Fate of Pharmaceuticals in the Environment and in Water Treatment Systems - Chapter 4 potx

104 4.2.2 Acute Toxicity Test of Dimeglumine Gadopentetate, Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset Trisodium with Fish .... 106 4.2.3 Acute Immobilization Test of Dimeglum

Contrast Agents for

Magnetic Resonance

Imaging (MRI)

Investigations on

the Environmental

Fate and Effects

Claudia Neubert, Reinhard Länge,

and Thomas Steger-Hartmann

Contents

4.1 Introduction 102 4.2 Methods 104 4.2.1 Biodegradability of Dimeglumine Gadopentetate, Gadobutrol, Gadoxetic Acid, Disodium, and Gadofosveset Trisodium 104 4.2.2 Acute Toxicity Test of Dimeglumine Gadopentetate,

Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset

Trisodium with Fish 106 4.2.3 Acute Immobilization Test of Dimeglumine Gadopentetate,

Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset

Trisodium with Daphnia magna 107

4.2.4 Growth Inhibition Test of Dimeglumine Gadopentetate,

Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset

Trisodium on Green Algae 107 4.2.5 Growth Inhibition Test of Dimeglumine Gadopentetate on

Different Microorganisms 108 4.3 Results 109 4.3.1 Biodegradability of Dimeglumine Gadopentetate, Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset Trisodium 109 4.3.2 Acute Toxicity of Dimeglumine Gadopentetate, Gadobutrol,

Gadoxetic Acid Disodium, and Gadofosveset Trisodium to Fish 109

4.1 INTRODUCTION

Mainly due to progress in analytical instrumentation, there has been an increased awareness of the presence of pharmaceutical compounds as environmental contami-nants in recent years.1,2Although concentrations of pharmaceuticals in the aquatic environment are usually only in the parts-per-billion or parts-per-trillion levels, there is growing concern over their release because of their biological activity, which

is not limited to human targets

As a result of that concern, specific ecological risk assessment procedures have been refined, which led to the introduction of guidelines in some of the major human pharmaceutical markets (Europe, United States) Essentially these procedures con-sist of an estimation of the environmental concentration, on the one hand, and the experimental determination of a no-effect concentration (NOEC) of the pharmaceu-tical on the other hand.3,4Because the aquatic environment represents the primary recipient of pharmaceuticals that are being discharged from wastewater treatment plant effluents, risk assessment has focussed on the aquatic ecosystem The Euro-pean risk assessment guideline3proposes a tiered system in which exposure estima-tion and risk screening are included, as well as the determinaestima-tion of physicochemical properties of new human pharmaceuticals and diagnostic agents

To assess the potential effects of contaminants on the aquatic environment, a battery of selected organisms, each representing a specific level of the aquatic eco-system (seeFigure 4.1), is investigated Furthermore, in order to assess persistence and thus temporal development of exposure, tests on biodegradation are conducted Screening tests for biodegradation allow a first qualitative assessment of the poten-tial of sewage treatment plants or natural surface waters to degrade the compound

of interest

Among the first pharmaceutical compounds that were analytically detected in the aquatic environment5and subsequently assessed for their ecotoxicological risk were iodinated X-ray contrast agents.6Fewer data are currently available for the sec-ond class of contrast agents used in MRI, even though those compounds have been detected in ground water as early as in 1996.7

4.3.3 Acute Immobilization Test of Dimeglumine Gadopentetate,

Gadobutrol, Gadoxetic Acid Disodium, and Gadofosveset

Trisodium with Daphnia magna 110

4.3.4 Growth Inhibition Test of Gadobutrol, Dimeglumine Gadopentetate, Gadoxetic Acid Disodium, and Gadofosveset Trisodium on Green Algae 110

4.3.5 Growth Inhibition Test of Dimeglumine Gadopentetate and Gadobutrol on Different Microorganisms 112

4.4 Discussion 112

4.4.1 Degradation Tests 113

4.4.2 Ecotoxicity Tests 115

4.4.3 Environmental Relevance 116

4.5 Summary and Outlook 118

References 118

This chapter reports the results of ecotoxicological studies and biodegradability tests of several gadolinium-containing contrast enhancing agents for MRI and pro-vides an environmental risk assessment based on the information obtained MRI is

an essential tool in the noninvasive diagnostics of various diseases, such as tumors,

to improve lesion identification and characterization In order to improve the sen-sitivity and specificity of diagnoses, several contrast enhancing agents have been developed in the last few decades by various pharmaceutical manufacturers and are marketed worldwide.8

Gadolinium (Gd), a lanthanide, is the most widely used metal in MRI contrast agents Its ion has paramagnetic properties (seven unpaired electrons) and a very long electronic relaxation time Due to the toxicity of free Gd, which is caused by

an interaction with calcium channels,9,10and a precipitation tendency above pH 6 with subsequent trapping in the liver,11–13clinical use is only possible in a complexed form Commonly used chelating agents are polyamino-polycarboxylic ligands such

as diethylenetriaminepentaacetic (DTPA) The complexes formed by the different chelates can be grouped, according to their size and structure, into:

macrocyclic chelates such as gadobutrol (Gadovist®) and

linear chelates such as dimeglumine gadopentetate (Gd–DTPA) (Magnev-ist®) or Gadodiamide, Gd–diethylenetriamine pentaacetate bismethylamide (Gd–DTPA–BMA) (Omniscan®)

Due to the exceptional stability of these highly hydrophilic chelates and the lack

of human metabolism, the contrast media are quantitatively excreted unchanged after administration within hours, and are subsequently emitted into the aquatic

•

•

Producers (photosyn.

organisms) Consumers(e.g.,

zooplankton)

Consumers (fish)

Consumers (predator fish)

Dead Material

Destruents (bacteria)

Inorganic Nutrients

Toxicity to Daphnia

Toxicity to Fish Degradation

by Sewage

Bacteria

Toxicity to

Unicellular Algae

Sediment Air

FIGURE 4.1 Interactions in an aquatic ecosystem and derived test systems (gray) on

dif-ferent trophic levels.

environment Several studies have shown notable increases in Gd concentrations in surface or groundwaters receiving sewage effluents, an observation which has been termed “Gd anomaly.”14–16

The Gd anomaly results from the use of MRI contrast agents for which the most significant entry route is the effluent from wastewater treatment works.16Relatively little information on the aquatic toxicity of Gd or Gd-chelates has been published Therefore, in a first step, the aquatic toxicity of these compounds was investigated in short-term tests on standard aquatic species at high concentrations Furthermore, the biological stability under the incubation with activated sludge bacteria was studied

in screening tests

4.2 METHODS

All described tests were performed according to internationally standardized guide-lines and in accordance with the good laboratory practice (GLP) principles Dime-glumine gadopentetate, gadobutrol, and gadoxetic acid disodium were manufactured

by Bayer Schering Pharma AG, Germany, gadofosveset trisodium by Mallinckrodt Inc., United States Table 4.1 shows the structures and selected physicochemical properties of the tested compounds

4.2.1 B IODEGRADABILITY OF D IMEGLUMINE G ADOPENTETATE , G ADOBUTROL ,

G ADOXETIC A CID , D ISODIUM , AND G ADOFOSVESET T RISODIUM

Test systems for ready biodegradability were originally established for household detergents and are required by the European Reserach Area (EU ERA) guideline

to assess the degradation of a human pharmaceutical The test compounds dime-glumine gadopentetate, gadoxetic acid disodium, and gadofosveset trisodium were investigated according to the test guideline of the Organization for Economic Coop-eration and Development (OECD), 301E.17Briefly, the compounds were incubated

in aqueous solutions including nutrients with microorganisms from a municipal sewage treatment plant for 62 days (test compound: dimeglumine gadopentetate, in duplicate) and 28 to 29 days (test compound: gadoxetic acid disodium, gadofosveset trisodium, in triplicate)

The test concentration for the substances was adjusted to 20 mg dissolved organic carbon (DOC) per liter corresponding to 56.7 mg dimeglumine gadopen-tetate, 52.5 mg gadoxetic acid disodium, and 51.65 mg gadofosveset trisodium Additionally, a reference substance (sodium acetate) was tested at the same DOC concentration in order to verify the viability and activity of the degrading microor-ganisms Furthermore, one flask containing both the test substance and the reference substance was tested as a toxicity control Three additional vessels without any test

or reference substances were used as blank (control)

The biological degradation of the test and reference substances was evaluated by the decrease of DOC in the solutions Total organic carbon (TOC) and DOC were measured by a TOC analyzer Additionally, for this specific case, the concentration

TABLE 4.1

Structure, International Union of Pure and Applied Chemistry (IUPAC) Names, Molecular Weight, and Water Solubility of the Tested Compounds IUPAC Names, Molecular

Weight, and Water

Compound:

Magnevist

Active agent: Dimeglumine

gadopentetate

Molecular weight: 938

IUPAC name:

Diethylenetriamine- pentaacetic acid, Gadolinium Complex, dimeglumine salt Water solubility: c469 g/L

N N O N

O

O – O –

O

O –

O

O –

O

O –

Gd 3+

H3C

NH2+

HO HO H

HO H OH

H H OH

2

Compound:

Gadovist

Active agent: Gadobutrol

Molecular weight: 604.7

IUPAC name:

10-[(1SR, 2RS) – 2,3 – Dihydroxy – 1 – hydroxymethylpropyl] – 1, 4,

7, 10 – tetraazacyclododecane – 1, 4, 7 – triacetic acid, Gadolinium – Complex Water solubility: 1081 ± g/L

N OH

OH N

N

OH

N O

O –

O

O –

O

O –

Gd 3+

Compound:

Primovist®

Active agent: Gadoxetic

acid disodium

Molecular weight: 725.7

IUPAC name:

(4S) – 4 – (4 – Ethoxybenzyl) – 3, 6, 9 – tris (carboxy-latomethyl) – 3, 6, 9 – triazaundecanedioic acid, Gadolinium – Complex, Disodium salt) Water solubility:1057 g/L

N N O

O –

O

O –

N O

O – O

O O –

O

O – Chiral

Gd 3+

Na +

Na +

Compound:

Vasovist®

Active agent:

Gadofosve-set trisodium

Molecular weight: 957.9

IUPAC name:

Trisodium {N – (2 – {bis[(carboxy-kappa O) methyl]amino-kappa N}ethyl) – N - [(R) – 2 – {bis[(carboxy-kappa O) methyl] amino – kappa N} –

3 – {[(4,4 – diphenylcyclohex yloxy)phosphinato – kappa O]oxy} propyl]glycinato(6 - ) – kappa N, kappa O}

gadolinate(3 -) Water solubility: c247 g/L

O P O O

O –

N N

O

O –

O

N O

O –

O O –

O

O –

Chiral

Gd 3+

Na +

Na +

Na +

O –

of dimeglumine gadopentetate was analyzed by high-performance liquid chroma-tography/ultraviolet (HPLC/UV) Specific concentration analysis of gadoxetic acid disodium and gadofosveset trisodium was not performed because it is not required

by the OECD guideline

Gadobutrol was tested for microbial degradation in agreement with the test guideline 3.11 of the Environmental Assessment Technical Assistance Handbook,18

which slightly differs from the OECD 301 E procedure in using an inoculum from

a municipal sewage treatment plant mixed with a filtered suspension of garden soil The inoculum was preadapted in an aqueous solution including nutrients with the test substance gadobutrol or the reference substance (glucose monohydrate) for 14 days Afterward, the test substance (10 mg/L), reference substance (10 mg/L), and the blank solution were incubated with the preadapted microorganisms for 27 days The biological degradation of the test and reference substances was evaluated

by the measurement of the carbon dioxide (CO2) produced during the test period

CO2was absorbed by Ba(OH)2 CO2production was determined by titration of the Ba(OH)2solution as described in the guideline

4.2.2 A CUTE T OXICITY T EST OF D IMEGLUMINE G ADOPENTETATE ,

G ADOBUTROL , G ADOXETIC A CID D ISODIUM ,

AND G ADOFOSVESET T RISODIUM WITH F ISH

Fish represent the nonmammalian consumer of an aquatic ecosystem (Figure 4.1)

In order to assess the toxicity of the test compound to representative species of this trophic level, the acute toxicity of gadobutrol and gadoxetic acid disodium was

determined with rainbow trout (Oncorhynchus mykiss) on the basis of the guideline

Freshwater Fish Acute Toxicity, Environmental Assessment Technical Assistance Handbook, Technical Assistance Document 4.1119with a test duration of 96 hours The acute toxicity of dimeglumine gadopentetate and gadofosveset trisodium to the

zebrafish (Danio rerio) was conducted in accordance with the test guideline OECD

20320and the EC Guideline Part 2—Testing Methods, Part C 1.21

Ten fish were used for each concentration of the test compound and for the con-trol group The fish were exposed for a period of 96 hours to the dilution water and

to various concentrations of the substances (0.1, 1.0, 10.0, 100.0, and 1000.0 mg/L in case of gadobutrol and gadoxetic acid disodium, 100 mg/L in case of dimeglumine gadopentetate, and 1000 mg/L in case of gadofosveset trisodium)

Mortalities and visual abnormalities, as well as pH value, oxygen concentration, and temperature, were recorded at approximately 3, 6, 24, 48, 72, and 96 hours Sam-ples for the concentration analysis by inductively coupled plasma/mass spectrometry (ICP/MS) (inductively coupled plasma/atomic emission spectrometry [ICP/AES] in the case of gadofosveset trisodium) were taken in regular intervals The analytical method determined the Gd concentration on the basis of which the test substance concentration was calculated

4.2.3 A CUTE I MMOBILIZATION T EST OF D IMEGLUMINE

WITH D APHNIA MAGNA

The crustacean Daphnia magna represents the primary feeder of an aquatic

ecosys-tem (Figure 4.1).In order to assess the toxicity of the test compound to representative species of this trophic level, the test compound gadobutrol was investigated in agree-ment with the test guideline: Daphnia Acute Toxicity, Environagree-mental Assessagree-ment Technical Assistance Handbook, Technical Assistance Document 4.08,22 whereas the test compounds dimeglumine gadopentetate, gadoxetic acid disodium, and gado-fosveset trisodium were investigated according to the guideline of the OECD 202 and the EC guideline part C.2.23,24Different guidelines were used for these tests because they were performed for the use in different regulatory regions

The test was performed with five juvenile daphnia in each vessel and four repli-cates for each concentration The crustaceans were exposed for a period of 48 hours under static conditions Immobilization was recorded at 24 and 48 hours The pH value, oxygen concentration, and temperature were measured at 0 and 48 hours The test solutions had nominal concentrations of 0.1, 1.0, 10.0, 100.0, and 1000.0 mg/L (test compound: gadobutrol); 100 mg/L (test compounds: dimeglumine gadopentetate and gadoxetic acid disodium); and 90 mg/L (test compound: gadofos-veset trisodium)

Samples for the concentration analysis of gadobutrol and gadoxetic acid diso-dium by ICP/MS were taken daily The method included a detection of Gd, and the final concentrations for gadobutrol and gadoxetic acid disodium were calculated accordingly

For dimeglumine gadopentetate and gadofosveset trisodium only nominal val-ues were available Since these compounds are very well soluble in water (≤469 g/L for dimeglumine gadopentetate and ≤247 g/L for gadofosveset trisodium, respec-tively) and are very stable, the actual concentration was assumed to be in agreement with the nominal

4.2.4 G ROWTH I NHIBITION T EST OF D IMEGLUMINE G ADOPENTETATE ,

G ADOBUTROL , G ADOXETIC A CID D ISODIUM , AND

G ADOFOSVESET T RISODIUM ON G REEN A LGAE

Green algae are the main primary producers in freshwater ecosystems Unicellu-lar green algae are established in ecotoxicity testing, since they represent the main part of the floral biomass The studies were conducted with an algae population of

Chlorella vulgaris (test compound: gadobutrol) and Desmodesmus subspicatus (test

compounds: dimeglumine gadopentetate, gadoxetic acid disodium, and gadofosveset trisodium) in agreement with the OECD guideline 201 and the EC guideline part C.3.25,26

The test substances were incubated in an aqueous solution including nutrients for the duration of approximately 72 hours The nutrient solution was made up of mainly nitrate, phosphates, and some trace elements Due to the long-time course of the experiments and to the changing guideline requirements, the tested concentrations

were not identical for the different contrast agents The nominal test concentrations were 0, 1.25, 2, 4, 10, 20, and 100 mg/L for the test compound dimeglumine gado-pentetate; 0, 40, 88, 194, 426, 937, and 2062 mg/L for the test compound gadobutrol;

0, 2, 4, 10, 20, 40, and 80 mg/L for the test compound gadofosveset trisodium; and

63, 125, 250, 500, and 1000 mg/L for the test compound gadoxetic acid disodium In

an additional test with gadoxetic acid disodium, solutions with nominal loadings of

1000, 5000, and 10,000 mg/L were prepared

The algae were exposed to each concentration in triplicate Six vessels were prepared for the control The algae were incubated in an incubator shaker under continuous light As a parameter for the growth of the algae population, the cell con-centrations of the test and control solutions were counted with an electronic particle counter (“Coulter Counter”) at approximately 24, 48, and 72 hours The pH value was measured at the beginning and at the end of the test

For the study with dimeglumine gadopentetate and gadofosveset trisodium, an incubating apparatus (Abimed Algen Test XT) was used In this case the cell number was determined via measurement of chlorophyll fluorescence The increase of biomass and the growth rate was calculated on the basis of the cell counts The calculated bio-mass and growth rate of each concentration were compared to those of the controls, and the inhibition was calculated Concentration analysis was not performed

4.2.5 G ROWTH I NHIBITION T EST OF D IMEGLUMINE

G ADOPENTETATE ON D IFFERENT M ICROORGANISMS

Microorganisms play a role as degraders in the aquatic environment, thus lowering the exposure with introduced contaminants Furthermore, some of the microorga-nisms (bluegreen algae) also represent the trophic level of producers

The growth inhibition test of dimeglumine gadopentetate was conducted in agreement with the standard DIN 38 412 L8.27It was incubated in an aqueous

solu-tion including nutrients, with a bacterial populasolu-tion containing Pseudomonas putida

for the test duration of approximately 16 hours

The test concentrations were 0.1, 1.0, 10.0, 100.0, and 1000.0 mg/L and a con-trol All test concentrations were incubated in duplicate As a parameter for the test growth of the bacterial population, the turbidity of the test and control solutions was analyzed photometrically at a wavelength of 436 nm A concentration analysis was not performed

The effect of gadobutrol on different microbes was studied in a growth inhibition test in agreement with the test guideline Microbial Growth Inhibition, Environmen-tal Assessment Technical Assistance Handbook, Technical Assistance Document 4.02.28Different bacterial, fungal, and algal microbes (Pseudomonas putida,

Azo-tobacter beijerinckii, Aspergillus niger, Caetomium globosom, and Nostoc ellipsos-porum) were exposed to graduated concentrations of gadobutrol The microbes were

incubated on agar plates containing nutrients and the test substance over periods of

20 hours (Pseudomonas putida), 48 hours (Azotobacter beijerinckii), 3 days

(Asper-gillus niger, Caetomium globosom), and 10 days (Nostoc ellipsosporum) under

appropriate conditions The concentrations of the test substance were 0.1, 1.0, 10.0, 100.0, and 1000.0 mg/L The growth of the microbes was assessed at the end of the respective incubation period Growth was defined as appearance of colonies Concentration analysis was not performed

4.3 RESULTS

4 3.1 B IODEGRADABILITY OF D IMEGLUMINE G ADOPENTETATE , G ADOBUTROL ,

G ADOXETIC A CID D ISODIUM , AND G ADOFOSVESET T RISODIUM

Figure4.2 summarizes the results of the degradation tests at the end of the incuba-tion period Microbial degradaincuba-tion was only observed in the test with dimeglumine gadopentetate, which was likely due to the degradation of meglumine (seeSection 4.4)

The individual degradation curves of dimeglumine gadopentetate and sodium acetate are depicted inFigure 4.3 Degradation of the test compound started between day 15 and day 21, and degradation values of approximately 40% were reached after

43 days

Figure 4.4shows the concentrations of dimeglumine gadopentetate [mg/L] mea-sured by HPLC/UV They varied between 53.6 and 62.1 mg/L The analysis for free

Gd was negative, indicating that no Gd was released from the chelate The results of the degradation of gadoxetic acid disodium, gadobutrol, and gadofosveset trisodium showed that none of these compounds was readily biodegradable and none of the compounds was toxic to the degrading bacteria

4.3.2 A CUTE T OXICITY OF D IMEGLUMINE G ADOPENTETATE , G ADOBUTROL ,

G ADOXETIC A CID D ISODIUM , AND G ADOFOSVESET T RISODIUM TO F ISH

The measured substance concentrations were approximately 90 to 120% of the nominal values The time course of the results demonstrates that the substance solutions were stable during the whole exposure period The results of the measured

0

10

20

30

40

50

60

70

80

90

100

Dimeglumine Gadopentetate

Gadobutrol Gadoxetic Acid

Disodium

Gadofosveset Trisodium

FIGURE 4.2 Biological degradation of dimeglumine gadopentetate, gadobutrol, gadoxetic

acid disodium, and gadofosveset trisodium at the end of the degradation tests [%].

concentrations of the test compounds of the studies on the acute toxicity to fish and waterflea are summarized inTable 4.2

No substance-related mortality or abnormal behavior was observed in the tests during the whole exposure time On the basis of the given results the LC50/96 hours for gadobutrol, gadoxetic acid disodium, and gadofosveset trisodium was >1000 mg/L, for dimeglumine gadopentetate >100 mg/L

4.3.3 A CUTE I MMOBILIZATION T EST OF D IMEGLUMINE

G ADOPENTETATE , G ADOBUTROL , G ADOXETIC A CID D ISODIUM ,

AND G ADOFOSVESET T RISODIUM WITHD APHNIA MAGNA

Immobilized daphnia were not observed in either the test or in the control solutions of dimeglumine gadopentetate, gadobutrol, and gadofosveset trisodium In the test with gadoxetic acid disodium, one daphnia was immobilized in the control.Table4.2 summa-rizes the results of the measured concentrations of the test compounds of the studies

4.3.4 G ROWTH I NHIBITION T EST OF G ADOBUTROL , D IMEGLUMINE

G ADOPENTETATE , G ADOXETIC A CID D ISODIUM , AND

G ADOFOSVESET T RISODIUM ON G REEN A LGAE

Figure 4.5gives the inhibition [%] of the growth of Chlorella vulgaris after 72 hours

exposure to gadobutrol on the basis of the biomass (integral) and the growth rate

In order to illustrate the data on which the inhibition [%] is calculated, cell numbers

Day of Sampling (d)

0

10

20

30

40

50

60

70

80

90

100

Reference (sodium acetate) Dimeglumine Gadopentetate

FIGURE 4.3 Biological degradation of dimeglumine gadopentetate and the reference

com-pound sodium acetate [%] in the modified OECD screening test.