78 67 242 2,7821996 511 845 169 141 27 177 1,8701997 587 1,677 335 132 16 181 2,928

a: Viuf (Personal communication), b: Data from 3 counties (Danske amter, Personal communication), Other:dimetridazole (antiprotozoal), florfenicol, metronidazole, sulfamerazine, and antimicrobials as such.

Compared with other Countries

mg/kg (fish) mg/L (effluent)Country 1987 1994 1997 1995Scotlanda - 40 8 naNorwayb 885 7 - naDenmarkc - 61 84 0.7USAd - - - 4a: Baird et al. (Personal communication), b: Markestad and Grave (1997), c: Viuf (Personal communication)and Danske amter (Personal communication), d: Vicari et al., (Personal communication), -: not available,na: not applicable.

Biotic Fate Low oral bioavailability (ca. 10%)a

Various degrees of biotransformationb

Mainly excreted as parent compoundsc

a: Bjørklund and Bylund (1991); b: Grondel et al. (1989), Ishida (1992), Tan and Wall (1995); c: Bergsjø et al. (1979), Rogstad et al. (1991), Ishida (1992), Tan and Wall (1995)

Preliminary Environmental Assessment

Low oral bioavailability and low biotransformation result mainly in excretion of a parent compound

The principal exposure is to the aquatic environment, however the distribution is widely pH-dependent

Environmental exposure of biological active chemicals may affect non-target organisms

Environmental Fate

Environmental Processes

Objectives Determine distribution coefficients (DDOC) of 4-quinolones

between dissolved organic carbon (DOC) and water using SPME-HPLC analysis

What influence does pH have?

pH?

DOC?

N

F

CH3

COOHO

N

OCOOH

CH3

O

ON

FO

COOH

NHN

F

Holten Lützhøft et al. (Accepted)

Principles of SPME Negligible depletion solid phase

microextraction (nd-SPME)

Kinetic extraction approach Measurement of the free concentration

MXMX

MXMXFibreXFibre

Interaction with DOC

Chemical Log KOW Log KOC from QSARa Log DDOC

FLU 1.7b 1.7 3.40.27c

OXA 1.0d 1.0 3.90.05c

SAF - - 4.80.01c

DDT 6.9e 6.8 5.60.18f

HxCBz 5.7e 5.6 5.00.05f

PeCBz 5.2e 5.1 4.50.03f

a: Di Toro (1985), b: Takács-Novák and Avdeef (1996), c: Holten Lützhøft et al. (Accepted) III, d: HoltenLützhøft et al. (2000) II, e: Bruijn et al. (1989), f: Urrestarazu Ramos et al. (1998).

pH-Dependent Interaction with DOC

N

F

CH3

COOHO

pKa=6.5

FLU

N

OCOOH

CH3

O

O pKa=6.9

OXA

2 3 4 5 6 7 83

4

5

6

SAF

OXAFLU

pH

Log

DDO

C

N

FO

COOH

NHN

F

pKa=6.8

pKa=4.1

SAF

pKa=8.6

Conclusions DDOC values for the investigated 4-quinolones appeared to

be remarkably high

DDOC values for the investigated 4-quinolones were shown to be pH dependent, suggesting electrostatic interactions to work in the system

Summary of Environmental Fate

Log DSED values of 2.3-2.7a

Similar physical chemical properties for sulphadiazine and amoxicillin suggest log DSED in the same range

Photodegradation is negligibleb

Non-biodegradablec

Oxytetracycline is biodegradable in freshwater sediment slurries (t½: 2-8 days)

a:Lai et al. (1995), Holten Lützhøft (Unpublished); b: Lunestad et al. (1995); c: Hektoen et al. (1995), Lai et al. (1995)

Environmental Effects

Objectives Investigate the suitability of using the standard test algae

Selenastrum capricornutum to represent micro-algae for evaluation of antimicrobials, e.g. ISO (1989) and EMEA (1998)

Establish toxicity data for antimicrobials towards algae

Holten Lützhøft et al. (1999)

Tested Algae Microcystis aeruginosa (freshwater cyanobacteria) Rhodomonas salina (marine cryptophycean) Selenastrum capricornutum (freshwater green algae)

BacteriaProcaryoticNo nucleus0.5-2.0 µm

No photosynthesis

Modified from Brock and Madigan (1991)

CyanobacteriaProcaryoticNo nucleus0.5-60 µm

Photosynthesis

AlgaeEucaryotic

Nucleus2-200 µm

Photosynthesis

Overview of Acute Effect Data

0,1

1

10

100

1000

AMX SAF FLU SDZ OXA OTC TMP

LC50

, mg/

L

a: Migliore et al. (1997); b: Andersen (1999), Andersen (Personal Communication); c: Andersen (1999), Wollenberger et al. (2000), Halling-Sørensen et al. (In press); d: Andersen (1999), Halling-Sørensen et al. (In press)

: NOEC

Artemia sp.a A. tonsab

B. reriodD. magnac

Overview of Chronic Effect Data

0,0001

0,001

0,01

0,1

1

10

100

1000

10000

AMX SAF FLU SDZ OXA OTC TMP

EC50

, mg/

L

: NOEC

a: Backhaus et al. (2000); b: Holten Lützhøft et al. (1999) IV; c: Wollenberger et al. (2000)

V. fischeria M. aeruginosab R. salinab

S. capricornutumb D. magnac

Conclusion Environmental evaluation of antimicrobials requires tests

of cyanobacteria

The effect concentrations are comparable to concentrations of antimicrobials found in the environment

Exposure scenarios

Overview of Exposure Scenarios

PECs

Environmental Risk Assessment

Procedure to Derive PNEC Constant assessment factor approach proposed by the

OECDa and EMEAb

Assessment factor depends on quality of data i.e. acute/chronic, NOEC/EC50, trophic levels

a: OECD (1992); b: EMEA (1998)

Factor AssessmentEC or NOECLowest PNEC 50

0

50

100

NOEC

Chronic Acute

EC50

AF=10

Log C

AF=100

NOEC

Effe

ct, %

EC50

Assessment FactorsThree trophic levels Less than three trophic levels

0

50

100

Log C

AF=1,000

NOEC

EC50

Effe

ct, %

PNECs

Risk Quotients

PNECPECRQ

Main Objective

Assess the aquatic environmental risk associated with application of antimicrobials in freshwater fish farming

Environmental Ranking

TMP OTC SDZ SAF OXA FLU AMX

Harmless understudied scenarios

More studiesrequired RQ

10-3 10-2 10-1 1 101 102 103

I would like to thank ... Sven Erik Jørgensen and Bent Halling-Sørensen for the

thorough supervision during my Ph.D. project the last 3-4 years