on effects of graded antibiotics on soil - MTT's Portal … 2.2. BONUS PROMISE 2 Phosphorus Recycling of Mixed Substances (BONUS PROMISE) DELIVERABLE 2.4 Report on effects of graded

BONUS PROMISE DELIVERABLE 2.4

Report on effects of graded antibiotics on soil

parameters

Authors

Elke Bloem

DEL 2.2. BONUS PROMISE

2

Phosphorus Recycling of Mixed Substances (BONUS PROMISE)

DELIVERABLE 2.4

Report on effects of graded antibiotics on soil

parameters

AUTHORS: Elke Bloem

DATE: December, 2016

BONUS PROMISE project has received funding from BONUS

(Art 185), funded jointly by the EU and Ministry of Agriculture

and Forestry, PTJ and VINNOVA


3

Contents

1. Summary ......................................................................................................................................... 4

2. Introduction .................................................................................................................................... 4

2.1. Sorption behavior of antibiotics in the soil ............................................................................. 5

2.2. Effect of antibiotics on soil microorganisms ........................................................................... 6

2.3. Development of antimicrobial resistance ............................................................................... 7

2.4. Evaluation of the risk to change the function and the soil microbial community by manure

application .......................................................................................................................................... 8

3. Conclusions ......................................................................................................................................... 9

References ............................................................................................................................................ 10


4

1. SummaryOrganic nutrient sources such as farmyard manure, sewage sludge, animal‐by‐products or digestates

can be valuable organic fertilizers delivering nutrients and organic matter to the soil but may pose

the risk to contaminate soils by organic xenobiotics such as pharmaceuticals. In the present study

antibiotics were chosen exemplary from the broad range of possible organic contaminants as they

are of high importance in animal derived manure samples and human waste products such as

sewage sludge. The fate of antibiotics in the soil is not well understood and it is the aim of the

current report to collect data about the effect of antibiotics on soil parameter. Antibiotics are

especially designed to unfold an effect on microorganisms with the target to kill cells or stop their

multiplication. The antimicrobial action involves the inhibition or regulation of enzymes involved in

cell wall biosynthesis, nucleic acid metabolism or protein biosynthesis. When antibiotics enter the

environment two processes are of major importance with regard to microorganisms, the

development of antibiotic resistance genes in the soil and the direct toxicity to soil microorganisms.

It is the effect on important microbial community functions (e.g. nitrification rates, degradation of

organic matter) which can be changed by antibiotics and which will change the ´transformation

power´ of soils.

Soil characteristics are of major importance for the extent of the antibiotic action such as

accumulation and binding in the soil, persistence and release into the soil solution. The different

processes in the soil that affect the antibiotic action are compiled in this report as well as knowledge

about the effects of antibiotics on soil functioning.

2. Introduction

The antibiotic consumption worldwide accounts for 100,000 to 200,000 tons per year (Jeong et al.,

2010). From this amount 65,000 tons are used in livestock farming (van Boeckel et al., 2015) causing

increasing contaminations of animal manures with antibiotics and their metabolites.

Tetracyclines (TCs) represent the most important pharmaceutical class in veterinary medicine

accounting for more than 30% of the antibiotics used in Germany (Wallmann et al., 2015) and for

about 66% of the antibiotic consumption in Europe (Folett, 2000). Very different consumption rates

were reported for sulfonamids with values of up to 23% in Germany (Folett, 2000) and 10% in the EU

in 2012 (EMA, 2014). Nowadays a shift from TCs to fluoroquinolones (FCs) takes place as FCs are

used in smaller amounts and during shorter time intervals (Wallmann et al., 2015).

Most antibiotics are excreted in great proportions of up to 90% with urine and feces (Sarmah et al.,

2006) with the potential to end up directly via grazing livestock or indirectly via slurry, digestate or

farmyard manure application on agricultural land and in water bodies. Water‐soluble antibiotics (e.g.

tetracyclines, sulphonamides and some ß‐lactam antibiotics) are excreted via the urine as the parent

compound (e.g. tetracyclines and ß‐lactam antibiotics) or metabolites (e.g. sulphonamides and

macrolides) while less soluble compounds such as macrolides and pleuromutilin derivatives are

excreted with feces (Halling‐Sørensen, 2000). A great proportion of 40 – 90% of the administered

antibiotics is excreted without prior metabolization (Thiele‐Bruhn, 2003; Kumar et al., 2005). While

being a valuable source of nutrients, organic substrates such as manure and sewage sludge may


5

pose a risk for contaminating farm sites with antibiotics and by this contribute to the development

of antibiotic resistant microorganisms in the soil.

The fate of antibiotics that enter the environment depend on different soil properties such as

organic matter content, soil texture, pH, cation speciation (Tolls, 2001; Zhou et al., 2010), the

contact time and sequestration in the soil (Förster at al., 2009) as well as the aboveground

vegetation (Chen et al., 2013; Migliore et al., 2010).

If antibiotics that enter the soil can unfold a negative impact on soil properties depend very much on

their concentration as well as on their bioaccessibility. Only compounds that are dissolved or can be

desorbed from the solid phase can affect microorganisms in that way, that unwanted effects on

microbial community functions (e.g. nitrification rates) or resistant gene formation are likely to

occur.

Common antibiotic concentrations in the soil are in the range of several µg/kg but also very high

concentrations of up to 1,44 mg/kg are reported in literature (Hamscher et al., 2002) which by far

exceed the recommended trigger value of 100 µg/kg soil (Guideline on environmental impact

assessment for veterinary medicinal products, 2007). When such high concentrations are reached

negative effects are very likely especially for compounds that are known to persist in soils, such as

tetracyclines, fluoroquinolones, and sulfonamides (Christian et al., 2003; Hamscher et al., 2002).

2.1. SorptionbehaviorofantibioticsinthesoilSorption‐desorption processes are regulating the concentration and thereby the bioavailability of

antibiotics in the soil solution (Singh et al., 2008). Antibiotics of different classes behave very diverse

in the soil according to their sorption characteristics. TCs are building strong complexes with divalent

metal cations and have a high affinity to bind to silanol groups. They adsorb strongly to the soil

matrix especially to clay minerals and to the organic matter (Stevens, 2009) and were classified as

immobile compounds. When they enter the soil they can be only found in the upper soil layers up to

40 cm soil depths. Moreover TCs are persistent and therefore they can accumulate in the soil profile

(Winckler and Grafe, 2000; Hamscher et al., 2005).

Fluoroquinolones are also bound strongly to the soil matrix and are rated as very immobile in the

soil with a low bioavailable fraction (Wetzstein, 2001).

Sulfonamides show a distinctly different behavior: they were leached down in the soil profile and

can be detected in the groundwater (Hamscher et al., 2005). Sorption of sulfonamids mainly takes

place on the organic matter and a high sequestration of sulfonamides is reported in literature

(Stevens, 2009). Nevertheless sulfonamides are classified as mobile antibiotics in the soil. The very

different sorption characteristics of the different classes of antibiotics become apparent when

comparing their adsorption coefficients (Kd‐values) on the soil matix. Kd values of 420‐1030 mL/g

were determined for oxytetracyclines, 260 – 5612 mL/g for enrofloxacin but only 0,6‐3,5 mL/g soil

for sulfadimidin (Gans et al., 2005).

Generally the soil structure is of major importance for the sorption of antibiotics. Fine‐textured soils

have a higher sorption capacity and the sorption of antibiotics increase with increasing clay content

(Scheffer and Schachtschabel, 2002). Therefore stronger antibiotic effects can be found on sandy

soils in comparison to loamy soils as a stronger adsorption led to a smaller and delayed antibiotic


6

effect (Thiele‐Bruhn and Beck, 2005). Some antibiotics were found to accumulate in soils when their

application rate via manure or other sources is higher than their degradation especially the immobile

antibiotics. Addition of organic matter to the soil such as manure application can significantly

influence the sorption‐desorption processes in soils (Thiele‐Bruhn and Aust, 2004). Especially for

sulfonamid sorption also the pH is of major relevance: it was shown that sulfamethazine (SMZ)

sorption decreased with increasing pH due to the ionisable character of the molecule (Vittoria Pinna

et al., 2012).

2.2. EffectofantibioticsonsoilmicroorganismsAcute or subacute toxicity of veterinary antibiotics on the soil macro‐ and mesofauna (earthworms,

springtails enchytraeids) are not very likely to occur because the concentrations that are found in

soils are much lower than the effect concentrations for those organisms (Höper et al., 2002; Thiele‐

Bruhn, 2003). This is different for soil microorganisms. Antibiotics are especially designed to have a

strong impact on microorganisms, making environmental effects very likely to occur. The

accumulation of antibiotics in the soil can affect microbial biomass as well as the functions and the

structure of the soil microbial communities for example the ratio between bacterial and fungal

biomass can be shifted. OTC application shifted the ratio in favor of the bacteria especially gram‐

negative soil bacteria (Chen et al., 2013). After sulfamethazine (SMZ) application a shift of the

culturable microbial populations toward a lower bacterial/fungal ration was reported by Vittoria

Pinna et al. (2012). A shift in the structure of the soil microbial community also affects the soil

enzyme activity and therefore the transformation properties of the soil. Antibiotics of all classes can

unfold a negative impact on soil microorganisms and reduce soil enzymatic activities.

The function of the microbial community is very often characterized by the activity of certain soil

enzymes. The most important soil enzyme activities, which are involved in C, N, P and S cycling are

the dehydrogenase (DHA), the urease (URE), the phosphatases (acid and alkaline) and the

arylsulfatase which are often determined to characterize the activity of the microbial soil

community. The DHA activity is representative for the oxidative activity of the soil microflora and is

responsible for the oxidation of organic compounds. The URE is closely related to the N cycle via the

hydrolysis of urea to ammonium. Phosphatases catalyse the hydrolysis of ester–phosphate bonds,

leading to the release of phosphates and arylsulfatase catalyze the hydrolysis of ester sulfates. All of

these enzymes play an integral role in the decomposition of complex organic molecules and they are

important for the cycling of nutrients in the soil

It was shown by different authors that the soil enzymes do not all behave in the same manner in

relation to antibiotics (Chen et al., 2013). In Table 1 the changes in enzyme activities in relation to

antibiotic application are summarized from literature. In most studies differences in relation to

antibiotics could only be determined after prior activation of the microbial growth by organic matter

application (Liu et al., 2014; Thiele‐Bruhn and Beck, 2005; Vittoria Pinna et al., 2012). Most tests

were conducted as incubation tests and the enzymatic activity was determined after different

incubation times. All of these studies have in common that the effect of organic matter application

had a stronger effect on the enzymatic activity than the application of antibiotics. Moreover the

negative effects of antibiotics on enzyme activity disappeared again with the time of incubation

(Chen et al., 2013; Vittoria Pinna et al., 2012).


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Table 1. Effect of graded antibiotic application on enzyme activity after activation with organic matter application.

Antibiotic Investigated concentrations [mg/kg soil]

Enzyme Change in enzyme activity Reference

OTC 0 – 1 – 15 – 200 DHA Decrease with increasing OTC Chen et al., 2013

ARS Decrease with increasing OTC

ALP Stimulated by 1 mg/kg OTC

URE Stimulated by 15 mg/kg OTC

CTC 0 – 10 ‐ 100 DHA Decrease with increasing CTC after 1, 6 and 12 days of incubation

Highest decrease after 1 day: 49.3% (10, ) + 74% (100)

Liu et al., 2014

ACP Concentration dependent decrease with CTC only after 12 days of

incubation

ALP No clear effect

URE Slight decrease at 100 mg CTC/kg after 1 and 45 days of incubation

SMZ 53.6 DHA Clear decrease in activity after 1 day of incubation (17‐41%) which disappeared after 1 week

Vittoria Pinna et al., 2012

URE Short‐term decrease without organic matter application which

disappeared after 1 week

SA mixture 0 – 0.9 – 9 – 90 ‐ 900 DHA Reduction of the activity but no dose‐response

Gutierrez et al., 2010

URE Dose‐response relationship after 163 h incubation

DHA –dehydrogenase; ARS‐arylsulfatase; ALP‐alkaline phosphatase; ACP‐ acid phosphatase; URE – urease; SA sulfonamide;

OTC oxytetracycline, CTC chlortetracycline, SMZ sulfamethazine

In different studies a reduction in the dehydrogenase (DHA) activity was found with tetracycline or

sulfonamide application. Urease activity was also reduced by higher antibiotic application, but Chen

et al. (2013) could demonstrate a stimulation of the urease activity at lower oxytetracyclin (OTC)

concentrations. For sulfonamides Gutierrez et al. (2010) could demonstrate that both the function

(enzyme activities) and structural diversity of the soil microbial community have been affected. To

obtain a clear effect on microbial activity it was always necessary to stimulate the growth by

application of a carbon source. Without activation Thiele‐Bruhn and Beck (2005) found no effect on

DHA activity and on basal respiration up to an OTC or sulfapyridin application rate of 1000 mg/kg

soil.

2.3. DevelopmentofantimicrobialresistanceThe application of antibiotics as feed additives in sub‐therapeutic doses (<100 mg/kg feed) bear the

risk of the development of multi‐resistant pathogenic organisms which are a serious threat for public

health (Richter et al., 1996). Moreover factors such as use of broad‐spectrum antibiotics, medication

of whole herds, prophylactic and metaphylactic antibiotic treatment (see Figure 1) increase the risk


8

for the development of antibiotic resistance in the environment (Stevens, 2009). Antibiotic residues

can provoke resistance either directly or indirectly via the transfer of plasmids (Wegener et al.,

1998). Once a resistance gene is present on a plasmid it can be spread to other bacteria quickly

(Gräfe, 1992). Though antibiotics used in human therapy were not allowed to be used as feed

additives, cross‐resistances can develop (Wegener et al., 1998). Some strains of human pathogens

such as Enterococcus faecalis are meanwhile resistant to more than 100 different drugs (Schmidt,

2002).

These antibiotic resistant bacteria can be spread into the environment when manure containing

such resistant bacteria is applied to land or they can be enriched when residual antibiotics in manure

or sewage wastes generated from households and hospitals provide an environment for the

selection of antibiotic resistant bacteria in soils (Chander et al., 2005). As a consequence of rising

problems with resistant or multi‐resistant bacteria in hospitals, the environmental risk of veterinary

medicinal products became a matter of increasing public scrutiny and legal requirements and several

antibiotics lost their permission as feed additives in the EU. Since 2006 antibiotic feed additives for

growth promotion have been banned in the EU (Hamscher and Mohring, 2012) but official statistics

for Germany show that the use of antibiotics in livestock production has further increased since then

because of higher livestock numbers and increasing prescriptions (Bloem and Kratz, 2016).

Figure 1. Factors that increase the risk of the development of antibiotic resistance (according to Stevens, 2009).

2.4. Evaluationoftherisktochangethefunctionandthesoilmicrobialcommunitybymanureapplication

Antibiotics show a high persistence in the soil and 100 days was estimated as the half life of

tetracyclines and fluoroquinolones while for sulfonamids a half life of 30 days was determined

(Halling‐Sørensen, 2001). In most experiments, where effects of antibiotics were observed on

microbial community as well as enzyme activities quite high concentrations of antibiotics were

tested (Chen et al., 2013). Antibiotic contaminations which were detected in soils are often in the

range of µg/kg after long‐term use of animal manures (Song et al., 2010). Much higher

concentrations were reported in the vicinity of feedlots were concentrations as high as 16.77 mg/kg

soil can be determined (Ji et al., 2012). Zhou et al. (2011) reported 1 mg OTC/kg soil to be a typical


9

concentration in ordinary agricultural operations in China and concentration between 119 – 307

mg/kg had been determined in soils irrigated with domestic waste water. In Austrian agricultural

soils CTC concentrations of in maximum 810 µg/kg were determined (Gans et al., 2005). Also for

enrofloxacin and its metabolite ciprofloxacin high concentrations were detected in soils (up to 0.2

and 0.37 mg/g soil, respectively) indicating to an enrichment of these persistent antibiotics in the

soils (Gans et al., 2005). Extreme contaminations of 1.435 mg/kg CTC were also found in a crust of

slurry which developed after dry weather (Hamscher et al., 2002). Therefore under certain

circumstances very high antibiotic contaminations can be found which can affect the soil microbial

community. Especially the persistent tetracyclines and fluoroquiniolones are critical contaminants as

they can enrich in the soil when regularly contaminated farm fertilizer were applied.

Soil test for microbial activity are not standardized. Therefore results are not always easy to

compare. For tetracyclines short‐term dose‐response relationships of toxicity on microorganisms

were obtained in a range of expected environmental concentrations (Thiele‐Bruhn, 2005; Schmitt et

al., 2006). Very diverse results were reported for different soils because of the differences in

sorption, pH, texture and organic matter content.

One major obstacle of organic matter activated soil tests is that very often materials for activation

are used such as manure which contains microorganisms itself. It is not clear from the results if the

original soil microorganisms were affected by the antibiotic application or the microorganisms that

are added via the organic amendment.

The risk for the development of antibiotic resistance in soil microorganisms is high, when antibiotics

were regularly applied to agricultural soils at low doses. Especially the very persistent antibiotics will

be available in low concentrations when they are regularly applied to the soil, which establish the

optimum conditions for the selection of resistance genes. As there is the serious danger that such

resistance is also transferred to humans, this can be assessed as the greatest danger antibiotics pose

in the environment.

3.ConclusionsIf negative effects of antibiotics on soil microbial activity can be determined depends very much on

soil characteristics, mainly the soil texture, organic matter content and soil pH and on characteristics

of the antibiotic by self. Nevertheless several studies underline that antibiotics can change the

structure of the soil microbial community (Thiele‐Bruhn and Beck, 2005; Vittoria Pinna et al., 2012)

and the microbial activity under certain circumstances. Veterinary antibiotics have the potential to

influence the soil microbial activity, the bacterial community function and they can cause a shift in

the ratio between bacteria and fungi (Liu et al., 2014; Thiele‐Bruhn and Beck, 2005). Moreover the

development of antibiotic resistance genes is a serious threat to human health. The high persistence

of several antibiotics is a further reason why it is so important to reduce the contamination of farm‐

derived fertilizers by antibiotics.

Today it is hardly possible to come up with consistent conclusions as not enough data are available

about the effects of antibiotics on soil life. The available trials differ in soil characteristics, incubation

times, activation of the trials and concentration range of the antibiotics and different aspects were

investigated to characterize the effects. But it can be concluded from the available data that also at


10

environmental relevant antibiotics concentrations effects on the soil microbial community can be

expected at least as a temporary effect.

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on effects of graded antibiotics on soil - MTT's Portal … 2.2. BONUS PROMISE 2 Phosphorus Recycling of Mixed Substances (BONUS PROMISE) DELIVERABLE 2.4 Report on effects of graded